Methods and compositions for using plasma cell depleting agents and / or b cell depleting agents to suppress host Anti-aav antibody response and enable aav transduction and re-dosing

Abstract

Provided herein are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, methods of treating an enzyme deficiency in a subject in need thereof, and methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof. Some methods, such as when a subject has preexisting against an immunogen to be administered, use plasma cell depleting agents or combinations comprising plasma cell depleting agents to mitigate immune response and facilitate redosing of nucleic acid constructs encoding a polypeptide of interest and nuclease agents targeting a target genomic locus to achieve, for example, a step-wise increase in expression of a polypeptide of interest in a subject following insertion of the nucleic acid construct without overshooting. Other methods, such as when a subject has no preexisting immunity against an immunogen to be administered, use B cell depleting agents (e.g., anti-CD20xCD3 antibody or functional fragment thereof) to mitigate immune response and facilitate redosing of nucleic acid constructs encoding a polypeptide of interest and nuclease agents targeting a target genomic locus to achieve, for example, a step-wise increase in expression of a polypeptide of interest in a subject following insertion of the nucleic acid construct without overshooting.

Description

Attorney Docket No.057766 / 624641 METHODS AND COMPOSITIONS FOR USING PLASMA CELL DEPLETING AGENTS AND / OR B CELL DEPLETING AGENTS TO SUPPRESS HOST ANTI-AAV ANTIBODY RESPONSE AND ENABLE AAV TRANSDUCTION AND RE-DOSING CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Application No.63 / 625,654, filed January 26, 2024, and US Application No.63 / 639,285, filed April 26, 2024, each of which is herein incorporated by reference in its entirety for all purposes. REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE

[0002] The Sequence Listing written in file 624641SEQLIST.xml is 1,816,797 bytes, was created on January 21, 2025, and is hereby incorporated by reference in its entirety. BACKGROUND

[0003] Adeno-associated virus (AAV)-based vectors hold tremendous promise to transform treatment of genetic diseases. Yet, the potential of AAV gene therapy has so far been limited by development of host antibodies (e.g., neutralizing antibodies (nAbs)) that block transduction or affect uptake on subsequent exposures. Clinically, the inability to re-dose AAVs presents challenges because efficacy cannot be restored if transgene expression is subtherapeutic or lost (e.g., due to cell division, silencing, or a cytotoxic immune response). Moreover, due to natural AAV exposure, many patients develop nAbs prior to treatment that render them ineligible for even a single dose. Therefore, strategies that prevent or attenuate anti-AAV nAb responses could vastly expand the utility and accessibility of existing AAV gene therapies, while safeguarding eligibility for future AAV-based advances.

[0004] Similarly, in AAV gene therapies, seronegative / naive patients can be dosed with AAV and develop antibody responses to the AAV capsid antigen. This antibody response prevents future re-dosing of AAV because the antibodies are neutralizing, and the antibody response is sustained for 10+ years..Attorney Docket No.057766 / 624641 SUMMARY

[0005] Provided herein are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, methods of treating an enzyme deficiency in a subject in need thereof, and methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof. Also provided are compositions, combination, or kits, e.g., for use in such methods.

[0006] In one aspect, provided are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In some such methods, provided are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus.

[0007] In another aspect, provided are methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the nuclease agent cleaves the nuclease targetAttorney Docket No.057766 / 624641 site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus. In some such methods, provided are methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus.

[0008] In another aspect, provided are methods of treating an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the polypeptide of interest comprises an enzyme to treat the enzyme deficiency; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the enzyme deficiency. In some such methods, provided are methods of treating an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the polypeptide of interest comprises an enzyme to treat the enzyme deficiency; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for theAttorney Docket No.057766 / 624641 nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the enzyme deficiency.

[0009] In another aspect, provided are methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the enzyme deficiency is characterized by a loss-of-function of the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the enzyme deficiency. In some such methods, provided are methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the enzyme deficiency is characterized by a loss-of-function of the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the enzyme deficiency.

[0010] In some such methods, the subject has a disease of a bleeding disorder characterized by the enzyme deficiency, a disease of an inborn error of metabolism characterized by theAttorney Docket No.057766 / 624641 enzyme deficiency, or a lysosomal storage disease characterized by the enzyme deficiency. Optionally, the disease is hemophilia B and the polypeptide of interest is a factor IX protein, the disease is hemophilia A and the polypeptide of interest is a factor VIII protein, or the disease is Pompe disease and the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase.

[0011] Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) the nuclease agent or the one or more nucleic acids encoding the nuclease agent; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject. Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject. Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject. Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a second coding sequence for the polypeptide of interest, wherein the second coding sequence is different from the first coding sequence; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets aAttorney Docket No.057766 / 624641 second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject. Some such methods comprise the following steps prior to the subsequent administration step: (i) measuring expression and / or activity of the polypeptide of interest in the subject; and (ii) determining the dose of the nucleic acid construct and the nuclease agent or the one or more nucleic acids encoding the nuclease agent for the subsequent administration step in order to achieve the desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

[0012] In some such methods, the polypeptide of interest is a factor IX protein, and the desired expression level of the factor IX protein in the subject is a serum level of at least about 3 ^g / mL or about 3-5 ^g / mL. In some such methods, the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase, and the desired expression level of the multidomain therapeutic protein in the subject is a serum level of at least about 2 ^g / mL or at least about 5 ^g / mL.

[0013] Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a coding sequence for a second polypeptide of interest that is different from the first polypeptide of interest; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the plasma cell depleting agent, wherein the second nuclease agent cleaves the second nuclease target site, and the second nucleic acid construct is inserted into the second target genomic locus.

[0014] In some such methods, the one or more subsequent administration steps is one subsequent administration step. In some such methods, the one or more subsequent administration steps is two subsequent administration steps or comprises at least two subsequentAttorney Docket No.057766 / 624641 administration steps. In some such methods, the plasma cell depleting agent is administered in the one or more subsequent administration steps if there is no preexisting plasma cell depleting agent in the subject or if preexisting plasma cell depleting agent expression and / or activity levels are below a desired threshold level. Optionally, the method comprises measuring the plasma cell depleting agent expression and / or activity levels prior to the one or more subsequent administration steps. In some such methods, the plasma cell depleting agent is capable of depleting long-lived plasma cells (LLPC).

[0015] In some such methods, the plasma cell depleting agent is a B cell maturation antigen (BCMA) targeting agent. In some such methods, the BCMA targeting agent is a chimeric antigen receptor against BCMA or an anti-BCMA antibody or a functional fragment thereof. In some such methods, the anti-BCMA antibody or functional fragment thereof is conjugated to a cytotoxic agent. In some such methods, the anti-BCMA antibody is a multispecific antibody or a functional fragment thereof. In some such methods, the multispecific anti-BCMA antibody or functional fragment thereof targets BCMA and CD3. In some such methods, the multispecific anti-BCMA antibody or functional fragment thereof is anti-BCMAxCD3 bispecific antibody or functional fragment thereof. In some such methods, the multispecific anti-BCMA antibody or functional fragment thereof is anti-BCMAxCD3 bispecific antibody or functional fragment thereof. In some such methods, the anti-BCMAxCD3 bispecific antibody is selected from linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ- 64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B.

[0016] In some such methods, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to BCMA comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In some such methods, the first antigen-binding domain that specifically binds to BCMA comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acidAttorney Docket No.057766 / 624641 sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0017] In some such methods, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 26 and 34, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In some such methods, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or 36, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 38, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 32 or 40, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0018] In some such methods, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 28, 30, and 32, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively. In some such methods, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 36, 38, and 40, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively. In some such methods, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or moreAttorney Docket No.057766 / 624641 modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc- gamma receptor (FcγR). In some such methods, the human IgG heavy chain constant region is isotype IgG4 or IgG1.

[0019] Some such methods further comprise administering to the subject an effective amount of a B cell depleting agent and / or an immunoglobulin depleting agent. Some such methods further comprise administering to the subject an effective amount of a B cell depleting agent and an immunoglobulin depleting agent. In some such methods, the B cell depleting agent is administered before, at the same time as, or after the plasma cell depleting agent. In some such methods, the B cell depleting agent is administered prior to and after the nucleic acid construct. In some such methods, the immunoglobulin depleting agent is administered prior to and after the nucleic acid construct. In some such methods, the immunoglobulin depleting agent is administered after the plasma cell depleting agent. In some embodiments, the immunoglobulin depleting agent is administered after an initial dose of the plasma cell depleting agent, or wherein the immunoglobulin depleting agent is administered after an initial dose of the plasma cell depleting agent and after an initial dose of the B cell depleting agent. In some such methods, the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA. In some such methods, the B cell depleting agent is an agent that binds to a B cell surface molecule. In some such methods, the B cell depleting agent is selected from an anti- CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD79 antibody, an anti- CD20xCD3 bispecific antibody, an anti-CD19xCD3 bispecific antibody, an anti-CD22xCD3 bispecific antibody, an anti-CD79xCD3 bispecific antibody, functional fragments of any of said antibodies, and any combinations thereof. In some such methods, the B cell depleting agent is selected from an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD19 antibody and an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD79 antibody, an anti-CD20xCD3 bispecific antibody, an anti-CD19xCD3 bispecific antibody, an anti-CD22xCD3 bispecific antibody, an anti-CD79xCD3 bispecific antibody, functional fragments of any of said antibodies, and any combinations thereof. In some such methods, the B cell depleting agent comprises an anti-CD20 antibody or a functional fragment thereof and an anti-CD19 antibody or a functional fragment thereof.

[0020] In some such methods, the B cell depleting agent is an anti-CD20 antibody or aAttorney Docket No.057766 / 624641 functional fragment thereof, wherein the anti-CD20 antibody is a multispecific antibody or a functional fragment thereof. In some such methods, the multispecific anti-CD20 antibody or functional fragment thereof targets CD20 and CD3. In some such methods, the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD20xCD3 bispecific antibody or functional fragment thereof.

[0021] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such methods, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0022] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such methods, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0023] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2,Attorney Docket No.057766 / 624641 and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively. In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc- gamma receptor (FcγR). In some such methods, the human IgG heavy chain constant region is isotype IgG4 or IgG1.

[0024] In some such methods, the B cell depleting agent is an agent targeting a B cell survival factor. In some such methods, the B cell depleting agent is a BLyS / BAFF inhibitor, an APRIL inhibitor, a BLyS receptor 3 / BAFF receptor inhibitor, or any combination thereof. In some such methods, the immunoglobulin depleting agent is capable of accelerating IgG clearance. In some such methods, the immunoglobulin depleting agent is a neonatal Fc receptor (FcRn) blocker. In some such methods, the FcRn blocker is selected from Efgartigimod (ARGX- 113), Rozanolixizumab (UCB7665), Batoclimab (RVT-1401), IMVT-1402, Nipocalimab (M281), Orilanolimab (SYNT001), and any combinations thereof.

[0025] Some such methods, further comprise plasmapheresis, therapeutic plasma exchange, or immunoadsorption. In some such methods, the plasma cell depleting agent is administered simultaneously with the nucleic acid construct. In some such methods, the plasma cell depleting agent is administered prior to the nucleic acid construct. In some such methods, the plasma cell depleting agent is administered prior to and after the nucleic acid construct. In some such methods, the plasma cell depleting agent is administered within about 6 months after the nucleic acid construct. Optionally, the nucleic acid construct is in a viral vector, and the plasma cell depleting agent is administered if the viral vector is still present in the subject. In some such methods, the nucleic acid construct is administered within about 3 months, within about 2 months, within about 7 weeks, within about 6 weeks, within about 5 weeks, within about 4 weeks, within about 3 weeks, or within about 2 weeks after an initial dose of the plasma cellAttorney Docket No.057766 / 624641 depleting agent, or the nucleic acid construct is administered at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 2 months, or at least about 3 months after an initial dose of the plasma cell depleting agent. In some such methods, the nucleic acid construct is administered about 2 weeks to about 7 weeks, about 3 weeks to about 6 weeks, or about 4 weeks to about 5 weeks after an initial dose of the plasma cell depleting agent. In some such methods, the plasma cell depleting agent is administered about 1 week prior to or within about 1 week prior to the nucleic acid construct. In some such methods, the nucleic acid construct is administered simultaneously with the nuclease agent or the one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered prior to or after the nuclease agent or the one or more nucleic acids encoding the nuclease agent.

[0026] In some such methods, the nucleic acid construct is in the nucleic acid vector. Optionally, the nucleic acid vector is a viral vector. Optionally, the viral vector is administered at a dose of about 3E11 vg / kg to about 5E13 vg / kg. In some such methods, the nucleic acid vector is an adeno-associated viral (AAV) vector. Optionally, the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 281. In some such methods, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such methods, the AAV vector is a recombinant AAV8 (rAAV8) vector.

[0027] In some such methods, the polypeptide of interest is a factor IX protein. In some such methods, the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO: 97. In some such methods, the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO: 61.

[0028] In some such methods, the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IXAttorney Docket No.057766 / 624641 protein coding sequence are different but encode the same factor IX protein sequence. In some such methods, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such methods, the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.

[0029] In some such methods, the nucleic acid construct is a unidirectional construct. In some such methods, the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

[0030] In some such methods, the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase. In some such methods, the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO: 296. In some such methods, the lysosomal alpha-glucosidase coding sequence comprises or consist of the sequence set forth in SEQ ID NO: 857. In some such methods, the delivery domain is a CD63-binding delivery domain. In some such methods, the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein. In some such methods, the CD63- binding delivery domain is a single-chain variable fragment (scFv). In some such methods, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 306. In some such methods, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 866. In some such methods, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 316. In some such methods, the coding sequence for the multidomainAttorney Docket No.057766 / 624641 therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884.

[0031] In some such methods, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0032] In some such methods, the delivery domain is a TfR-binding delivery domain. In some such methods, the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein. In some such methods, the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof). In some such methods, the anti-TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof). In some such methods, the anti-TfR antigen-binding protein comprises a HCVRAttorney Docket No.057766 / 624641 that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

[0033] In some such methods, the TfR-binding delivery domain comprises a single-chain variable fragment (scFv). In some such methods, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 672. In some such methods, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 713. In some such methods, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 691. In some such methods, the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871.

[0034] In some such methods, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0035] In some such methods, the polypeptide of interest is a factor VIII protein. In some such methods, the polypeptide of interest is an antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such methods, the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene. In some such methods, the nuclease target site is in intron 1 of the albumin gene.

[0036] In some such methods, the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or aAttorney Docket No.057766 / 624641 nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0037] In some such methods, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0038] In some such methods, the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164. In some such methods, the DNA-targeting segment consists of any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164. In some such methods, the guide RNA comprises any one of SEQ ID NOS: 185-248, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228. In some such methods, the DNA-targeting segment comprises or consists of SEQ ID NO: 159. In some such methods, the guide RNA comprises SEQ ID NO: 191 or 223. Some such methods comprise administering the guide RNA in the form of RNA.

[0039] In some such methods, the guide RNA comprises at least one modification. In some such methods, the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. Some such methods comprise administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.Attorney Docket No.057766 / 624641

[0040] In some such methods, the Cas protein is a Cas9 protein. Optionally, the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. In some such methods, the Cas protein comprises the sequence set forth in SEQ ID NO: 134. Some such methods comprise administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such methods, the mRNA encoding the Cas protein comprises at least one modification. In some such methods, the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine. In some such methods, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125. Some such methods comprise administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

[0041] Some such methods comprise administering the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125. Some such methods comprise administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

[0042] In some such methods, the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such methods, the lipid nanoparticle comprises a cationic lipid, aAttorney Docket No.057766 / 624641 neutral lipid, a helper lipid, and a stealth lipid. In some such methods, the cationic lipid is Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and / or the helper lipid is cholesterol, and / or the stealth lipid is 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000. In some such methods, the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such methods, the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0043] In some such methods, the cell is a liver cell or a hepatocyte, or the population of cells is a population of liver cells or hepatocytes. In some such methods, the subject is a human subject. In some such methods, the subject is a neonatal subject. In some such methods, the subject has preexisting AAV immunity. In some such methods, the nucleic acid vector is in an adeno-associated viral (AAV) vector, and the subject has preexisting AAV immunity. In some such methods, the method further comprises determining whether the subject has immunity against the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or the delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent prior to the administering. Optionally, the determining comprises determining the presence of neutralizing antibodies against the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or the delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent.

[0044] In another aspect, provided are compositions or combinations comprising an effective amount of a plasma cell depleting agent in combination with: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus.

[0045] In some such compositions or combinations, the plasma cell depleting agent is capable of depleting long-lived plasma cells (LLPC). In some such compositions or combinations, the plasma cell depleting agent is a B cell maturation antigen (BCMA) targetingAttorney Docket No.057766 / 624641 agent. In some such compositions or combinations, the BCMA targeting agent is a chimeric antigen receptor against BCMA or an anti-BCMA antibody or a functional fragment thereof. In some such compositions or combinations, the anti-BCMA antibody or functional fragment thereof is conjugated to a cytotoxic agent. In some such compositions or combinations, the anti- BCMA antibody is a multispecific antibody or a functional fragment thereof. In some such compositions or combinations, the multispecific anti-BCMA antibody or functional fragment thereof targets BCMA and CD3. In some such compositions or combinations, the multispecific anti-BCMA antibody or functional fragment thereof is anti-BCMAxCD3 bispecific antibody or functional fragment thereof. In some such compositions or combinations, the anti-BCMAxCD3 bispecific antibody is selected from linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B.

[0046] In some such compositions or combinations, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to BCMA comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In some such compositions or combinations, the first antigen-binding domain that specifically binds to BCMA comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0047] In some such compositions or combinations, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 26 and 34, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In some such compositions orAttorney Docket No.057766 / 624641 combinations, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or 36, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 38, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 32 or 40, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.

[0048] In some such compositions or combinations, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 28, 30, and 32, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively. In some such compositions or combinations, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 36, 38, and 40, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively. In some such compositions or combinations, the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc- gamma receptor (FcγR). In some such compositions or combinations, the human IgG heavy chain constant region is isotype IgG4 or IgG1.

[0049] In some such compositions or combinations, the plasma cell depleting agent is further in combination with an effective amount of a B cell depleting agent and / or an immunoglobulin depleting agent. In some such compositions or combinations, the plasma cell depleting agent isAttorney Docket No.057766 / 624641 further in combination with an effective amount of a B cell depleting agent and an immunoglobulin depleting agent. In some such compositions or combinations, the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA. In some such compositions or combinations, the B cell depleting agent is an agent that binds to a B cell surface molecule. In some such compositions or combinations, the B cell depleting agent is selected from an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD79 antibody, an anti-CD20xCD3 bispecific antibody, an anti-CD19xCD3 bispecific antibody, an anti-CD22xCD3 bispecific antibody, an anti-CD79xCD3 bispecific antibody, functional fragments of any of said antibodies, and any combinations thereof. In some such compositions or combinations, the B cell depleting agent is selected from an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD19 antibody and an anti-CD20 antibody, an anti- CD22 antibody, an anti-CD79 antibody, an anti-CD20xCD3 bispecific antibody, an anti- CD19xCD3 bispecific antibody, an anti-CD22xCD3 bispecific antibody, an anti-CD79xCD3 bispecific antibody, functional fragments of any of said antibodies, and any combinations thereof. In some such compositions or combinations, the B cell depleting agent comprises an anti-CD20 antibody or a functional fragment thereof and an anti-CD19 antibody or a functional fragment thereof. In some such compositions or combinations, the B cell depleting agent is an anti-CD20 antibody or a functional fragment thereof, wherein the anti-CD20 antibody is a multispecific antibody or a functional fragment thereof. In some such compositions or combinations, the multispecific anti-CD20 antibody or functional fragment thereof targets CD20 and CD3. In some such compositions or combinations, the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD20xCD3 bispecific antibody or functional fragment thereof.

[0050] In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such compositions or combinations, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2Attorney Docket No.057766 / 624641 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0051] In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such compositions or combinations, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0052] In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively. In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR). In some such compositions or combinations, the human IgG heavy chain constant region is isotype IgG4 or IgG1.

[0053] In some such compositions or combinations, the B cell depleting agent is an agentAttorney Docket No.057766 / 624641 targeting a B cell survival factor. In some such compositions or combinations, the B cell depleting agent is a BLyS / BAFF inhibitor, an APRIL inhibitor, a BLyS receptor 3 / BAFF receptor inhibitor, or any combination thereof. In some such compositions or combinations, the immunoglobulin depleting agent is capable of accelerating IgG clearance. In some such compositions or combinations, the immunoglobulin depleting agent is a neonatal Fc receptor (FcRn) blocker. In some such compositions or combinations, the FcRn blocker is selected from Efgartigimod (ARGX-113), Rozanolixizumab (UCB7665), Batoclimab (RVT-1401), IMVT- 1402, Nipocalimab (M281), Orilanolimab (SYNT001), and any combinations thereof.

[0054] In some such compositions or combinations, the nucleic acid construct is in the nucleic acid vector. Optionally, the nucleic acid vector is a viral vector. In some such compositions or combinations, the nucleic acid vector is an adeno-associated viral (AAV) vector. Optionally, the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 281. In some such compositions or combinations, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such compositions or combinations, the AAV vector is a recombinant AAV8 (rAAV8) vector.

[0055] In some such compositions or combinations, the polypeptide of interest is a factor IX protein. In some such compositions or combinations, the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO: 97. In some such compositions or combinations, the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or the factor IX protein coding sequence comprises or consists of SEQ ID NO: 61.

[0056] In some such compositions or combinations, the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence. In some such compositions or combinations, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a firstAttorney Docket No.057766 / 624641 polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions or combinations, the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.

[0057] In some such compositions or combinations, the nucleic acid construct is a unidirectional construct. In some such compositions or combinations, the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

[0058] In some such compositions or combinations, the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha- glucosidase. In some such compositions or combinations, the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO: 296. In some such compositions or combinations, the lysosomal alpha-glucosidase coding sequence comprises or consist of the sequence set forth in SEQ ID NO: 857. In some such compositions or combinations, the delivery domain is a CD63-binding delivery domain. In some such compositions or combinations, the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein. In some such compositions or combinations, the CD63-binding delivery domain is a single-chain variable fragment (scFv). In some such compositions or combinations, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 306. In some such compositions or combinations, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 866. In some such compositions or combinations, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 316. In some such compositions or combinations, theAttorney Docket No.057766 / 624641 coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884.

[0059] In some such compositions or combinations, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0060] In some such compositions or combinations, the delivery domain is a TfR-binding delivery domain. In some such compositions or combinations, the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein. In some such compositions or combinations, the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof). In some such compositions or combinations, the anti-TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof). In someAttorney Docket No.057766 / 624641 such compositions or combinations, the anti-TfR antigen-binding protein comprises a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

[0061] In some such compositions or combinations, the TfR-binding delivery domain comprises a single-chain variable fragment (scFv). In some such compositions or combinations, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 672. In some such compositions or combinations, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 713. In some such compositions or combinations, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 691. In some such compositions or combinations, the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871.

[0062] In some such compositions or combinations, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0063] In some such compositions or combinations, the polypeptide of interest is a factor VIII protein. In some such compositions or combinations, the polypeptide of interest is an antigen-binding protein, optionally wherein the antigen-binding protein is an antibody. In some such compositions or combinations, the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene. In some such compositions or combinations,Attorney Docket No.057766 / 624641 the nuclease target site is in intron 1 of the albumin gene.

[0064] In some such compositions or combinations, the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0065] In some such compositions or combinations, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0066] In some such compositions or combinations, the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164. In some such compositions or combinations, the DNA- targeting segment consists of any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164. In some such compositions or combinations, the guide RNA comprises any one of SEQ ID NOS: 185-248. Optionally, the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228. In some such compositions or combinations, the DNA-targeting segment comprises or consists of SEQ ID NO: 159. In some such compositions or combinations, the guide RNA comprises SEQ ID NO: 191 or 223. In some such compositions or combinations, the composition or combination comprises the guide RNA in the form of RNA.

[0067] In some such compositions or combinations, the guide RNA comprises at least one modification. In some such compositions or combinations, the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. In some such compositions or combinations, the composition or combination comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ IDAttorney Docket No.057766 / 624641 NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

[0068] In some such compositions or combinations, the Cas protein is a Cas9 protein. Optionally, the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. In some such compositions or combinations, the Cas protein comprises the sequence set forth in SEQ ID NO: 134. In some such compositions or combinations, the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such compositions or combinations, the mRNA encoding the Cas protein comprises at least one modification. In some such compositions or combinations, the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine. In some such compositions or combinations, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

[0069] In some such compositions or combinations, the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail. In some such compositions or combinations, the composition or combination comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and the composition or combination comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

[0070] In some such compositions or combinations, the composition or combination comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotidesAttorney Docket No.057766 / 624641 at the 3’ end of the guide RNA, wherein the composition or combination comprises the nucleic acid encoding the Cas protein, and wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl- pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

[0071] In some such compositions or combinations, the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such compositions or combinations, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such compositions or combinations, the cationic lipid is Lipid A ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or the neutral lipid is distearoylphosphatidylcholine or 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), and / or the helper lipid is cholesterol, and / or the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In some such compositions or combinations, the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such compositions or combinations, the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k- DMG.

[0072] Some such compositions or combinations are provided for use in a method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject.

[0073] Some such compositions or combinations are provided for use in a method of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject.

[0074] Some such compositions or combinations are provided for use in a method of treating an enzyme deficiency in a subject in need thereof.

[0075] Some such compositions or combinations are provided for use in a method of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof.

[0076] In another aspect, provided are kits comprising any such compositions orAttorney Docket No.057766 / 624641 combinations described herein.

[0077] In another aspect, provided are plasma cell depleting agents for use in any such methods described herein.

[0078] In another aspect, provided are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus. In some such methods, provided are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus.

[0079] In another aspect, provided are methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus. In some such methods, provided are methodsAttorney Docket No.057766 / 624641 of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus.

[0080] In another aspect, provided are methods of treating an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the polypeptide of interest comprises an enzyme to treat the enzyme deficiency; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the enzyme deficiency. In some such methods, provided are methods of treating an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the polypeptide of interest comprises an enzyme to treat the enzyme deficiency; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nucleaseAttorney Docket No.057766 / 624641 agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the enzyme deficiency.

[0081] In another aspect, provided are methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the enzyme deficiency is characterized by a loss-of-function of the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the enzyme deficiency. In some such methods, provided are methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the enzyme deficiency is characterized by a loss-of-function of the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the enzyme deficiency.

[0082] In some such methods, the subject has a disease of a bleeding disorder characterized by the enzyme deficiency, a disease of an inborn error of metabolism characterized by theAttorney Docket No.057766 / 624641 enzyme deficiency, or a lysosomal storage disease characterized by the enzyme deficiency. Optionally, the disease is hemophilia B and the polypeptide of interest is a factor IX protein, the disease is hemophilia A and the polypeptide of interest is a factor VIII protein, or the disease is Pompe disease and the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase.

[0083] Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) the nuclease agent or the one or more nucleic acids encoding the nuclease agent; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

[0084] Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

[0085] Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

[0086] Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a second coding sequence for the polypeptide of interest, wherein the second coding sequence is different from the first coding sequence; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets aAttorney Docket No.057766 / 624641 second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

[0087] Some such methods further comprise the following steps prior to the subsequent administration step: (i) measuring expression and / or activity of the polypeptide of interest in the subject; and (ii) determining the dose of the nucleic acid construct and the nuclease agent or the one or more nucleic acids encoding the nuclease agent for the subsequent administration step in order to achieve the desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

[0088] In some such methods, the polypeptide of interest is a factor IX protein, and the desired expression level of the factor IX protein in the subject is a serum level of at least about 3 ^g / mL or about 3-5 ^g / mL. In some such methods, the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase, and the desired expression level of the multidomain therapeutic protein in the subject is a serum level of at least about 2 ^g / mL or at least about 5 ^g / mL.

[0089] Some such methods further comprise a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a coding sequence for a second polypeptide of interest that is different from the first polypeptide of interest; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the secondAttorney Docket No.057766 / 624641 nuclease agent cleaves the second nuclease target site, and the second nucleic acid construct is inserted into the second target genomic locus.

[0090] In some such methods, the one or more subsequent administration steps is one subsequent administration step. In some such methods, the one or more subsequent administration steps is two subsequent administration steps or comprises at least two subsequent administration steps.

[0091] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered in the one or more subsequent administration steps if there is no preexisting anti-CD20xCD3 bispecific antibody or functional fragment thereof in the subject or if the preexisting the expression and / or activity levels of the anti-CD20xCD3 bispecific antibody or functional fragment thereof are below a desired threshold level. Optionally, the method comprises measuring the expression and / or activity levels of the anti-CD20xCD3 bispecific antibody or functional fragment thereof prior to the one or more subsequent administration steps.

[0092] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such methods, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0093] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such methods, theAttorney Docket No.057766 / 624641 second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0094] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[0095] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region. Optionally, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR). In some such methods, the human IgG heavy chain constant region is isotype IgG4 or IgG1.

[0096] In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the nucleic acid construct. In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered prior to the nucleic acid construct. In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered prior to and after the nucleic acid construct. In some such methods, the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered about 1 week prior to or within about 1 week prior to the nucleic acid construct. In some such methods, the nucleic acid construct is administered within about 3 months, within about 2 months, within about 7 weeks, within about 6 weeks, within about 5 weeks, within about 4 weeks, within about 3 weeks, within about 2 weeks, or within about 1 week after an initial dose of the anti-CD20xCD3 bispecific antibody or functional fragmentAttorney Docket No.057766 / 624641 thereof, or the nucleic acid construct is administered at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 2 months, or at least about 3 months after an initial dose of the anti-CD20xCD3 bispecific antibody or functional fragment thereof. In some such methods, the nucleic acid construct is administered simultaneously with the nuclease agent or the one or more nucleic acids encoding the nuclease agent. In some such methods, the nucleic acid construct is administered prior to or after the nuclease agent or the one or more nucleic acids encoding the nuclease agent.

[0097] In some such methods, the nucleic acid construct is in the nucleic acid vector. Optionally, the nucleic acid vector is a viral vector. Optionally, the viral vector is administered at a dose of about 3E11 vg / kg to about 5E13 vg / kg. In some such methods, the nucleic acid vector is an adeno-associated viral (AAV) vector. Optionally, the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 281. In some such methods, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such methods, the AAV vector is a recombinant AAV8 (rAAV8) vector.

[0098] In some such methods, the polypeptide of interest is a factor IX protein. In some such methods, the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO: 97. In some such methods, the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or the factor IX protein coding sequence comprises or consists of SEQ ID NO: 61.

[0099] In some such methods, the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence. In some such methods, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a first polyadenylation signal, a reverse complement of aAttorney Docket No.057766 / 624641 second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such methods, the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.

[0100] In some such methods, the nucleic acid construct is a unidirectional construct. In some such methods, the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

[0101] In some such methods, the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase. In some such methods, the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO: 296. In some such methods, the lysosomal alpha-glucosidase coding sequence comprises or consist of the sequence set forth in SEQ ID NO: 857.

[0102] In some such methods, the delivery domain is a CD63-binding delivery domain. In some such methods, the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein. In some such methods, the CD63-binding delivery domain is a single-chain variable fragment (scFv). In some such methods, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 306. In some such methods, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 866.

[0103] In some such methods, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 316. In some such methods, the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900Attorney Docket No.057766 / 624641 or 884. In some such methods, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal. Optionally, the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859. Optionally, the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0104] In some such methods, the delivery domain is a TfR-binding delivery domain. In some such methods, the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein. In some such methods, the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof). In some such methods, the anti-TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof). In some such methods, the anti-TfR antigen-binding protein comprises a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).Attorney Docket No.057766 / 624641

[0105] In some such methods, the TfR-binding delivery domain comprises a single-chain variable fragment (scFv). In some such methods, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 672. In some such methods, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 713.

[0106] In some such methods, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 691. In some such methods, the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871. In some such methods, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal. Optionally, the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859. Optionally, the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0107] In some such methods, the polypeptide of interest is a factor VIII protein.

[0108] In some such methods, the polypeptide of interest is an antigen-binding protein. Optionally, the antigen-binding protein is an antibody.

[0109] In some such methods, the target genomic locus is an albumin gene. Optionally, the albumin gene is a human albumin gene. In some such methods, the nuclease target site is in intron 1 of the albumin gene.

[0110] In some such methods, the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guideAttorney Docket No.057766 / 624641 RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0111] In some such methods, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such methods, the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164, or the DNA-targeting segment consists of any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164. In some such methods, the guide RNA comprises any one of SEQ ID NOS: 185-248. Optionally, the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228. In some such methods, the DNA-targeting segment comprises or consists of SEQ ID NO: 159. In some such methods, the guide RNA comprises SEQ ID NO: 191 or 223.

[0112] Some such methods comprise administering the guide RNA in the form of RNA. In some such methods, the guide RNA comprises at least one modification. In some such methods, the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. Some such methods comprise administering the guide RNA in the form of RNA, wherein the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

[0113] In some such methods, the Cas protein is a Cas9 protein. Optionally, the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. In some such methods, the Cas protein comprises the sequence set forth in SEQ ID NO: 134.Attorney Docket No.057766 / 624641

[0114] Some such methods comprise administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such methods, the mRNA encoding the Cas protein comprises at least one modification. In some such methods, the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine. In some such methods, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125. Some such methods comprise administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

[0115] Some such methods comprise administering the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and also comprise administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125. Some such methods comprise administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and also comprise administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

[0116] In some such methods, the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such methods, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such methods, the cationic lipid is Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or the neutralAttorney Docket No.057766 / 624641 lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and / or the helper lipid is cholesterol, and / or the stealth lipid is 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000. In some such methods, the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such methods, the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

[0117] In some such methods, the cell is a liver cell or a hepatocyte, or the population of cells is a population of liver cells or hepatocytes. In some such methods, the subject is a human subject. In some such methods, the subject is a neonatal subject. In some such methods, the nucleic acid vector is in an adeno-associated viral (AAV) vector, and the subject does not have preexisting AAV immunity.

[0118] In some such methods, the method does not comprise administering a plasma cell depleting agent. In some such methods, the nucleic acid vector is in an adeno-associated viral (AAV) vector, the subject does not have preexisting AAV immunity, and the method does not comprise administering a plasma cell depleting agent.

[0119] In another aspect, provided are compositions or combinations comprising an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof in combination with: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus.

[0120] In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such compositions or combinations, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50,Attorney Docket No.057766 / 624641 a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0121] In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In some such compositions or combinations, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0122] In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[0123] In some such compositions or combinations, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region. Optionally, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR). In some such compositions or combinations, the human IgG heavy chain constant region is isotype IgG4 or IgG1.

[0124] In some such compositions or combinations, the nucleic acid construct is in the nucleic acid vector. Optionally, the nucleic acid vector is a viral vector. In some suchAttorney Docket No.057766 / 624641 compositions or combinations, the nucleic acid vector is an adeno-associated viral (AAV) vector. Optionally, the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283. Optionally, the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281. Optionally, the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 281. In some such compositions or combinations, the AAV vector is a single-stranded AAV (ssAAV) vector. In some such compositions or combinations, the AAV vector is a recombinant AAV8 (rAAV8) vector.

[0125] In some such compositions or combinations, the polypeptide of interest is a factor IX protein. In some such compositions or combinations, the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO: 97. In some such compositions or combinations, the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or the factor IX protein coding sequence comprises or consists of SEQ ID NO: 61.

[0126] In some such compositions or combinations, the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence. In some such compositions or combinations, the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms. In some such compositions or combinations, the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.Attorney Docket No.057766 / 624641

[0127] In some such compositions or combinations, the nucleic acid construct is a unidirectional construct. In some such compositions or combinations, the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

[0128] In some such compositions or combinations, the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha- glucosidase. In some such compositions or combinations, the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO: 296. In some such compositions or combinations, the lysosomal alpha-glucosidase coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 857.

[0129] In some such compositions or combinations, the delivery domain is a CD63-binding delivery domain. In some such compositions or combinations, the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein. In some such compositions or combinations, the CD63-binding delivery domain is a single-chain variable fragment (scFv). In some such compositions or combinations, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 306. In some such compositions or combinations, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 866.

[0130] In some such compositions or combinations, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 316. In some such compositions or combinations, the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884. In some such compositions or combinations, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGHAttorney Docket No.057766 / 624641 polyadenylation signal and a unidirectional SV40 late polyadenylation signal. Optionally, the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859. Optionally, the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0131] In some such compositions or combinations, the delivery domain is a TfR-binding delivery domain. In some such compositions or combinations, the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein. In some such compositions or combinations, the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof). In some such compositions or combinations, the anti-TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof). In some such compositions or combinations, the anti-TfR antigen-binding protein comprises a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

[0132] In some such compositions or combinations, the TfR-binding delivery domain comprises a single-chain variable fragment (scFv). In some such compositions or combinations, the scFv comprises or consists of the sequence set forth in SEQ ID NO: 672. In some suchAttorney Docket No.057766 / 624641 compositions or combinations, the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 713.

[0133] In some such compositions or combinations, the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 691. In some such compositions or combinations, the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871. In some such compositions or combinations, the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852. Optionally, the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal. Optionally, the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859. Optionally, the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

[0134] In some such compositions or combinations, the polypeptide of interest is a factor VIII protein.

[0135] In some such compositions or combinations, the polypeptide of interest is an antigen- binding protein. Optionally, the antigen-binding protein is an antibody.

[0136] In some such compositions or combinations, the target genomic locus is an albumin gene. Optionally, the albumin gene is a human albumin gene. In some such compositions or combinations, the nuclease target site is in intron 1 of the albumin gene.

[0137] In some such compositions or combinations, the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segmentAttorney Docket No.057766 / 624641 that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

[0138] In some such compositions or combinations, the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence. In some such compositions or combinations, the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164, or the DNA-targeting segment consists of any one of SEQ ID NOS: 153-184. Optionally, the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164. In some such compositions or combinations, the guide RNA comprises any one of SEQ ID NOS: 185-248. Optionally, the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228. In some such compositions or combinations, the DNA-targeting segment comprises or consists of SEQ ID NO: 159. In some such compositions or combinations, the guide RNA comprises SEQ ID NO: 191 or 223.

[0139] Some such compositions or combinations comprise the guide RNA in the form of RNA. In some such compositions or combinations, the guide RNA comprises at least one modification. In some such compositions or combinations, the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA. Some such compositions or combinations comprise the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.Attorney Docket No.057766 / 624641

[0140] In some such compositions or combinations, the Cas protein is a Cas9 protein. Optionally, the Cas protein is derived from a Streptococcus pyogenes Cas9 protein. In some such compositions or combinations, the Cas protein comprises the sequence set forth in SEQ ID NO: 134.

[0141] Some such compositions or combinations comprise the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein. In some such compositions or combinations, the mRNA encoding the Cas protein comprises at least one modification. In some such compositions or combinations, the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine. In some such compositions or combinations, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125. Some such compositions or combinations comprise the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

[0142] Some such compositions or combinations comprise the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and the composition or combination comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125. Some such compositions or combinations comprise the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.Attorney Docket No.057766 / 624641

[0143] In some such compositions or combinations, the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle. In some such compositions or combinations, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid. In some such compositions or combinations, the cationic lipid is Lipid A ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or the neutral lipid is distearoylphosphatidylcholine or 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), and / or the helper lipid is cholesterol, and / or the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In some such compositions or combinations, the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG. In some such compositions or combinations, the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k- DMG.

[0144] In some such compositions or combinations, the composition or combination does not comprise a plasma cell depleting agent.

[0145] In another aspect, the compositions or combinations described herein are provided for use in a method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject.

[0146] In another aspect, the compositions or combinations described herein are provided for use in a method of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject.

[0147] In another aspect, the compositions or combinations described herein are provided for use in a method of treating an enzyme deficiency in a subject in need thereof.

[0148] In another aspect, the compositions or combinations described herein are provided for use in a method of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof.

[0149] In another aspect, provided are kits comprising the compositions or combinations described herein.

[0150] In another aspect, provided is an anti-CD20xCD3 bispecific antibody or functional fragment thereof for use in the methods described herein.Attorney Docket No.057766 / 624641 BRIEF DESCRIPTION OF THE FIGURES

[0151] Figure 1 shows an experimental timeline for the study described in Examples 1, 2, 3, and 11.

[0152] Figure 2 shows the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 and anti-CD20 antibodies (anti-CD19 / CD20 antibodies), or combination thereof, on anti-AAV8 capsid IgG titers over time in mice previously treated with recombinant AAV8 vector.

[0153] Figure 3 shows the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 / CD20 antibodies, or combination thereof, on liver transduction 10 days following administration of a second AAV8 vector in mice previously treated with recombinant AAV8 vector, as measured by Taqman quantitative real-time polymerase chain reaction (PCR) of green fluorescent protein (GFP) transgene DNA.

[0154] Figure 4 shows the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 / CD20 antibodies, or combination thereof, on liver transduction 10 days following administration of a second recombinant AAV8 vector in mice previously treated with a first recombinant AAV8 vector, as measured by Taqman quantitative real-time reverse-transcription PCR of GFP transgene RNA.

[0155] Figures 5A-5B show the effect of plasma cell depletion with anti-BCMAxCD3 bispecific antibody, FcRn blockade via efgartigimod alfa, B cell depletion with anti-CD19 / CD20 antibodies, or combination thereof, on liver transduction 10 days following administration of a second recombinant AAV8 vector in mice previously treated with a first recombinant AAV8 vector, as measured by GFP immunohistochemical (IHC) staining of formalin-fixed paraffin embedded liver sections. Figure 5A shows GFP-positive area quantified using HALO software (Indica labs). Figure 5B shows representative images.

[0156] Figures 6A-6J show flow cytometry analysis of B cell and plasma cell frequencies and counts in bone marrow and spleen following treatment with anti-BCMAxCD3 bispecific antibody, FcRn blockade, anti-CD19 / CD20 antibodies, or combinations thereof. Figure 6A shows bone marrow plasma cell frequencies, Figure 6B shows spleen plasma cell frequencies,Attorney Docket No.057766 / 624641 Figure 6C shows spleen naïve B cell frequencies, Figure 6D shows spleen total memory B cell frequencies, Figure 6E shows spleen AAV-specific memory B cell frequencies, Figure 6F shows bone marrow plasma cell counts, Figure 6G shows spleen plasma cell counts, Figure 6H shows spleen naïve B cell counts, Figure 6I shows spleen total memory B cell counts, and Figure 6J shows spleen AAV-specific memory B cell counts.

[0157] Figure 7 shows the effect of efgartigimod on serum drug concentration of REGN5458 (BCMAxCD3).

[0158] Figure 8 shows an experimental timeline for the study described in Example 12.

[0159] Figures 9A-9B show the effect of plasma cell depletion, B cell depletion, neonatal Fc receptor blockade, and combinations thereof, on naturally-occurring anti-AAV antibody titers in cynomolgus macaques. AAV8 neutralizing antibody (NAb) titer levels are presented for each treatment group over the duration of the study (Figure 9A) and specifically at Study Day 29 (Figure 9B).

[0160] Figure 10 shows an experimental timeline for the study described in Examples 13 and 14.

[0161] Figures 11A-11C show a comparison of the effect of CD20xCD3-mediated versus anti-CD20-mediated B cell depletion on the development of anti-AAV IgM antibody titers (Figure 11A) and anti-AAV IgG antibody titers (Figures 11B-11C) in mice.

[0162] Figures 12A-12C show a comparison of the effect of CD20xCD3-mediated versus anti-CD20-mediated B cell depletion on AAV transduction (Figure 12A) and transgene expression (Figures 12B-12C) following vector re-administration in mice.

[0163] Figure 13 shows an experimental timeline for the study described in Examples 15 and 16.

[0164] Figures 14A-14F show the effect of prophylactic CD20xCD3-mediated B cell depletion on serum anti-AAV8 IgM (Figure 14A and Figure 14D), IgG (Figure 14B and Figure 14E), and neutralizing antibody (nAb) (Figure 14C and Figure 14F) titers in cynomolgus macaques.

[0165] Figures 15A-15C show the effect of prophylactic CD20xCD3-mediated B cell depletion on AAV transduction (Figure 15A) and transgene expression (Figures 15B-15C) following AAV vector re-administration in cynomolgus macaques.Attorney Docket No.057766 / 624641 DEFINITIONS

[0166] The terms “protein,” “polypeptide,” and “peptide,” used interchangeably herein, include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term “domain” refers to any part of a protein or polypeptide having a particular function or structure.

[0167] The terms “nucleic acid” and “polynucleotide,” used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. They include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0168] The term “expression vector” or “expression construct” or “expression cassette” refers to a recombinant nucleic acid containing a desired coding sequence operably linked to appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell or organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, as well as other sequences. Eukaryotic cells are generally known to utilize promoters, enhancers, and termination and polyadenylation signals, although some elements may be deleted and other elements added without sacrificing the necessary expression.

[0169] The term “viral vector” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and / or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either ex vivo or in vivo. Numerous forms of viral vectors are known.

[0170] The term “isolated” with respect to proteins, nucleic acids, and cells includes proteins, nucleic acids, and cells that are relatively purified with respect to other cellular or organism components that may normally be present in situ, up to and including a substantially pure preparation of the protein, nucleic acid, or cell. The term “isolated” may include proteins and nucleic acids that have no naturally occurring counterpart or proteins or nucleic acids thatAttorney Docket No.057766 / 624641 have been chemically synthesized and are thus substantially uncontaminated by other proteins or nucleic acids. The term “isolated” may include proteins, nucleic acids, or cells that have been separated or purified from most other cellular components or organism components with which they are naturally accompanied (e.g., but not limited to, other cellular proteins, nucleic acids, or cellular or extracellular components).

[0171] The term “wild type” or “wild-type” includes entities having a structure and / or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles).

[0172] The term “endogenous sequence” refers to a nucleic acid sequence that occurs naturally within a cell or animal. For example, an endogenous ALB sequence of an animal refers to a native ALB sequence that naturally occurs at the ALB locus in the animal.

[0173] “Exogenous” molecules or sequences include molecules or sequences that are not normally present in a cell in that form or that are introduced into a cell from an outside source. Normal presence includes presence with respect to the particular developmental stage and environmental conditions of the cell. An exogenous molecule or sequence, for example, can include a mutated version of a corresponding endogenous sequence within the cell, such as a humanized version of the endogenous sequence, or can include a sequence corresponding to an endogenous sequence within the cell but in a different form (i.e., not within a chromosome). In contrast, endogenous molecules or sequences include molecules or sequences that are normally present in that form in a particular cell at a particular developmental stage under particular environmental conditions.

[0174] The term “heterologous” when used in the context of a nucleic acid or a protein indicates that the nucleic acid or protein comprises at least two segments that do not naturally occur together in the same molecule. For example, the term “heterologous,” when used with reference to segments of a nucleic acid or segments of a protein, indicates that the nucleic acid or protein comprises two or more sub-sequences that are not found in the same relationship to each other (e.g., joined together) in nature. As one example, a “heterologous” region of a nucleic acid vector is a segment of nucleic acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid vector could include a coding sequence flanked by a heterologous promoter not found in association with the coding sequence in nature. Likewise, a “heterologous” region of aAttorney Docket No.057766 / 624641 protein is a segment of amino acids within or attached to another peptide molecule that is not found in association with the other peptide molecule in nature (e.g., a fusion protein, or a protein with a tag). Similarly, a nucleic acid or protein can comprise a heterologous label or a heterologous secretion or localization sequence.

[0175] “Codon optimization” takes advantage of the degeneracy of codons, as exhibited by the multiplicity of three-base pair codon combinations that specify an amino acid, and generally includes a process of modifying a nucleic acid sequence for enhanced expression in particular host cells by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the genes of the host cell while maintaining the native amino acid sequence. For example, a nucleic acid encoding a protein can be modified to substitute codons having a higher frequency of usage in a given prokaryotic or eukaryotic cell, including a bacterial cell, a yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, a hamster cell, or any other host cell, as compared to the naturally occurring nucleic acid sequence. Codon usage tables are readily available, for example, at the “Codon Usage Database.” These tables can be adapted in a number of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, herein incorporated by reference in its entirety for all purposes. Computer algorithms for codon optimization of a particular sequence for expression in a particular host are also available (see, e.g., Gene Forge).

[0176] A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013 / 176772, herein incorporated by reference in its entirety for all purposes.

[0177] A constitutive promoter is one that is active in all tissues or particular tissues at allAttorney Docket No.057766 / 624641 developing stages. Examples of constitutive promoters include the human cytomegalovirus immediate early (hCMV), mouse cytomegalovirus immediate early (mCMV), human elongation factor 1 alpha (hEF1α), mouse elongation factor 1 alpha (mEF1α), mouse phosphoglycerate kinase (PGK), chicken beta actin hybrid (CAG or CBh), SV40 early, and beta 2 tubulin promoters..

[0178] Examples of inducible promoters include, for example, chemically regulated promoters and physically-regulated promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline (tet)-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid-regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal-regulated promoters (e.g., a metalloprotein promoter). Physically regulated promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter or a light-repressible promoter).

[0179] Tissue-specific promoters can be, for example, neuron-specific promoters or glial- specific promoters or muscle-specific promoters or liver-specific promoters.

[0180] Developmentally regulated promoters include, for example, promoters active only during an embryonic stage of development, or only in an adult cell.

[0181] “Operable linkage” or being “operably linked” includes juxtaposition of two or more components (e.g., a promoter and another sequence element) such that both components function normally and allow the possibility that at least one of the components can mediate a function that is exerted upon at least one of the other components. For example, a promoter can be operably linked to a coding sequence if the promoter controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence).

[0182] The term “in vitro” includes artificial environments and to processes or reactions that occur within an artificial environment (e.g., a test tube or an isolated cell or cell line). The term “in vivo” includes natural environments (e.g., a cell, organism, or body) and to processes or reactions that occur within a natural environment. The term “ex vivo” includes cells that haveAttorney Docket No.057766 / 624641 been removed from the body of an individual and processes or reactions that occur within such cells..

[0183] The term “antigen-binding molecule” includes antibodies and antigen-binding fragments of antibodies, including multispecific antibodies, e.g., bispecific antibodies.

[0184] The term “antibody,” as used herein, refers to an antigen-binding molecule or molecular complex comprising a set of complementarity determining regions (CDRs) that specifically bind to or interact with a particular antigen (e.g., BCMA, CD20, CD3). The term “antibody,” as used herein, includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). In a typical antibody, each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody (or antigen-binding portion thereof) may be identical to the human germline sequences, or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs.

[0185] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition (enhanced Chothia or Martin), the IMGT definition, and the Honneger definition (AHo). In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of the structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda,Attorney Docket No.057766 / 624641 Md. (1991); Chothia et al., J Mol Biol (1987), 4:901–17; Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989); see also, Dondelinger et al., Front. Immunol. (2018), 9:2278, doi:10.3389 / fimmu.2018.02278. Public databases are also available for identifying CDR sequences within an antibody.

[0186] The term “antibody,” as used herein, also includes antigen-binding fragments of full antibody molecules. The terms “antigen-binding portion” of an antibody, “antigen-binding fragment” of an antibody, “antigen-binding domain,” and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add, or delete amino acids, etc.

[0187] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed within the expression "antigen-binding fragment," as used herein.

[0188] An antigen-binding fragment of an antibody will typically comprise at least one variable domain. The variable domain may be of any size or amino acid composition and will generally comprise at least one CDR which is adjacent to or in frame with one or moreAttorney Docket No.057766 / 624641 framework sequences. In antigen-binding fragments having a VH domain associated with a VL domain, the VH and VL domains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VLor VL- VL dimers. Alternatively, the antigen-binding fragment of an antibody may contain a monomeric VH or VL domain.

[0189] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting, exemplary configurations of variable and constant domains that may be found within an antigen- binding fragment of an antibody include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1- CH2; (v) VH-CH1-CH2-CH3; (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2; (x) VL- CH3; (xi) VL-CH1-CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, an antigen-binding fragment of an antibody may comprise a homo-dimer or hetero- dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VHor VLdomain (e.g., by disulfide bond(s)).

[0190] The term “antibody,” as used herein, also includes multispecific (e.g., bispecific) antibodies. A multispecific antibody or antigen-binding fragment of an antibody will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen.

[0191] Any multispecific antibody format may be adapted for use in the context of an antibody or antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art. For example, the present disclosure includes bispecific antibodies wherein one arm of an immunoglobulin is specific for an epitope of BCMA or CD20 and the other arm of the immunoglobulin is specific for an epitope of CD3. Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv- based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig,Attorney Docket No.057766 / 624641 Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2bispecific formats (see, e.g., Klein et al.2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency, and geometry. See, e.g., Kazane et al., J. Am. Chem. Soc. (Epub: Dec.4, 2012).

[0192] The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may nonetheless include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site- specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3. However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0193] The term “recombinant antibody,” as used herein, is intended to include all antibodies that are prepared, expressed, created, or isolated by recombinant means. The term includes, but is not limited to, antibodies expressed using a recombinant expression vector transfected into a host cell (e.g., Chinese hamster ovary (CHO) cell) or cellular expression system, antibodies isolated from a recombinant, combinatorial human antibody library, and antibodies isolated from a non- human animal (e.g., a mouse, such as a mouse that is transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res.20:6287-6295). In some embodiments, the recombinant antibody is a recombinant human antibody. In some embodiments, recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derived from and related to human germline VHand VLsequences, may not naturally exist within the human antibody germline repertoire in vivo.Attorney Docket No.057766 / 624641

[0194] An “isolated antibody” refers to an antibody that has been identified and separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which the antibody naturally exists or is naturally produced, is an “isolated antibody.” An isolated antibody also includes an antibody in situ within a recombinant cell. Isolated antibodies are antibodies that have been subjected to at least one purification or isolation step. According to certain embodiments, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0195] The term “specifically binds,” or the like, means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiologic conditions. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10-6M or less, e.g., 10-7M, 10-8M, 10-9M, 10-10M, 10-11M, or 10-12M (a smaller KDdenotes a tighter binding). Methods for determining whether an antibody specifically binds to an antigen are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., BIACORE™), bio-layer interferometry assay (e.g., Octet® HTX biosensor), solution-affinity ELISA, and the like. In some embodiments, specific binding is measured in a surface plasmon resonance assay, e.g., at 25°C or 37°C. An antibody or antigen-binding fragment that specifically binds an antigen from one species may or may not have cross- reactivity to other antigens, such as an orthologous antigen from another species.

[0196] The term “KD,” as used herein, refers to the equilibrium dissociation constant of a particular antibody-antigen interaction.

[0197] The term “surface plasmon resonance,” as used herein, refers to an optical phenomenon that allows for the analysis of real-time biomolecular interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIACORE™ system (Cytiva, Marlborough, MA).

[0198] The term “epitope,” as used herein, refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be either linear or discontinuous (e.g.,Attorney Docket No.057766 / 624641 conformational). A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. Epitopes may also be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. An epitope typically includes at least 3, and more usually, e.g., at least 5 or at least 8-10 amino acids in a unique spatial conformation.

[0199] Methods for determining the epitope of an antigen-binding protein, e.g., an antibody or antigen-binding fragment, include alanine scanning mutational analysis, peptide blot analysis (Reineke, Methods Mol Biol 2004, 248:443-463), peptide cleavage analysis, crystallographic studies, and nuclear magnetic resonance (NMR) analysis. In addition, methods such as epitope exclusion, epitope extraction, and chemical modification of antigens can be employed (Tomer, Prot Sci 2000, 9:487-496). Another method that can be used to identify the amino acids within a polypeptide with which an antigen-binding protein (e.g., an antibody or antigen-binding fragment) interacts is hydrogen / deuterium exchange detected by mass spectrometry (HDX). See, e.g., Ehring, Analytical Biochemistry 1999, 267:252-259; Engen and Smith, Anal Chem 2001, 73:256A-265A.

[0200] The term “competes,” as used in reference to competing for binding, refers to an antigen-binding protein (e.g., antibody or antigen-binding fragment) that binds to an antigen and inhibits or blocks the binding of another antigen-binding protein (e.g., antibody or antigen- binding fragment) to the antigen. Unless otherwise stated, the term also includes competition between two antigen-binding proteins (e.g., antibodies) in both orientations, i.e., a first antigen that binds an antigen and blocks binding of the antigen by a second antibody, and vice versa. Thus, in some embodiments, competition occurs in one such orientation. In some embodiments, the first antigen-binding protein (e.g., antibody) and second antigen-binding protein (e.g., antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different epitopes, which may be overlapping or non- overlapping, wherein binding of one antigen-binding protein inhibits or blocks the binding of theAttorney Docket No.057766 / 624641 second antigen-binding protein, e.g., via steric hindrance. Competition between antigen-binding proteins may be measured by methods known in the art, e.g., by a real-time, label-free bio-layer interferometry assay.

[0201] In the context of the present disclosure, the term “neutralizing antibody” or “nAb” refers to an antibody that binds to a pathogen (e.g., a virus) and interferes with its ability to infect a cell. Non-limiting examples of neutralizing antibodies include antibodies that bind to a viral particle and inhibit successful transduction, e.g., one or more steps selected from binding, entry, trafficking to the nucleus, and transcription of the viral genome. Some neutralizing antibodies may block a virus at the post-entry step.

[0202] The term “immune response” refers to a response of a cell of the immune system (e.g., a B-cell, T-cell, macrophage or polymorphonucleocyte) to a stimulus such as an immunogen, e.g., antigen (e.g., a viral antigen). Active immune responses can involve differentiation and proliferation of immunocompetent cells, which leads to synthesis of antibodies or the development of cell-mediated reactivity, or both. An active immune response can be mounted by the host after exposure to an antigen (e.g., by infection or by vaccination). Active immune response can be contrasted with passive immunity, which can be acquired through the transfer of substances such as, e.g., an antibody, transfer factor, thymic graft, and / or cytokines from an actively immunized host to a non-immune host.

[0203] The term “T cell” is used herein in its broadest sense to refer to all types of immune cells expressing CD3, including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), T- regulatory cells (Treg), and natural killer (NK)-T cells.

[0204] The terms “substantial identity” and “substantially identical,” as used with reference to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.Attorney Docket No.057766 / 624641

[0205] As applied to polypeptides, the terms “substantial identity” and “"substantially identical” mean that two peptide sequences, when optimally aligned, share at least about 90% sequence identity, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, residue positions that are not identical differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein.

[0206] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 2000 supra). Another preferred algorithm when comparing a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al., 1990, J. Mol. Biol.215: 403-410 and 1997 Nucleic Acids Res.25:3389-3402.such an immunoglobulin, VH, VL, heavy chain, light chain, or CDR comprising an amino acid sequence specifically set forth herein, refers to a polypeptide comprising an amino acid sequence that is at least about 70%-99.9% (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, or 99.9%) identical to the reference polypeptide sequence (e.g., as set forth in the sequence listing below), when the comparison is performed by a BLAST algorithm wherein the parameters of the algorithm are selected to give the largest match between the respective sequences over the entire length of the respective reference sequences. In some embodiments, a variant of a polypeptide includes a polypeptideAttorney Docket No.057766 / 624641 having the amino acid sequence of a reference polypeptide sequence (e.g., as set forth in the sequence listing below) but for one or more (e.g., 1 to 10, or less than 20, or less than 10) missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions.

[0208] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode of administration, and the like.

[0209] The phrase “pharmaceutically acceptable,” as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0210] The terms “treat” or “treatment” of a state, disorder or condition include: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder or condition developing in a subject that may be afflicted with or predisposed to the state, disorder or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0211] An “individual” or “subject” or “animal” refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of diseases (e.g., mice, rats). In a preferred embodiment, the subject is a human.Attorney Docket No.057766 / 624641

[0212] Compositions or methods “comprising” or “including” one or more recited elements may include other elements not specifically recited. For example, a composition that “comprises” or “includes” a protein may contain the protein alone or in combination with other ingredients. The transitional phrase “consisting essentially of” means that the scope of a claim is to be interpreted to encompass the specified elements recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of” when used in a claim of this invention is not intended to be interpreted to be equivalent to “comprising.”

[0213] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which the event or circumstance does not.

[0214] Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0215] At least 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing an upper limit even if one is not specifically provided as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is not specifically provided. When “at least,” “up to,” or other similar language modifies a number, it can be understood to modify each number in the series.

[0216] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0217] As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition) that the value is limited by the method of detection. For example, 100% inhibition is understood as inhibition to a level below the level of detection of the assay.Attorney Docket No.057766 / 624641

[0218] Unless otherwise apparent from the context, the term “about” encompasses values ± 5% of a stated value. In certain embodiments, the term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement, or a percent of a value as tolerated in the art, e.g., with age. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0219] The term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0220] The term “or” refers to any one member of a particular list and also includes any combination of members of that list.

[0221] The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof.

[0222] Statistically significant means p ≤0.05.

[0223] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates. DETAILED DESCRIPTION I. Overview

[0224] Provided herein are methods of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, methods of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, methods of treating an enzyme deficiency (e.g., FIX deficiency or GAA deficiency) in a subject in need thereof, and methods of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof.

[0225] Gene transfer technologies generally rely on one or more protein components for the encapsulation, transport, and / or transmission of genetic material. Frequently, these components contain antigenic or immunogenic regions that upon transfer into a living organism can beAttorney Docket No.057766 / 624641 recognized by the host immune system, leading to immune responses that can impact the initial or long-term effectiveness of gene transfer.

[0226] For adeno-associated virus (AAV)-based vectors, which consist of a single-stranded DNA genome packaged in a protein capsid, presence of pre-existing antibodies against the capsid can lead to significantly reduced efficiency of transduction. These antibody responses develop following exposure to either wild type or recombinant AAVs, are usually neutralizing at relatively low titers, and can persist for at least a decade (>10 years), especially following exposure to the high doses required for existing AAV gene therapy products.

[0227] Thus, the development and persistence of high-titer neutralizing antibody responses following AAV exposure means that most systemic AAV-based gene therapies are expected to be a once per lifetime treatment, regardless of treatment outcome. This once-per-lifetime treatment paradigm poses several challenges in the clinic. Strategies to mitigate antibody responses to AAV therefore have the potential to greatly benefit patients due to both improved effectiveness and durability of treatment, as well as expanded access to existing and future AAV gene therapies.

[0228] Other broad-spectrum immunosuppression methodologies, including broad spectrum immunosuppression (e.g., calcineurin inhibitors [tacrolimus, cyclosporine], rapamycin, MMF, corticosteroids, methotrexate, proteasome inhibitors, costimulation blockade [CTLA4-Ig], Src kinase inhibitors, Btk inhibitors), B cell depletion (rituximab), IgG degrading enzymes (IdeS), IgG half-life reducers (FcRn blockers), or combinations thereof, have not been shown to be effective at enabling AAV vector re-administration at levels equivalent to naïve individuals.

[0229] In one aspect, the present disclosure provides a distinct B cell immunosuppression approach that enables AAV vector re-transduction at levels equal to seronegative animals, by depleting pre-existing nAbs (e.g., via combined plasma cell and immunoglobulin depletion). Long-lived plasma cells (LLPC) mediate constitutive antibody production to most antigens and are the likely reservoir of persistent anti-AAV antibody immunity. The present disclosure was made in part based on the discovery that pre-existing AAV nAbs could be directly eliminated in vivo by LLPC depletion with linvoseltamab, a fully-human T cell-bridging bispecific antibody targeting B cell maturation antigen and CD3 (anti-BCMAxCD3 bispecific antibody), either alone or in combination with B cell depletion (to eliminate potential non-LLPC sources of anti-AAV nAbs) and / or FcRn blockade (to accelerate serum IgG clearance).Attorney Docket No.057766 / 624641

[0230] The methods described herein use plasma cell depleting agents or combinations comprising plasma cell depleting agents to mitigate immune response and facilitate redosing of nucleic acid constructs encoding a polypeptide of interest and nuclease agents targeting a target genomic locus. Optionally, the plasma cell depleting agents (e.g., BCMAxCD3 antigen-binding molecules) are used in combination with other immunosuppression methodologies, such as immunoglobulin depleting agents (e.g., FcRn blockers or IgG degrading enzymes), B cell depleting agents, plasmapheresis, therapeutic plasma exchange, immunoadsorption, broad spectrum immunosuppression, or combinations thereof. In one example, plasma cell depleting agents (e.g., BCMAxCD3 bispecific antigen-binding molecules) are used in combination with immunoglobulin depleting agents (e.g., IgG half-life reducers, such as FcRn blockers). In another example, plasma cell depleting agents (e.g., BCMAxCD3 bispecific antigen-binding molecules) are used in combination with B cell depleting agents (e.g., CD20xCD3 antigen- binding molecules). In another example, BCMAxCD3 bispecific antigen-binding molecules are used in combination with immunoglobulin depleting agents (e.g., FcRn blockers) and B cell depleting agents (e.g., CD20xCD3 antigen-binding molecules). This allows re-dosing of any AAV gene therapy product. For example, for CRISPR-mediated gene insertion platforms consisting of an AAV and LNP, the AAV and / or LNP can be re-dosed multiple times when plasma cell depleting agents or combinations comprising plasma cell depleting agents is co- administered.

[0231] Using plasma cell depleting agents or combinations comprising plasma cell depleting agents to mitigate an anti-AAV antibody response can allow for repeated dosing of an identical gene insertion therapeutic cargo. This allows targeted cells in a subject to produce a polypeptide of interest in a step-wise, increasing fashion due to increased gene insertion in additional targeted cells following repeated dosing until a desired level of expression and / or activity of the polypeptide of interest is achieved in a subject without overshooting. This can be particularly advantageous in situations in which overshooting (i.e., achieving higher than desired levels of expression and / or activity of the polypeptide of interest) would result in undesired side effects (e.g., toxicity). Likewise, using plasma cell depleting agents or combinations comprising plasma cell depleting agents to mitigate an anti-AAV antibody response can allow for gene insertion of an AAV template into two separate genomic locations from two discrete dosings (e.g., two discrete dosings of AAV and LNP). Similarly, using plasma cell depleting agents orAttorney Docket No.057766 / 624641 combinations comprising plasma cell depleting agents to mitigate an anti-AAV antibody response can allow for gene insertion of two different AAV templates (encoding different polypeptides of interest or the same polypeptide of interest) from two discrete dosings (e.g., two discrete dosings of AAV and LNP).

[0232] Also provided are compositions or combinations or kits comprising a plasma cell depleting agent or combination comprising a plasma cell depleting agent in combination with: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus. As used herein, the term “in combination with” a plasma cell depleting agent means that additional component(s) may be administered prior to, concurrent with, or after the administration of the plasma cell depleting agent. The different components of the combination can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit including each component, for example, wherein the further agent is in a separate formulation).

[0233] In another aspect, the present disclosure provides use of B cell depleting agents such as anti-CD20xCD3 bispecific antibodies or functional fragments thereof to mitigate immune response and facilitate redosing of nucleic acid constructs encoding a polypeptide of interest and nuclease agents targeting a target genomic locus. The B cell depleting agents (e.g., anti- CD20xCD3 bispecific antibodies or functional fragments thereof) are able to suppress host B cell responses to new antigens. In AAV gene therapies, seronegative / naive patients are dosed with AAV and develop antibody responses to the AAV capsid antigen. This antibody response prevents future re-dosing of AAV because the antibodies are neutralizing, and the antibody response is sustained for 10+ years. When AAV is co-administered with B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof), the B cell response is suppressed and anti-AAV IgM and IgG responses are significantly suppressed. This allows re- dosing of any AAV gene therapy product. For example, for CRISPR-mediated gene insertion platforms consisting of an AAV and LNP, the AAV and / or LNP can be re-dosed multiple times when B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) are co-administered. The B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) prevent antibody formation against the AAV. The BAttorney Docket No.057766 / 624641 cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) can also prevent antibody formation against certain LNP components (e.g., anti-PEG IgG) or Cas proteins, which can improve efficacy of LNP redosing. In such contexts in which the patient is seronegative / naïve with respect AAV immunity or another immunogen to be administered, the B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) can be used in methods in which a plasma cell depleting agent is not administered. Likewise, in such contexts in which the patient is seronegative / naïve with respect AAV immunity or another immunogen to be administered, the B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) can be used in methods in which an immunoglobulin depleting agent is not administered.

[0234] Using B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) to mitigate an anti-AAV antibody response can allow for repeated dosing of an identical gene insertion therapeutic cargo. This allows targeted cells in a subject to produce a polypeptide of interest in a step-wise, increasing fashion due to increased gene insertion in additional targeted cells following repeated dosing until a desired level of expression and / or activity of the polypeptide of interest is achieved in a subject without overshooting. This can be particularly advantageous in situations in which overshooting (i.e., achieving higher than desired levels of expression and / or activity of the polypeptide of interest) would result in undesired side effects (e.g., toxicity). Likewise, using B cell depleting agents (e.g., anti- CD20xCD3 bispecific antibodies or functional fragments thereof) to mitigate an anti-AAV antibody response can allow for gene insertion of an AAV template into two separate genomic locations from two discrete dosings (e.g., two discrete dosings of AAV and LNP). Similarly, using B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) to mitigate an anti-AAV antibody response can allow for gene insertion of two different AAV templates (encoding different polypeptides of interest or the same polypeptide of interest) from two discrete dosings (e.g., two discrete dosings of AAV and LNP).

[0235] Other broad-spectrum immunosuppression methodologies have not been shown to be effective at enabling AAV vector re-administration at levels equivalent to naïve individuals. The B cell depleting agents (e.g., anti-CD20xCD3 bispecific antibodies or functional fragments thereof) disclosed herein, can achieve levels of re-transduction similar to naïve animals.Attorney Docket No.057766 / 624641

[0236] Also provided are compositions or combinations or kits comprising a B cell depleting agent (e.g., anti-CD20xCD3 bispecific antibody or functional fragment thereof) in combination with: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus. As used herein, the term “in combination with” a B cell depleting agent (e.g., anti-CD20xCD3 bispecific antibody or functional fragment thereof) means that additional component(s) may be administered prior to, concurrent with, or after the administration of the B cell depleting agent (e.g., anti-CD20xCD3 bispecific antibody or functional fragment thereof). The different components of the combination can be formulated into a single composition, e.g., for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit including each component, for example, wherein the further agent is in a separate formulation). II. Plasma Cell Depleting Agents

[0237] In some embodiments, the compositions disclosed herein comprise or the methods disclosed herein include administering a therapeutically effective amount of a plasma cell depleting agent to a subject in need thereof. As used herein, a “plasma cell depleting agent” refers to any molecule capable of specifically binding to a surface antigen on plasma cells and killing or depleting the plasma cells.

[0238] The plasma cell depleting agents can be administered to a subject in need thereof either alone, or in combination with, a B cell depleting agent and / or an immunoglobulin depleting agent. In various aspects, a plasma cell depleting agent may be combined or administered in combination with a B cell depleting agent, an immunoglobulin depleting agent, plasmapheresis, therapeutic plasma exchange, immunoadsorption, and / or an immunogen (e.g., nucleic acid construct, nuclease agent or CRISPR / Cas system, e.g., in an immunogenic delivery vehicle such as, e.g., AAV) disclosed herein. Suitable combinations comprising a plasma cell depleting agent are described in more detail elsewhere herein. In some embodiments, the plasma cell depleting agent of the present disclosure is capable of depleting plasma cells including, without limitation, long-lived plasma cells (LLPCs). In some embodiments, a plasma cell depleting agent is administered to a subject having a pre-existing immunity against an immunogen (e.g., an immunogenic delivery vehicle such as, e.g., AAV (e.g., AAV comprising aAttorney Docket No.057766 / 624641 nucleic acid construct described herein)). In some embodiments, a plasma cell depleting agent is administered to a subject having a pre-existing immunity against a nucleic acid construct described herein, a polypeptide of interest encoded by a nucleic acid construct described herein, a nuclease agent or one or more nucleic acids encoding the nuclease agent as described herein, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent as described herein. In some embodiments, a plasma cell depleting agent is administered to a subject having a pre-existing immunity against an AAV vector comprising a nucleic acid construct described herein.

[0239] As used herein, the term “immunogen” refers to any molecule that is capable of eliciting an immune response. Non-limiting examples of immunogens include immunogenic delivery vehicles such as viral vectors also termed herein “viral particles” (e.g., viral vectors derived from adeno-associated viruses (AAV), adenoviruses, retroviruses [e.g., lentiviruses], or oncolytic viruses [e.g., an adenovirus, a rhabdovirus, a herpes virus, a measles virus, a coxsackievirus, a poliovirus, a reovirus, a poxvirus, a parvovirus, Maraba virus, or Newcastle disease virus]) or portions thereof (e.g., capsid proteins), virus-like particles (VLPs), non-viral vectors (e.g., bacteriophages [such as lambda (X) bacteriophage, EMBL bacteriophage; bacterial vectors such as pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a; pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5]; eukaryotic vectors [such as pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG and pSVL]; transposons [such as Sleeping Beauty transposon and PiggyBac transposon]; bacterial vectors, fungal vectors, and protozoal vectors), liposomes, lipid nanoparticles (LNPs), non-lipid nanoparticles, mammalian cells (e.g., allogeneic cells), and other carriers. Non-limiting examples of immunogens also include polypeptide molecules (e.g., proteins [e.g., therapeutic proteins or antibodies or fragments thereof], peptides), polynucleotide molecules (e.g., mRNAs, interfering nucleic acid molecules [RNAi, siRNA, shRNA], miRNAs, antisense oligonucleotides, ribozymes, aptamers, mixmers, or multimers), antigen-binding molecules fused to a payload, as well as naturally occurring or modified bacteria, fungi, protozoa, parasites, helminths, ectoparasites, or other microorganisms (including bacteria, fungi and other microorganisms found in microbiota). In some embodiments, the immunogen is an immunogenic delivery vehicle, a polypeptide, or a polynucleotide. In some embodiments, the immunogen is an immunogenic delivery vehicle (e.g., AAV) or a polypeptide or polynucleotide encoded by a nucleic acid construct or transgene withinAttorney Docket No.057766 / 624641 the immunogenic delivery vehicle. In some embodiments, the immunogen is an immunogenic delivery vehicle. In some embodiments, the immunogenic delivery vehicle is a viral vector. In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non-lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, a protozoal vector, or a mammalian cell. In some embodiments, the immunogenic delivery vehicle is a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a non- lipid nanoparticle, a liposome, a bacterial vector, a fungal vector, or a protozoal vector. Glycans and lipids are further encompassed by the term immunogen as used herein. In some embodiments, an immunogen can be a nucleic acid construct described herein, a polypeptide of interest encoded by a nucleic acid construct described herein, a nuclease agent or one or more nucleic acids encoding the nuclease agent as described herein, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent as described herein.

[0240] In some embodiments, the plasma cell depleting agent can be an antibody, a small molecule compound, a nucleic acid, a polypeptide, or a functional fragment or variant thereof. Non-limiting examples of suitable plasma cell depleting agents include B cell maturation antigen (BCMA) targeting agents (described elsewhere herein), proteasome inhibitors [e.g., bortezomib (Velcade), carfilzomib (Kyprolis), ixazomib (Niniaro)], histone deacetylase inhibitors [e.g., panobinostat (Farydak)], B-cell activating factor (BAFF; also referred to as BLyS, TALL-1, or CD257) inhibitors (e.g., anti-BAFF antibodies such as belimumab, tabalumab, AMG570; or anti- BAFF receptor antibodies such as ianalumab), proliferation-inducing ligand (APRIL; also referred to as TNFSF13 or CD256) inhibitors (e.g., anti-APRIL antibodies such as BION-1301 or VIS624), G protein–coupled receptor, class C, group 5, member D (GPRC5D) inhibitors (e.g., anti-GPRC5D antibodies, anti-GPRC5DxCD3 bispecific antibodies such as talquetamab), Fc receptor homolog 5 (FcRH5; also referred to as FcRL5, IRTA2, or CD307) inhibitors (e.g., anti- FcRH5 antibodies, anti-FcRH5 xCD3 bispecific antibodies such as Cevostamab), and cluster of differentiation 38 (CD38; also referred to as CADPR1 or ADPRC1) inhibitors (e.g., anti-CD38 antibodies).

[0241] In some embodiments, the plasma cell depleting agents used in the compositions and methods disclosed herein are BCMA targeting agents. As used herein, the term “BCMA targeting agent” refers to any molecule capable of binding specifically to BCMA that isAttorney Docket No.057766 / 624641 expressed on the surface of a cell, e.g., a cell in a subject, thus targeting the cell for destruction. BCMA is expressed exclusively in B-cell lineage cells, particularly in the interfollicular region of the germinal center as well as on plasmablasts and differentiated plasma cells. BCMA is selectively induced during plasma cell differentiation and is required for optimal survival of long-lived plasma cells (LLPCs) in the bone marrow. Thus, a BCMA targeting agent binds to BCMA expressed on a plasma cell surface and mediates killing or depletion of cells that express BCMA (plasma cell depletion). In some embodiments, a BCMA targeting agent comprises a binding moiety that binds to plasma cell-surface-expressed BCMA (an antigen-binding moiety or antigen-binding fragment thereof) and a moiety that facilitates killing of said plasma cell. In some embodiments, the plasma cell-surface-expressed BCMA-binding moiety is an antibody or antigen-binding fragment thereof that binds specifically to BCMA. Such a BCMA-binding moiety can be linked (e.g., covalently bound) to a moiety that facilitates killing or destruction of the targeted plasma cell. The moiety that facilitates targeted killing of the bound plasma cell may be a molecule that directly kills the targeted cell (e.g., a cytotoxic agent) or may be a protein or fragment thereof that mediates killing of the targeted cell, e.g., by an immune cell, e.g., a T-cell. In the context of the present disclosure, the term “BCMA targeting agent” includes, but is not limited to, anti-BCMA antibodies that are conjugated to a therapeutic agent such as a cytotoxic drug (“BCMA ADC” or “anti-BCMA ADC,” e.g., Belantamab Mafodotin / GSK2857916, MEDI2228, HDP-101), chimeric antigenic receptors (CARs) that bind specifically to BCMA, (“BCMA CAR” or “anti-BCMA CAR”) and anti-BCMAxCD3 bispecific antibodies (e.g., linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ- 64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B ).

[0242] In some embodiments, the BCMA targeting agent used in the context of the disclosed methods is an antibody-drug conjugate (ADC) comprising an anti-BCMA antibody and a cytotoxic drug. In some embodiments, the anti-BCMA antibody or antigen-binding fragment thereof and the cytotoxic agent are covalently attached via a linker. In general terms, the ADCs comprise: A-[L-P]y, in which A is an antigen-binding molecule, e.g., an anti-BCMA antibody, or a fragment thereof, L is a linker, P is the payload or therapeutic moiety (e.g., cytotoxic agent), and y is an integer from 1 to 30. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming ADCs are known in the art. Non-limiting examples of suitable cytotoxicAttorney Docket No.057766 / 624641 agents that can be conjugated to anti-BCMA antibodies for use in the disclosed methods are auristatin such as monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), a tubulysin such as TUB-OH or TUB-OMOM, a tomaymycin derivative, a dolastatin derivative, or a maytansinoid such as DM1 or DM4. In some exemplary embodiments, an anti-BCMA ADC used in the present methods comprises the HCVR, LCVR and / or CDR amino acid sequences of any of the anti-BCMA antigen-binding molecules disclosed herein.

[0243] Other anti-BCMA ADCs that can be used in the context of the methods of the present disclosure include, e.g., the ADCs referred to and known in the art as Belantamab Mafodotin (GSK2857916), AMG224, HDP-101, MEDI2228, and TBL-CLN1, or any of the anti-BCMA ADCs set forth, e.g., in International Patent Publications WO2011 / 108008, WO2014 / 089335, WO2017 / 093942, WO2017 / 143069, or WO2019 / 025983. The portions of the publications cited herein that identify anti-BCMA ADCs are hereby incorporated by reference.

[0244] In some embodiments, the BCMA targeting agent used in the context of the disclosed methods is a chimeric antigen receptor (CAR) that binds specifically to BCMA (“BCMA CAR”). Generally, a “chimeric antigen receptor” (CAR) exhibits a specific anti-target cellular immune activity and comprises a binding domain against a component present on the target cell, for example an antibody-based specificity for a desired antigen (e.g., BCMA on plasma cell), and a T cell receptor-activating intracellular domain. CARs typically comprise an extracellular single chain antibody-binding domain (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and have the ability, when expressed in T cells, to redirect antigen recognition based on the monoclonal antibody's specificity. In certain embodiments, the BCMA CAR or antigen-binding fragment thereof comprises a HCVR, LCVR, and / or CDRs comprising the amino acid sequences of any of the antibodies set forth in US Patent Publication No. US 2020 / 0023010, which is hereby incorporated by reference in its entirety. In some exemplary embodiments, an anti-BCMA CAR used in the present methods comprises the HCVR, LCVR and / or CDR amino acid sequences of any of the anti-BCMA antigen-binding molecules disclosed herein.

[0245] Other anti-BCMA CARs that can be used in the context of the methods of the present disclosure include, e.g., the CARs referred to and known in the art as bb2121, LCAR-B38M, and 4C8A, or any of the anti-BCMA CARs set forth, e.g., in WO 2015 / 052538, WO 2015 / 052536, WO 2016 / 094304, WO 2016 / 166630, WO 2016 / 151315, WO 2016 / 130598, WO 2017 / 183418,Attorney Docket No.057766 / 624641 WO 2017 / 173256, WO 2017211900, WO 2017 / 130223, WO 2018 / 229492, WO 2018 / 085690, WO 2018 / 151836, WO 2018 / 028647, WO 2019 / 006072. The portions of the publications cited herein that identify anti-BCMA CARs are hereby incorporated by reference.

[0246] In some exemplary embodiments, the BCMA targeting agent used in the disclosed methods is a multispecific (e.g., bispecific) antibody, or a functional fragment thereof, that specifically binds B cell maturation antigen (BCMA) and CD3 (e.g., an anti-BCMA×CD3 bispecific antibody). The anti-BCMAxCD3 multispecific (e.g., bispecific) antibodies are useful for specific targeting and T-cell-mediated killing of cells that express BCMA. The terms “antibody,” “antigen-binding fragment,” “human antibody,” “recombinant antibody,” and other related terminology are defined above. In the context of anti-BCMAxCD3 antibodies and antigen-binding fragments thereof, the present disclosure includes the use of bispecific antibodies wherein one arm of an immunoglobulin is specific for BCMA or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target (e.g., CD3 on T-cells). Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mabe bispecific formats (see, e.g., Klein et al.2012, mAbs 4(6):653-663, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. See, e.g., Kazane et al., J. Am. Chem. Soc., 2013, 135(1):340-46.

[0247] An anti-BCMAxCD3 bispecific antibody, or functional fragment thereof, may comprise any of various anti-BCMAxCD3 bispecific antibodies, or functional fragments thereof, disclosed herein, or any other such anti-BCMAxCD3 bispecific antibodies, or functional fragments thereof, known to persons of ordinary skill in the art (e.g., linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B ). In a specific embodiment, the anti-BCMAxCD3 bispecific antibody isAttorney Docket No.057766 / 624641 REGN5458. In another specific embodiment, the anti-BCMAxCD3 bispecific antibody is REGN5459. A. BCMAxCD3 Antigen-Binding Molecules

[0248] In some embodiments, the present disclosure provides antigen-binding molecules including multispecific (e.g., bispecific) antibodies that specifically bind B cell maturation antigen (BCMA) and CD3 (e.g., an anti-BCMAxCD3 bispecific antibody). In some embodiments, the antigen-binding molecule is a multispecific (e.g., bispecific) antibody. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, e.g., Tutt et al., 1991, J. Immunol.147:60-69; Kufer et al., 2004, Trends Biotechnol.22:238-244. In some embodiments, the multispecific antibodies of the present disclosure can be linked to or co- expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment to produce a bispecific or a multispecific antibody with a second binding specificity. In some embodiments, the multispecific antibody contains an antigen-binding domain that is specific for BCMA and an antigen-binding domain that is specific for CD3.

[0249] The term “CD3,” as used herein, refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the dimeric association of two of four receptor chains: CD3-epsilon, CD3-delta, CD3-zeta, and CD3-gamma (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta). CD3 is required for T cell activation.

[0250] As used herein, “an antibody that binds CD3” or an “anti-CD3 antibody” includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., epsilon, delta, gamma or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). Antibodies against CD3 have been shown to cluster CD3 on T cells, thereby causing T cell activation in a manner similar to the engagement of the TCR by peptide-loaded major histocompatibility complex (MHC) molecules. Thus, bispecificAttorney Docket No.057766 / 624641 antigen-binding molecules that are capable of binding both CD3 and another antigen (e.g., CD20 or BCMA) would be useful in settings in which specific targeting and T cell-mediated killing of cells that express the non-CD3 antigen (e.g., CD20 or BCMA) is desired.

[0251] The antibodies and antigen-binding fragments of the present invention may bind soluble CD3 and / or cell surface-expressed CD3. Soluble CD3 includes natural CD3 proteins as well as recombinant CD3 protein variants such as, e.g., monomeric and dimeric CD3 constructs, that lack a transmembrane domain or are otherwise unassociated with a cell membrane.

[0252] As used herein, the expression “cell surface-expressed CD3” means one or more CD3 protein(s) that is / are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a CD3 protein is exposed to the extracellular side of the cell membrane and is accessible to an antigen-binding portion of an antibody. “Cell surface-expressed CD3” includes CD3 proteins contained within the context of a functional T cell receptor in the membrane of a cell. The expression “cell surface-expressed CD3” includes CD3 protein expressed as part of a homodimer or heterodimer on the surface of a cell (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers). The expression “cell surface-expressed CD3” also includes a CD3 chain (e.g., CD3-epsilon, CD3-delta or CD3-gamma) that is expressed by itself, without other CD3 chain types, on the surface of a cell. A “cell surface-expressed CD3” can comprise or consist of a CD3 protein expressed on the surface of a cell which normally expresses CD3 protein. Alternatively, “cell surface-expressed CD3” can comprise or consist of CD3 protein expressed on the surface of a cell that normally does not express human CD3 on its surface but has been artificially engineered to express CD3 on its surface.

[0253] As used herein, the expression “anti-CD3 antibody” includes both monovalent antibodies with a single specificity, as well as bispecific antibodies comprising one arm that binds CD3 and another arm that binds a different antigen, wherein the anti-CD3 arm comprises any of the HCVR / LCVR or CDR sequences, or functional fragments thereof, as set forth in Table 1 or Table 2 herein. Examples of anti-CD3 bispecific antibodies are described elsewhere herein. Exemplary anti-CD3 antibodies are also described in PCT International Application No. PCT / US2013 / 060511, which is herein incorporated by reference in its entirety.

[0254] The present disclosure includes bispecific antibodies and functional fragments thereof that bind human CD3 with high affinity. The present disclosure also includes bispecific antibodies and functional fragments thereof that bind human CD3 with medium or low affinity,Attorney Docket No.057766 / 624641 depending on the therapeutic context and particular targeting properties that are desired. For example, in the context of a bispecific antigen-binding molecule, wherein one arm binds CD3 and a second arm binds another antigen (e.g., CD20 or BCMA), it may be desirable for the second arm to bind the non-CD3 (e.g., CD20 or BCMA) antigen with high affinity while the anti-CD3 arm binds CD3 with only moderate or low affinity. In this manner, preferential targeting of the antigen-binding molecule to cells expressing the non-CD3 (e.g., CD20 or BCMA) antigen may be achieved while avoiding general / untargeted CD3 binding and the consequent adverse side effects associated therewith.

[0255] In certain embodiments, the anti-CD3 antibodies induce T cell proliferation with an EC50value of less than about 0.33 pM, as measured by an in vitro T cell proliferation assay (e.g., assessing the proliferation of Jurkat cells or PBMCs in the presence of anti-CD3 antibodies). In certain embodiments, the anti-CD3 antibodies induce T cell proliferation (e.g., Jurkat cell proliferation and / or PBMC proliferation) with an EC50value of less than about 0.32 pM, less than about 0.31 pM, less than about 0.30 pM, less than about 0.28 pM, less than about 0.26 pM, less than about 0.24 pM, less than about 0.22 pM, or less than about 0.20 pM, as measured by an in vitro T cell proliferation assay.

[0256] In some embodiments, the anti-BCMAxCD3 bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) that binds an epitope of BCMA (e.g., human BCMA), and a second antigen-binding domain (D2) that binds an epitope of CD3 (e.g., human CD3).

[0257] In some exemplary embodiments, the anti-BCMAxCD3 bispecific antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (HCVR), light chain variable region (LCVR), and / or complementarity determining regions (CDRs) comprising the amino acid sequences of any of the anti-BCMAxCD3 antibodies set forth in US 11,384,153 and US 2020 / 0345843, which are hereby incorporated by reference in their entireties.

[0258] In some exemplary embodiments, an anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof that can be used in the context of the present disclosure comprising a HCVR, a LCVR, and / or CDRs comprising the amino acid sequences of REGN5458 or REGN5459 as set forth in Table 1 below.Attorney Docket No.057766 / 624641

[0259] Table 1. Amino Acid Sequences of Exemplary Anti-BCMA×CD3 Bispecific Antibodies. Anti-BCMA Anti-CD3 Common First Antigen-Binding Second Antigen-Binding Light Chain Variable Domain Domain Region R3GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGG ATTCACCTTTAGTAACTTTTGGATGACCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGGCCAACATGA ACCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAGC TCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATCGGGAATATTG TATTAGTACCAGCTGCTATGATGACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 2 - Anti-BCMA HCVR Protein Sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFWMTWVRQAPGKGLEWVANMNQDGSEKYYVDSVKGRFTISRDNAKS SLYLQMNSLRAEDTAVYYCARDREYCISTSCYDDFDYWGQGTLVTVSS SEQ ID NO: 3 - Anti-BCMA HCDR1 DNA Sequence GGATTCACCTTTAGTAACTTTTGG SEQ ID NO: 4 - Anti-BCMA HCDR1 Protein Sequence GFTFSNFW SEQ ID NO: 5 - Anti-BCMA HCDR2 DNA Sequence ATGAACCAAGATGGAAGTGAGAAA SEQ ID NO: 6 - Anti-BCMA HCDR2 Protein Sequence MNQDGSEK SEQ ID NO: 7 - Anti-BCMA HCDR3 DNA Sequence GCGAGAGATCGGGAATATTGTATTAGTACCAGCTGCTATGATGACTTTGACTAC SEQ ID NO: 8 - Anti-BCMA HCDR3 Protein Sequence ARDREYCISTSCYDDFDYAttorney Docket No.057766 / 624641 SEQ ID NO: 9 - Anti-BCMA LCVR DNA Sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAG TCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAT CCAGTTTGCATAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT CTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGAC ACGACTGGAGATTAAA SEQ ID NO: 10 - Anti-BCMA LCVR Protein Sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLHSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSYSTPPITFGQGTRLEIK SEQ ID NO: 11 - Anti-BCMA LCDR1 DNA Sequence CAGAGCATTAGCAGCTAT SEQ ID NO: 12 - Anti-BCMA LCDR1 Protein Sequence QSISSY SEQ ID NO: 13 - Anti-BCMA LCDR2 DNA Sequence GCTGCATCC SEQ ID NO: 14 - Anti-BCMA LCDR2 Protein Sequence AAS SEQ ID NO: 15 - Anti-BCMA LCDR3 DNA Sequence CAACAGAGTTACAGTACCCCTCCGATCACC SEQ ID NO: 16 - Anti-BCMA LCDR3 Protein Sequence QQSYSTPPIT SEQ ID NO: 17 - Common LCVR DNA Sequence GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAG TCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCAT CCAGTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGT CTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGATCACCTTCGGCCAAGGGAC ACGACTGGAGATTAAAAttorney Docket No.057766 / 624641 SEQ ID NO: 18 - Common LCVR Protein Sequence DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSYSTPPITFGQGTRLEIK SEQ ID NO: 19 - Common LCDR1 DNA Sequence CAGAGCATTAGCAGCTAT SEQ ID NO: 20 - Common LCDR1 Protein Sequence QSISSY SEQ ID NO: 21 - Common LCDR2 DNA Sequence GCTGCATCC SEQ ID NO: 22 - Common LCDR2 Protein sequence AAS SEQ ID NO: 23 - Common LCDR3 DNA sequence CAACAGAGTTACAGTACCCCTCCGATCACC- Sequence QQSYSTPPIT SEQ ID NO: 25 - Anti-CD3 HCVR DNA Sequence – REGN5458 GAAGTACAGCTTGTAGAATCCGGCGGAGGACTGGTACAACCTGGAAGAAGTCTTAGACTGAGTTGCGCAGCTAGTGG GTTTACATTCGACGATTACAGCATGCATTGGGTGAGGCAAGCTCCTGGTAAAGGATTGGAATGGGTTAGCGGGATAT CATGGAACTCAGGAAGCAAGGGATACGCCGACAGCGTGAAAGGCCGATTTACAATATCTAGGGACAACGCAAAAAAC TCTCTCTACCTTCAAATGAACTCTCTTAGGGCAGAAGACACAGCATTGTATTATTGCGCAAAATACGGCAGTGGTTA TGGCAAGTTTTATCATTATGGACTGGACGTGTGGGGACAAGGGACAACAGTGACAGTGAGTAGC SEQ ID NO: 26 - Anti-CD3 HCVR Protein Sequence – REGN5458 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSKGYADSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYYCAKYGSGYGKFYHYGLDVWGQGTTVTVSS SEQ ID NO: 27 - Anti-CD3 HCDR1 DNA Sequence – REGN5458 GGGTTTACATTCGACGATTACAGC SEQ ID NO: 28 - Anti-CD3 HCDR1 Protein Sequence – REGN5458 GFTFDDYSAttorney Docket No.057766 / 624641 SEQ ID NO: 29 - Anti-CD3 HCDR2 DNA Sequence – REGN5458 ATATCATGGAACTCAGGAAGCAAG SEQ ID NO: 30 - Anti-CD3 HCDR2 Protein Sequence – REGN5458 ISWNSGSK SEQ ID NO: 31 - Anti-CD3 HCDR3 DNA Sequence – REGN5458 GCAAAATACGGCAGTGGTTATGGCAAGTTTTATCATTATGGACTGGACGTG SEQ ID NO: 32 - Anti-CD3 HCDR3 Protein Sequence – REGN5458 AKYGSGYGKFYHYGLDV SEQ ID NO: 33 - Anti-CD3 HCVR DNA Sequence – REGN5459 GAAGTACAGCTTGTAGAATCCGGCGGAGGACTGGTACAACCTGGAAGAAGTCTTAGACTGAGTTGCGCAGCTAGTGG GTTTACATTCGACGATTACAGCATGCATTGGGTGAGGCAAGCTCCTGGTAAAGGATTGGAATGGGTTAGCGGGATAT CATGGAACTCAGGAAGCATCGGATACGCCGACAGCGTGAAAGGCCGATTTACAATATCTAGGGACAACGCAAAAAAC TCTCTCTACCTTCAAATGAACTCTCTTAGGGCAGAAGACACAGCATTGTATTATTGCGCAAAATACGGCAGTGGTTA TGGCAAGTTTTATTATTATGGAATGGACGTGTGGGGACAAGGGACAACAGTGACAGTGAGTAGC SEQ ID NO: 34 - Anti-CD3 HCVR Protein Sequence – REGN5459 EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYYCAKYGSGYGKFYYYGMDVWGQGTTVTVSS SEQ ID NO: 35 - Anti-CD3 HCDR1 DNA Sequence – REGN5459 GGGTTTACATTCGACGATTACAGC SEQ ID NO: 36 - Anti-CD3 HCDR1 Protein Sequence – REGN5459 GFTFDDYS SEQ ID NO: 37 - Anti-CD3 HCDR2 DNA Sequence – REGN5459 ATATCATGGAACTCAGGAAGCATC SEQ ID NO: 38 - Anti-CD3 HCDR2 Protein Sequence – REGN5459 ISWNSGSI SEQ ID NO: 39 - Anti-CD3 HCDR3 DNA Sequence – REGN5459 GCAAAATACGGCAGTGGTTATGGCAAGTTTTATTATTATGGAATGGACGTGAttorney Docket No.057766 / 624641 SEQ ID NO: 40 - Anti-CD3 HCDR3 Protein Sequence – REGN5459 AKYGSGYGKFYYYGMDV SEQ ID NO: 41 - Anti-BCMA Heavy Chain Protein Sequence (IgG4 Heavy Chain Constant Region) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFWMTWVRQAPGKGLEWVANMNQDGSEKYYVDSVKGRFTISRDNAKS SLYLQMNSLRAEDTAVYYCARDREYCISTSCYDDFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 42 - Anti-CD3 Heavy Chain Protein Sequence (IgG4 Heavy Chain Constant Region with H435R / Y436F) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYSMHWVRQAPGKGLEWVSGISWNSGSKGYADSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYYCAKYGSGYGKFYHYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 43 – Common Anti-BCMA and Anti-CD3 Light Chain Protein Sequence (Kappa Light Chain Constant Region) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0260] In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof that can be used the present disclosure comprises: (a) a first antigen binding domain that binds specifically to BCMA; and (b) a second antigen-binding domain that binds specifically to CD3. In one embodiment, the anti-BCMA antigen-binding domain comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the first antigen-Attorney Docket No.057766 / 624641 binding domain comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 6; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 8; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 20; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 22; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24. In one embodiment, the second antigen-binding domain comprises the heavy chain complementarity determining regions (HCDRs) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26 or 34 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the second antigen-binding domain comprises three HCDRs (HCDR1, HCDR2 and HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 28 or 36; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 30 or 38; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 32 or 40; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 20; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 22; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 24.

[0261] In one embodiment, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24; and (b) a second antigen binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 28, 30, and 32, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24. In one embodiment, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 2 and a LCVR comprising the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 26 and a LCVR comprising the amino acid sequence of SEQ ID NO: 18.Attorney Docket No.057766 / 624641

[0262] In one embodiment, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24; and (b) a second antigen binding domain that comprises HCDR1, HCDR2, and HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 36, 38, and 40, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24. In one embodiment, the anti-BCMA / anti-CD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 2 and a LCVR comprising the amino acid sequence of SEQ ID NO:18; and (b) a second antigen-binding domain that comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 34 and a LCVR comprising the amino acid sequence of SEQ ID NO: 18.

[0263] Exemplary anti-BCMAxCD3 bispecific antibodies include the fully human bispecific antibodies known as REGN5458 and REGN5459. See, e.g., WO 2020 / 018820, US 2020 / 0024356, US 2022 / 0306758, and US 11,384,153, each of which is herein incorporated by reference. According to certain exemplary embodiments, the methods of the present disclosure comprise the use of REGN5458 or REGN5459, or a bioequivalent thereof. As used herein, the term “bioequivalent” with respect to anti-BCMAxCD3 antibodies refers to antibodies or BCMAxCD3 binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives having a rate and / or extent of absorption that does not show a significant difference with that of a reference antibody (e.g., REGN5458 or REGN5459) when administered at the same molar dose under similar experimental conditions, either single dose or multiple dose; the term “bioequivalent” also includes antigen-binding proteins that bind to BCMA / CD3 and do not have clinically meaningful differences with the reference antibody (e.g., REGN5458 or REGN5459) with respect to safety, purity and / or potency.

[0264] In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%,Attorney Docket No.057766 / 624641 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NOS: 28, 30, and 32, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 26, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0265] In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-BCMAxCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3)Attorney Docket No.057766 / 624641 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18; and (b) a second antigen-binding domain that comprises three HCDRs (HCDR1, HCDR2 and HCDR3) comprising the amino acid sequences of SEQ ID NOS: 36, 38, and 40, respectively, and a HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 34, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 18.

[0266] The present disclosure also includes variants of the anti-BCMAxCD3 antibodies described herein comprising any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein with one or more conservative amino acid substitutions. For example, the present disclosure includes use of anti-BCMAxCD3 antibodies having HCVR, LCVR and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. In some embodiments, the disclosure includes use of an anti- BCMAxCD3 antibody having HCVR, LCVR and / or CDR amino acid sequences with 1, 2, 3, or 4 conservative amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein.

[0267] Other anti-BCMAxCD3 antibodies that can be used in the methods of the present disclosure include, e.g., the antibodies referred to and known in the art as pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B, or any of the anti- BCMAxCD3 antibodies set forth, e.g., in WO 2013 / 072415, WO 2014 / 140248, WO 2014 / 122144, WO 2016 / 166629, WO 2016 / 079177, WO 2016 / 020332, WO 2017 / 031104, WO 2017 / 223111, WO 2017 / 134134, WO 2018 / 083204, or WO 2018 / 201051. The portions of the publications cited herein that identify anti-BCMAxCD3 antibodies are hereby incorporated byAttorney Docket No.057766 / 624641 reference.

[0268] In some embodiments, the CDRs disclosed herein are identified according to the Kabat definition. In some embodiments, the CDRs are identified according to the Chothia definition. In some embodiments, the CDRs are identified according to the AbM definition. In some embodiments, the CDRs are identified according to the IMGT definition.

[0269] The bispecific antigen-binding molecules disclosed herein may be bispecific antibodies. In some cases, the bispecific antibody comprises a human IgG heavy chain constant region. In some cases, the human IgG heavy chain constant region is isotype IgG1. In some cases, the human IgG heavy chain constant region is isotype IgG4. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn). In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR).

[0270] In some embodiments, the heavy chain constant region attached to the HCVR of the first antigen-binding domain or the heavy chain constant region attached to the HCVR of the second antigen-binding domain, but not both, contains an amino acid modification that reduces Protein A binding relative to a heavy chain of the same isotype without the modification. In some cases, the modification comprises a H435R substitution (EU numbering) in a heavy chain of isotype IgG1 or IgG4. In some cases, the modification comprises a H435R substitution and a Y436F substitution (EU numbering) in a heavy chain of isotype IgG1 or IgG4.

[0271] In some embodiments, the antibody comprises a first heavy chain containing the HCVR of the first antigen-binding domain and a second heavy chain containing the HCVR of the second antigen-binding domain, wherein the first heavy chain comprises residues 1-450 of the amino acid sequence of SEQ ID NO: 41 and the second heavy chain comprises residues 1- 449 of the amino acid sequence of SEQ ID NO: 42.

[0272] In some embodiments, the antibody comprises a common light chain containing the LCVR of the first and second antigen-binding domains, wherein the common light chain comprises the amino acid sequence of SEQ ID NO: 43.

[0273] In some embodiments, the anti-BCMAxCD3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO: 41, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 42, and a common light chain comprisingAttorney Docket No.057766 / 624641 the amino acid sequence of SEQ ID NO: 43. In some cases, the mature form of the antibody may not include the C-terminal lysine residues of SEQ ID NOS: 41 and 42. Thus, in some cases the anti-BCMA binding arm comprises a heavy chain comprising residues 1-450 of SEQ ID NO: 41, and the anti-CD3 binding arm comprises a heavy chain comprising residues 1-449 of SEQ ID NO: 42.

[0274] The first antigen-binding domain and the second antigen-binding domain may be directly or indirectly connected to one another to form a bispecific antigen-binding molecule of the present invention. Alternatively, the first antigen-binding domain and the second antigen- binding domain may each be connected to a separate multimerizing domain. The association of one multimerizing domain with another multimerizing domain facilitates the association between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. For example, a multimerizing domain may be a polypeptide comprising an immunoglobulin CH3 domain. A non-limiting example of a multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0275] In some embodiments, a bispecific antigen-binding molecule of the present disclosure comprises two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain. The first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0276] In some embodiments, the multimerizing domain is an Fc fragment or an amino acid sequence of from 1 to about 200 amino acids in length containing at least one cysteine residue. In other embodiments, the multimerizing domain is a cysteine residue, or a short cysteine- containing peptide. Other multimerizing domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix-loop motif, or a coiled-coil motif.Attorney Docket No.057766 / 624641 III. B Cell Depleting Agents

[0277] In some embodiments, the methods disclosed herein include administering a therapeutically effective amount of a B cell depleting agent to a subject in need thereof. As used herein, a “B cell depleting agent” refers to any molecule capable of specifically binding to a surface antigen on B cells and killing or depleting said B cell. Thus, in general, a B cell depleting agent can be any agent that binds to a B cell surface molecule. In some embodiments, the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA.

[0278] In various aspects, the present disclosure provides B cell depleting agents, which may be administered to a subject in need thereof, e.g., either alone or combined with, or administered in combination with, a plasma cell depleting agent (e.g., an anti-BCMAxCD3 bispecific antibody, or a functional fragment thereof), an immunoglobulin depleting agent (e.g., an FcRn blocker such as, e.g., efgartigimod), and / or an immunogen (e.g., a nucleic acid construct described herein, a polypeptide of interest encoded by a nucleic acid construct described herein, a nuclease agent or one or more nucleic acids encoding the nuclease agent as described herein, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent as described herein such as, e.g., AAV (e.g., AAV comprising a nucleic acid construct described herein). In some embodiments, plasmapheresis, therapeutic plasma exchange, and / or immunoadsorption may be further combined with the administering of the B cell depleting agent, the plasma cell depleting agent, the immunoglobulin depleting agent, and / or the immunogen. In some embodiments, the subject does not have a pre-existing immunity against the immunogen. For example, in some embodiments, the subject does not have a pre- existing immunity against a nucleic acid construct described herein, a polypeptide of interest encoded by a nucleic acid construct described herein, a nuclease agent or one or more nucleic acids encoding the nuclease agent as described herein, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent as described herein. In some such methods in which the subject does not have a pre-existing immunity against the immunogen (e.g., immunogenic delivery vehicle such as, e.g., AAV), the methods do not comprise administering a plasma cell depleting agent (the B cell depleting agent is not used in combination with a plasma cell depleting agent). In some such methods in which the subject does not have a pre-existing immunity against the immunogen (e.g., immunogenicAttorney Docket No.057766 / 624641 delivery vehicle such as, e.g., AAV), the methods do not comprise administering an immunoglobulin depleting agent (the B cell depleting agent is not used in combination with an immunoglobulin depleting agent).

[0279] In some embodiments, a B cell depleting agent may be administered alone (e.g., as a monotherapy, in the absence of the administration of any other additional immunomodulators [e.g., plasma cell depleting agents, immunoglobulin depleting agents], and optionally, combined with, or administered in combination with, an immunogen) to a subject in need thereof, e.g., a subject without a pre-existing immunity against an immunogen (e.g., an immunogen to be administered to the subject e.g., an immunogenic delivery vehicle such as, e.g., AAV). For example, the subject may be a subject without a pre-existing immunity against a nucleic acid construct described herein, a polypeptide of interest encoded by a nucleic acid construct described herein, a nuclease agent or one or more nucleic acids encoding the nuclease agent as described herein, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent as described herein. In some embodiments, the B cell depleting agent may be administered alone to a subject who is immunologically naïve to an immunogen to be administered to the subject (e.g., AAV). In some embodiments, the B cell depleting agent may be administered alone to an AAV seronegative subject, and the subject is further administered an immunogen (e.g., AAV). In some embodiments, a B cell depleting agent may be useful as a prophylactic treatment to prevent or suppress an immune response (e.g., an anti-AAV IgG, IgM, and / or nAb response) to an immunogen (e.g., AAV) in a subject in need thereof (e.g., a subject without a pre-existing immunity to the immunogen).

[0280] In some embodiments, the suppression or prevention of an immune response (e.g., an anti-AAV IgG, IgM, and / or nAb response) to an immunogen in a subject (e.g., a subject without a pre-existing immunity to the immunogen) can be achieved by administering a B cell depleting agent described herein (e.g., an anti-CD20xCD3 bispecific antibody or a functional fragment thereof). Administration of the B cell depleting agent to the subject can suppress or prevent the immune response in the subject following the initial dosing and / or re-dosing of an immunogen (e.g., post-AAV dosing and / or re-dosing). In some embodiments, an immune response may be suppressed in a subject following AAV dosing and / or re-dosing. The immune response may be suppressed by about 1%, about 2%, about 3%, about 4%, about 5%, about 7% about 8%, aboutAttorney Docket No.057766 / 624641 9%, about 10%, from about 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 40%, from about 40% to about 50% or more, e.g., relative to an immune response in a subject receiving no immunomodulation treatment or treatment with a conventional anti-CD20 therapeutic alone (e.g., rituximab, or derivatives or equivalents thereof). The immune response may be suppressed by from about 50% to about 60%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, more than 60%, from about 60% to about 70%, from about 60% to about 80%, from about 60% to about 90%, more than about 70%, from about 70% to about 80%, from about 70% to about 90%, more than about 80%, from about 80% to about 90%, more than 90%, from about 90% to about 95%, from about 90% to about 98%, more than 95%, from about 95% to about 98%, more than about 98%, or more than about 99%. The immune response may be suppressed by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or even 100%. In some embodiments, the immune response is prevented. In some embodiments, an immune response may be suppressed or prevented in a subject following AAV dosing and / or re-dosing in the subject to achieve levels equivalent to, or even below, those of an AAV-naïve subject. In some embodiments, a B cell depleting agent is sufficient to enable effective re-dosing of an immunogen to a subject. The B cell depleting agent can be administered to the subject prior to the re-dosing of the immunogen any number of times and can be used to maintain a suppressed immune response to the immunogen in the subject for any period of time thereafter.

[0281] In some embodiments, a B cell depleting agent is capable of suppressing an anti- immunogen response (e.g., an anti-AAV response) in a subject, and the anti-immunogen response is mounted by the subject in response to repeated doses of the immunogen (e.g., AAV).

[0282] It is contemplated that a B cell depleting agent may be used in the suppression or prevention of an anti-immunogen antibody response (e.g., an anti-AAV antibody response) in a subject, and the suppression or prevention of the anti-immunogen antibody response involves B cell depletion in primary and / or secondary lymphoid tissues and non-lymphoid tissue as well, such as B cell aggregates forming in liver and muscle after AAV administration. Non-limiting examples of primary lymphoid tissues include bone marrow and thymus. In some embodiments, the compositions and methods of the disclosure encompass B cell depletion in secondaryAttorney Docket No.057766 / 624641 lymphoid tissues, for example, and without limitation, spleen and / or lymph nodes. In some embodiments, the compositions and methods of the disclosure relate to B cell depletion in lymph nodes, which is achieved by a B cell depleting agent described herein.

[0283] In some embodiments, the present disclosure provides B cell depleting agents combined with, or administered in combination with, plasma cell depleting agents (e.g., an anti- BCMAxCD3 bispecific antibody, or a functional fragment thereof) described herein to subjects, e.g., subjects with or without a pre-existing immunity against an immunogen (i.e., an immunogen administered to the subject, e.g., an immunogenic delivery vehicle such as, e.g., AAV). In some embodiments, the B cell depleting agent may be administered in combination with a plasma cell depleting agent, an immunoglobulin depleting agent, plasmapheresis, therapeutic plasma exchange, immunoadsorption, and / or an immunogen (e.g., an immunogenic delivery vehicle) disclosed herein. In some embodiments, the B cell depleting agent may be administered to subjects without a pre-existing immunity against an immunogen (i.e., an immunogen to be administered to the subject, e.g., an immunogenic delivery vehicle such as, e.g., AAV) not only alone, but also in combination with a plasma cell depleting agent, an immunoglobulin depleting agent, plasmapheresis, therapeutic plasma exchange, or immunoadsorption, and / or an immunogen (e.g., nucleic acid construct, nuclease agent or CRISPR / Cas system, e.g., in an immunogenic delivery vehicle) (e.g., an immunogenic delivery vehicle such as, e.g., AAV) disclosed herein. In some embodiments, the B cell depleting agent may be administered to subjects with a pre-existing immunity against an immunogen (i.e., an immunogen to be administered to the subject, e.g., an immunogenic delivery vehicle such as, e.g., AAV) in combination with a plasma cell depleting agent, an immunoglobulin depleting agent, plasmapheresis, therapeutic plasma exchange, or immunoadsorption, and / or an immunogen (e.g., nucleic acid construct, nuclease agent or CRISPR / Cas system, e.g., in an immunogenic delivery vehicle) (e.g., an immunogenic delivery vehicle such as, e.g., AAV) disclosed herein. Suitable combinations comprising a plasma cell depleting agent are described in more detail elsewhere herein.

[0284] In some embodiments, the B cell depleting agent is an agent that directly targets a B cell, e.g., an agent that binds to a B cell surface molecule. In some embodiments, the B cell depleting agent causes a reduction in the number of B cells in a subject (e.g., in a blood sample taken from the subject). In some embodiments, a B cell depleting agent may be useful for, e.g.,Attorney Docket No.057766 / 624641 eliminating non-plasma cell (e.g., non-long-lived plasma cell [LLPC] sources of immunogen (e.g., anti-AAV) nAbs. In some embodiments, a B cell depleting agent may be useful for, e.g., preventing formation of non-plasma cell (e.g., non-long-lived plasma cell [LLPC] sources of immunogen (e.g., anti-AAV) nAbs (e.g., in AAV-naïve patients). In some embodiments, the B cell depleting agent may capture a wider range of AAV-specific B cells and plasma cells that may not express high levels of BCMA (e.g., committed memory B cells and early plasmablasts).

[0285] In some embodiments, the B cell depleting agent comprises an anti-CD19 antibody (e.g., MEDI-551, tefasitamab, Inebilizumab, loncastuximab), an anti-CD20 antibody (e.g., rituximab, ocrelizumab, obinutuzumab, ublituximab, or ofatumumab), an anti-CD22 antibody (e.g., epratuzumab), an anti-CD79 antibody (e.g., polatuzumab), a bispecific anti-CD20xCD3 B cell depleting antibody (e.g. odronextamab, glofitamab, mosunetuzumab, epcoritamab), a bispecific anti-CD19xCD3 antibody (e.g., blinatumomab), a bispecific anti-CD22xCD3 antibody (e.g., inotuzumab), or functional fragments thereof, or any combination thereof.

[0286] In some embodiments, the B cell depleting agent is an agent that indirectly targets a B cell, e.g., by targeting a B cell survival factor. In some embodiments, the B cell depleting agent is a BLyS / BAFF inhibitor (e.g., belimumab, lanalumab, BR3-Fc, AMG-570, or AMG-623), an APRIL inhibitor (e.g., telitacicept, atacicept), or a BLyS receptor 3 / BAFF receptor inhibitor (e.g., anti-BR3), or any combination thereof.

[0287] In some embodiments, the B cell depleting agent is selected from anti-CD19 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD79 antibodies, multispecific antibodies combining two or more of any of said antibody specificities, multispecific antibodies combining any of said antibody specificities with anti-CD3 antibodies, functional fragments of any of said antibodies, and any combinations thereof. In certain embodiments, the B cell depleting agent is an anti-CD20 antibody or a functional fragment thereof. In some embodiments, a multispecific anti-CD20 antibody or functional fragment thereof of the present disclosure targets CD20 and CD19. In some embodiments, the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD19xCD20 bispecific antibody, or functional fragment thereof. In some embodiments, the B cell depleting agent comprises an anti-CD19 antibody and an anti-CD20 antibody.Attorney Docket No.057766 / 624641

[0288] In some embodiments, the B cell depleting agent comprises anti-CD19 and anti-CD20 antibodies (also referred to as “anti-CD19 / CD20 antibodies” herein), or functional fragments thereof, disclosed herein.

[0289] In a specific embodiment, the B cell depleting agent comprises a bispecific antibody that specifically binds CD3 and CD19. Such antibodies may be referred to herein as, e.g., “anti- CD19 / anti-CD3,” or “anti-CD19×CD3” or “CD19×CD3” bispecific antibodies, or other similar terminology.

[0290] In a specific embodiment, the B cell depleting agent comprises a bispecific antibody that specifically binds CD3 and CD20. Such antibodies may be referred to herein as, e.g., “anti- CD20 / anti-CD3,” or “anti-CD20×CD3” or “CD20×CD3” bispecific antibodies, or other similar terminology.

[0291] As used herein, the expression “bispecific antibody” refers to an immunoglobulin protein comprising at least a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain specifically binds a first antigen (e.g., CD20), and the second antigen-binding domain specifically binds a second, distinct antigen (e.g., CD3). Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR), each comprising three CDRs. In the context of a bispecific antibody, the CDRs of the first antigen-binding domain may be designated with the prefix “A” and the CDRs of the second antigen-binding domain may be designated with the prefix “B.” Thus, the CDRs of the first antigen-binding domain may be referred to herein as A-HCDR1, A-HCDR2, and A-HCDR3; and the CDRs of the second antigen-binding domain may be referred to herein as B-HCDR1, B-HCDR2, and B-HCDR3.

[0292] The first antigen-binding domain and the second antigen-binding domain can each be connected to a separate multimerizing domain. As used herein, a “multimerizing domain” is any macromolecule, protein, polypeptide, peptide, or amino acid that has the ability to associate with a second multimerizing domain of the same or similar structure or constitution. In the context of the present invention, the multimerizing component is an Fc portion of an immunoglobulin (comprising a CH2-CH3 domain), e.g., an Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0293] Bispecific antibodies of the present invention typically comprise two multimerizing domains, e.g., two Fc domains that are each individually part of a separate antibody heavy chain.Attorney Docket No.057766 / 624641 The first and second multimerizing domains may be of the same IgG isotype such as, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4. Alternatively, the first and second multimerizing domains may be of different IgG isotypes such as, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0294] Any bispecific antibody format or technology may be used to make the bispecific antigen-binding molecules of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity to produce a bispecific antigen-binding molecule. Specific exemplary bispecific formats that can be used in the context of the present invention include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into- holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab2bispecific formats (see, e.g., Klein et al.2012, mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats).

[0295] In the context of bispecific antibodies of the present invention, Fc domains may comprise one or more amino acid changes (e.g., insertions, deletions or substitutions) as compared to the wild-type, naturally occurring version of the Fc domain. For example, the invention includes bispecific antigen-binding molecules comprising one or more modifications in the Fc domain that results in a modified Fc domain having a modified binding interaction (e.g., enhanced or diminished) between Fc and FcRn. In one embodiment, the bispecific antigen- binding molecule comprises a modification in a CH2 or a CH3 region, wherein the modification increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Non-limiting examples of such Fc modifications are disclosed in US 2015 / 0266966, incorporated herein in its entirety.

[0296] The present invention also includes bispecific antibodies comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from one another by at least one amino acid, and wherein at least one amino acid difference reduces binding of the bispecific antibody to Protein A as compared to a bi-specific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds Protein A and the second Ig CH3 domain contains a mutation that reduces or abolishes Protein A binding such as anAttorney Docket No.057766 / 624641 H95R modification (by IMGT exon numbering; H435R by EU numbering). The second CH3 may further comprise a Y96F modification (by IMGT; Y436F by EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V821 (by IMGT; D356E, L358M, N384S, K392N, V397M, and V4221 by EU) in the case of IgG1 antibodies; N44S, K52N, and V821 (IMGT; N384S, K392N, and V4221 by EU) in the case of IgG2 antibodies; and Q15R, N44S, K52N, V57M, R69K, E79Q, and V821 (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V4221 by EU) in the case of IgG4 antibodies.

[0297] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences derived from more than one immunoglobulin isotype. For example, a chimeric Fc domain can comprise part or all of a CH2 sequence derived from a human IgG1, human IgG2 or human IgG4 CH2 region, and part or all of a CH3 sequence derived from a human IgG1, human IgG2 or human IgG4. A chimeric Fc domain can also contain a chimeric hinge region. For example, a chimeric hinge may comprise an “upper hinge” sequence, derived from a human IgG1, a human IgG2 or a human IgG4 hinge region, combined with a “lower hinge” sequence, derived from a human IgG1, a human IgG2 or a human IgG4 hinge region. A particular example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgG4 CH1]-[IgG4 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2]-[IgG4 CH3]. Another example of a chimeric Fc domain that can be included in any of the antigen-binding molecules set forth herein comprises, from N- to C-terminus: [IgG1 CH1]-[IgG1 upper hinge]-[IgG2 lower hinge]-[IgG4 CH2] [IgG1 CH3]. These and other examples of chimeric Fc domains that can be included in any of the antigen-binding molecules of the present invention are described in US Patent Publication No.2014 / 0243504, which is herein incorporated in its entirety. Chimeric Fc domains having these general structural arrangements, and variants thereof, can have altered Fc receptor binding, which in turn affects Fc effector function. A. CD20xCD3 Antigen-Binding Molecules

[0298] The term “CD20,” as used herein, refers to an antigen which is expressed on B cells and which consists of a non-glycosylated phosphoprotein expressed on the cell membranes of mature B cells. The human CD20 protein can have the amino acid sequence as in NCBI Reference Sequence NP_690605.1. As used herein, the expression “anti-CD20 antibody” includes monovalent antibodies with a single specificity, such as RITUXAN® (rituximab), asAttorney Docket No.057766 / 624641 described in U.S. Pat. No.7,879,984. Exemplary anti-CD20 antibodies are also described in U.S. Pat. No.7,879,984 and PCT International Application No. PCT / US2013 / 060511, filed on Sep. 19, 2013, each incorporated by reference herein.

[0299] In some exemplary embodiments, the CD20 targeting agent used in the disclosed methods is a multispecific (e.g., bispecific) antibody, or a functional fragment thereof, that specifically binds CD20 and CD3 (e.g., an anti-CD20×CD3 bispecific antibody). The anti- CD20xCD3 multispecific (e.g., bispecific) antibodies are useful for specific targeting and T-cell- mediated killing of cells that express CD20. The terms “antibody,” “antigen-binding fragment,” “human antibody,” “recombinant antibody,” and other related terminology are defined above. In the context of anti-CD20xCD3 antibodies and antigen-binding fragments thereof, the present disclosure includes the use of bispecific antibodies wherein one arm of an immunoglobulin is specific for CD20 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target (e.g., CD3 on T-cells). Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, Quadroma, knobs-into- holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mabe bispecific formats (see, e.g., Klein et al.2012, mAbs 4(6):653-663, and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc., 2013, 135(1):340-46).

[0300] The anti-CD20×CD3 bispecific antibodies are capable of simultaneously binding to human CD3 and human CD20. According to certain embodiments, the anti-CD20×CD3 bispecific antibodies specifically interact with cells that express CD3 and / or CD20. The extent to which the anti-CD20×CD3 bispecific antibodies binds cells that express CD3 and / or CD20 can be assessed by fluorescence activated cell sorting (FACS). In certain embodiments, the anti- CD20×CD3 bispecific antibodies specifically bind human T-cell lines which express CD3 (e.g., Jurkat), human B-cell lines which express CD20 (e.g., Raji), and primate T-cells (e.g., cynomolgus peripheral blood mononuclear cells [PBMCs]).Attorney Docket No.057766 / 624641

[0301] In some embodiments, the anti-CD20xCD3 bispecific antigen-binding molecule comprises a first antigen-binding domain (D1) that binds an epitope of CD20 (e.g., human CD20), and a second antigen-binding domain (D2) that binds an epitope of CD3 (e.g., human CD3).

[0302] According to certain exemplary embodiments of the present invention, the bispecific anti-CD20xCD3 antibody, or antigen-binding fragment thereof comprises heavy chain variable regions (A-HCVR and B-HCVR), light chain variable region (LCVR), and / or complementarity determining regions (CDRs) comprising any of the amino acid sequences of the bispecific anti- CD20xCD3 antibodies as set forth in US Patent Publication No.20150266966, incorporated herein by reference in its entirety for all purposes. In certain exemplary embodiments, the bispecific anti-CD20xCD3 antibody or antigen-binding fragment thereof that can be used in the context of the methods of the present invention comprises: (a) a first antigen-binding arm comprising the heavy chain complementarity determining regions (A-HCDR1, A-HCDR2 and A-HCDR3) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO: 44 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding arm comprising the heavy chain CDRs (B-HCDR1, B-HCDR2 and B- HCDR3) of a HCVR (B-HCVR) comprising the amino acid sequence of SEQ ID NO: 46 and the light chain CDRs of a LCVR comprising the amino acid sequence of SEQ ID NO: 45. According to certain embodiments, the A-HCDR1 comprises the amino acid sequence of SEQ ID NO: 47; the A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 48; the A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 49; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 50; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 51; the LCDR3 comprises the amino acid sequence of SEQ ID NO: 52; the B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 53; the B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 54; and the B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 55. In yet other embodiments, the bispecific anti-CD20xCD3 antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding arm comprising a HCVR (A-HCVR) comprising SEQ ID NO: 44 and a LCVR comprising SEQ ID NO: 45; and (b) a second antigen-binding arm comprising a HCVR (B-HCVR) comprising SEQ ID NO: 46 and a LCVR comprising SEQ ID NO: 45.Attorney Docket No.057766 / 624641

[0303] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0304] In some embodiments, the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0305] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.

[0306] In some embodiments, the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.

[0307] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, andAttorney Docket No.057766 / 624641 LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

[0308] Other bispecific anti-CD20xCD3 antibodies that can be used in the context of the methods of the present invention include, e.g., any of the antibodies as set forth in US 2014 / 0088295, US 2015 / 0166661, and US 2017 / 0174781, each of which is herein incorporated by reference in its entirety for all purposes. An exemplary bispecific anti-CD20xCD3 antibody that can be used in the context of the methods of the present invention is the bispecific anti- CD20xCD3 antibody known as REGN1979 or bsAB1.

[0309] In some exemplary embodiments, an anti-CD20xCD3 bispecific antibody or antigen- binding fragment thereof that can be used in the context of the present disclosure comprising a HCVR, a LCVR, and / or CDRs comprising the amino acid sequences of REGN1979 as set forth in Table 2 below.

[0310] Table 2. Amino Acid Sequences of Exemplary Anti-CD20×CD3 Bispecific Antibodies. Anti-CD20 Anti-CD3 Common First Antigen-Binding Second Antigen-Binding Light Chain Variable D i D i R i R3EVQLVESGGGLVQPGRSLRLSCVASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVKGRFTISRDNAKN SLYLQMHSLRAEDTALYYCAKDNHYGSGSYYYYQYGMDVWGQGTTVTVSS SEQ ID NO: 45 – Common LCVR Protein Sequence EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISS LQSEDFAVYYCQHYINWPLTFGGGTKVEIKR SEQ ID NO: 46 – Anti-CD3 HCVR Protein Sequence EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYTMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKK SLYLQMNSLRAEDTALYYCAKDNSGYGHYYYGMDVWGQGTTVTVAS SEQ ID NO: 47 – Anti-CD20 HCDR1 Protein Sequence GFTFNDYAAttorney Docket No.057766 / 624641 SEQ ID NO: 48 – Anti-CD20 HCDR2 Protein Sequence ISWNSDSI SEQ ID NO: 49 – Anti-CD20 HCDR3 Protein Sequence AKDNHYGSGSYYYYQYGMDV SEQ ID NO: 50 – Common LCDR1 Protein Sequence QSVSSN SEQ ID NO: 51 – Common LCDR2 Protein Sequence GAS SEQ ID NO: 52 – Common LCDR3 Protein Sequence QHYINWPLT SEQ ID NO: 53 – Anti-CD3 HCDR1 Protein Sequence GFTFDDYT SEQ ID NO: 54 – Anti-CD3 HCDR2 Protein Sequence ISWNSGSI SEQ ID NO: 55 – Anti-CD3 HCDR3 Protein Sequence AKDNSGYGHYYYGMDV

[0311] In some embodiments, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof that can be used in the present disclosure comprises: (a) a first antigen binding domain that binds specifically to CD20; and (b) a second antigen-binding domain that binds specifically to CD3. In one embodiment, the anti-CD20 antigen-binding domain comprises the heavy chain complementarity determining regions (A-HCDRs) of a heavy chain variable region (A-HCVR) comprising the amino acid sequence of SEQ ID NO: 44 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the first antigen- binding domain comprises three HCDRs (A-HCDR1, A-HCDR2 and A-HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the A-HCDR1 comprises the amino acidAttorney Docket No.057766 / 624641 sequence of SEQ ID NO: 47; the A-HCDR2 comprises the amino acid sequence of SEQ ID NO: 48; the A-HCDR3 comprises the amino acid sequence of SEQ ID NO: 49; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 50; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 51; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 52.

[0312] In one embodiment, the second antigen-binding domain comprises the heavy chain complementarity determining regions (B-HCDRs) of a heavy chain variable region (B-HCVR) comprising the amino acid sequence of SEQ ID NO: 46 and the light chain complementarity determining regions (LCDRs) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45. In one embodiment, the second antigen-binding domain comprises three HCDRs (B-HCDR1, B-HCDR2 and B-HCDR3) and three LCDRs (LCDR1, LCDR2 and LCDR3), wherein the B-HCDR1 comprises the amino acid sequence of SEQ ID NO: 53; the B-HCDR2 comprises the amino acid sequence of SEQ ID NO: 54; the B-HCDR3 comprises the amino acid sequence of SEQ ID NO: 55; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 50; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 51; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 52.

[0313] In one embodiment, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises A-HCDR1, A- CDR2, and A-HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52; and (b) a second antigen binding domain that comprises B-HCDR1, B-HCDR2, and B-HCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 53, 54, and 55, and LCDR1, LCDR2, and LCDR3 domains, respectively, comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52. In one embodiment, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a A-HCVR comprising the amino acid sequence of SEQ ID NO: 44 and a LCVR comprising the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding domain that comprises a B-HCVR comprising the amino acid sequence of SEQ ID NO: 46 and a LCVR comprising the amino acid sequence of SEQ ID NO: 45.

[0314] Exemplary anti-CD20xCD3 bispecific antibodies include the fully human bispecific antibody known as REGN1979. See, e.g., US 2014 / 0088295, US 2015 / 0166661, and USAttorney Docket No.057766 / 624641 2017 / 0174781, each of which is herein incorporated by reference. According to certain exemplary embodiments, the methods of the present disclosure comprise the use of REGN1979, or a bioequivalent thereof. As used herein, the term “bioequivalent” with respect to anti- CD20xCD3 antibodies refers to antibodies or CD20xCD3 binding proteins or fragments thereof that are pharmaceutical equivalents or pharmaceutical alternatives having a rate and / or extent of absorption that does not show a significant difference with that of a reference antibody (e.g., REGN1979) when administered at the same molar dose under similar experimental conditions, either single dose or multiple dose; the term “bioequivalent” also includes antigen-binding proteins that bind to CD20 / CD3 and do not have clinically meaningful differences with the reference antibody (e.g., REGN1979) with respect to safety, purity, and / or potency.

[0315] In some embodiments, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises a A-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding domain that comprises a B-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 46 and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the anti-CD20xCD3 bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding domain that comprises three HCDRs (A-HCDR1, A- HCDR2 and A-HCDR3) comprising the amino acid sequences of SEQ ID NOs: 47, 48, and 49, respectively, and an A-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 44, and comprises three LCDRs (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 45; and (b) a second antigen-binding domain that comprises three HCDRs (B-HCDR1, B-HCDR2 and B-HCDR3) comprising the amino acid sequences of SEQ ID NOs: 53, 54, and 55, respectively, and a B-HCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 46, and comprises three LCDRsAttorney Docket No.057766 / 624641 (LCDR1, LCDR2 and LCDR3) comprising the amino acid sequences of SEQ ID NOs: 50, 51, and 52, respectively, and a LCVR having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:45.

[0316] The present disclosure also includes variants of the anti-CD20xCD3 antibodies described herein comprising any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein with one or more conservative amino acid substitutions. For example, the present disclosure includes use of anti-CD20xCD3 antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, the disclosure includes use of an anti- CD20xCD3 antibody having HCVR, LCVR, and / or CDR amino acid sequences with 1, 2, 3, or 4 conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0317] In some embodiments, the CDRs disclosed herein are identified according to the Kabat definition. In some embodiments, the CDRs are identified according to the Chothia definition. In some embodiments, the CDRs are identified according to the AbM definition. In some embodiments, the CDRs are identified according to the IMGT definition.

[0318] In some embodiments, the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region. In some embodiments, the human IgG heavy chain constant region is isotype IgG4 or IgG1. In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn). In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR). IV. Sequence Variants

[0319] The antigen-binding molecules of the present disclosure may comprise one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and / or light chain variable domains as compared to the corresponding germline sequences from which the individual antigen-binding domains were derived. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to germ lineAttorney Docket No.057766 / 624641 sequences available from, for example, public antibody sequence databases. The antigen-binding molecules of the present disclosure may comprise antigen binding fragments which are derived from any of the exemplary amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which the antibody was derived, or to the corresponding residue(s) of another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are referred to herein collectively as “germline mutations”). A person of ordinary skill in the art, starting with the heavy and light chain variable region sequences disclosed herein, can easily produce numerous antibodies and antigen-binding fragments which comprise one or more individual germline mutations or combinations thereof. In certain embodiments, all of the framework and / or CDR residues within the VH and / or VL domains are mutated back to the residues found in the original germline sequence from which the antigen-binding domain was originally derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., only the mutated residues found within the first 8 amino acids of FR1 or within the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residue(s) are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that is different from the germ line sequence from which the antigen-binding domain was originally derived). Furthermore, the antigen-binding domains may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., wherein certain individual residues are mutated to the corresponding residue of a particular germ line sequence while certain other residues that differ from the original germ line sequence are maintained or are mutated to the corresponding residue of a different germline sequence. Once obtained, antigen- binding domains that contain one or more germline mutations can be easily tested for one or more desired property such as, improved binding specificity, increased binding affinity, improved, or enhanced antagonistic or agonistic biological properties, reduced immunogenicity, etc. Bispecific antigen-binding molecules comprising one or more antigen-binding domains obtained in this general manner are encompassed within the present disclosure.

[0320] The present disclosure also includes antigen-binding molecules wherein one or both antigen-binding domains comprise variants of any of the HCVR, LCVR, and / or CDR amino acidAttorney Docket No.057766 / 624641 sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes antigen-binding molecules comprising an antigen-binding domain having HCVR, LCVR, and / or CDR amino acid sequences with, e.g., 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative amino acid substitution(s) relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, the disclosure includes use of an antibody having HCVR, LCVR and / or CDR amino acid sequences with 1, 2, 3, or 4 conservative amino acid substitutions relative to any of the HCVR, LCVR and / or CDR amino acid sequences disclosed herein. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine- glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0321] The present disclosure also includes antigen-binding molecules comprising an antigen binding domain with a HCVR, LCVR, and / or CDR amino acid sequence that is substantially identical to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein. In some embodiments, an antigen-binding molecule comprises a HCVR, LCVR, and / or CDR amino acid sequence having at least 85% sequence identity, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to a sequence disclosed in Table 1. In some embodiments, an antigen- binding molecule comprises a HCVR, LCVR, and / or CDR amino acid sequence having at leastAttorney Docket No.057766 / 624641 85% sequence identity, e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to a sequence disclosed in Table 1, wherein the differences in the amino acid residue(s) relative to the sequence disclosed in Table 1 are conservative substitutions or moderately conservative substitutions. V. Antigen-Binding Proteins Comprising Fc Modifications

[0322] In some embodiments, an antigen-binding molecule as disclosed herein (e.g., a BCMAxCD3 bispecific antigen-binding molecule such as an anti-BCMAxCD3 bispecific antibody or a CD20xCD3 bispecific antigen-binding molecule such as an anti-CD20xCD3 bispecific antibody) comprises an Fc domain comprising one or more modifications or mutations that enhance or diminish antibody binding to the FcRn receptor. For example, the present disclosure includes antigen-binding molecules comprising one or more mutations in the CH2 and / or CH3 region of the Fc domain, wherein the mutation(s) increases the affinity of the Fc domain to FcRn in an acidic environment (e.g., in an endosome where pH ranges from about 5.5 to about 6.0). Such mutations may result in an increase in serum half-life of the antibody when administered to an animal.

[0323] Non-limiting examples of such Fc modifications include, e.g., a modification at position 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or a modification at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., H / F or Y); or a modification at position 250 and / or 428; or a modification at position 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modification comprises a 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modification; a 433K (e.g., H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g., T250Q and M428L); and a 307 and / or 308 modification (e.g., 308F or 308P). See, e.g., Ko et al., BioDrugs 2021, 35:147-157.

[0324] In certain embodiments, a BCMAxCD3 bispecific antigen-binding molecule or a CD20xCD3 bispecific antigen-binding molecule comprises an Fc domain comprising one or more pairs or groups of mutations selected from the group consisting of: 250Q and 248L (e.g., T250Q and M248L); 252Y, 254T and 256E (e.g., M252Y, S254T and T256E); 428L and 434SAttorney Docket No.057766 / 624641 (e.g., M428L and N434S); and 433K and 434F (e.g., H433K and N434F). In some embodiments, the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn). For example, in some embodiments the human IgG heavy chain constant region comprises M252Y, S254T, and T256E mutations.

[0325] In some embodiments, the BCMAxCD3 bispecific antigen-binding molecules or the CD20xCD3 bispecific antigen-binding molecules of the present disclosure comprise a modified Fc domain having reduced effector function. As used herein, a “modified Fc domain having reduced effector function” means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc., relative to a wild-type, naturally occurring Fc domain such that a molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis and opsonization, relative to a comparator molecule comprising the wild-type, naturally occurring version of the Fc portion. In certain embodiments, a “modified Fc domain having reduced effector function” is an Fc domain with reduced or attenuated binding to an Fc receptor (e.g., FcγR).

[0326] In certain embodiments, a modified Fc domain having reduced binding to an Fc receptor, such as an Fc-gamma receptor (e.g., Fcγ receptor, e.g., FcγRI, FcγRIIA, FcγRIIB, or FcγRIIIA), is a variant IgG1 Fc or a variant IgG4 Fc comprising one or more substitutions or modifications in the hinge region and / or a CH region (e.g., CH2). For example, a modified Fc domain may comprise a variant IgG1 Fc wherein at least one amino acid of an IgG1 Fc hinge region and / or CH region is replaced with the corresponding amino acid from an IgG2 Fc hinge region and / or CH region. In certain embodiments, the modified Fc domain is a variant IgG1 Fc or a variant IgG4 Fc comprising one or more substitutions or modifications in the hinge region. For example, a modified Fc domain may comprise a variant IgG1 Fc wherein at least one amino acid of the IgG1 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. In one example, the variant IgG1 Fc can comprise a human IgG2 lower hinge amino acid sequence or can comprise both a human IgG2 lower hinge amino acid sequence and a human IgG4 CH2 amino acid sequence. For example, in some embodiments, the heavy chain constant region can comprise a variant IgG1 Fc in which positions 233-236 by EU numbering are occupied by PVA. See, e.g., US 10,988,537, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the heavy chain constant region can compriseAttorney Docket No.057766 / 624641 a variant IgG1 Fc in which the IgG1 CH2 region is replaced with the corresponding amino acids from the IgG4 CH2 region and in which positions 233-236 by EU numbering are occupied by PVA. Alternatively, a modified Fc domain may comprise a variant IgG4 Fc wherein at least one amino acid of an IgG4 Fc hinge region and / or CH region is replaced with the corresponding amino acid from an IgG2 Fc hinge region and / or CH region. Alternatively, a modified Fc domain may comprise a variant IgG4 Fc wherein at least one amino acid of the IgG4 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. In one example, the variant IgG4 Fc can comprise a human IgG2 lower hinge amino acid sequence. For example, in some embodiments, the heavy chain constant region can comprise a variant IgG4 Fc in which positions 233-236 by EU numbering are occupied by PVA. See, e.g., US 10,988,537, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, a modified Fc domain comprises a modified hinge region in which each of positions 233-236 by EU numbering is occupied by G or is unoccupied. In some embodiments, a modified Fc domain comprises modifications in which each of positions 233-236 by EU numbering is occupied by G or is unoccupied. For example, in some embodiments, a modified Fc domain can comprise a modified hinge region in which positions 233-236 by EU numbering are occupied by GGG. See, e.g., US 11,518,807, the disclosure of which is hereby incorporated by reference in its entirety. In some embodiments, the heavy chain constant region can comprise a variant IgG1 Fc in which the IgG1 CH2 region is replaced with the corresponding amino acids from the IgG4 CH2 region and in which positions 233-236 by EU numbering are occupied by GGG. Non-limiting, exemplary modified Fc regions that can be used in the context of the present disclosure are set forth in US Patent No.11,518,807, the disclosure of which is hereby incorporated by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions set forth therein. Other modified Fc domains and Fc modifications that can be used in the context of the present disclosure include any of the modifications as set forth in US 8,697,396, US 10,988,537, US 2014 / 0171623, US 2014 / 0134162, US 2014 / 0243504, and WO 2014 / 043361, the disclosures of each of which are incorporated by reference herein.

[0327] All possible combinations of the foregoing Fc domain mutations, and other mutations within the antibody variable domains disclosed herein, are contemplated within the scope of the present disclosure.Attorney Docket No.057766 / 624641 VI. Polynucleotides, Vectors, and Host Cells

[0328] In another aspect, the present disclosure provides nucleic acid molecules comprising one or more polynucleotide sequences encoding the antigen-binding molecules disclosed herein, as well as vectors (e.g., expression vectors) encoding such polynucleotide sequences and host cells into which such vectors have been introduced.

[0329] Polynucleotides, as disclosed herein, may encode all or a portion of an antigen- binding molecule, antibody, or antigen-binding fragment as disclosed throughout the present disclosure. In some cases, a single polynucleotide may encode both a HCVR and a LCVR (e.g., defined with reference to the CDRs contained within the respective amino acid sequence-defined HCVR and LCVR, defined with reference to the amino acid sequences of the CDRs of the HCVR and LCVR, respectively, or defined with reference to the amino acid sequences of the HCVR and LCVR, respectively) of an antibody or antigen-binding fragment, or the HCVR and LCVR may be encoded by separate polynucleotides (i.e., a pair of polynucleotides). In the latter case, in which the HCVR and LCVR are encoded by separate polynucleotides, the polynucleotides may be combined in a single vector or may be contained in separate vectors (i.e., a pair of vectors). In any case, a host cell used to express the polynucleotide(s) or vector(s) may contain the full complement of component parts to generate the antibody or antigen-binding fragment thereof. For example, a host cell may comprise separate vectors, each encoding a HCVR and a LCVR, respectively, of an antibody or antigen-binding fragment thereof as discussed above or herein. Similarly, the polynucleotide or polynucleotides, and the vector or vectors, may be used to express the full-length heavy chain and full-length light chain of an antibody as discussed above or herein. For example, a host cell may comprise a single vector with polynucleotides encoding both a heavy chain and a light chain of an antibody, or the host cell may comprise separate vectors with polynucleotides encoding, respectively, a heavy chain and a light chain of an antibody as disclosed above or herein.

[0330] In some embodiments, the nucleic acid molecule comprises one or more polynucleotide sequences encoding an antigen-binding molecule disclosed in Table 1.

[0331] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-BCMA HCVR comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 4, 6, and 8, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-BCMA HCVR comprising orAttorney Docket No.057766 / 624641 consisting of the sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 1, or a polynucleotide sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to SEQ ID NO: 1.

[0332] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD3 HCVR comprising the HCDR1, HCDR2, and HCDR3 of SEQ ID NOS: 28, 30, and 32, respectively; or of SEQ ID NOS: 36, 38, and 40, respectively. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an anti-CD3 HCVR comprising or consisting of the sequence of SEQ ID NO: 26 or SEQ ID NO: 34. In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence of SEQ ID NO: 25 or 33, or a polynucleotide sequence having at least 70% sequence identity, e.g., at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity, to SEQ ID NO: 25 or 33.

[0333] In some embodiments, the nucleic acid molecule comprises a polynucleotide sequence that encodes an LCVR comprising an LCDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 20, an LCDR2 comprising the amino acid sequence AAS (SEQ ID NO: 22), and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid molecule...

Claims

Attorney Docket No.057766 / 624641 We claim:

1. A method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus.

2. A method of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus.Attorney Docket No.057766 / 624641 3. A method of treating an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the polypeptide of interest comprises an enzyme to treat the enzyme deficiency; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the enzyme deficiency.

4. A method of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the enzyme deficiency is characterized by a loss-of-function of the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of a plasma cell depleting agent, wherein the subject has preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the enzyme deficiency.Attorney Docket No.057766 / 624641 5. The method of claim 3 or 4, wherein the subject has a disease of a bleeding disorder characterized by the enzyme deficiency, a disease of an inborn error of metabolism characterized by the enzyme deficiency, or a lysosomal storage disease characterized by the enzyme deficiency, optionally wherein the disease is hemophilia B and the polypeptide of interest is a factor IX protein, the disease is hemophilia A and the polypeptide of interest is a factor VIII protein, or the disease is Pompe disease and the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase.

6. The method of any preceding claim, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) the nuclease agent or the one or more nucleic acids encoding the nuclease agent; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

7. The method of any one of claims 1-5, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

8. The method of any one of claims 1-5, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct;Attorney Docket No.057766 / 624641 (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

9. The method of any one of claims 1-5, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a second coding sequence for the polypeptide of interest, wherein the second coding sequence is different from the first coding sequence; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the plasma cell depleting agent, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

10. The method of any one of claims 6-9, further comprising the following steps prior to the subsequent administration step: (i) measuring expression and / or activity of the polypeptide of interest in the subject; and (ii) determining the dose of the nucleic acid construct and the nuclease agent or the one or more nucleic acids encoding the nuclease agent for the subsequent administrationAttorney Docket No.057766 / 624641 step in order to achieve the desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

11. The method of any one of claims 6-10, wherein the polypeptide of interest is a factor IX protein, and the desired expression level of the factor IX protein in the subject is a serum level of at least about 3 ^g / mL or about 3-5 ^g / mL.

12. The method of any one of claims 6-10, wherein the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha- glucosidase, and the desired expression level of the multidomain therapeutic protein in the subject is a serum level of at least about 2 ^g / mL or at least about 5 ^g / mL.

13. The method of any one of claims 1-5, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a coding sequence for a second polypeptide of interest that is different from the first polypeptide of interest; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the plasma cell depleting agent, wherein the second nuclease agent cleaves the second nuclease target site, and the second nucleic acid construct is inserted into the second target genomic locus.

14. The method of any one of claims 6-13, wherein the one or more subsequent administration steps is one subsequent administration step.Attorney Docket No.057766 / 624641 15. The method of any one of claims 6-13, wherein the one or more subsequent administration steps is two subsequent administration steps or comprises at least two subsequent administration steps.

16. The method of any one of claims 6-15, wherein the plasma cell depleting agent is administered in the one or more subsequent administration steps if there is no preexisting plasma cell depleting agent in the subject or if preexisting plasma cell depleting agent expression and / or activity levels are below a desired threshold level, optionally wherein the method comprises measuring the plasma cell depleting agent expression and / or activity levels prior to the one or more subsequent administration steps.

17. The method of any preceding claim, wherein the plasma cell depleting agent is capable of depleting long-lived plasma cells (LLPC).

18. The method of any preceding claim, wherein the plasma cell depleting agent is a B cell maturation antigen (BCMA) targeting agent.

19. The method of claim 18, wherein the BCMA targeting agent is a chimeric antigen receptor against BCMA or an anti-BCMA antibody or a functional fragment thereof.

20. The method of claim 19, wherein the anti-BCMA antibody or functional fragment thereof is conjugated to a cytotoxic agent.

21. The method of claim 19 or 20, wherein the anti-BCMA antibody is a multispecific antibody or a functional fragment thereof.

22. The method of claim 21, wherein the multispecific anti-BCMA antibody or functional fragment thereof targets BCMA and CD3.

23. The method of claim 22, wherein the multispecific anti-BCMA antibody or functional fragment thereof is anti-BCMAxCD3 bispecific antibody or functional fragment thereof.

24. The method of claim 23, wherein the anti-BCMAxCD3 bispecific antibody is selected from linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420),Attorney Docket No.057766 / 624641 teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B.

25. The method of claim 23, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to BCMA comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18.

26. The method of claim 25, wherein the first antigen-binding domain that specifically binds to BCMA comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

24.

27. The method of claim 25 or 26, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 26 and 34, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18.

28. The method of claim 27, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or 36, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 38, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 32 or 40, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 24.Attorney Docket No.057766 / 624641 29. The method of any one of claims 25-28, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 28, 30, and 32, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively.

30. The method of claim 29, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 36, 38, and 40, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively.

31. The method of any one of claims 23-30, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc- gamma receptor (FcγR).

32. The method of claim 31, wherein the human IgG heavy chain constant region is isotype IgG4 or IgG1.Attorney Docket No.057766 / 624641 33. The method of any preceding claim, further comprising administering to the subject an effective amount of a B cell depleting agent and / or an immunoglobulin depleting agent, optionally further comprising administering to the subject an effective amount of a B cell depleting agent and an immunoglobulin depleting agent.

34. The method of claim 33, wherein the B cell depleting agent is administered before, at the same time as, or after the plasma cell depleting agent.

35. The method of claim 33 or 34, wherein the B cell depleting agent is administered prior to and after the nucleic acid construct.

36. The method of any one of claims 33-35, wherein the immunoglobulin depleting agent is administered prior to and after the nucleic acid construct.

37. The method of any one of claims 33-36, wherein the immunoglobulin depleting agent is administered after an initial dose of the plasma cell depleting agent, or wherein the immunoglobulin depleting agent is administered after an initial dose of the plasma cell depleting agent and after an initial dose of the B cell depleting agent.

38. The method of any one of claims 33-37, wherein the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA.

39. The method of any one of claims 33-38, wherein the B cell depleting agent is an agent that binds to a B cell surface molecule.

40. The method of claim 39, wherein the B cell depleting agent is selected from an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD19 antibody and an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD79 antibody, an anti-CD20xCD3 bispecific antibody, an anti-CD19xCD3 bispecific antibody, an anti-CD22xCD3 bispecific antibody, an anti-CD79xCD3 bispecific antibody, functional fragments of any of said antibodies, and any combinations thereof.

41. The method of any one of claims 33-40, wherein the B cell depleting agent is an anti-CD20 antibody or a functional fragment thereof, wherein the anti-CD20 antibody is a multispecific antibody or a functional fragment thereof.Attorney Docket No.057766 / 624641 42. The method of claim 41, wherein the multispecific anti-CD20 antibody or functional fragment thereof targets CD20 and CD3.

43. The method of claim 42, wherein the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD20xCD3 bispecific antibody or functional fragment thereof.

44. The method of claim 43, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

45.

45. The method of claim 44, wherein the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

52.

46. The method of claim 44 or 45, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

45.

47. The method of claim 46, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acidAttorney Docket No.057766 / 624641 sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

52.

48. The method of any one of claims 44-47, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

49. The method of any one of claims 43-48, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc- gamma receptor (FcγR).

50. The method of claim 49, wherein the human IgG heavy chain constant region is isotype IgG4 or IgG1.

51. The method of any one of claims 33-38, wherein the B cell depleting agent is an agent targeting a B cell survival factor.

52. The method of any one of claims 33-38, wherein the B cell depleting agent is a BLyS / BAFF inhibitor, an APRIL inhibitor, a BLyS receptor 3 / BAFF receptor inhibitor, or any combination thereof.

53. The method of any one of claims 33-52, wherein the immunoglobulin depleting agent is capable of accelerating IgG clearance.Attorney Docket No.057766 / 624641 54. The method of any one of claims 33-53, wherein the immunoglobulin depleting agent is a neonatal Fc receptor (FcRn) blocker.

55. The method of claim 54, wherein the FcRn blocker is selected from Efgartigimod (ARGX-113), Rozanolixizumab (UCB7665), Batoclimab (RVT-1401), IMVT- 1402, Nipocalimab (M281), Orilanolimab (SYNT001), and any combinations thereof.

56. The method of any preceding claim, wherein the method further comprises plasmapheresis, therapeutic plasma exchange, or immunoadsorption.

57. The method of any preceding claim, wherein the plasma cell depleting agent is administered simultaneously with the nucleic acid construct.

58. The method of any one of claims 1-56, wherein the plasma cell depleting agent is administered prior to the nucleic acid construct.

59. The method of any one of claims 1-56, wherein the plasma cell depleting agent is administered prior to and after the nucleic acid construct.

60. The method of claim 59, wherein the plasma cell depleting agent is administered within about 6 months after the nucleic acid construct, optionally wherein the nucleic acid construct is in a viral vector, and the plasma cell depleting agent is administered if the viral vector is still present in the subject.

61. The method of any one of claims 58-60, wherein the nucleic acid construct is administered within about 3 months, within about 2 months, within about 7 weeks, within about 6 weeks, within about 5 weeks, within about 4 weeks, within about 3 weeks, or within about 2 weeks after an initial dose of the plasma cell depleting agent, or wherein the nucleic acid construct is administered at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 2 months, or at least about 3 months after an initial dose of the plasma cell depleting agent.

62. The method of any one of claims 58-61, wherein the plasma cell depleting agent is administered about 1 week prior to or within about 1 week prior to the nucleic acid construct.Attorney Docket No.057766 / 624641 63. The method of any preceding claim, wherein the nucleic acid construct is administered simultaneously with the nuclease agent or the one or more nucleic acids encoding the nuclease agent.

64. The method of any one of claims 1-62, wherein the nucleic acid construct is administered prior to or after the nuclease agent or the one or more nucleic acids encoding the nuclease agent.

65. The method of any preceding claim, wherein the nucleic acid construct is in the nucleic acid vector, optionally wherein the nucleic acid vector is a viral vector, and optionally wherein the viral vector is administered at a dose of about 3E11 vg / kg to about 5E13 vg / kg.

66. The method of claim 65, wherein the nucleic acid vector is an adeno- associated viral (AAV) vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO:

281.

67. The method of claim 66, wherein the AAV vector is a single-stranded AAV (ssAAV) vector.

68. The method of claim 66 or 67, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector.

69. The method of any preceding claim, wherein the polypeptide of interest is a factor IX protein.

70. The method of claim 69, wherein the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO: 97.Attorney Docket No.057766 / 624641 71. The method of claim 69 or 70, wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO:

61.

72. The method of any one of claims 69-71, wherein the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence.

73. The method of claim 72, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

74. The method of claim 72 or 73, wherein the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.

75. The method of any one of claims 69-71, wherein the nucleic acid construct is a unidirectional construct.

76. The method of claim 75, wherein the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence,Attorney Docket No.057766 / 624641 wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

77. The method of any one of claims 1-68, wherein the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha- glucosidase.

78. The method of claim 77, wherein the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO:

296.

79. The method of claim 77 or 78, wherein the lysosomal alpha-glucosidase coding sequence comprises or consist of the sequence set forth in SEQ ID NO:

857.

80. The method of any one of claims 77-79, wherein the delivery domain is a CD63-binding delivery domain.

81. The method of claim 80, wherein the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein.

82. The method of claim 80 or 81, wherein the CD63-binding delivery domain is a single-chain variable fragment (scFv).

83. The method of claim 82, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO:

306.

84. The method of claim 82 or 83, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO:

866.

85. The method of any one of claims 80-84, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 316.Attorney Docket No.057766 / 624641 86. The method of any one of claims 80-85, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884.

87. The method of any one of claims 80-86, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

88. The method of any one of claims 77-79, wherein the delivery domain is a TfR-binding delivery domain.

89. The method of claim 88, wherein the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein.

90. The method of claim 89, wherein the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).Attorney Docket No.057766 / 624641 91. The method of claim 89 or 90, wherein the anti-TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof).

92. The method of any one of claims 89-91, wherein the anti-TfR antigen- binding protein comprises a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

93. The method of any one of claims 89-91, wherein the TfR-binding delivery domain comprises a single-chain variable fragment (scFv).

94. The method of claim 93, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO:

672.

95. The method of claim 93 or 94, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO:

713.

96. The method of any one of claims 88-95, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO:

691.

97. The method of any one of claims 88-96, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871.Attorney Docket No.057766 / 624641 98. The method of any one of claims 88-97, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

99. The method of any one of claims 1-68, wherein the polypeptide of interest is a factor VIII protein.

100. The method of any one of claims 1-68, wherein the polypeptide of interest is an antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

101. The method of any preceding claim, wherein the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene.

102. The method of claim 101, wherein the nuclease target site is in intron 1 of the albumin gene.

103. The method of any preceding claim, wherein the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; andAttorney Docket No.057766 / 624641 (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

104. The method of any one of claims 1-102, wherein the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

105. The method of claim 104, wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164, or wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 153- 184, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164.

106. The method of claim 104 or 105, wherein the guide RNA comprises any one of SEQ ID NOS: 185-248, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228.

107. The method of any one of claims 104-106, wherein the DNA-targeting segment comprises or consists of SEQ ID NO:

159.

108. The method of any one of claims 104-107, wherein the guide RNA comprises SEQ ID NO: 191 or 223.

109. The method of any one of claims 104-108, wherein the method comprises administering the guide RNA in the form of RNA.Attorney Docket No.057766 / 624641 110. The method of any one of claims 104-109, wherein the guide RNA comprises at least one modification.

111. The method of claim 110, wherein the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

112. The method of any one of claims 104-111, wherein the method comprises administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

113. The method of any one of claims 104-112, wherein the Cas protein is a Cas9 protein, optionally wherein the Cas protein is derived from a Streptococcus pyogenes Cas9 protein.

114. The method of any one of claims 104-113, wherein the Cas protein comprises the sequence set forth in SEQ ID NO:

134.

115. The method of any one of claims 104-114, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein.

116. The method of claim 115, wherein the mRNA encoding the Cas protein comprises at least one modification.

117. The method of claim 116, wherein the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine.Attorney Docket No.057766 / 624641 118. The method of any one of claims 115-117, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

119. The method of any one of claims 104-118, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

120. The method of any one of claims 104-119, wherein the method comprises administering the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

121. The method of any one of claims 104-120, wherein the method comprises administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

122. The method of any one of claims 104-121, wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle.Attorney Docket No.057766 / 624641 123. The method of claim 122, wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid.

124. The method of claim 123, wherein the cationic lipid is Lipid A ((9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or wherein the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), and / or wherein the helper lipid is cholesterol, and / or wherein the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

125. The method of claim 124, wherein the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG.

126. The method of any one of claims 123-125, wherein the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

127. The method of any preceding claim, wherein the cell is a liver cell or a hepatocyte, or the population of cells is a population of liver cells or hepatocytes.

128. The method of any preceding claim, wherein the subject is a human subject.

129. The method of any preceding claim, wherein the subject is a neonatal subject.

130. The method of any preceding claim, further comprising determining whether the subject has immunity against the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or the delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent prior to the administering,Attorney Docket No.057766 / 624641 optionally wherein the determining comprises determining the presence of neutralizing antibodies against the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or the delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent.

131. The method of any preceding claim, wherein the nucleic acid vector is in an adeno-associated viral (AAV) vector, and wherein the subject has preexisting AAV immunity.

132. A composition or combination comprising an effective amount of a plasma cell depleting agent in combination with: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus.

133. The composition or combination of claim 132, wherein the plasma cell depleting agent is capable of depleting long-lived plasma cells (LLPC).

134. The composition or combination of claim 132 or 133, wherein the plasma cell depleting agent is a B cell maturation antigen (BCMA) targeting agent.

135. The composition or combination of claim 134, wherein the BCMA targeting agent is a chimeric antigen receptor against BCMA or an anti-BCMA antibody or a functional fragment thereof.

136. The composition or combination of claim 135, wherein the anti-BCMA antibody or functional fragment thereof is conjugated to a cytotoxic agent.

137. The composition or combination of claim 135 or 136, wherein the anti- BCMA antibody is a multispecific antibody or a functional fragment thereof.

138. The composition or combination of claim 137, wherein the multispecific anti-BCMA antibody or functional fragment thereof targets BCMA and CD3.Attorney Docket No.057766 / 624641 139. The composition or combination of claim 138, wherein the multispecific anti-BCMA antibody or functional fragment thereof is anti-BCMAxCD3 bispecific antibody or functional fragment thereof.

140. The composition or combination of claim 139, wherein the anti- BCMAxCD3 bispecific antibody is selected from linvoseltamab (REGN5458), REGN5459, pacanalotamab (AMG420), teclistamab (JNJ-64007957), AMG701, alnuctamab (CC-93269), EM801, EM901, elranatamab (PF-06863135), TNB383B (ABBV-383), and TNB384B .

141. The composition or combination of claim 139, wherein the anti- BCMAxCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to BCMA comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

18.

142. The composition or combination of claim 141, wherein the first antigen- binding domain that specifically binds to BCMA comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 6, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 8, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

24.

143. The composition or combination of claim 141 or 142, wherein the anti- BCMAxCD3 bispecific antibody or functional fragment thereof comprises a second antigen- binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 26 and 34, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 18.Attorney Docket No.057766 / 624641 144. The composition or combination of claim 143, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or 36, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 30 or 38, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 32 or 40, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

24.

145. The composition or combination of any one of claims 141-144, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 28, 30, and 32, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively.

146. The composition or combination of claim 145, wherein the anti- BCMAxCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 4, 6, and 8, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 36, 38, and 40, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 20, 22, and 24, respectively.

147. The composition or combination of any one of claims 139-146, wherein the anti-BCMAxCD3 bispecific antibody or functional fragment thereof comprises a human IgGAttorney Docket No.057766 / 624641 heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR).

148. The composition or combination of claim 147, wherein the human IgG heavy chain constant region is isotype IgG4 or IgG1.

149. The composition or combination of any one of claims 132-148, wherein the plasma cell depleting agent is further in combination with an effective amount of a B cell depleting agent and / or an immunoglobulin depleting agent, optionally wherein the plasma cell depleting agent is further in combination with an effective amount of a B cell depleting agent and an immunoglobulin depleting agent.

150. The composition or combination of claim 149, wherein the B cell depleting agent is capable of depleting B cells and plasma cells that express low levels of BCMA.

151. The composition or combination of claim 149 or 150, wherein the B cell depleting agent is an agent that binds to a B cell surface molecule.

152. The composition or combination of claim 151, wherein the B cell depleting agent is selected from an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD19 antibody and an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD79 antibody, an anti- CD20xCD3 bispecific antibody, an anti-CD19xCD3 bispecific antibody, an anti-CD22xCD3 bispecific antibody, an anti-CD79xCD3 bispecific antibody, functional fragments of any of said antibodies, and any combinations thereof.

153. The composition or combination of any one of claims 149-152, wherein the B cell depleting agent is an anti-CD20 antibody or a functional fragment thereof, wherein the anti-CD20 antibody is a multispecific antibody or a functional fragment thereof.

154. The composition or combination of claim 153, wherein the multispecific anti-CD20 antibody or functional fragment thereof targets CD20 and CD3.Attorney Docket No.057766 / 624641 155. The composition or combination of claim 154, wherein the multispecific anti-CD20 antibody or functional fragment thereof is anti-CD20xCD3 bispecific antibody or functional fragment thereof.

156. The composition or combination of claim 155, wherein the anti- CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

45.

157. The composition or combination of claim 156, wherein the first antigen- binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

52.

158. The composition or combination of claim 156 or 157, wherein the anti- CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen- binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

45.

159. The composition or combination of claim 158, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acidAttorney Docket No.057766 / 624641 sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

52.

160. The composition or combination of any one of claims 156-159, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

161. The composition or combination of any one of claims 155-160, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR).

162. The composition or combination of claim 161, wherein the human IgG heavy chain constant region is isotype IgG4 or IgG1.

163. The composition or combination of claim 149 or 150, wherein the B cell depleting agent is an agent targeting a B cell survival factor.

164. The composition or combination of claim 149 or 150, wherein the B cell depleting agent is a BLyS / BAFF inhibitor, an APRIL inhibitor, a BLyS receptor 3 / BAFF receptor inhibitor, or any combination thereof.

165. The composition or combination of any one of claims 149-164, wherein the immunoglobulin depleting agent is capable of accelerating IgG clearance.Attorney Docket No.057766 / 624641 166. The composition or combination of any one of claims 149-165, wherein the immunoglobulin depleting agent is a neonatal Fc receptor (FcRn) blocker.

167. The composition or combination of claim 166, wherein the FcRn blocker is selected from Efgartigimod (ARGX-113), Rozanolixizumab (UCB7665), Batoclimab (RVT- 1401), IMVT-1402, Nipocalimab (M281), Orilanolimab (SYNT001), and any combinations thereof.

168. The composition or combination of any one of claims 132-167, wherein the nucleic acid construct is in the nucleic acid vector, optionally wherein the nucleic acid vector is a viral vector.

169. The composition or combination of claim 168, wherein the nucleic acid vector is an adeno-associated viral (AAV) vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO:

281.

170. The composition or combination of claim 169, wherein the AAV vector is a single-stranded AAV (ssAAV) vector.

171. The composition or combination of claim 169 or 170, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector.

172. The composition or combination of any one of claims 132-171, wherein the polypeptide of interest is a factor IX protein.

173. The composition or combination of claim 172, wherein the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO: 97.Attorney Docket No.057766 / 624641 174. The composition or combination of claim 172 or 173, wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO:

61.

175. The composition or combination of any one of claims 172-174, wherein the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence.

176. The composition or combination of claim 175, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

177. The composition or combination of claim 175 or 176, wherein the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.

178. The composition or combination of any one of claims 172-174, wherein the nucleic acid construct is a unidirectional construct.

179. The composition or combination of claim 178, wherein the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence,Attorney Docket No.057766 / 624641 wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

180. The composition or combination of any one of claims 132-171, wherein the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase.

181. The composition or combination of claim 180, wherein the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO:

296.

182. The composition or combination of claim 180 or 181, wherein the lysosomal alpha-glucosidase coding sequence comprises or consist of the sequence set forth in SEQ ID NO:

857.

183. The composition or combination of any one of claims 180-182, wherein the delivery domain is a CD63-binding delivery domain.

184. The composition or combination of claim 183, wherein the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein.

185. The composition or combination of claim 183 or 184, wherein the CD63- binding delivery domain is a single-chain variable fragment (scFv).

186. The composition or combination of claim 185, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO:

306.

187. The composition or combination of claim 185 or 186, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 866.Attorney Docket No.057766 / 624641 188. The composition or combination of any one of claims 183-187, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO:

316.

189. The composition or combination of any one of claims 183-188, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884.

190. The composition or combination of any one of claims 183-189, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

191. The composition or combination of any one of claims 180-182, wherein the delivery domain is a TfR-binding delivery domain.

192. The composition or combination of claim 191, wherein the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein.Attorney Docket No.057766 / 624641 193. The composition or combination of claim 192, wherein the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

194. The composition or combination of claim 192 or 193, wherein the anti- TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof).

195. The composition or combination of any one of claims 192-194, wherein the anti-TfR antigen-binding protein comprises a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

196. The composition or combination of any one of claims 192-195, wherein the TfR-binding delivery domain comprises a single-chain variable fragment (scFv).

197. The composition or combination of claim 196, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO:

672.

198. The composition or combination of claim 196 or 197, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO:

713.

199. The composition or combination of any one of claims 191-198, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 691.Attorney Docket No.057766 / 624641 200. The composition or combination of any one of claims 191-199, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871.

201. The composition or combination of any one of claims 191-200, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

202. The composition or combination of any one of claims 132-171, wherein the polypeptide of interest is a factor VIII protein.

203. The composition or combination of any one of claims 132-171, wherein the polypeptide of interest is an antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

204. The composition or combination of any one of claims 132-203, wherein the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene.Attorney Docket No.057766 / 624641 205. The composition or combination of claim 204, wherein the nuclease target site is in intron 1 of the albumin gene.

206. The composition or combination of any one of claims 132-205, wherein the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

207. The composition or combination of any one of claims 132-205, wherein the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

208. The composition or combination of claim 207, wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164, or wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 153- 184, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164.

209. The composition or combination of claim 207 or 208, wherein the guide RNA comprises any one of SEQ ID NOS: 185-248, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228.Attorney Docket No.057766 / 624641 210. The composition or combination of any one of claims 207-209, wherein the DNA-targeting segment comprises or consists of SEQ ID NO:

159.

211. The composition or combination of any one of claims 207-210, wherein the guide RNA comprises SEQ ID NO: 191 or 223.

212. The composition or combination of any one of claims 207-211, wherein the composition or combination comprises the guide RNA in the form of RNA.

213. The composition or combination of any one of claims 207-212, wherein the guide RNA comprises at least one modification.

214. The composition or combination of claim 213, wherein the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

215. The composition or combination of any one of claims 207-214, wherein the composition or combination comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

216. The composition or combination of any one of claims 207-215, wherein the Cas protein is a Cas9 protein, optionally wherein the Cas protein is derived from a Streptococcus pyogenes Cas9 protein.

217. The composition or combination of any one of claims 207-216, wherein the Cas protein comprises the sequence set forth in SEQ ID NO: 134.Attorney Docket No.057766 / 624641 218. The composition or combination of any one of claims 207-217, wherein the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein.

219. The composition or combination of claim 218, wherein the mRNA encoding the Cas protein comprises at least one modification.

220. The composition or combination of claim 219, wherein the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine.

221. The composition or combination of any one of claims 218-220, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

222. The composition or combination of any one of claims 207-221, wherein the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

223. The composition or combination of any one of claims 207-222, wherein the composition or combination comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and wherein the composition or combination comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

224. The composition or combination of any one of claims 207-223, wherein the composition or combination comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotidesAttorney Docket No.057766 / 624641 at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and wherein the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

225. The composition or combination of any one of claims 207-224, wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle.

226. The composition or combination of claim 225, wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid.

227. The composition or combination of claim 226, wherein the cationic lipid is Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or wherein the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), and / or wherein the helper lipid is cholesterol, and / or wherein the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

228. The composition or combination of claim 227, wherein the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k- DMG.

229. The composition or combination of any one of claims 226-228, wherein the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.Attorney Docket No.057766 / 624641 230. The composition or combination of any one of claims 132-229, for use in a method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject.

231. The composition or combination of any one of claims 132-229, for use in a method of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject.

232. The composition or combination of any one of claims 132-229, for use in a method of treating an enzyme deficiency in a subject in need thereof.

233. The composition or combination of any one of claims 132-229, for use in a method of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof.

234. A kit comprising the composition or combination of any one of claims 132-233.

235. A plasma cell depleting agent for use in a method according to any one of claims 1-131.

236. A method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, andAttorney Docket No.057766 / 624641 wherein the nuclease agent cleaves the nuclease target site, and the nucleic acid construct is inserted into the target genomic locus.

237. A method of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in the target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus.

238. A method of treating an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the polypeptide of interest comprises an enzyme to treat the enzyme deficiency; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, andAttorney Docket No.057766 / 624641 wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby treating the enzyme deficiency.

239. A method of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof, comprising administering to the subject: (a) a nucleic acid construct comprising a coding sequence for a polypeptide of interest, wherein the enzyme deficiency is characterized by a loss-of-function of the polypeptide of interest; (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus; and (c) an effective amount of an anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the subject does not have preexisting immunity to the nucleic acid construct, the polypeptide of interest, the nuclease agent, the one or more nucleic acids encoding the nuclease agent, or a delivery vehicle for the nucleic acid construct, the nuclease agent, or the one or more nucleic acids encoding the nuclease agent, and wherein the nuclease agent cleaves the nuclease target site, the nucleic acid construct is inserted into the target genomic locus to create a modified target genomic locus, and the polypeptide of interest is expressed from the modified target genomic locus, thereby preventing or reducing the onset of the sign or symptom of the enzyme deficiency.

240. The method of claim 238 or 239, wherein the subject has a disease of a bleeding disorder characterized by the enzyme deficiency, a disease of an inborn error of metabolism characterized by the enzyme deficiency, or a lysosomal storage disease characterized by the enzyme deficiency, optionally wherein the disease is hemophilia B and the polypeptide of interest is a factor IX protein, the disease is hemophilia A and the polypeptide of interest is a factor VIII protein, or the disease is Pompe disease and the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase.Attorney Docket No.057766 / 624641 241. The method of any one of claims 236-240, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) the nuclease agent or the one or more nucleic acids encoding the nuclease agent; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

242. The method of any one of claims 236-240, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

243. The method of any one of claims 236-240, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) the nucleic acid construct; (b) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.Attorney Docket No.057766 / 624641 244. The method of any one of claims 236-240, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a second coding sequence for the polypeptide of interest, wherein the second coding sequence is different from the first coding sequence; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, until a desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

245. The method of any one of claims 241-244, further comprising the following steps prior to the subsequent administration step: (i) measuring expression and / or activity of the polypeptide of interest in the subject; and (ii) determining the dose of the nucleic acid construct and the nuclease agent or the one or more nucleic acids encoding the nuclease agent for the subsequent administration step in order to achieve the desired level of expression and / or activity of the polypeptide of interest is achieved in the subject.

246. The method of any one of claims 241-245, wherein the polypeptide of interest is a factor IX protein, and the desired expression level of the factor IX protein in the subject is a serum level of at least about 3 ^g / mL or about 3-5 ^g / mL.Attorney Docket No.057766 / 624641 247. The method of any one of claims 241-245, wherein the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase, and the desired expression level of the multidomain therapeutic protein in the subject is a serum level of at least about 2 ^g / mL or at least about 5 ^g / mL.

248. The method of any one of claims 236-240, further comprising a subsequent administration step comprising administering to the subject at one or more subsequent times: (a) a second nucleic acid construct comprising a coding sequence for a second polypeptide of interest that is different from the first polypeptide of interest; (b) (i) the first nuclease agent or the one or more nucleic acids encoding the first nuclease agent; (ii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in the target genomic locus, wherein the second nuclease target site is different from the first nuclease target site; or (iii) a second nuclease agent or one or more nucleic acids encoding the second nuclease agent, wherein the second nuclease agent targets a second nuclease target site in a second target genomic locus that is different from the first target genomic locus; and optionally (c) the anti-CD20xCD3 bispecific antibody or functional fragment thereof, wherein the second nuclease agent cleaves the second nuclease target site, and the second nucleic acid construct is inserted into the second target genomic locus.

249. The method of any one of claims 241-248, wherein the one or more subsequent administration steps is one subsequent administration step.

250. The method of any one of claims 241-248, wherein the one or more subsequent administration steps is two subsequent administration steps or comprises at least two subsequent administration steps.

251. The method of any one of claims 241-250, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered in the one or more subsequent administration steps if there is no preexisting anti-CD20xCD3 bispecific antibody or functionalAttorney Docket No.057766 / 624641 fragment thereof in the subject or if the preexisting the expression and / or activity levels of the anti-CD20xCD3 bispecific antibody or functional fragment thereof are below a desired threshold level, optionally wherein the method comprises measuring the expression and / or activity levels of the anti-CD20xCD3 bispecific antibody or functional fragment thereof prior to the one or more subsequent administration steps.

252. The method of any one of claims 236-251, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

45.

253. The method of claim 252, wherein the first antigen-binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

52.

254. The method of claim 252 or 253, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen-binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

45.

255. The method of claim 254, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO: 52.Attorney Docket No.057766 / 624641 256. The method of any one of claims 252-255, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

257. The method of any one of claims 236-256, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc- gamma receptor (FcγR).

258. The method of claim 257, wherein the human IgG heavy chain constant region is isotype IgG4 or IgG1.

259. The method of any one of claims 236-258, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered simultaneously with the nucleic acid construct.

260. The method of any one of claims 236-259, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered prior to the nucleic acid construct.

261. The method of any one of claims 236-260, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof is administered prior to and after the nucleic acid construct.Attorney Docket No.057766 / 624641 262. The method of claim 260 or 261, wherein the nucleic acid construct is administered within about 3 months, within about 2 months, within about 7 weeks, within about 6 weeks, within about 5 weeks, within about 4 weeks, within about 3 weeks, within about 2 weeks, or within about 1 week after an initial dose of the anti-CD20xCD3 bispecific antibody or functional fragment thereof, or wherein the nucleic acid construct is administered at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 2 months, or at least about 3 months after an initial dose of the anti-CD20xCD3 bispecific antibody or functional fragment thereof.

263. The method of any one of claims 236-262, wherein the nucleic acid construct is administered simultaneously with the nuclease agent or the one or more nucleic acids encoding the nuclease agent.

264. The method of any one of claims 236-262, wherein the nucleic acid construct is administered prior to or after the nuclease agent or the one or more nucleic acids encoding the nuclease agent.

265. The method of any one of claims 236-264, wherein the nucleic acid construct is in the nucleic acid vector, optionally wherein the nucleic acid vector is a viral vector, and optionally wherein the viral vector is administered at a dose of about 3E11 vg / kg to about 5E13 vg / kg.

266. The method of claim 265, wherein the nucleic acid vector is an adeno- associated viral (AAV) vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 281.Attorney Docket No.057766 / 624641 267. The method of claim 266, wherein the AAV vector is a single-stranded AAV (ssAAV) vector.

268. The method of claim 266 or 267, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector.

269. The method of any one of claims 236-268, wherein the polypeptide of interest is a factor IX protein.

270. The method of claim 269, wherein the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO:

97.

271. The method of claim 269 or 270, wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO:

61.

272. The method of any one of claims 269-271, wherein the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence.

273. The method of claim 272, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, andAttorney Docket No.057766 / 624641 wherein the nucleic acid construct does not comprise homology arms.

274. The method of claim 272 or 273, wherein the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.

275. The method of any one of claims 269-271, wherein the nucleic acid construct is a unidirectional construct.

276. The method of claim 275, wherein the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

277. The method of any one of claims 236-268, wherein the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase.

278. The method of claim 277, wherein the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO:

296.

279. The method of claim 277 or 278, wherein the lysosomal alpha-glucosidase coding sequence comprises or consist of the sequence set forth in SEQ ID NO:

857.

280. The method of any one of claims 277-279, wherein the delivery domain is a CD63-binding delivery domain.

281. The method of claim 280, wherein the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein.Attorney Docket No.057766 / 624641 282. The method of claim 280 or 281, wherein the CD63-binding delivery domain is a single-chain variable fragment (scFv).

283. The method of claim 282, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO:

306.

284. The method of claim 282 or 283, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO:

866.

285. The method of any one of claims 280-284, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO:

316.

286. The method of any one of claims 280-285, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884.

287. The method of any one of claims 280-286, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.Attorney Docket No.057766 / 624641 288. The method of any one of claims 277-279, wherein the delivery domain is a TfR-binding delivery domain.

289. The method of claim 288, wherein the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein.

290. The method of claim 289, wherein the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

291. The method of claim 289 or 290, wherein the anti-TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof).

292. The method of any one of claims 289-291, wherein the anti-TfR antigen- binding protein comprises a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

293. The method of any one of claims 289-292, wherein the TfR-binding delivery domain comprises a single-chain variable fragment (scFv).

294. The method of claim 293, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO: 672.Attorney Docket No.057766 / 624641 295. The method of claim 293 or 294, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO:

713.

296. The method of any one of claims 288-295, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO:

691.

297. The method of any one of claims 288-296, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871.

298. The method of any one of claims 288-297, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

299. The method of any one of claims 236-268, wherein the polypeptide of interest is a factor VIII protein.Attorney Docket No.057766 / 624641 300. The method of any one of claims 236-268, wherein the polypeptide of interest is an antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.

301. The method of any one of claims 236-300, wherein the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene.

302. The method of claim 301, wherein the nuclease target site is in intron 1 of the albumin gene.

303. The method of any one of claims 236-302, wherein the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

304. The method of any one of claims 236-302, wherein the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

305. The method of claim 304, wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164, orAttorney Docket No.057766 / 624641 wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 153- 184, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164.

306. The method of claim 304 or 305, wherein the guide RNA comprises any one of SEQ ID NOS: 185-248, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228.

307. The method of any one of claims 304-306, wherein the DNA-targeting segment comprises or consists of SEQ ID NO:

159.

308. The method of any one of claims 304-307, wherein the guide RNA comprises SEQ ID NO: 191 or 223.

309. The method of any one of claims 304-308, wherein the method comprises administering the guide RNA in the form of RNA.

310. The method of any one of claims 304-309, wherein the guide RNA comprises at least one modification.

311. The method of claim 310, wherein the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

312. The method of any one of claims 304-311, wherein the method comprises administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.Attorney Docket No.057766 / 624641 313. The method of any one of claims 304-312, wherein the Cas protein is a Cas9 protein, optionally wherein the Cas protein is derived from a Streptococcus pyogenes Cas9 protein.

314. The method of any one of claims 304-313, wherein the Cas protein comprises the sequence set forth in SEQ ID NO:

134.

315. The method of any one of claims 304-314, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein.

316. The method of claim 315, wherein the mRNA encoding the Cas protein comprises at least one modification.

317. The method of claim 316, wherein the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine.

318. The method of any one of claims 315-317, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

319. The method of any one of claims 304-318, wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

320. The method of any one of claims 304-319, wherein the method comprises administering the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

321. The method of any one of claims 304-320, wherein the method comprises administering the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223,Attorney Docket No.057766 / 624641 and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and wherein the method comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

322. The method of any one of claims 304-321, wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle.

323. The method of claim 322, wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid.

324. The method of claim 323, wherein the cationic lipid is Lipid A ((9Z,12Z)- 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or wherein the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), and / or wherein the helper lipid is cholesterol, and / or wherein the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

325. The method of claim 324, wherein the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k-DMG.

326. The method of any one of claims 323-325, wherein the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.Attorney Docket No.057766 / 624641 327. The method of any one of claims 236-326, wherein the cell is a liver cell or a hepatocyte, or the population of cells is a population of liver cells or hepatocytes.

328. The method of any one of claims 236-327, wherein the subject is a human subject.

329. The method of any one of claims 236-328, wherein the subject is a neonatal subject.

330. The method of any one of claims 236-329, wherein the nucleic acid vector is in an adeno-associated viral (AAV) vector, and wherein the subject does not have preexisting AAV immunity.

331. The method of any one of claims 236-330, wherein the method does not comprise administering a plasma cell depleting agent.

332. The method of any one of claims 236-331, wherein the nucleic acid vector is in an adeno-associated viral (AAV) vector, wherein the subject does not have preexisting AAV immunity, and wherein the method does not comprise administering a plasma cell depleting agent.

333. A composition or combination comprising an effective amount of an anti- CD20xCD3 bispecific antibody or functional fragment thereof in combination with: (a) a nucleic acid construct comprising a coding sequence for the polypeptide of interest; and (b) a nuclease agent or one or more nucleic acids encoding the nuclease agent, wherein the nuclease agent targets a nuclease target site in a target genomic locus.

334. The composition or combination of claim 333, wherein the anti- CD20xCD3 bispecific antibody or functional fragment thereof comprises a first antigen-binding domain that specifically binds to CD20 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 44, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 45.Attorney Docket No.057766 / 624641 335. The composition or combination of claim 334, wherein the first antigen- binding domain that specifically binds to CD20 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 48, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 49, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

52.

336. The composition or combination of claim 334 or 335, wherein the anti- CD20xCD3 bispecific antibody or functional fragment thereof comprises a second antigen- binding domain that specifically binds to CD3 comprising three heavy chain CDRs (HCDR1, HCDR2 and HCDR3) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 46, and three light chain CDRs (LCDR1, LCDR2 and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

45.

337. The composition or combination of claim 336, wherein the second antigen-binding domain that specifically binds to CD3 comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 53, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 54, a HCDR3 comprising the amino acid sequence of SEQ ID NO: 55, a LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, a LCDR2 comprising the amino acid sequence of SEQ ID NO: 51, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:

52.

338. The composition or combination of any one of claims 334-337, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises: (a) a first antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 47, 48, and 49, respectively, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively; and (b) a second antigen-binding domain that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 53, 54, and 55, respectively, andAttorney Docket No.057766 / 624641 LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50, 51, and 52, respectively.

339. The composition or combination of any one of claims 333-338, wherein the anti-CD20xCD3 bispecific antibody or functional fragment thereof comprises a human IgG heavy chain constant region, optionally wherein the human IgG heavy chain constant region comprises one or more modifications that increase binding to a neonatal Fc receptor (FcRn) and / or the human IgG heavy chain constant region comprises one or more modifications that decrease binding to an Fc-gamma receptor (FcγR).

340. The composition or combination of claim 339, wherein the human IgG heavy chain constant region is isotype IgG4 or IgG1.

341. The composition or combination of any one of claims 333-340, wherein the nucleic acid construct is in the nucleic acid vector, optionally wherein the nucleic acid vector is a viral vector.

342. The composition or combination of claim 341, wherein the nucleic acid vector is an adeno-associated viral (AAV) vector, optionally wherein the nucleic acid construct is flanked by inverted terminal repeats (ITRs) on each end, optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 283, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO: 283, or optionally wherein the ITR on at least one end comprises, consists essentially of, or consists of SEQ ID NO: 281, and optionally wherein the ITR on each end comprises, consists essentially of, or consists of SEQ ID NO:

281.

343. The composition or combination of claim 342, wherein the AAV vector is a single-stranded AAV (ssAAV) vector.

344. The composition or combination of claim 342 or 343, wherein the AAV vector is a recombinant AAV8 (rAAV8) vector.Attorney Docket No.057766 / 624641 345. The composition or combination of any one of claims 333-344, wherein the polypeptide of interest is a factor IX protein.

346. The composition or combination of claim 345, wherein the factor IX protein coding sequence encodes a factor IX protein comprising SEQ ID NO:

97.

347. The composition or combination of claim 345 or 346, wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO: 68, or wherein the factor IX protein coding sequence comprises or consists of SEQ ID NO:

61.

348. The composition or combination of any one of claims 345-347, wherein the nucleic acid construct is a bidirectional construct, wherein the factor IX protein coding sequence is a first factor IX protein coding sequence, and the bidirectional construct further comprises a reverse complement of a second factor IX protein coding sequence, wherein the first factor IX protein coding sequence and the second factor IX protein coding sequence are different but encode the same factor IX protein sequence.

349. The composition or combination of claim 348, wherein the nucleic acid construct comprises from 5’ to 3’: a first splice acceptor, the first factor IX protein coding sequence, a first polyadenylation signal, a reverse complement of a second polyadenylation signal, the reverse complement of the second factor IX protein coding sequence, and a reverse complement of a second splice acceptor, wherein: (i) the first factor IX protein coding sequence comprises SEQ ID NO: 61 and the second factor IX protein coding sequence comprises SEQ ID NO: 68; or (ii) the first factor IX protein coding sequence comprises SEQ ID NO: 68 and the second factor IX protein coding sequence comprises SEQ ID NO: 61, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

350. The composition or combination of claim 348 or 349, wherein the nucleic acid construct comprises SEQ ID NO: 109 or 82 or the reverse complement thereof.Attorney Docket No.057766 / 624641 351. The composition or combination of any one of claims 345-347, wherein the nucleic acid construct is a unidirectional construct.

352. The composition or combination of claim 351, wherein the nucleic acid construct is a unidirectional construct comprising the factor IX protein coding sequence, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the factor IX protein coding sequence, and a polyadenylation signal, wherein the factor IX protein coding sequence comprises SEQ ID NO: 61 or SEQ ID NO: 68, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the factor IX protein, and wherein the nucleic acid construct does not comprise homology arms.

353. The composition or combination of any one of claims 333-344, wherein the polypeptide of interest is a multidomain therapeutic protein comprising a delivery domain fused to a lysosomal alpha-glucosidase.

354. The composition or combination of claim 353, wherein the lysosomal alpha-glucosidase comprises or consists of the sequence set forth in SEQ ID NO:

296.

355. The composition or combination of claim 353 or 354, wherein the lysosomal alpha-glucosidase coding sequence comprises or consists of the sequence set forth in SEQ ID NO:

857.

356. The composition or combination of any one of claims 353-355, wherein the delivery domain is a CD63-binding delivery domain.

357. The composition or combination of claim 356, wherein the CD63-binding delivery domain comprises an anti-CD63 antigen-binding protein.

358. The composition or combination of claim 356 or 357, wherein the CD63- binding delivery domain is a single-chain variable fragment (scFv).

359. The composition or combination of claim 358, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO: 306.Attorney Docket No.057766 / 624641 360. The composition or combination of claim 358 or 359, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO:

866.

361. The composition or combination of any one of claims 356-360, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO:

316.

362. The composition or combination of any one of claims 356-361, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884.

363. The composition or combination of any one of claims 356-362, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 863, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 900 or 884, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

364. The composition or combination of any one of claims 353-355, wherein the delivery domain is a TfR-binding delivery domain.Attorney Docket No.057766 / 624641 365. The composition or combination of claim 364, wherein the TfR-binding delivery domain comprises an anti-TfR antigen-binding protein.

366. The composition or combination of claim 365, wherein the anti-TfR antigen-binding protein comprises a HCVR comprising the HCDR1, HCDR2 and HCDR3 of a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR comprising the LCDR1, LCDR2 and LCDR3 of a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

367. The composition or combination of claim 365 or 366, wherein the anti- TfR antigen-binding protein comprises a HCVR that comprises: an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 556 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 557 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 558 (or a variant thereof); and a LCVR that comprises: an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 561 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 562 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 563 (or a variant thereof).

368. The composition or combination of any one of claims 365-367, wherein the anti-TfR antigen-binding protein comprises a HCVR that comprises the amino acid sequence set forth in SEQ ID NO: 555 (or a variant thereof); and a LCVR that comprises the amino acid sequence set forth in SEQ ID NO: 560 (or a variant thereof).

369. The composition or combination of any one of claims 365-368, wherein the TfR-binding delivery domain comprises a single-chain variable fragment (scFv).

370. The composition or combination of claim 369, wherein the scFv comprises or consists of the sequence set forth in SEQ ID NO:

672.

371. The composition or combination of claim 369 or 370, wherein the scFv coding sequence comprises or consists of the sequence set forth in SEQ ID NO: 713.Attorney Docket No.057766 / 624641 372. The composition or combination of any one of claims 364-371, wherein the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO:

691.

373. The composition or combination of any one of claims 364-372, wherein the coding sequence for the multidomain therapeutic protein comprises or consists of the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871.

374. The composition or combination of any one of claims 364-373, wherein the nucleic acid construct comprises from 5’ to 3’: a splice acceptor, the coding sequence for the multidomain therapeutic protein, and a polyadenylation signal or sequence, wherein the coding sequence for the multidomain therapeutic protein comprises the sequence set forth in SEQ ID NO: 852, optionally wherein the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 887 or 871, wherein the polyadenylation signal comprises a BGH polyadenylation signal and a unidirectional SV40 late polyadenylation signal, optionally wherein the BGH polyadenylation signal comprises the sequence set forth in SEQ ID NO: 858 and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 859, optionally wherein the polyadenylation signal comprising the BGH polyadenylation signal and the unidirectional SV40 late polyadenylation signal comprises the sequence set forth in SEQ ID NO: 902, wherein the nucleic acid construct does not comprise a promoter that drives the expression of the multidomain therapeutic protein, and wherein the nucleic acid construct does not comprise a homology arm.

375. The composition or combination of any one of claims 333-344, wherein the polypeptide of interest is a factor VIII protein.

376. The composition or combination of any one of claims 333-344, wherein the polypeptide of interest is an antigen-binding protein, optionally wherein the antigen-binding protein is an antibody.Attorney Docket No.057766 / 624641 377. The composition or combination of any one of claims 333-376, wherein the target genomic locus is an albumin gene, optionally wherein the albumin gene is a human albumin gene.

378. The composition or combination of claim 377, wherein the nuclease target site is in intron 1 of the albumin gene.

379. The composition or combination of any one of claims 333-378, wherein the nuclease agent comprises: (a) a zinc finger nuclease (ZFN); (b) a transcription activator-like effector nuclease (TALEN); or (c) (i) a Cas protein or a nucleic acid encoding the Cas protein; and (ii) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

380. The composition or combination of any one of claims 333-378, wherein the nuclease agent comprises: (a) a Cas protein or a nucleic acid encoding the Cas protein; and (b) a guide RNA or one or more DNAs encoding the guide RNA, wherein the guide RNA comprises a DNA-targeting segment that targets a guide RNA target sequence, and wherein the guide RNA binds to the Cas protein and targets the Cas protein to the guide RNA target sequence.

381. The composition or combination of claim 380, wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 153-184, optionally wherein the DNA-targeting segment comprises any one of SEQ ID NOS: 159, 153, 156, and 164, or wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 153- 184, optionally wherein the DNA-targeting segment consists of any one of SEQ ID NOS: 159, 153, 156, and 164.Attorney Docket No.057766 / 624641 382. The composition or combination of claim 380 or 381, wherein the guide RNA comprises any one of SEQ ID NOS: 185-248, optionally wherein the guide RNA comprises any one of SEQ ID NOS: 191, 223, 185, 217, 188, 220, 196, and 228.

383. The composition or combination of any one of claims 380-382, wherein the DNA-targeting segment comprises or consists of SEQ ID NO:

159.

384. The composition or combination of any one of claims 380-383, wherein the guide RNA comprises SEQ ID NO: 191 or 223.

385. The composition or combination of any one of claims 380-384, wherein the composition or combination comprises the guide RNA in the form of RNA.

386. The composition or combination of any one of claims 380-385, wherein the guide RNA comprises at least one modification.

387. The composition or combination of claim 386, wherein the at least one modification comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

388. The composition or combination of any one of claims 380-387, wherein the composition or combination comprises the guide RNA in the form of RNA, the guide RNA comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA.

389. The composition or combination of any one of claims 380-388, wherein the Cas protein is a Cas9 protein, optionally wherein the Cas protein is derived from a Streptococcus pyogenes Cas9 protein.Attorney Docket No.057766 / 624641 390. The composition or combination of any one of claims 380-389, wherein the Cas protein comprises the sequence set forth in SEQ ID NO:

134.

391. The composition or combination of any one of claims 380-390, wherein the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein.

392. The composition or combination of claim 391, wherein the mRNA encoding the Cas protein comprises at least one modification.

393. The composition or combination of claim 392, wherein the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine.

394. The composition or combination of any one of claims 391-393, wherein the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

395. The composition or combination of any one of claims 380-394, wherein the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

396. The composition or combination of any one of claims 380-395, wherein the composition or combination comprises the guide RNA in the form of RNA, and the guide RNA comprises SEQ ID NO: 191 or 223, and wherein the composition or combination comprises administering the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, and the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125.

397. The composition or combination of any one of claims 380-396, wherein the composition or combination comprises the guide RNA in the form of RNA, the guide RNAAttorney Docket No.057766 / 624641 comprises SEQ ID NO: 223, and the guide RNA comprises: (i) phosphorothioate bonds between the first four nucleotides at the 5’ end of the guide RNA; (ii) phosphorothioate bonds between the last four nucleotides at the 3’ end of the guide RNA; (iii) 2’-O-methyl-modified nucleotides at the first three nucleotides at the 5’ end of the guide RNA; and (iv) 2’-O-methyl-modified nucleotides at the last three nucleotides at the 3’ end of the guide RNA, and wherein the composition or combination comprises the nucleic acid encoding the Cas protein, wherein the nucleic acid comprises an mRNA encoding the Cas protein, the mRNA encoding the Cas protein comprises the sequence set forth in SEQ ID NO: 124 or 125, and the mRNA encoding the Cas protein is fully substituted with N1-methyl-pseudouridine, comprises a 5’ cap, and comprises a poly(A) tail.

398. The composition or combination of any one of claims 380-397, wherein the Cas protein or the nucleic acid encoding the Cas protein and the guide RNA or the one or more DNAs encoding the guide RNA are associated with a lipid nanoparticle.

399. The composition or combination of claim 398, wherein the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a helper lipid, and a stealth lipid.

400. The composition or combination of claim 399, wherein the cationic lipid is Lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate), and / or wherein the neutral lipid is distearoylphosphatidylcholine or 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), and / or wherein the helper lipid is cholesterol, and / or wherein the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

401. The composition or combination of claim 400, wherein the cationic lipid is Lipid A, the neutral lipid is DSPC, the helper lipid is cholesterol, and the stealth lipid is PEG2k- DMG.Attorney Docket No.057766 / 624641 402. The composition or combination of any one of claims 399-401, wherein the lipid nanoparticle comprises four lipids at the following molar ratios: about 50 mol% Lipid A, about 9 mol% DSPC, about 38 mol% cholesterol, and about 3 mol% PEG2k-DMG.

403. The composition or combination of any one of claims 333-402, wherein the composition or combination does not comprise a plasma cell depleting agent.

404. The composition or combination of any one of claims 333-403, for use in a method of inserting a nucleic acid encoding a polypeptide of interest into a target genomic locus in a cell or a population of cells in a subject.

405. The composition or combination of any one of claims 333-403, for use in a method of expressing a polypeptide of interest from a target genomic locus in a cell or a population of cells in a subject.

406. The composition or combination of any one of claims 333-403, for use in a method of treating an enzyme deficiency in a subject in need thereof.

407. The composition or combination of any one of claims 333-403, for use in a method of preventing or reducing the onset of a sign or symptom of an enzyme deficiency in a subject in need thereof.

408. A kit comprising the composition or combination of any one of claims 333-407.

409. An anti-CD20xCD3 bispecific antibody or functional fragment thereof for use in a method according to any one of claims 236-332.

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