Vectors and methods for in vivo antibody production

Abstract

The present invention relates to compositions and methods that can target B cells and / or hematopoietic stem cells (HSCs), manipulate these cells, and express specific antibodies ex vivo or in vivo to become part of the host's long-term immune repertoire.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 339,665, filed May 9, 2022, the disclosure of which is incorporated by reference in its entirety into this application. [Technical Field]

[0002] The present invention relates to compositions and methods by which B cells and / or hematopoietic stem cells (HSCs) can be targeted and manipulated to express specific antibodies ex vivo or in vivo, which become part of the host's long-term immune repertoire. [Background technology]

[0003] In general, successful immunization depends on the host's ability to generate an appropriate response to a given immunogen. In certain populations (e.g., children, the elderly, immunocompromised individuals, etc.), vaccines may not adequately induce the desired response. Designing immunogens to generate sufficiently broad and strong immune responses has been unsuccessful, even in normal, healthy populations. Furthermore, for some pathogens (e.g., dengue fever), depending on the individual vaccine response, such as the antibody isotype induced, vaccination may actually result in enhanced infection (ADE) rather than protection. While monoclonal antibodies can be selected or engineered to overcome many of these problems, passively delivered antibodies have a short lifespan compared to vaccines, necessitating frequent re-administration for long-term immunity. Over the past few years, approaches to express monoclonal antibodies in vivo have been pursued. Summary of the Invention [Problem to be solved by the invention]

[0004] As noted in the "Background" section, there is a need in the art for the in vivo expression of monoclonal antibodies. The present invention addresses this and other needs by providing compositions and methods that target B cells and / or hematopoietic stem cells (HSCs) and manipulate these cells to express specific antibodies ex vivo or in vivo, which can become part of the host's long-term immune repertoire. [Means for solving the problem]

[0005] In one aspect, the present disclosure provides a system for producing an antibody or antigen-binding fragment thereof in a subject, the system comprising: a) a first component comprising a polynucleotide molecule, said polynucleotide molecule comprising a sequence encoding said antibody or antigen-binding fragment thereof; and b) a second component comprising a gene editing molecule or a polynucleotide molecule comprising a sequence encoding said gene editing molecule; Includes.

[0006] In some embodiments, administration of the first component and the second component to a subject causes the sequence encoding the antibody or antigen-binding fragment thereof to be incorporated into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), thereby causing production of the antibody or antigen-binding fragment in the subject's body.

[0007] In some embodiments, the first component and the second component are administered ex vivo to B cells and / or hematopoietic stem cells (HSCs), whereby a sequence encoding the antibody or antigen-binding fragment thereof is incorporated into the DNA of the cells to produce modified B cells or modified HSCs, which, when administered to a subject, result in the production of the antibody or antigen-binding fragment thereof in the subject.

[0008] In some embodiments, the B cells are B1 B cells. In some embodiments, the B cells are B2 B cells.

[0009] In some embodiments, the first component and / or the second component are independently selected from a viral vector, a virus-like particle (VLP), a liposome, a lipid nanoparticle (LNP), and a ribonuclear protein (RNP) complex.

[0010] In some embodiments, the first component and the second component are both viral vectors. In some embodiments, the viral vectors are derived from the same viral species. In other embodiments, the viral vectors are derived from different viral species.

[0011] In some embodiments, the first component or the second component further comprises a guide RNA (gRNA) molecule or a sequence encoding the gRNA molecule.

[0012] In some embodiments, the first component comprises the polynucleotide molecule comprising the sequence encoding the antibody or antigen-binding fragment thereof and the sequence encoding a gRNA.

[0013] In some embodiments, the first component comprises: (i) a first polynucleotide molecule comprising the above-described sequence encoding the antibody or antigen-binding fragment thereof; and (ii) a second polynucleotide molecule comprising a sequence encoding a gRNA.

[0014] In some embodiments, the first component comprises: (i) a first polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof; and (ii) a gRNA molecule.

[0015] In some embodiments, the second component comprises a gRNA molecule or a sequence as described above that encodes a gRNA molecule.

[0016] In one aspect, the present disclosure provides a vector system for generating a cell population capable of producing an antibody or antigen-binding fragment thereof in vivo, the vector system comprising: a first viral vector comprising a sequence encoding a target antibody or fragment thereof and a sequence encoding a guide RNA (gRNA); and a second viral vector comprising a sequence encoding a gene-editing molecule, wherein the vector system integrates the sequence encoding the target antibody or antigen-binding fragment thereof into the DNA of the cell, thereby causing the cell to produce the antibody or antigen-binding fragment thereof.

[0017] In some embodiments, the cell population is a human cell population. In some embodiments, the cell population is a B cell population (e.g., comprising B1 B cells and / or comprising B2 B cells). In some embodiments, the cell population is a hematopoietic stem cell (HSC) population.

[0018] In some embodiments, one or both of the viral vectors used in the systems of the present disclosure are adeno-associated virus (AAV) vectors. In some embodiments, the AAV vector is derived from AAV1, AAV2, AAV6, AAV9, or AAV9.PHP. In some embodiments, the AAV vector capsid comprises one or more mutations that abolish the natural tropism of the AAV vector. In some embodiments, the AAV vector capsid is derived from AAV1 or AAV6 and comprises the mutation Y445F and / or V473D. In some embodiments, the AAV vector capsid is derived from AAV9 and comprises the mutation W503A.

[0019] In some embodiments, one or both of the viral vectors used in the systems of the present disclosure are retroviral vectors, such as lentiviral vectors.

[0020] In some embodiments, the viral vector used in the system of the present disclosure further comprises a targeting moiety. In some embodiments, the targeting moiety is expressed on the outer surface of the viral capsid. In some embodiments, the targeting moiety is attached to the outer surface of the viral capsid by a linker.

[0021] In some embodiments, the viral vector is an AAV vector, and the targeting moiety is inserted into or covalently or non-covalently attached to a protein forming the viral capsid. In some embodiments, the targeting moiety is attached to the viral capsid via a first member and a second member of a binding pair. The first member and the second member may form an isopeptide bond.

[0022] In some embodiments, the viral vector is a lentiviral vector and the targeting moiety is covalently or non-covalently attached to a fusogen.

[0023] In some embodiments, the targeting moiety is attached to the outer surface of the viral capsid via the SpyTag:SpyCatcher system. In some embodiments, the targeting moiety is a targeting antibody or antigen-binding fragment thereof. Non-limiting examples of antibodies that can be used include, for example, anti-CD19, anti-CD20, anti-CD34, anti-CD38, anti-CD40, anti-CD117, anti-CD22, anti-CD79, anti-CD180, anti-CD5, anti-B cell receptor (BCR) (e.g., IgM, IgD, IgG), anti-B cell activating factor (BAFF), and anti-Sca-1 antibodies, or antigen-binding fragments thereof.

[0024] In some embodiments, the gene editing molecule is a Cas nuclease, such as a Cas9 nuclease.

[0025] In various embodiments, the gRNA is complementary to a sequence at the IgH locus, the J chain locus, or the Igκ locus. In some embodiments, the gRNA is complementary to a sequence at the J chain locus. In one embodiment, the gRNA is complementary to a sequence within the fourth exon of the J chain locus. In one embodiment, the gRNA is complementary to a sequence within the first intron of the J chain locus.

[0026] In some embodiments, the sequence encoding the gRNA encodes a gRNA complementary to a sequence encoding the V13 region of an antibody, hi some embodiments, the gRNA is selected from gRNA1, gRNA2, gRNA3, gRNA4, gRNA5, gRNA6, gRNA7, gRNA8, gRNA9, gRNA10, and gRNA12.

[0027] In some embodiments, the sequence encoding the antibody or antigen-binding fragment thereof comprises a sequence encoding a light chain variable region, and optionally a light chain constant region, of the antibody. In some embodiments, the sequence encoding the antibody or fragment thereof comprises a sequence encoding a heavy chain variable sequence of the antibody.

[0028] In some embodiments, the sequence encoding the antibody or antigen-binding fragment thereof is integrated into the IgH locus in the genomic region downstream of the last J gene and upstream of the Eμ enhancer.

[0029] In some embodiments, the kappa light chain constant region is disrupted by incorporating sequences encoding the antibody or antigen-binding fragment thereof into the DNA of a B cell or HSC.

[0030] In some embodiments, the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a 5' IgH homology region, a splice acceptor, a 2A sequence with a 5' furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence with a 5' furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a splice donor sequence, and a 3' IgH homology region.

[0031] In some embodiments, the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a 5' J chain exon 4 homology region, a 2A sequence with a 5' furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence with a 5' furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a sequence encoding the heavy chain constant region of the antibody, and a 3' J chain exon 4 homology region, and the heavy chain and light chain sequences may be arranged in any order.

[0032] In some embodiments, the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof comprises, from 5' to 3', a sequence encoding a guide RNA (gRNA) sequence, a splice acceptor sequence, a 2A sequence, a sequence encoding the light chain of the target antibody, a 2A sequence, a sequence encoding the heavy chain variable sequence of the target antibody, and a splice donor sequence.

[0033] In some embodiments, the sequence encoding the antibody or antigen-binding fragment thereof does not comprise a promoter sequence. The sequence encoding the antibody or antigen-binding fragment thereof, when incorporated into the DNA of a B cell or HSC, can be under the transcriptional control of an endogenous promoter. In one embodiment, when the sequence encoding the antibody or antigen-binding fragment thereof is incorporated into the DNA of a B cell or HSC, the sequence is under the transcriptional control of an endogenous heavy chain promoter in the B cell or HSC. In one embodiment, when the sequence encoding the antibody or antigen-binding fragment thereof is incorporated into the DNA of a B cell or HSC, the sequence is under the transcriptional control of an endogenous J chain promoter in the B cell or HSC.

[0034] In some embodiments, the sequence encoding the antibody or antigen-binding fragment thereof comprises a promoter sequence. In some embodiments, the promoter is a B cell-specific promoter or an HSC-specific promoter. Non-limiting examples of B cell-specific promoters or HSC-specific promoters include the Hg38-mCP promoter and the spleen focus forming virus (SFFV) promoter, or fragments thereof.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof binds to an antigen associated with a disease or disorder, including but not limited to, an infectious disease, cancer, an autoimmune disease, a cardiovascular disease, a musculoskeletal disease, or a neurodegenerative disease. In some embodiments, the infectious disease is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection. In some embodiments, the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a tumor-associated antigen (TAA).

[0036] In various embodiments of the systems described herein, the subject is a human.

[0037] In various embodiments of the systems described herein, the subject is a laboratory animal, such as a mouse or a rat.

[0038] In a related aspect, there is provided a modified B cell or modified hematopoietic stem cell (HSC) comprising the system of any one of the embodiments described herein.

[0039] In another aspect, there is provided a pharmaceutical composition comprising the system of any one of the embodiments described herein and a pharmaceutically acceptable carrier or excipient.

[0040] In another aspect, a kit is provided that includes (i) a system of any one of the embodiments described herein, and optionally (ii) a container and / or instructions for use.

[0041] In another aspect, a method for generating modified B cells or modified hematopoietic stem cells (HSCs) that produce an antibody or antigen-binding fragment thereof is provided. The method may comprise transducing B cells or HSCs ex vivo with an effective amount of the system of any one of the embodiments described herein, wherein a first component and a second component of the system are administered to the cells simultaneously or sequentially in any order, and wherein administration of the first component and the second component integrates a sequence encoding the antibody or antigen-binding fragment thereof into the DNA of the cells, causing the cells to become modified cells.

[0042] In some embodiments of the ex vivo methods described above, the first and second components of the system are administered to the cells simultaneously as two separate compositions.

[0043] In some embodiments of the ex vivo methods described above, the first and second components of the system are administered to the cells simultaneously as a single composition.

[0044] In various embodiments of the ex vivo methods described above, the B cells or HSCs are present in a heterogeneous cell population during transduction.

[0045] In various embodiments of the ex vivo methods described above, the B cells are isolated from the spleen, peritoneum, or peripheral blood.

[0046] In some embodiments, the B cells are primary B cells.

[0047] In various embodiments of the ex vivo methods described above, the B cells are B2 B cells.

[0048] In various embodiments of the ex vivo methods described above, the B cells are B1 B cells. In some embodiments, the B cells are B1a B cells (CD19+ / CD5+ / CD23-) or B1b B cells (CD19+ / CD5- / CD23-).

[0049] In various embodiments of the ex vivo methods described above, the B cells are cultured under stimulatory conditions before and / or after transduction.

[0050] In some embodiments, the stimulatory conditions promote B cell activation without differentiation.

[0051] In various embodiments of the ex vivo methods described above, the B cells are cultured in the presence of a CD40 agonist and / or a CD180 agonist before and / or after transduction. In some embodiments, the B cells are cultured in the presence of a CD40 agonist and a CD180 agonist before and / or after transduction. In some embodiments, the CD40 agonist is CD40 ligand (CD40L) or an anti-CD40 antibody, or an antigen-binding fragment thereof. In some embodiments, the CD180 agonist is an anti-CD180 antibody, or an antigen-binding fragment thereof.

[0052] In some embodiments, the B cells are cultured in the presence of about 20 ng / ml or less of a CD40 agonist (e.g., CD40L) and / or about 100 ng / ml or less of a CD180 agonist (e.g., an anti-CD180 antibody) before and / or after transduction. In some embodiments, the CD40 agonist (e.g., CD40L) used in culturing B cells is about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 ng / ml, or about 0-5 ng / ml, about 4-8 ng / ml, about 5-10 ng / ml, about 6-12 ng / ml, about 8-15 ng / ml, about 10-15 ng / ml, about 12-18 ng / ml, or about 15-20 ng / ml. In some embodiments, the CD180 agonist (e.g., anti-CD180 antibody) used in culturing B cells is at about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 85, about 90, about 95, about 100 ng / ml, or about 0-5 ng / ml. ng / ml, about 4-8 ng / ml, about 5-10 ng / ml, about 6-12 ng / ml, about 8-15 ng / ml, about 10-15 ng / ml, about 12-18 ng / ml, about 15-20 ng / ml, about 20-25 ng / ml, about 20-30 ng / ml, about 25-40 ng / ml, about 30-50 ng / ml, about 40-60 ng / ml, about 50-75 ng / ml, about 60-80 ng / ml, about 70-90 ng / ml, or about 80-100 ng / ml. In some embodiments, B cells are cultured in the presence of about 20 ng / ml of a CD40 agonist (e.g., CD40L) and about 20 ng / ml of a CD180 agonist (e.g., an anti-CD180 antibody) before and / or after transduction. In some embodiments, the B cells are cultured in the presence of a CD40 agonist (eg, CD40L) and / or a CD180 agonist (eg, an anti-CD180 antibody) for about 4 days or less prior to transduction.In some embodiments, B cells are cultured in the presence of a CD40 agonist (e.g., CD40L) and / or a CD180 agonist (e.g., an anti-CD180 antibody) for about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 1 day, about 36 hours, about 2 days, about 60 hours, about 3 days, about 84 hours, or about 4 days prior to transduction. In some embodiments, B cells are cultured in the presence of a CD40 agonist (e.g., CD40L) and / or a CD180 agonist (e.g., an anti-CD180 antibody) for about 2 days prior to transduction. In some embodiments, B cells are cultured in the presence of a CD40 agonist (e.g., CD40L) and / or a CD180 agonist (e.g., an anti-CD180 antibody) for about 2 days after transduction.

[0053] In some embodiments of the ex vivo methods described above, the methods further comprise culturing the modified B cells or modified HSCs under differentiation conditions to promote differentiation of the modified B cells or modified HSCs into modified plasma cells.

[0054] In various embodiments of the ex vivo methods described above, the method further comprises introducing the modified B cells, HSCs, or plasma cells into a subject. In some embodiments, the modified B cells, HSCs, or plasma cells are introduced into the subject intraperitoneally. In some embodiments, the subject is depleted of CD20+ cells prior to the introduction of the modified B cells or HSCs. In some embodiments, after the modified B cells, HSCs, or plasma cells are introduced into the subject, the modified B cells, HSCs, or plasma cells are expanded in vivo by administering to the subject an antigen recognized by an antibody, or antigen-binding fragment thereof, produced by the B cells, HSCs, or plasma cells. In some embodiments, the modified B cells, HSCs, or plasma cells are autologous to the subject. In some embodiments, the modified B cells, HSCs, or plasma cells are allogeneic to the subject. In some embodiments, the subject is a human. In some embodiments, the subject is an experimental animal.

[0055] In a related aspect, there is provided a modified B cell or modified HSC, or population thereof, produced by the method of any one of the above-described embodiments. In a related aspect, there is provided a modified plasma cell produced by the above-described method.

[0056] In another aspect, the disclosure provides methods for producing an antibody or antigen-binding fragment thereof in vivo in a subject in need thereof, the method comprising the step of ex vivo transducing B cells and / or hematopoietic stem cells (HSCs) isolated from a subject or donor with an effective amount of any of the above-described systems, wherein a first component (e.g., a viral vector) and a second component (e.g., a viral vector) are administered simultaneously or sequentially in any order, and then the transduced cells are reintroduced into the subject. In some embodiments, the B cells comprise B1 B cells. In some embodiments, the B cells comprise B2 B cells. In some embodiments, the B cells comprise primary B cells.

[0057] In a related aspect, the present disclosure provides methods for producing an antibody or antigen-binding fragment thereof in vivo in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the above-described systems, wherein the first component (e.g., a viral vector) and the second component (e.g., a viral vector) are administered simultaneously or sequentially in any order. In some embodiments, administering the first component and the second component to the subject causes a sequence encoding the antibody or antigen-binding fragment thereof to be integrated into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), thereby causing production of the antibody or antigen-binding fragment thereof in the subject.

[0058] In some embodiments of the in vivo methods described above, the first and second components of the system are administered simultaneously to the subject as two separate compositions.

[0059] In some embodiments of the in vivo methods described above, the first and second components of the system are administered to the subject simultaneously as a single composition.

[0060] In some embodiments of the in vivo methods described above, the first component and / or the second component of the system are administered intraperitoneally to the subject.

[0061] In some embodiments of the in vivo methods described above, the method further comprises administering to the subject an effective amount of a CD180 agonist and / or a CD40 agonist prior to administering the system to the subject. In some embodiments, the CD40 agonist is a CD40 ligand (CD40L) or an anti-CD40 antibody, or an antigen-binding fragment thereof. In some embodiments, the CD180 agonist is an anti-CD180 antibody or an antigen-binding fragment thereof.

[0062] In some embodiments, the method comprises administering to the subject an effective amount of a CD180 agonist (e.g., an anti-CD180 antibody) and a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 250 μg or less of a CD180 agonist (e.g., an anti-CD180 antibody) and / or about 50 μg or less of a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method further comprises administering to the subject, prior to administering the system, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 610, about 620, about 630, about 640, about 650, about 660 The method includes administering about 20, about 230, about 240, about 250, or about 0 to 5, about 4 to 8, about 5 to 10, about 6 to 12, about 8 to 15, about 10 to 15, about 12 to 18, about 15 to 20, about 20 to 25, about 20 to 30, about 25 to 40, about 30 to 50, about 40 to 60, about 50 to 75, about 60 to 80, about 70 to 90, about 80 to 100, about 100 to 120, about 120 to 150, about 140 to 160, about 150 to 180, about 175 to 200, about 200 to 225, or about 225 to 250 μg of a CD180 agonist (e.g., an anti-CD180 antibody). In some embodiments, the method includes administering about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, or about 0-5, about 4-8, about 5-10, about 6-12, about 8-15, about 10-15, about 12-18, about 15-20, about 20-25, about 20-30, about 25-35, about 30-40, or about 40-50 μg of a CD40 agonist (e.g., an anti-CD40 antibody) to the subject prior to administering the system to the subject.In one embodiment, the method comprises administering to the subject about 12.5 μg of a CD180 agonist (e.g., an anti-CD180 antibody) and no CD40 agonist (e.g., an anti-CD40 antibody). In one embodiment, the method comprises administering to the subject about 12.5 μg of a CD40 agonist (e.g., an anti-CD40 antibody) without administering to the subject a CD180 agonist (e.g., an anti-CD180 antibody).

[0063] In some embodiments, the method comprises administering to the subject effective amounts of a CD180 agonist (e.g., an anti-CD180 antibody) and a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 8.5 mg / kg or less of body weight of a CD180 agonist (e.g., an anti-CD180 antibody) and / or about 1.8 mg / kg or less of body weight of a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In some embodiments, the method includes administering to the subject about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.33, about 8.5, or about 0-0.5, about 0.4-0.8, about 0.5-1, about 1-2, about 1.5-2.5, about 2-4, about 3-5, about 4-6, about 5-7, about 6-8, or about 7.5-8.5 mg / kg body weight of a CD180 agonist (e.g., an anti-CD180 antibody) prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, or about 0-0.5, about 0.2-0.6, about 0.4-0.8, about 0.5-1, about 0.6-1.2, about 0.8-1.5, about 1-1.5, about 1.2-1.6, or about 1.5-1.8 mg / kg body weight of a CD40 agonist (e.g., an anti-CD40 antibody) prior to administering the system to the subject. In one embodiment, the method comprises administering to the subject about 0.4 mg / kg body weight of a CD180 agonist (e.g., an anti-CD180 antibody). In one embodiment, the method comprises administering to the subject about 0.4 mg / kg body weight of a CD40 agonist (e.g., an anti-CD40 antibody). In some embodiments, the method comprises administering to the subject a CD180 agonist without a CD40 agonist. In some embodiments, the method comprises administering to the subject a CD40 agonist without a CD180 agonist.

[0064] In some embodiments, the method comprises administering to the subject a CD180 agonist (e.g., an anti-CD180 antibody) and / or a CD40 agonist (e.g., an anti-CD40 antibody) about 7 days or less prior to administering the system to the subject. In some embodiments, the method comprises administering to the subject a CD180 agonist (e.g., an anti-CD180 antibody) and / or a CD40 agonist (e.g., an anti-CD40 antibody) about 4 hours, about 8 hours, about 12 hours, about 16 hours, about 20 hours, about 1 day, about 36 hours, about 2 days, about 60 hours, about 3 days, about 84 hours, about 4 days, about 5 days, about 6 days, or about 7 days prior to administering the system to the subject. In some embodiments, the method includes administering to the subject a CD180 agonist (e.g., an anti-CD180 antibody) and / or a CD40 agonist (e.g., an anti-CD40 antibody) about 2-3 days prior to administering the system to the subject.

[0065] In some embodiments of the in vivo method described above, the method further comprises administering to a subject an effective amount of an antigen recognized by the antibody or antigen-binding fragment thereof, wherein the antigen is administered before and / or after administration of the first and / or second components of the system. In some embodiments, the antigen has low affinity for the antibody or antigen-binding fragment thereof. In some embodiments, the antigen has high affinity (e.g., in the picomolar range) for the antibody or antigen-binding fragment thereof. In some embodiments, the method comprises administering an antigen having low affinity for the antibody or antigen-binding fragment thereof before administering the first and second components of the system, and administering an antigen having high affinity for the antibody or antigen-binding fragment thereof (e.g., in the picomolar range) after administering the first and second components of the system.

[0066] In some embodiments of the in vivo methods described above, the methods include: administering to a subject an effective amount of a first antigen, wherein the first antigen has a low affinity for the antibody or antigen-binding fragment thereof, and the first antigen is administered before administering the first and second components of the system; and administering to a subject an effective amount of a second antigen, wherein the second antigen has a high affinity for the antibody or antigen-binding fragment thereof, and the second antigen is administered after administering the first and second components of the system.

[0067] In some embodiments of the in vivo methods described above, the subject is a human.

[0068] In some embodiments of the in vivo methods described above, the subject is an experimental animal.

[0069] In a further aspect, the present disclosure provides a method for treating or reducing the likelihood of a disease or disorder in a subject in need thereof, said method comprising performing the ex vivo method of any one of the above-mentioned embodiments or the in vivo method of any one of the above-mentioned embodiments, wherein said method results in production in the subject of an effective amount of an antibody or antigen-binding fragment thereof.

[0070] In yet another aspect, the present disclosure provides a method for treating or reducing the likelihood of a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of any of the above-described systems, wherein a first component (e.g., a viral vector) and a second component (e.g., a viral vector) are administered simultaneously or sequentially in any order, and wherein administration of the system causes in vivo production in the subject of an effective amount of an antibody or antigen-binding fragment thereof.

[0071] In yet another aspect, the disclosure provides methods of treating a disease in a subject in need thereof, the method comprising ex vivo transducing B cells and / or hematopoietic stem cells (HSCs) isolated from the subject or donor with an effective amount of any of the above-described systems, wherein a first component (e.g., a viral vector) and a second component (e.g., a viral vector) are administered simultaneously or sequentially in either order, after which the transduced cells are reintroduced into the subject, wherein administration of the system results in in vivo production in the subject of an effective amount of an antibody or antigen-binding fragment thereof. The produced antibody or antigen-binding fragment thereof binds to an antigen associated with the disease or disorder. In some embodiments, the B cells comprise primary B cells. In some embodiments, the B cells comprise B1 B cells. In some embodiments, the B cells comprise B2 B cells.

[0072] In some embodiments of any of the above methods of treatment, the disease is an infectious disease, cancer, an autoimmune disease, a cardiovascular disease, a musculoskeletal disease, or a neurodegenerative disease, hi some embodiments, the infectious disease is a viral infection, a bacterial infection, a fungal infection, or a parasitic infection.

[0073] In some embodiments of any of the above-described methods of treatment, the first component (eg, viral vector) and the second component (eg, viral vector) are administered as a single composition.

[0074] In some embodiments of any of the above methods, the subject is a human.

[0075] In some embodiments of any of the above methods, the subject is a laboratory animal, such as a mouse or rat.

[0076] These and other aspects described herein will become apparent to those skilled in the art in light of the following description, claims, and drawings. [Brief explanation of the drawings]

[0077] [Figure 1] FIG. 1 shows an overview of AAV-mediated delivery of transgenes to mice for episomal expression. [Figure 2] FIG. 2 shows an overview of AAV+Cas9 / guide RNA (gRNA)-mediated insertion of a transgene into a genomic locus in mouse liver. [Figure 3] Figure 3 shows results demonstrating that in vitro neutralization by episomal and liver-inserted anti-PcrV monoclonal antibodies (mAbs) from mouse serum was within 2- to 5-fold of CHO-purified mAbs. [Figure 4] FIG. 4 shows results demonstrating that episomal and liver-inserted anti-PcrV mAbs provide protection from lethal infection in an in vivo challenge model with P. aeruginosa. [Figure 5A-1] FIG. 5A outlines the ex vivo strategy for adaptive antibody vaccination in mice. [Figure 5A-2] Continued from Figure 5A. [Figure 5B] FIG. 5B shows an exemplary AAV vector carrying antibody heavy and light chain genes. [Figure 6] FIG. 6 shows the results showing the percentage of B cells expressing the transduced B-cell receptor (BCR) of interest for mock control, RNP control, and RNP+AAV1. [Figure 7A] Figures 7A-7C show an overview of engineering B cell specificity by inserting antibody genes into the heavy chain locus of peripheral B cells. Figure 7A shows depletion of mouse B cells and their modification with AAV-delivered Cas9 / gRNA. [Figure 7B] Figure 7B shows the VI3 heavy chain, gRNA cleavage site, and BCR insert. [Figure 7C] FIG. 7C shows the BCR insert spliced ​​into the VI3 heavy chain. [Figure 8A] Figures 8A-8B show ULC pairing and full-length BCR insertions. Figure 8A shows the two BCR variants used: ULC pairing anti-BCMA (top) and anti-PcrV (bottom). [Figure 8B] Figure 8B shows the results for both variants in the AAV-only and RNP-only controls and the AAV+RNP experimental condition. Antigen binding is given as a percentage for each condition. [Figure 9A] Figures 9A-9B show the insertion of mCherry constructs to test template designs. Figure 9A shows two mCherry variants, T2A-mCherry (top) and pVh3-23-mCherry (bottom). [Figure 9B] Figure 9B shows the results for both variants, along with no virus and no RNP controls. Results were analyzed 3 days post-infection. Positive mCherry expression in both variants with 150 pmol Cas9 and 400 pmol gRNA3. [Figure 10A] Figures 10A-10B show the use of multiple gRNA targeting sites for VI3 insertion. Figure 10A shows the insertion locations of the eight different gRNAs used. [Figure 10B] Figure 10B shows the results of T2A-mCherry expression using each of the eight different gRNAs. [Figure 11A] Figures 11A-11B show multiple gRNA targeting sites available in the Igκ locus to disrupt endogenous light chain expression and support insertion of a full-length antibody. Figure 11A shows seven different gRNAs used for comparison: gRNA4, gRNA6, gRNA7, gRNA8, gRNA9, gRNA10, and gRNA4+6. [Figure 11B] Figure 11B shows results showing that gRNA7, which cleaves at the splice acceptor site and does not require recoding of the kappa constant region of the AAV template, reduced expression of mlgλ and mlgκ by 91.7%, along with gRNA10 (92.2) and gRNA4+6 (92.0).Other results: mock control (5.42), gRNA4 (82.4), gRNA6 (85.7), gRNA8 (37.3), and gRNA9 (81.7). [Figure 12A]Figures 12A-12B show results for mouse splenic B cells cultured with the following growth factors: 1) CD40L-HA, anti-HA, and IL-4 (described above), 2) anti-CD180, 3) CD40L-HA, anti-HA, and BAFF, 4) anti-CD180 and BAFF, and 5) CD40L-HA, anti-HA, anti-CD180, and BAFF. At 24 hours, 3 million cells were nucleofected with 150 pmol of Cas9, 400 pmol of gRNA, and AAV6-VI3-gRNA1-T2A-mCherry (Figure 12A). [Figure 12B] 500,000 cells were infected with AAV6 at 2.5e5vg / cell and analyzed on day 3 post-infection. As shown in Figure 12B, mCherry expression was strongest in conditions 1 (19.5) and 5 (10.8). [Figure 13A] Figures 13A-13B show the results of mouse splenic B cells cultured with 1) CD40L-HA, anti-HA, and IL-4, or 2) CD40L-HA, anti-HA, and anti-CD180. Three million cells were nucleofected with 150 pmol of Cas9, 400 pmol of total gRNA (BCR-gRNA1 and mlgK-gRNA7), and the full-length H1H29338 antibody (Figure 13A). [Figure 13B] 500,000 cells were infected with AAV1 at 2e5vg / cell and analyzed on day 2 post-infection. Both conditions were functional, with a yield of 8.24% for condition 1 and 3.64% for condition 2, as shown in Figure 13B. [Figure 14A] Figures 14A-14B show the results of transfer and immunization experiments with anti-PcrV-edited B cells. B cells from CHC WT mice were grown in CD40L-HA, anti-HA, and anti-CD180, followed by RNP nucleofection and AAV1 infection with the h1h29339 anti-PcrV full-length antibody 24 hours later (Figure 14A). [Figure 14B]Compared to standard, uninserted VI3 / ULC, antibodies from CHC WT littermate performed better (9.42 and 13.7, respectively, Figure 14B). Overall results for PcrV binding for 1) CHC WT littermate: mock control (0.28), RNP only (0.13), RNP + AAV1 (13.7); and 2) VI3 / ULC: mock control (0.25), RNP only (0.54), RNP + AAV1 (9.42). [Figure 15A] Figures 15A-15B show that B cells edited to express anti-PcrV BCR can mature and produce anti-PcrV antibodies both in vitro and in vivo after adaptive transfer and immunization into mice. In vitro, supernatant analysis of PcrV antibodies from B cells edited for PcrV BCR and cultured in LPS for 7 days demonstrated antibody production (Figure 15A). [Figure 15B] In vivo, B cells were edited for the PcrV BCR as described above and transferred into Flu-CHC mice, and serum analysis was performed approximately 1 week after immunization, demonstrating that the mice again produced antibodies (Figure 15B). [Figure 16] Figure 16 shows adoptive transfer of donor B cells from HA antigen-immunized mice into CD20 cell-depleted naive mouse recipients, and the donor's in vitro-activated B cells appear to expand significantly one week after transfer but do not persist one month later. [Figure 17A] Figure 17A shows the SFFV promoter with subsequences 1, 2, 3, 4, "B cell core", and the putative core promoter (predicted based on the position of the TATA box) indicated. [Figure 17B] Figure 17B shows the expression and cell type specificity of enriched transcription factors (TFs). This expression data is used to select B cell-specific transcription factors. [Figure 17C-1] Figure 17C shows consensus sites that can be incorporated into enhancer scaffolds to enhance expression. [Figure 17C-2] Continued from Figure 17C. [Figure 18A]Figures 18A-18B show an overview and results of reporter construct generation. Figure 18A shows an overview of a representative reporter construct in the AAV context. SFFV subsequences were paired with MLP and cloned upstream of the eGFP coding sequence. This overview shows only the sequence between the AAV ITRs that is paired with bacterial sequences (such as ampicillin resistance) for propagation in Stbl2 cells. [Figure 18B] Figure 18B shows GFP expression (x-axis) after infection of primary mouse B cells with AAV encoding five SFFV subsequences as promoter constructs, all of which are paired with the adenovirus major late promoter. [Figure 19] Figure 19 shows the results of transfecting full-length SFFV-eGFP and variants including SFFV-core-mCP-GFP, SFFV1-mCP-eGFP, SFFV2-mCP-eGFP, SFFV3-mCP-eGFP, and SFFV4-mCP-eGFP into Ramos and HEK293-HZ cells. SFFV4 is 121 bp in length and shows activity in Ramos and HEK cells. [Figure 20] Figure 20 shows results demonstrating that HS-B is a 180-bp B-cell-specific Pax5 enhancer, as indicated by luciferase expression in mouse B-cell lines. Note that GFP in the top row is shifted to the right for pro-B, pre-B, immature, and mature B cells compared to the control. [Figure 21A] Figure 21A shows the results of AAV-GFP testing of 120-170 base pair promoters in primary B cells and HEK293-HZ cells. Three promoters were used with mCP-eGFP: 1) HS-B, 2) hg38HS-B, and 3) SFFV4. Cells were cultured and transfected with 5e5 per cell of AAV6 crude virus preparation, as well as CD40L-HA, anti-HA, and IL-4. [Figure 21B]Figure 21B shows the ranking of HS-B based on B cell enrichment for the ATAC-seq dataset from Corces et al.: #286 / 589,844 (#5344 when ranked by B cell signal). [Figure 22] FIG. 22 shows a schematic of SpyTag:SpyCatcher used to attach mAbs to the surface of AAV capsids for retargeting purposes. [Figure 23A] FIG. 23A shows that AAV2 and AAV6 can be complexed to hCD20 mAb at levels comparable to the ASGR1 control antibody. [Figure 23B-1] Figure 23B shows results demonstrating that AAV2 (top) and AAV6 (bottom) can be targeted to CD20-expressing HEK293 cells. AAV2 or AAV6 with anti-hCD20 mAb attached via SpyTag:SpyCatcher precisely targets HEK293-hCD20 cells. [Figure 23B-2] Continued from Figure 23B. [Figure 24] Figure 24 shows results demonstrating that AAV2 (top) and AAV6 (bottom) can be targeted to CD20-expressing Ramos cells. AAV2 or AAV6 with anti-hCD20 mAb attached via SpyTag:SpyCatcher precisely targets Ramos3-hCD20 cells. The resulting graph (bottom) shows minimal off-targeting with AAV6, but no off-targeting with AAV2. [Figure 25A] Figure 25A shows human B cells cultured under various stimulatory conditions. CD19+ B cells were isolated from human peripheral blood and cultured under various stimulatory conditions: 1) IL-4 alone, 2) IL-4, CD40L-HA, and anti-HA mAb, and 3) IL-4 and anti-CD40 mAb. Cells were infected with AAV2 / CD20 or AAV6 / CD20, and virally delivered eGFP was measured by flow cytometry 4 days after infection. [Figure 25B]Figure 25B shows results demonstrating that AAV2 (top) and AAV6 (bottom) can target CD20-expressing human B cells. These results demonstrate that although both AAV2 and AAV6 can target primary human B cells via CD20, AAV6 / CD20 shows dramatically enhanced transduction. [Figure 26] Figure 26 shows AAV1 WT, AAV1 detargeted mutants, and AAV1-hCD20, all packaged with SSF-eGFP and retargeted to HEK 293 CD20(-), HEK 293 CD20(+), Jurkat T cells, and the Duadi B cell line. These results demonstrate that the AAV1 detargeted mutants still achieve transduction, and that although antibody conjugation slightly reduces off-target transduction, the retargeted viruses are equivalent to WT in the Duadi cell line. [Figure 27] Figure 27 shows AAV2 WT, AAV2 detargeted mutants, and AAV2-hCD20, all packaged with SSF-eGFP and retargeted to HEK 293 CD20(-), HEK 293 CD20(+), Jurkat T cells, and Duadi B cell lines. These results demonstrate that AAV2-CD20 exhibits gain of function in the Duadi cell line. [Figure 28] Figure 28 shows AAV6 WT, AAV6 detargeting mutants, and AAV6-hCD20, all packaged with SSF-eGFP and retargeted to HEK 293 CD20(-), HEK 293 CD20(+), Jurkat T cells, and Duadi B cell lines. These results demonstrate that the AAV6 retargeting mutants are not completely detargeted, that the non-binding mAb reduces off-target transduction, and that AAV6-CD20 exhibits gain of function in the 293 hCD20(+) cell line. [Figure 29]Figure 29 shows AAV9 WT, AAV9 detargeted mutants, and AAV9-hCD20, all packaged with SSF-eGFP and retargeted to HEK 293 CD20(-), HEK 293 CD20(+), Jurkat T cells, and Duadi B cell lines. These results demonstrate that AAV9-CD20 exhibits gain of function in hCD20(+) cell lines and exhibits little off-target transduction. [Figure 30] Figure 30 shows a schematic diagram of a method for translating ex vivo B cell targeting and editing technology into in vivo use by delivering viral vectors in vivo to mediate BCR insertion. [Figure 31] FIG. 31 shows a schematic diagram illustrating that human stem cells are upstream of immune cells and are targets for transduction by a variety of viruses, including AAV and lentiviruses. [Figure 32] Figure 32 shows the results demonstrating that AAV6 attached to anti-hCD34 (My10) antibody via the SpyTag:SpyCatcher system, along with various promoters attached to GFP, was used to infect human umbilical cord blood cells and primary mouse B cells. The results showed that SFFV was a preferred promoter over CAG and EF1. [Figure 33] Figure 33 shows AAV2-hCD34 packaged with SFFV-eGFP and retargeted to HSCs. The results demonstrate that natural tropism abolishes retargeting antibodies to human cord blood cells, while non-binding mAbs reduce off-target transduction, and anti-CD34 mAbs can retarget AAV2 HBM mutants in 293 / hCD34 and human cord blood cells. [Figure 34] Figure 34 shows AAV9-hCD34 packaged with SFFV-eGFP and retargeted to HSCs. The results demonstrate gain of function in the 293 hCD34+ cell line in the presence of CD34 antibody; low off-target transduction; and poor transduction of human umbilical cord blood cells with AAV9+ / - anti-hCD34 antibody. [Figure 35]Figure 35 shows AAV6-hCD34 packaged with SFFV-eGFP and retargeted to HSCs. The results show that anti-CD34 mAb can potently retarget the AAV6 HBM mutant in 293 / hCD34 and moderately in human cord blood cells, although natural tropism again abolishes the retargeting antibody to human cord blood cells. [Figure 36-1] Figure 36 shows results demonstrating that lentiviral vectors complexed with an anti-CD34 comparator mAb specifically retarget CD34-expressing cells with mAb-dependent transduction efficacy. 10,000 cells were seeded per plate (96-well plate), and 2E+08VG of LV-SINmuZZ EF1a-FLuc was mixed with 2-fold serially diluted CHOt supe (starting at 100 μl) in DMEM. After 30 minutes of incubation at 37°C, the LV-CHOt mixture was added to the cells and incubated at 37°C. Fluc readings were performed 4 days post-transduction. Results are shown for the following nine conditions: 1) 9C5(CD34)-SpyC; 2) My1C(CD34)-SpyC; 3) 563(CD34)-SpyC; 4) CD20-SpyC; 5) 9C5; 6) CD20; 7) BSTpro MOCK; 8) VLP only; and 9) NT. The above experiment was also repeated with HEK293, 293-hCD20, and 293-hCD34 cells. [Figure 36-2] Continued from Figure 36. [Figure 37-1]Figure 37 shows results demonstrating that SpyTagged AAV2 complexed with the anti-CD34-SpyCatcher comparator mAb can also specifically retarget CD34-expressing cells, with mAb-dependent transduction efficacy. Results are shown for the following nine conditions: 1) 9C5(CD34)-SpyC; 2) My1C(CD34)-SpyC; 3) 563(CD34)-SpyC; 4) CD20-SpyC; 5) 9C5; 6) CD20; 7) BSTpro MOCK; 8) VLP only; and 9) NT. The above experiment was also repeated with HEK293, 293-hCD20, and 293-hCD34 cells. [Figure 37-2] Continued from Figure 37. [Figure 38] Figure 38 shows results demonstrating that optimizing the mosaic ratio reveals that AAV2 HBM-mixer 1 / 4 leads to relatively high transduction in the HEK293T / hCD34 cell line. Screening for platform gene delivery to CD34 begins with seeding 10,000 cells per well in a 96-well clear-bottom black plate using three cell types (293, 293-hCD20, and 293-hCD34). Next, 5E+09VG of AAV2 1 / 8 SpyTag / HBM SFFV-Fluc is mixed with two-fold serially diluted CHOt super (starting at 100 μl) in DMEM and incubated at 37°C for 1.5 hours. The AAV2-CHOt mixture is then added to the cells and incubated at 37°C. After three days, cells are harvested for flow analysis. The results shown in Figure 38 are for different types of HBM mixers. AAV2 HBM-mixer 1 / 4 showed relatively high transduction in 293-CD34 cells, followed by AAV2 HBM-mixer 1 / 2. [Figure 39A]Figures 39A-39C show the results of in vitro transduction of cell lines expressing the respective antigen receptors with lentiviral vectors retargeted with anti-CD117 and anti-Sca-1. 1E+04 cells were seeded in 100 μl of DCM containing 4 μg / ml polybrene in a 96-well clear-bottom black plate. The cells were transduced with 2E+04VG per cell in 100 μl aliquots of DCM containing 4 μg / ml polybrene. Fluorescence imaging was performed after 2 days (Figure 39A). [Figure 39B-1] Analysis of GFP by flow cytometry after 2 days (Figure 39B) [Figure 39B-2] Continued from Figure 39B. [Figure 39B-3] Continued from Figure 39B. [Figure 39B-4] Continued from Figure 39B. [Figure 39C] Analysis of GFP by flow cytometry after 2 days (Figure 39C). The results show that the vectors successfully retargeted cell lines expressing their respective target antigens by imaging and flow cytometry. [Figure 40] Figure 40 shows results demonstrating that surface expression of CD117 and Sca-1 is detected on mouse HSPCs. On day 0, mouse HSPCs are isolated from harvested bone marrow. Cells are cultured in a precursor medium containing SFEM+SCF (100 ng / mL), TPO (100 ng / mL), Flt3L (100 ng / mL), IL-6 (50 ng / mL), and IL-3 (30 ng / mL). Two days after inoculation, cells are examined for CD117 and Sca-1 and subsequently transduced with pseudoparticles containing the SFFV-GFP reporter. Two days after transduction (four days after inoculation), GFP expression is read by FACS. The results show the surface expression of CD117 and Sca-1 on mouse HSPCs. [Figure 41-1]Figure 41 shows results demonstrating the transduction of mouse HSPCs using lentiviral vectors pseudotyped with anti-mouse CD117 mAb and SINmu. Conditions were no additive, vectofusin-1, or lentiboost (left to right). Cells were either untransduced or transduced with LV-VSVg, LV-ahASGR1+SINmu, or LV-amCD117+SINmu (top to bottom). LVs pseudotyped with α-CD117+SINmu can transduce expanded mouse primary HSPCs with very low efficiency. This is an entry issue, as LVs pseudotyped with VSVg can efficiently transduce expanded mouse HSPCs. [Figure 41-2] Continued from Figure 41. [Figure 41-3] Continued from Figure 41. [Figure 41-4] Continued from Figure 41. [Figure 42A] Figures 42A-42B show results demonstrating that SpyTagged AAV2 is efficiently retargeted in vitro to cell lines expressing CD117 or Sca-1. AAV2-HBM 1 / 8 mosaic retargeted with CD117, Sca-1, hCD34, or hCD20 was successfully retargeted to HEK293 cell lines expressing these markers at 5E+05VG per cell, as shown by fluorescent imaging (Figure 42A) and analysis of GFP by flow cytometry (Figure 42B). [Figure 42B-1] Analysis of GFP by flow cytometry (Figure 42B) [Figure 42B-2] Continued from Figure 42B. [Figure 42B-3] Continued from Figure 42B. [Figure 42B-4] Continued from Figure 42B. [Figure 42B-5] Continued from Figure 42B. [Figure 42B-6] Continued from Figure 42B. [Figure 42B-7] Continued from Figure 42B. [Figure 42B-8] Continued from Figure 42B. [Figure 42B-9]Continued from Figure 42B. [Figure 42C] Figures 42C-42D show the design rationale for SpyTagged AAV2 containing an anti-ScaI antibody. [Figure 42D] The rationale for the design of SpyTagged AAV2 containing an anti-ScaI antibody is presented. [Figure 43] FIG. 43 shows a proposed strategy for antibody engineering in B1 B cells. [Figure 44] Figure 44 shows a proposed strategy for ectopically engineered antibody expression in B1 B cells. [Figure 45-1] FIG. 45 shows that B1a B cells activated with CD40L / aCD180 and transferred intraperitoneally show enhanced recovery at 14 and 32 days. [Figure 45-2] Continued from Figure 45. [Figure 46-1] FIG. 46 shows that CD180 stimulation of B1a cells leads to proliferation without differentiation into plasmablasts / plasma cells (PCs). [Figure 46-2] Continued from Figure 46. [Figure 47] FIG. 47 demonstrates that transduction efficiency differs between B1 and B2 peritoneal cavity (PerC) B cell subsets. [Figure 48] Figure 48 shows that peritoneal-derived Pan B cells can be edited, but less efficiently than B2 splenocytes. [Figure 49A] Figures 49A-49B show results demonstrating in vitro culture conditions favorable for the re-engraftment of ex vivo cultured mouse B cells. In vitro B cell culture with low levels of aCD40 and / or aCD180 promoted cell re-engraftment in mice, while high B cell activation did not contribute to long-term re-engraftment (Figure 49A). [Figure 49B] CD45.1 B cells cultured ex vivo for 3 days were adoptively transferred into CD45.2 mice (Figure 49B). [Figure 50A]Figures 50A-50D show results demonstrating ex vivo AAV transduction / editing of cultured undifferentiated Cas9 mouse B cells and transfer into SRG mice. Figure 50A shows a summary of ex vivo AAV transduction / editing of cultured undifferentiated Cas9 mouse B cells and transfer into SRG mice. [Figure 50B] Figure 50B shows the J chain locus exon 4 insertion. [Figure 50C] Figure 50C shows the ROSA insertion. [Figure 50D] FIG. 50D shows the luciferase signal measured in vivo over time using IVIS technology. [Figure 51A] Figures 51A-51B show exemplary editing strategies for different mouse loci for different modalities. Figure 51A shows an exemplary "immune repertoire enhancement" modality. [Figure 51B] FIG. 51B shows an exemplary "protein factory" modality. [Figure 52A] Figures 52A-52B show exemplary mouse J chain locus insertion strategies for high expression of a protein of interest in plasma cells. Figure 51A shows an exemplary strategy that uses the endogenous J chain promoter and preserves the J chain. [Figure 52B] Figure 52B shows an exemplary strategy using the endogenous J chain promoter and removing the J chain. [Figure 53A] Figures 53A-53B show that the generation of memory B cells is key to successful in vivo B cell editing for both adaptive antibody and protein factory modalities. Figure 53A shows an exemplary scheme from the expansion of BCR-edited B cells via the new BCR. [Figure 53B] Figure 53B shows an exemplary scheme from the expansion of non-BCR-edited B cells via "linked specificity" for a priming Ag. [Figure 54A]Figures 54A-54D show results demonstrating that modulating "Pan B cell" stimulation of Cas9 mice enables AAV editing of B cells and Ab production. Figure 54A shows the VI3 heavy chain, gRNA cleavage site, and BCR insert. [Figure 54B] Figure 54B shows results demonstrating the synergistic effect of in vivo stimulation of aCD40 and aCD180 on B cell editing and antibody expression. [Figure 54C] Figure 54C shows results demonstrating that Ab1 Ab expression is short-lived in "pan-B" primed / edited mice, but shows evidence of Ab recall after subsequent Ag challenge. [Figure 54D] Figure 54D shows results demonstrating that in vivo B cell editing of "Pan B" stimulated mice results in edited B cells that can be mobilized in response to subsequent Ag challenge. [Figure 55A] Figures 55A-55D show results demonstrating that priming and boosting with suboptimal BCR:Ag interactions stimulates Ab1 memory B cells over Ab-producing PCs. Figure 55A shows that suboptimal BCR:Ag interactions ("low affinity") are predicted to bias edited B cells toward the memory compartment. A "high affinity" BCR results in high B cell activation, and increased Tfh interactions drive cells toward Ab-producing plasma cells. B cells with "low affinity" BCRs are relatively less activated and fail to adequately engage Tfh cells. This neglect results in a bias toward memory B cells. [Figure 55B] Figure 55B shows the results demonstrating that Ab1 reduces neutralization of F490L Spike by 90-fold. [Figure 55C] Figure 55C shows results demonstrating that priming and boosting with F490L Spike Ag fails to induce Ab1 Ab production from Ab1-edited B cells. [Figure 55D]Figure 55D shows results demonstrating that editing of the Ab1 BCR in mice primed with "low affinity" Ag results in reproducible induction of Ab expression after boosting with WT "high affinity" Ag on day 28. [Figure 56A] Figures 56A-56C show results demonstrating that adding aCD180 to Ag prime increases the number of edited B cells that can be recalled 1 and 3 months after editing. Figures 56A-56B show an exemplary experimental workflow. [Figure 56B] 1 illustrates an exemplary experimental workflow. [Figure 56C] Figure 56C shows that in the Ag prime only group, Ab levels after the "high affinity" Ag boost were lower compared to the aCD180-treated group, and the increase in Ab titer in the aCD180 group after the Ab boost is evidence that more edited B cells were initially generated. [Figure 57A] Figures 57A-57C show results demonstrating the long-term persistence of in vivo edited B cells (non-IgH locus) in Ag-primed mice. Figure 57A shows an exemplary workflow for B cell-specific promoter-driven luciferase editing into the Rosa locus in Ag-primed Cas9 mice. [Figure 57B] Figure 57B shows the results of IVIS imaging of B cell-specific luciferase expression in edited mice. [Figure 57C] Figure 57C shows longitudinal analysis of luciferase signals demonstrating the durability of in vivo edited B cells in mice. [Figure 58A] Figures 58A-58B show results demonstrating that AAV "Nluc-Abl" editing of the IgH locus allows for in vivo tracking of BCR-edited cells over time. Figure 58A shows an exemplary workflow. [Figure 58B] Figure 58B shows results showing tracking of BCR-edited cells over time. [Figure 59A]Figures 59A-59C show results demonstrating peritoneal B cell editing achieved by IP delivery of AAV into unprimed Cas9Ready mice. Figure 59A shows an exemplary workflow. [Figure 59B] Figure 59B shows that luciferase signals were readily observed in all draining lymph nodes of the peritoneal cavity of Cas9Ready mice edited with B cell-specific luciferase. [Figure 59C] FIG. 59C shows that nLuc-positive signals are observed primarily in B1b and B1a B cells in the peritoneal cavity. DETAILED DESCRIPTION OF THE INVENTION

[0078] definition Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0079] The singular forms "a," "an," and "the" include plural referents unless the context dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0080] 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 amino acids that are chemically or biochemically modified or derivatized. These terms also include modified polymers, such as polypeptides having modified peptide backbones.

[0081] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, include polymeric forms of nucleotides of any length, containing ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. This includes single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other naturally occurring, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0082] Nucleic acids are described as having a "5' end" and a "3' end." This is because oligonucleotides are made by reacting mononucleotides such that the 5' phosphate of one mononucleotide pentose ring is unidirectionally linked to the 3' oxygen of the adjacent mononucleotide pentose ring via a phosphodiester bond. The terminus of an oligonucleotide is called the "5' end" if its 5' phosphate is not linked to the 3' oxygen of the mononucleotide pentose ring. The terminus of an oligonucleotide is called the "3' end" if its 3' oxygen is not linked to the 5' oxygen of another mononucleotide pentose ring. Nucleic acid sequences can be described as having 5' and 3' ends, even when they are internal to a larger oligonucleotide. In either a linear or circular DNA molecule, individual elements are said to be "upstream" or 5' of the "downstream" or 3' element.

[0083] The term "wild type" (WT) includes entities having a structure and / or activity found in a normal state or context (as opposed to a mutant, diseased, altered, etc.). Wild-type genes and polypeptides often exist in many alternative forms (e.g., alleles).

[0084] As used herein, the term "B cell" refers to a cell of the B cell lineage. In some embodiments, B cells used in the compositions and methods of the disclosure include, but are not limited to, B1 B cells, B2 B cells, memory B cells, plasmablasts, or plasma cells, or a combination thereof. In some embodiments, B cells used in the compositions and methods of the disclosure may be primary B cells.

[0085] The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) and two light (L) chains, interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (V H ) and heavy chain constant region (C H The heavy chain constant region comprises at least three domains: C H 1. C H 2. C H 3, and optionally C H 4. Each light chain contains a light chain variable domain (C H ) and light chain constant region (C L ). The heavy and light chain variable domains can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). The heavy and light chain variable domains each contain three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3). A typical tetrameric antibody structure contains two identical antigen-binding domains, each of which is designated V H Domains and V L domains, and C H Domain and C LThe domains together form the antibody Fv region. Single domain antibodies contain a single antigen-binding domain, e.g., V H or V L As used herein, the term "antibody" specifically encompasses B cell receptor (BCR) and secreted antibodies. The term "antibody" also encompasses monoclonal antibodies, multispecific (e.g., bispecific) antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFvs), single-chain antibodies, single-domain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFvs), intrabodies, minibodies, diabodies, and anti-idiotypic (anti-Id) antibodies (e.g., anti-Id antibodies against antigen-specific TCRs), as well as epitope-binding fragments of any of these. The terms "antibody and antibodies" also refer to covalent diabodies, such as those disclosed in U.S. Patent Application Publication No. 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication No. 2009 / 0060910. Antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0086] The antigen-binding domain of an antibody, e.g., the portion of an antibody that recognizes and binds to an epitope of an antigen, is also called the "paratope." This is a small region (5-10 amino acids) of the Fv region of an antibody, which is part of the antigen-binding fragment (Fab region), and may contain part of the heavy and / or light chains of the antibody. A paratope specifically binds to an epitope if it binds to that epitope with high affinity. The term "high affinity" antibody refers to an antibody that has a high affinity for its target epitope. D is about 10 -9 M or less (for example, about 1 × 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, or approximately 1 x 10-12 In one embodiment, K D is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K D is measured by ELISA.

[0087] The phrase "complementarity-determining region," or "CDR," includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin gene, which typically (i.e., in a wild-type animal) occurs between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T-cell receptor). CDRs can be encoded, for example, by germline sequences, or by rearranged or unrearranged sequences, and by, for example, naive or mature B or T cells. CDRs can be somatically mutated (e.g., different from the sequence encoded in the animal's germline), humanized, and / or modified by amino acid substitution, addition, or deletion. In some situations (e.g., in the case of a CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B-cell nucleic acid sequence, for example, as a result of splicing or joining of sequences (e.g., VDJ recombination to form a heavy chain CDR3).

[0088] An "epitope" is a portion of a macromolecule that is recognized by the immune system, specifically by antibodies, B cells, or cytotoxic T cells. Epitopes are usually considered to be derived from non-self proteins, but recognizable host-derived sequences are also classified as epitopes. Epitopes are at least 4 amino acids in length, preferably 4 to 30 amino acids, more preferably 5 to 20 amino acids, and especially 5 to 15 amino acids. Epitopes can typically be formed linearly or three-dimensionally by amino acids that are distant from each other in the primary protein structure but closely related in the secondary and / or tertiary structure. Epitopes that are specifically recognized by B cells are called B cell epitopes.

[0089] The term "light chain" includes immunoglobulin molecule light chain sequences from any organism, including human kappa and lambda light chains and VpreB, as well as surrogate light chains, unless otherwise specified. Unless otherwise specified, a light chain variable domain typically includes three light chain CDRs and four framework (FR) regions. Generally, a full-length light chain includes, from the amino terminus to the carboxy terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. The light chain variable domain is encoded by a light chain variable region gene sequence, which generally includes a V and J segment derived from a repertoire of V and J segments present in the germline. L Segment and J L The sequences, locations, and nomenclature of V and J light chain segments from various organisms can be found in the IMGT database, imgt.org. Light chains include, for example, light chains that do not selectively bind to the first or second epitopes selectively bound by the epitope-binding protein in which they appear. Light chains also include those that bind to and recognize one or more epitopes selectively bound by the epitope-binding protein in which they appear, or that assist a heavy chain or another light chain in binding to and recognizing said one or more epitopes. Common or universal light chains include those derived from the human Vκ1-39Jκ gene or the human Vκ3-20Jκ gene, and somatically mutated (e.g., affinity matured) versions thereof. Exemplary human Vκ1-39Jκ gene and Vκ3-20Jκ gene are also included. LSegments include a human Vκ1-39 gene segment, a human Vκ3-20 gene segment, a human Vλ1-40 gene segment, a human Vλ1-44 gene segment, a human Vλ2-8 gene segment, a human Vλ2-14 gene segment, and a human Vλ3-21 gene segment, including somatically mutated (e.g., affinity matured) versions thereof. Light chains can be engineered to contain variable domains from one organism (e.g., a human or rodent, such as a rat or mouse; or a bird, such as a chicken) and constant regions from the same or a different organism (e.g., a human or rodent, such as a rat or mouse; or a bird, such as a chicken).

[0090] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences, including immunoglobulin heavy chain constant region sequences, from any organism. Unless otherwise specified, a heavy chain variable domain contains three heavy chain CDRs and four FR regions. Fragments of heavy chains contain CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain contains (from the N-terminus to the C-terminus) the variable domain followed by a C H 1 domain, hinge, C H 2 domain, and C H A functional fragment of the heavy chain can specifically recognize an epitope (e.g., a K in the micromolar, nanomolar, or picomolar range). D The heavy chain variable domain is encoded by a variable region nucleotide sequence, and this variable region nucleotide sequence is generally a V region present in the germline. H , D H and J H V derived from segmental repertoire H , D H Segment and J HThe sequences, locations, and nomenclature of the V, D, and J heavy chain segments of various organisms can be found in the IMGT database, accessible via the Internet on the World Wide Web (www) at the URL "imgt.org."

[0091] The terms "heavy chain only antibody," "heavy chain only antigen binding protein," "single domain antigen binding protein," "single domain binding protein," and the like refer to antibodies that contain a functional C domain in the heavy chain constant region. H The term "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," etc., refers to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, which is unable to bind to a light chain because it typically lacks one domain. Thus, the terms "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," etc. refer to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, which is usually unable to bind to a light chain because it lacks one domain. H a monomeric single-domain antigen-binding protein comprising one immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region lacking one domain; or (ii) a functional C H In various embodiments, the homodimeric single domain antigen binding protein includes both a functional C α -α , a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains each comprising a variable domain operably linked to a heavy chain constant region lacking one domain, and a homodimeric single domain antigen binding protein comprising two immunoglobulin-like chains each comprising a variable domain operably linked to a heavy chain constant region lacking one domain. H The single domain antigen binding protein comprises two identical immunoglobulin-like chains, each comprising an identical variable domain operably linked to an identical heavy chain constant region lacking one domain. Furthermore, each immunoglobulin-like chain of a single domain antigen binding protein comprises a C-terminal fragment of a heavy chain constant region gene (e.g., IgG, IgA, IgE, IgD, or a combination thereof). H 1. A heavy chain constant region (C) containing deletions or inactivating mutations in the coding sequence (and optionally the hinge region)H ) gene sequence, linked to a heavy chain variable region gene segment (e.g., V H , D H , J H ), light chain gene segments (e.g., V L , J L ), or a combination thereof. Single domain antigen binding proteins comprising variable domains derived from heavy chain gene segments are referred to as "V H -single domain antibody" or "V H Single-domain antigen binding proteins comprising a variable domain derived from a light chain gene segment are sometimes referred to as "V-single-domain antigen binding proteins." See, e.g., U.S. Patent No. 8,754,287; U.S. Patent Application Publication No. 20140289876; U.S. Patent Application Publication No. 20150197553; U.S. Patent Application Publication No. 20150197554; U.S. Patent Application Publication No. 20150197555; U.S. Patent Application Publication No. 20150196015; U.S. Patent Application Publication No. 20150197556; and U.S. Patent Application Publication No. 20150197557, each of which is incorporated herein by reference in its entirety. ... L -single domain antibody" or "V L They are also sometimes referred to as "single-domain antigen-binding proteins." See, e.g., U.S. Patent Application Publication No. 20150289489, which is incorporated herein by reference in its entirety.

[0092] The terms "about" or "approximately" include within a statistically significant range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the terms "about" or "approximately" depends on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0093] The term "affinity tag" includes a polypeptide sequence that is a member of a specific binding pair, e.g., that specifically binds with high affinity to another polypeptide sequence, e.g., an antibody paratope. Exemplary, non-limiting affinity tags include hexahistidine tags, FLAG tags, Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP) tags, glutathione S-transferase (GST), maltose-binding protein (MBP), S tags, HA tags, and c-Myc tags (reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8, incorporated herein by reference).

[0094] The term "capsid protein" includes proteins that are part of a viral capsid. In the case of adeno-associated virus (AAV), the capsid proteins are commonly referred to as VP1, VP2, and / or VP3, each encoded by a single cap gene. In the case of AAV, the three AAV capsid proteins are produced redundantly from the cap open reading frame (ORF) by alternative mRNA splicing and / or alternative translation initiation codon usage, but all three proteins share a common stop codon. Warrington et al. (2004) J. Virol. 78:6595 (incorporated herein by reference in its entirety). AAV2 VP1 is generally translated from the ATG start codon (amino acid M1) on the 2.4 kb mRNA, while AAV2 VP2 and VP3 arise from a smaller 2.3 kb mRNA, using a weaker ACG start codon (amino acid T138) for production of VP2 and read-through translation to the next available ATG codon (amino acid M203) for production of the most abundant capsid protein, VP3. (Warrington, supra; Rutledge et al. (1998) J. Virol. 72:309-19, incorporated herein by reference in its entirety). The amino acid sequences of adeno-associated virus capsid proteins are known in the art and are generally conserved, particularly among dependoparvoviruses. See Rutledge et al., supra. For example, Rutledge et al. (1998), supra, provide in Figure 4B an amino acid sequence alignment of the VP1, VP2, and VP3 capsid proteins of AAV2, AAV3, AAV4, and AAV6, in which the start sites of each of the VP1, VP2, and VP3 capsid proteins are indicated by arrows and the variable domains are boxed.Thus, although the amino acid positions provided herein may be provided relative to the VP1 capsid protein of AAV, one skilled in the art can readily determine the same amino acid positions within the VP2 and / or VP3 capsid proteins of AAV, as well as corresponding amino acid positions between different serotypes. Furthermore, one skilled in the art can exchange domains between capsid proteins of different AAV serotypes to form "chimeric capsid proteins."

[0095] Swapping domains between two AAV capsid protein constructs to generate a "chimeric AAV capsid protein" has been previously described; see, e.g., Shen et al. (2007) Mol. Therapy 15(11):1955-1962, which is incorporated herein by reference in its entirety. A "chimeric AAV capsid protein" includes an AAV capsid protein that contains amino acid sequences, e.g., domains, from two or more different AAV serotypes and that can and / or does form an AAV-like viral capsid / viral particle. A chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., nucleotides, that contains a plurality of, e.g., at least two, nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a distinct AAV serotype, and which together encode a functional chimeric AAV capsid protein. Reference to a chimeric capsid protein associated with a particular AAV serotype indicates that the capsid protein comprises one or more domains from a capsid protein of that serotype and one or more domains from a capsid protein of a different serotype. For example, an AAV2 chimeric capsid protein comprises a capsid protein comprising one or more domains from an AAV2 VP1, VP2, and / or VP3 capsid protein and one or more domains from a VP1, VP2, and / or VP3 capsid protein of a different AAV.

[0096] A "mosaic capsid" contains at least two sets of VP1, VP2, and / or VP3 proteins, each set encoded by a different cap gene.

[0097] In some embodiments, the mosaic capsids described herein comprise recombinant VP1, VP2, and / or VP3 proteins encoded by a cap gene that has been genetically modified by the insertion of a nucleic acid sequence encoding a heterologous epitope, and further comprise VP1, VP2, and / or VP3 proteins encoded by a reference cap gene, such as: a wild-type reference cap gene encoding wild-type VP1, VP2, and / or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins; a control reference cap gene encoding VP1, VP2, and / or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins except for the absence of the heterologous epitope; or a mutant wild-type reference cap gene encoding substantially wild-type VP1, VP2, and / or VP3 proteins of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 proteins except for mutations (e.g., insertions, substitutions, deletions), which preferably reduce the tropism of the wild-type VP1, VP2, and VP3 proteins. In some embodiments, the reference capsid protein is a chimeric reference protein that includes at least one domain of a VP1, VP2, and / or VP3 protein of the same AAV serotype as the recombinant VP1, VP2, and / or VP3 protein described above. In some embodiments, the reference cap gene encodes a chimeric VP1, VP2, and / or VP3 protein.

[0098] The term "recombinant capsid protein" includes a capsid protein having at least one mutation compared to the corresponding capsid protein of a wild-type virus, which can serve as a reference and / or control virus for comparative studies. Recombinant capsid proteins include capsid proteins containing heterologous epitopes that can be inserted into and / or displayed by the capsid protein. "Heterologous" in this context means heterologous compared to the virus from which the capsid protein is derived. The inserted amino acid may simply be inserted between two given amino acids of the capsid protein. The insertion of an amino acid may also involve the deletion of a given amino acid of the capsid protein at the insertion site, e.g., one or more capsid protein amino acids are replaced with five or more heterologous amino acids.

[0099] "Retargeting" or "redirecting" may include scenarios in which a wild-type vector targets multiple cells within a tissue and / or multiple organs within an organism, where global targeting of the tissue or organ is reduced or eliminated by insertion of a heterologous epitope, and further retargeting to specific cells within the tissue or specific organs within the organism is achieved by a targeting ligand that binds to a marker expressed by said specific cells. Such retargeting or redirection may also include scenarios in which a wild-type vector targets a tissue, where tissue targeting is reduced or eliminated by insertion of a heterologous epitope, and retargeting to an entirely different tissue is achieved by a targeting ligand.

[0100] The term "inverted terminal repeat" or "ITR" refers to symmetrical nucleic acid sequences within the genome of an adeno-associated virus (AAV) that are necessary for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs function as origins of replication for viral DNA synthesis and are essential cis components for the generation of AAV integrating vectors.

[0101] "Codon optimization" takes advantage of codon degeneracy, as indicated by the variety of three-base-pair codon combinations that specify amino acids, and generally involves the process of modifying a nucleic acid sequence by replacing at least one codon of a native sequence with a codon that is relatively or most frequently used in the genes of a particular host cell (e.g., packaging cell) and / or target cell, while maintaining the native amino acid sequence, in order to enhance expression in the host cell and / or target cell. For example, a nucleic acid encoding a Cas9 protein can be modified to replace a codon that is more frequently used relative to the native nucleic acid sequence in a given prokaryotic or eukaryotic cell, including bacterial cells, yeast cells, human cells, non-human cells, mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, or any other host and / or target cell. Codon usage tables are readily available, for example, from the "Codon Usage Database." These tables can be adapted in a variety of ways. See Nakamura et al. (2000) Nucleic Acids Research 28:292, incorporated herein by reference in its entirety for all purposes. Computer algorithms are also available for codon optimization of a particular sequence for expression in a particular host and / or target (see, e.g., Gene Forge).

[0102] A "promoter" is a regulatory region of DNA that typically contains a TATA box that can direct RNA polymerase to begin RNA synthesis at the appropriate transcription start site for a particular polynucleotide sequence. A promoter may also contain other regions that affect the rate of transcription initiation. As used herein, the term "promoter" encompasses enhancers. The promoter sequences disclosed herein regulate the transcription of an operably linked polynucleotide. The promoter can be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The 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). RNA Pol III promoters are frequently used to express small interfering RNA (siRNA) / short hairpin RNA (shRNA) and guide RNA sequences used in the CRISPR-Cas9 system. Examples of RNA Pol III promoters that can be used in the present invention include, but are not limited to, the human U6 promoter, rat U6 polymerase III promoter, or mouse U6 polymerase III promoter, and the H1 promoter, as described, for example, in Goomer and Kunkel, Nucl. Acids Res., 20 (18): 4903-4912 (1992), and Myslinski et al., Nucleic Acids Res., 29 (12): 2502-9 (2001). Examples of promoters can be found, for example, in WO 2013 / 176772, which is incorporated herein by reference in its entirety for all purposes.

[0103] Examples of inducible promoters include chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include alcohol-regulated promoters (e.g., alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., tetracycline-responsive promoters, tetracycline operator sequence (tetO), tet-On promoters, tet-Off promoters), steroid-regulated promoters (e.g., rat glucocorticoid receptor, estrogen receptor promoter, or ecdysone receptor promoter), or metal-regulated promoters (e.g., metalloprotein promoters). Physically regulated promoters include temperature-regulated promoters (e.g., heat shock promoters) and light-regulated promoters (e.g., light-inducible promoters or light-repressible promoters).

[0104] Examples of tissue-specific promoters include neuron-specific promoters, glial-specific promoters, muscle cell-specific promoters, cardiac cell-specific promoters, kidney cell-specific promoters, bone cell-specific promoters, endothelial cell-specific promoters, or immune cell-specific promoters (e.g., B cell promoters or T cell promoters).

[0105] Developmentally-regulated promoters include, for example, promoters that are active only during embryonic stages of development or only in mature cells.

[0106] A "self-cleaving peptide" or "self-cleaving sequence" encoding a self-cleaving domain is a peptide or coding sequence, respectively, that induces ribosomal skipping during protein translation, resulting in cleavage. Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al. (1997) J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites are the cleavage sites for potyvirus NIa protease (e.g., tobacco etch virus protease), potyvirus HC protease, potyvirus P1 (P35) protease, bymovirus NIa protease, bymovirus RNA-2-encoded protease, aphthovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picornavirus 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleck virus) 3C-like protease, thrombin, factor Xa, and enterokinase. The TEV (tobacco etch virus) protease cleavage site is particularly preferred due to its high cleavage stringency. In some embodiments, the isolated nucleic acid comprises a self-cleaving peptidyl sequence encoding a self-cleaving peptidyl domain between the heavy and light chain sequences. A preferred self-cleaving peptide (also called a "cis-acting hydrolytic element," CHYSEL; see deFelipe (2002) Curr. Gene Ther. 2, 355-378) is derived from the potyvirus and cardiovirus 2A peptide.In some embodiments, the self-cleaving peptide is selected from 2A peptides derived from FMDV (foot and mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine teschovirus.

[0107] In some embodiments, a self-cleaving peptidyl linker sequence used herein is a 2A sequence. In some embodiments, the self-cleaving peptidyl linker sequence is a T2A sequence, a P2A sequence, an E2A sequence, or an F2A sequence. In some embodiments, the self-cleaving peptidyl linker sequence is a foot and mouth disease virus sequence. In some embodiments, the self-cleaving peptidyl linker sequence is PVKQLLNFDLLKLAGDVESNPGP (SEQ ID NO: 6). In some embodiments, the self-cleaving peptidyl linker sequence is an equine rhinitis A virus sequence. In some embodiments, the self-cleaving peptidyl linker sequence is QCTNYALLKLAGDVESNPGP (SEQ ID NO: 7). In several embodiments, the self-cleaving peptidyl linker sequence is a porcine teschovirus 1 sequence. In several embodiments, the self-cleaving peptidyl linker sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO: 8). In some embodiments, the self-cleaving peptidyl linker sequence is a Thosea asigna virus sequence. In some embodiments, the self-cleaving peptidyl linker sequence is EGRGSLLTCGDVESNPGP (SEQ ID NO: 9). In some embodiments, the light chain sequence is 3' to the heavy chain sequence. In some embodiments, the light chain sequence is 5' to the heavy chain sequence.

[0108] As used herein, the phrase "operably linked" includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other or otherwise cooperate with each other to participate in biological events, which juxtaposition achieves or enables such interaction and / or cooperation. By way of example, a regulatory sequence (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when it is positioned relative to the coding sequence such that its presence or absence affects the expression and / or activity of the coding sequence. In many embodiments, "operable linkage" involves the covalent attachment of the associated components or elements to each other. However, those skilled in the art will readily appreciate that in some embodiments, covalent attachment is not required to achieve effective operable linkage. For example, in some embodiments, a nucleic acid regulatory sequence operably linked to a coding sequence to be regulated is contiguous with the nucleotide of interest. Alternatively, or in addition, in some embodiments, one or more such regulatory sequences act in trans or remotely to regulate the coding sequence of interest. In some embodiments, the term "expression control sequence," as used herein, refers to polynucleotide sequences necessary and / or sufficient to effect the expression and processing of linked coding sequences. In some embodiments, expression control sequences may be or include: appropriate transcription initiation, termination, promoter, and / or enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and / or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more control sequences are preferentially or exclusively active in a particular host cell or organism, or type thereof.For example, in prokaryotes, control sequences typically include a promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include a promoter, enhancer, and / or transcription termination sequence. Those skilled in the art will understand from the context that in many embodiments the term "control sequence" refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0109] A "specific binding pair," "protein:protein binding pair," and the like, include two proteins (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a covalent isopeptide bond under conditions that allow or promote isopeptide bond formation, where the term "cognate" refers to components that function together, i.e., react together to form an isopeptide bond. Thus, two proteins that react together efficiently to form an isopeptide bond under conditions that allow or promote isopeptide bond formation can also be said to be a "complementary" pair of peptide linkers. Specific binding pairs that can interact to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506 and include peptide:peptide binding pairs such as SpyTag:SpyCatcher; SpyTag002:SpyCatcher002; SpyTag003:SpyCatcher003; SpyTag:KTag; Isopeptag:Pyrin C; SnoopTag:SnoopCatcher; SnoopTagJr:DogTag. In general, a peptide tag refers to a member of a protein:protein binding pair that is generally less than 30 amino acids in length and forms a covalent isopeptide bond with a second cognate protein, which is generally relatively large but may also be less than 30 amino acids in length, as in the SpyTag:KTag system.

[0110] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, where at least one of the carboxyl or carboxamide group and the amino group is not considered to be derived from the protein backbone or part of the protein skeleton. Isopeptide bonds can occur within a single protein, between two peptides, or between a peptide and a protein. Thus, isopeptide bonds can be formed intramolecularly within a single protein or intermolecularly, i.e., between two peptide / protein molecules, e.g., between two peptide linkers. Typically, isopeptide bonds can occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or the terminal carboxyl group of a protein or peptide chain, or between the alpha-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid. Each residue in a pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, an isopeptide bond can be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, an isopeptide bond can occur between the side chain amine of a lysine and the carboxamide group of an asparagine or the carboxyl group of an aspartic acid.

[0111] The SpyTag:SpyCatcher system, described in U.S. Pat. No. 9,547,003, Zakeri et al. (2012) PNAS 109:E690-E697, and WO 2019006046 (each of which is incorporated herein by reference in its entirety), is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein FbaB. By splitting this domain, Zakeri et al. obtained the peptide "SpyTag" with the sequence AHIVMVDAYKPTK (SEQ ID NO: 13), which forms an amide bond with its cognate protein, "SpyCatcher" (Zakeri (2012) supra). Another specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase (Fierer (2014) PNAS 111:E1176-1181). SpyLigase was engineered by cleaving out a beta chain from SpyCatcher containing a reactive lysine, resulting in KTag, a 10-residue peptide tag with the amino acid sequence ATHIKFSKRD (SEQ ID NO: 14). The SpyTag002:SpyCatcher002 system is described in Keeble et al (2017) Angew Chem Int Ed Engl 56:16521-25, which is incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK (SEQ ID NO: 15) and binds to SpyCatcher002.

[0112] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113:1202-07. SnoopTag (residues 734-745) and SnoopCatcher (residues 749-860) were formed by splitting the D4 Ig-like domain of RrgA, an adhesion molecule from Streptococcus pneumoniae. Incubation of SnoopTag and SnoopCatcher results in specific, spontaneous isopeptide bonds between the complementary proteins. Veggiani (2016), supra.

[0113] The isopeptag:pilin C specific binding pair was derived from the major pilin protein SpyO128 from Streptococcus pyogenes (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). The isopeptag has the amino acid sequence TDKDMTITFTNKKDAE (SEQ ID NO: 16) and binds to pilin C (residues 18-299 of SpyO128). Incubation of SnoopTag and SnoopCatcher results in a specific, spontaneous isopeptide bond between the complementary proteins. Zakeir and Howarth (2010), supra.

[0114] The term "peptide tag" includes a polypeptide that (1) is heterologous to the protein to which it is attached, (2) is a member of a specific protein:protein binding pair capable of forming an isopeptide bond, and (3) is 50 amino acids or less in length.

[0115] The term "detectable label" includes a polypeptide sequence that is a member of a specific binding pair and specifically binds with high affinity to another polypeptide sequence, such as an antibody paratope, e.g., by non-covalent bonding. Exemplary, non-limiting detectable labels include hexahistidine tags, FLAG tags, Strep II tags, streptavidin-binding peptide (SBP) tags, calmodulin-binding peptide (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), S tags, HA tags, and c-myc (reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8, incorporated herein by reference). A common detectable label for primate AAV is the B1 epitope. Non-primate AAV capsid proteins of the present invention that do not naturally contain the B1 epitope may be modified herein to contain the B1 epitope. Generally, a non-primate AAV capsid protein can contain a sequence substantially homologous to the B1 epitope within the last 10 amino acids of the capsid protein. Thus, in some embodiments, a non-primate AAV capsid protein of the invention can be modified by one to fewer than five point mutations within the last 10 amino acids of the capsid protein such that the AAV capsid protein contains the B1 epitope.

[0116] In various embodiments, the Fc domain can be modified to have altered Fc receptor binding, thereby affecting effector function. In some embodiments, the engineered heavy chain constant region (CH) comprising the Fc domain is chimeric. Thus, a chimeric CH region combines CH domains from two or more immunoglobulin isotypes. For example, a chimeric CH region may comprise a portion or all of a CH2 domain from a human IgG1, human IgG2, or human IgG4 molecule in combination with a portion or all of a CH3 domain from a human IgG1, human IgG2, or human IgG4 molecule. In some embodiments, the chimeric CH region includes a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acids 216-227 according to EU numbering; amino acids 226-240 according to Kabat numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" (amino acids 228-236 according to EU numbering; amino acids 241-249 according to Kabat numbering) derived from a human IgG1, human IgG2, or human IgG4 hinge region. In some embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge.

[0117] In some embodiments, the Fc domain may be engineered to activate all, some, or none of the normal Fc effector functions without affecting the desired pharmacokinetic properties of the Fc-containing protein (e.g., an antibody). See WO2014022540 (incorporated herein in its entirety) for examples of proteins containing chimeric CH regions and with altered effector functions.

[0118] The terms "transduction," "transfection," or "infection," as used interchangeably herein, refer to the introduction of nucleic acid into target cells, for example, by a viral vector. The term "efficiency," e.g., "transduction efficiency," related to transduction, refers to the proportion (e.g., percentage) of cells that express a nucleotide of interest after incubation with a set number of viral vectors containing the nucleotide of interest. Known methods for determining transduction efficiency include fluorescence-activated cell sorting of cells transduced with a fluorescent reporter gene, PCR for expression of the nucleotide of interest, and the like.

[0119] As used herein, the term "wild-type" includes entities having a structure and / or activity found in nature in a "normal" state or context (as opposed to mutant, diseased, altered, etc.). Those skilled in the art will understand that a wild-type viral vector, e.g., an AAV vector comprising a wild-type capsid protein, can be used as a reference viral vector in comparative studies. Generally, the reference viral capsid protein / capsid / vector is identical to the test viral capsid protein / capsid / vector except for the alteration whose effect is being tested. For example, to determine the effect of inserting a heterologous epitope into a test viral vector, e.g., on transduction efficiency, the transduction efficiency of the test viral vector (in the absence or presence of an appropriate binding molecule) can be compared to the transduction efficiency of a reference viral vector (in the absence or presence of an appropriate binding molecule, as needed) that is identical to the test viral vector in all respects (e.g., additional mutations, nucleotides of interest, number of viral vectors and target cells, etc.) except for the presence of the heterologous epitope.

[0120] "Complementarity" or "complementary" with respect to nucleic acids means that the nucleotide sequence of one strand of a nucleic acid forms hydrogen bonds with another sequence on an opposing nucleic acid strand due to the orientation of its nucleobase groups. Complementary bases in DNA are typically A and T and C and G. In RNA, they are typically C and G and U and A. Complementarity can be perfect or significant / sufficient. Perfect complementarity between two nucleic acids means that the two nucleic acids can form a duplex in which all bases in the duplex are bound to complementary bases by Watson-Crick pairing. "Substantial" or "sufficient" complementarity means that the sequence in one strand is not completely and / or perfectly complementary to the sequence in the opposing strand, but there is sufficient binding between the bases on the two strands to form a stable hybrid complex under a set of hybridization conditions (e.g., salt concentration and temperature). Such conditions can be predicted using sequences and standard mathematical calculations to predict the Tm (melting temperature) of hybridized strands, or by empirical determination of the Tm using conventional methods. The Tm comprises the temperature at which the population of hybridization complexes formed between two nucleic acid strands becomes 50% denatured (i.e., the population of double-stranded nucleic acid molecules becomes half-dissociated into single strands). Temperatures below the Tm promote the formation of hybridization complexes, while temperatures above the Tm promote the melting or separation of strands within the hybridization complexes. The Tm can be estimated, for example, using Tm = 81.5 + 0.41 (% G + C) for a nucleic acid of known G + C content in 1 M aqueous NaCl, although other known Tm calculations take into account structural properties of nucleic acids.

[0121] "Hybridization conditions" include the cumulative environment in which one nucleic acid strand binds to a second nucleic acid strand through complementary strand interaction and hydrogen bonding to form a hybridization complex. Such conditions include the chemical components and concentrations (e.g., salts, chelating agents, formamide) of the aqueous or organic solution containing the nucleic acid, as well as the temperature of the mixture. Other factors, such as the length of incubation time or the dimensions of the reaction chamber, may also contribute to the environment. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed., pp. 1.90-1.91, 9.47-9.51, 11.47-11.57 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (incorporated herein by reference in its entirety for all purposes).

[0122] Hybridization requires that two nucleic acids contain complementary sequences, although mismatches between bases can occur. Suitable conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, and these variables are well known. The higher the degree of complementarity between two nucleotide sequences, the higher the melting temperature (Tm) of hybrids of nucleic acids having those sequences. For hybridization between nucleic acids with short stretches of complementarity (e.g., complementarity over 35 or fewer, 30 or fewer, 25 or fewer, 22 or fewer, 20 or fewer, or 18 or fewer nucleotides), the position of mismatches becomes important (see Sambrook et al., supra, 11.7-11.8). Typically, the length of a hybridizable nucleic acid is at least about 10 nucleotides. Exemplary minimum lengths for a hybridizable nucleic acid include at least about 15 nucleotides, at least about 20 nucleotides, at least about 22 nucleotides, at least about 25 nucleotides, and at least about 30 nucleotides. Additionally, the temperature and salt concentration of the wash solution may be adjusted as needed depending on factors such as the length of the region of complementarity and the degree of complementarity.

[0123] To be specifically hybridizable, the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid / target locus. Furthermore, a polynucleotide can hybridize across one or more segments (e.g., a loop or hairpin structure) such that intervening or adjacent segments are not involved in the hybridization event. A polynucleotide (e.g., a gRNA) can have at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% sequence complementarity to a target region within the targeted target nucleic acid / target locus sequence. For example, a gRNA in which 18 of 20 nucleotides are complementary to the target region and thus specifically hybridizes would exhibit 90% complementarity. In this example, the remaining non-complementary nucleotides can be clustered or interspersed with complementary nucleotides and need not be contiguous with each other or with complementary nucleotides.

[0124] The percent complementarity between specific stretches of nucleic acid sequences within a nucleic acid can be determined conventionally using the BLAST (basic local alignment search tool) and PowerBLAST programs (Altschul et al. (1990) J. Mol. Biol. 215:403-410; Zhang and Madden (1997) Genome Res. 7:649-656), or the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison, Wisconsin) using the Smith-Waterman algorithm with default settings (Adv. Appl. Math., 1981, 2, 482-489).

[0125] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues that are identical in the two sequences when aligned for maximum correspondence over a specified comparison window. When percentages of sequence identity are used with respect to proteins, non-identical residue positions often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), thus not altering the functional properties of the molecule. When conservative substitutions result in sequences that differ, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making such adjustments are known. This typically involves increasing the percent sequence identity by scoring conservative substitutions as partial rather than complete mismatches. Thus, for example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score of 0 to 1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, CA).

[0126] "Percentage of sequence identity" includes a value determined by comparing two optimally aligned sequences over a comparison window (the maximum number of perfectly matched residues), where the portion of the polynucleotide sequence that falls within the comparison window may contain additions or deletions (i.e., gaps) when compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise specified (e.g., including heterologous sequences to which the shorter sequence is concatenated), the comparison window is the entire length of the shorter of the two sequences being compared.

[0127] Unless otherwise specified, sequence identity / similarity values ​​include values ​​obtained using GAP Version 10 with the following parameters: % identity and % similarity for nucleotide sequences using a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix; or equivalent programs. "Equivalent program" includes any sequence comparison program that produces alignments that have identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences at issue when compared to corresponding alignments produced by GAP Version 10.

[0128] The term "conservative amino acid substitution" refers to the replacement of an amino acid normally occurring in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Furthermore, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of an acidic residue such as aspartic acid or glutamic acid for another, are further examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, or methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue with a non-polar residue. Exemplary amino acid categories are summarized below. TIFF2025516527000001.tif141169

[0129] The term "in vitro" includes an artificial environment and processes or reactions that occur within an artificial environment (e.g., a test tube). The term "in vivo" includes a natural environment (e.g., a cell or organism or body) and processes or reactions that occur within a natural environment. The term "ex vivo" includes cells removed from an individual's body and processes or reactions that occur within such cells.

[0130] A composition or method that "comprising" or "including" one or more recited elements may contain 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 components. The transitional phrase "consisting essentially of" means that a claim should be construed to include the specified elements recited in the claim plus elements that do not materially affect one or more of the basic and novel characteristics of the claimed invention. Thus, the term "consisting essentially of," when used in the claims of the present invention, is not intended to be synonymous with "comprising."

[0131] "Individual," "subject," or "animal" refers to humans, domestic animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.), and experimental animal models of disease (e.g., mice, rats). In some embodiments, the subject is a human.

[0132] The term "treat" a state, disorder, or condition, or "treatment" thereof, includes: (1) preventing, delaying, or reducing the occurrence and / or likelihood of at least one clinical or subclinical symptom of the condition, disorder, or condition occurring in a subject who may be suffering from or predisposed to the condition, disorder, or condition, but who has not yet experienced or displayed clinical or subclinical symptoms of the condition, disorder, or condition; or (2) inhibiting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the progression of the disease or its recurrence, or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of the condition, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to a treated subject is statistically significant or at least perceptible to the patient or physician.

[0133] The term "effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that was effective when 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(s) employed, the mode of administration, etc.

[0134] The phrase "pharmaceutically acceptable," when used in connection with the compositions described herein, refers to molecular entities and other components of such compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias, for use in mammals, more particularly humans.

[0135] In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art, such techniques being fully explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989 (herein referred to as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984);Nucleic Acid Hybridization [BD Hames & SJ Higgins eds. (1985)];Transcription And Translation [BD Hames & SJ Higgins, eds. (1984)];Animal Cell Culture [RI Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)];B. Perbal, A Practical Guide To Molecular Cloning (1984);Ausubel, FM et al. (eds.). Current See Protocols in Molecular Biology. John Wiley & Sons, Inc., 1994. These techniques include those described in Kunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985); U.S. Pat. No. 5,071,743; Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360 (1999); Kim and Maas, BioTech.28: 196-198 (2000); Parikh and Guengerich, BioTech. 24: 428-431 (1998); Ray and Nickoloff, BioTech. 13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang and Malcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641 (1999), U.S. Patent Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14. 3: 74-75 (2001), U.S. Patent Nos. 5,702,931, 5,780,270, and 6,242,222; Angag and Schutz, Biotech. 30: 486-488 (2001); Wang and Wilkinson, Biotech. 29: 976-978 (2000); Kang et al., Biotech. 20: 44-46 (1996); Ogel and McPherson, Protein Engineer. 5: 467-468 (1992); Kirsch and Joly, Nucl. Acids. Res. 26: 1848-1850 (1998); Rhem and Hancock, J. Bacteriol. 178: 3346-3349 (1996); Boles and Miogsa, Curr. Genet. 28: 197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993), Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al., Meth. Molec. Biol. 67: 209-218.

[0136] "Optional" or "optionally" means that the event or circumstance described thereafter may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not.

[0137] In particular, the present disclosure provides a system for producing an antibody or antigen-binding fragment thereof in a subject, the system comprising: a) a first component comprising a polynucleotide molecule, the polynucleotide molecule comprising a sequence encoding the antibody or antigen-binding fragment thereof; and b) a second component comprising a gene editing molecule, or a polynucleotide molecule comprising a sequence encoding the gene editing molecule.

[0138] In some embodiments, administration of the first component and the second component to a subject causes the sequence encoding the antibody or antigen-binding fragment thereof to be incorporated into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), thereby causing production of the antibody or antigen-binding fragment in the subject's body.

[0139] In some embodiments, the first component and the second component are administered ex vivo to B cells and / or hematopoietic stem cells (HSCs) isolated from a subject, whereby a sequence encoding the antibody or antigen-binding fragment thereof is incorporated into the DNA of the cells to produce modified B cells or modified HSCs, thereby causing the production of the antibody or antigen-binding fragment thereof in the subject when the modified B cells or modified HSCs are administered to a subject.

[0140] In some embodiments, the first component and / or the second component are independently selected from a viral vector, a virus-like particle (VLP), a lipid nanoparticle (LNP), a liposome, and a ribonucleoprotein (RNP) complex.

[0141] Recombinant viral capsid proteins, viral vectors, and nucleic acids In some embodiments, the first component and the second component are both viral vectors. In some embodiments, the viral vectors are derived from the same viral species. In other embodiments, the viral vectors are derived from different viral species.

[0142] Viral vectors that can be used in the compositions and methods of the present application include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviruses (e.g., lentiviruses), baculoviral vectors, herpesvirus vectors, cytomegalovirus (CMV), Epstein-Barr virus (EBV), mouse mammary tumor virus (MMTV), human polyomavirus 2 (JC virus, i.e., John Cunningham virus), hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus 1 (HIV-1), influenza virus, norovirus, measles virus, polyomavirus, rhabdovirus (e.g., vesicular stomatitis virus), or variants thereof.

[0143] In some embodiments, one or both of the viral vectors used in the disclosed system are adeno-associated viral (AAV) vectors. "AAV" is an abbreviation for adeno-associated virus and may refer to the virus itself or its derivatives. AAV is a small, non-enveloped, single-stranded DNA virus. Generally, the wild-type AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames (ORFs), rep and cap. The wild-type rep reading frame encodes four proteins with molecular weights of 78 kD ("Rep78"), 68 kD ("Rep68"), 52 kD ("Rep52"), and 40 kD ("Rep40"). Rep78 and Rep68 are transcribed from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. These proteins primarily function to regulate the transcription and replication of the AAV genome. The wild-type cap reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83-85 kD (VP1), 72-73 kD (VP2), and 61-62 kD (VP3). VP3 accounts for more than 80% of the total protein in the AAV virion (capsid); in mature virions, VP1, VP2, and VP3 are found in a relative abundance ratio of approximately 1:1:10, although ratios as high as 1:1:8 have also been reported. (Padron et al. (2005) J. Virology 79:5047-58)

[0144] The genomic sequences of the various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank, see, e.g., GenBank Accession Nos. NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8), the disclosures of which are incorporated by reference into this application for their teaching of AAV nucleic acid and amino acid sequences. Also, for example, Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al. (1999) J. Virology 73: 1309; Bantel-Schaal et al. (1999) J. Virology 73:939; Xiao et al. (1999) J. Virology 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854; Moris et al. (2004) Virology 33:375-383; U.S. Patent Application Publication No. 20170130245; WO 2000 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303, each of which is incorporated by reference in its entirety. Table 2 herein provides sequences for various non-primate AAVs.

[0145] "AAV" includes all subtypes known in the art and both naturally occurring and modified forms. AAVs include primate AAVs (e.g., type 1 AAV (AAV1), type 2 primate AAV (AAV2), type 3 primate AAV (AAV3B), type 4 primate AAV (AAV4), type 5 primate AAV (AAV5), type 6 primate AAV (AAV6), type 7 primate AAV (AAV7), type 8 primate AAV (AAV8), type 9 primate AAV (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, rh10 primate AAV (AAV rh10), h10 AAV (AAV h10), hu11 AAV (AAV hu11), rh32, rh33 AAV (AAV rh32.33), AAV retro, AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAV2 / 8, etc.), non-primate AAV (e.g., avian AAV (AAAV)), and other non-primate AAV, such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, and ovine AAV), squamate AAV (e.g., snake AAV, bearded dragon AAV), etc. "Primate AAV" generally refers to AAV isolated from primates. Similarly, "non-primate animal AAV" refers to AAV isolated from non-primate animals.

[0146] In some embodiments, the AAV vector is derived from AAV1, AAV2, AAV6, AAV9, or AAV9.PHP.

[0147] Also included herein are recombinant viral particles genetically engineered to display a heterologous amino acid sequence comprising a first member of a specific binding pair, the amino acid sequence being less than 50 amino acids in length, and the recombinant viral capsid / particle protein exhibits reduced or abolished natural tropism. In some embodiments, the viral particle further comprises a second cognate member of the specific binding pair, wherein the first and second members are covalently linked and the second member is fused to a targeting ligand.

[0148] In some embodiments, the heterologous amino acid sequence comprises a first member of a specific binding pair and one or more linkers. In some embodiments, the heterologous amino acid sequence comprises the first member of a specific binding pair flanked by linkers, e.g., the heterologous amino acid sequence comprises, from N-terminus to C-terminus, a first linker, a first member of a specific binding pair, and a second linker. In some embodiments, the first linker and the second linker are each independently at least one amino acid in length. In some embodiments, the first linker and the second linker are identical.

[0149] Generally, heterologous amino acid sequences described herein, e.g., comprising a first member of a specific binding pair, alone or in combination with one or more linkers, are from about 5 amino acids to about 50 amino acids in length. In some embodiments, the heterologous amino acid sequence is at least 5 amino acids in length. In some embodiments, the heterologous amino acid sequence is 6 amino acids in length. In some embodiments, the heterologous amino acid sequence is 7 amino acids in length. In some embodiments, the heterologous amino acid sequence is 8 amino acids in length. In some embodiments, the heterologous amino acid sequence is 9 amino acids in length. In some embodiments, the heterologous amino acid sequence is 10 amino acids in length. In some embodiments, the heterologous amino acid sequence is 11 amino acids in length. In some embodiments, the heterologous amino acid sequence is 12 amino acids in length. In some embodiments, the heterologous amino acid sequence is 13 amino acids in length. In some embodiments, the heterologous amino acid sequence is 14 amino acids in length. In some embodiments, the heterologous amino acid sequence is 15 amino acids in length. In some embodiments, the heterologous amino acid sequence is 16 amino acids in length. In some embodiments, the heterologous amino acid sequence is 17 amino acids in length. In some embodiments, the heterologous amino acid sequence is 18 amino acids in length. In some embodiments, the heterologous amino acid sequence is 19 amino acids in length. In some embodiments, the heterologous amino acid sequence is 20 amino acids in length. In some embodiments, the heterologous amino acid sequence is 21 amino acids in length. In some embodiments, the heterologous amino acid sequence is 22 amino acids in length. In some embodiments, the heterologous amino acid sequence is 23 amino acids in length. In some embodiments, the heterologous amino acid sequence is 24 amino acids in length. In some embodiments, the heterologous amino acid sequence is 25 amino acids in length. In some embodiments, the heterologous amino acid sequence is 26 amino acids in length. In some embodiments, the heterologous amino acid sequence is 27 amino acids in length. In some embodiments, the heterologous amino acid sequence is 28 amino acids in length. In some embodiments, the heterologous amino acid sequence is 29 amino acids in length. In some embodiments, the heterologous amino acid sequence is 30 amino acids in length.In some embodiments, the heterologous amino acid sequence is 31 amino acids in length. In some embodiments, the heterologous amino acid sequence is 32 amino acids in length. In some embodiments, the heterologous amino acid sequence is 33 amino acids in length. In some embodiments, the heterologous amino acid sequence is 34 amino acids in length. In some embodiments, the heterologous amino acid sequence is 35 amino acids in length. In some embodiments, the heterologous amino acid sequence is 36 amino acids in length. In some embodiments, the heterologous amino acid sequence is 37 amino acids in length. In some embodiments, the heterologous amino acid sequence is 38 amino acids in length. In some embodiments, the heterologous amino acid sequence is 39 amino acids in length. In some embodiments, the heterologous amino acid sequence is 40 amino acids in length. In some embodiments, the heterologous amino acid sequence is 41 amino acids in length. In some embodiments, the heterologous amino acid sequence is 42 amino acids in length. In some embodiments, the heterologous amino acid sequence is 43 amino acids in length. In some embodiments, the heterologous amino acid sequence is 44 amino acids in length. In some embodiments, the heterologous amino acid sequence is 45 amino acids in length. In some embodiments, the heterologous amino acid sequence is 46 amino acids in length. In some embodiments, the heterologous amino acid sequence is 47 amino acids in length. In some embodiments, the heterologous amino acid sequence is 48 amino acids in length. In some embodiments, the heterologous amino acid sequence is 49 amino acids in length. In some embodiments, the heterologous amino acid sequence is 50 amino acids in length.

[0150] In some embodiments, the specific binding pair is a SpyTag:SpyCatcher binding pair, wherein the first member is SpyTag and the second cognate member is SpyCatcher. In some embodiments, the specific binding pair is a SpyTag:KTag, wherein the first member is SpyTag and the second cognate member is KTag. In some embodiments, the specific binding pair is a SpyTag:KTag, wherein the first member is KTag and the second cognate member is SpyTag. In some embodiments, the specific binding pair is an Isopeptag:Pilin C, wherein the first member is an Isopeptag and the second cognate member is Pilin C or a portion thereof. In some embodiments, the specific binding pair is a SnoopTag:SnoopCatcher, wherein the first member is a SnoopTag and the second cognate member is SnoopCatcher.

[0151] In some embodiments, the recombinant viral capsid protein described herein is derived from an adeno-associated virus (AAV) capsid gene, e.g., a genetically modified capsid protein of an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, an AAV6 capsid gene, an AAV1 capsid gene, or an AAV9 capsid gene. In some embodiments, the recombinant viral capsid protein is derived from an AAV2 capsid gene, e.g., a genetically modified AAV2 VP1 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from an AAV1 capsid gene, e.g., a genetically modified AAV1 VP1 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from the AAV9 capsid gene, for example, a genetically modified AAV9 VP1 capsid protein. In some embodiments, the recombinant viral capsid protein is derived from the AAV6 capsid gene, for example, a genetically modified AAV6 VP1 capsid protein. In some embodiments, the heterologous epitope is inserted into I-453 of the AAV9 capsid protein.

[0152] Generally, the recombinant viral capsid proteins described herein comprise a heterologous epitope inserted into and / or displayed by the capsid protein, such that the heterologous epitope reduces and / or abolishes the natural tropism of the capsid protein or capsids comprising the same. In some embodiments, the heterologous epitope is inserted into a region of the capsid protein that is responsible for the natural tropism of the wild-type reference capsid protein, e.g., a region of the capsid protein that is involved in a cellular receptor. In some embodiments, the heterologous epitope is inserted into and / or displayed by the knob domain of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted into and / or displayed by the HI loop of the Ad fiber protein. In some embodiments, the heterologous epitope is inserted after an amino acid position selected from the group consisting of G453 of AAV2 capsid protein VP1, N587 of AAV2 capsid protein VP1, Q585 of AAV6 capsid protein VP1, G453 of AAV9 capsid protein VP1, and A589 of AAV9 capsid protein VP1. In some embodiments, the heterologous epitope is inserted and / or displayed between amino acids N587 and R588 of the AAV2 VP1 capsid. Additional suitable insertion sites identified using AAV2 are known in the art (Wu et al. (2000) J. Virol. 74:8635-8647) and include I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716.The recombinant viral capsid protein described herein can be an AAV2 capsid protein comprising a heterologous epitope inserted at a position selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. Additional suitable insertion sites identified with additional AAV serotypes are known in the art, including I-587(AAV1), I-589(AAV1), I-585(AAV3), I-585(AAV4), and I-585(AAV5). In some embodiments, the recombinant viral capsid protein described herein may be an AAV2 capsid protein comprising a heterologous epitope inserted at a position selected from the group consisting of I-587(AAV1), I-589(AAV1), I-585(AAV3), I-585(AAV4), I-585(AAV5), and combinations thereof.

[0153] The nomenclature I-### used herein refers to the insertion site, with ### representing the amino acid number relative to the VP1 protein of the AAV capsid proteins, although such insertions may be located directly N- or C-terminal to one amino acid, preferably C-terminal, in the sequence 5 amino acids N- or C-terminal to a given amino acid, preferably 3, more preferably 2, and especially 1 amino acid N- or C-terminal to a given amino acid. Furthermore, the positions referred to herein are with respect to the VP1 protein encoded by the AAV capsid gene; corresponding positions (and mutations thereof) with respect to the VP2 and VP3 capsid proteins encoded by capsid genes can be readily identified by performing sequence alignments of the VP1, VP2, and VP3 proteins encoded by reference AAV capsid genes.

[0154] Thus, insertion of the coding nucleic acid at the corresponding position of one of these sites in the cap gene leads to an insertion in VP1, VP2, and / or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with offset start codons. Thus, for example, with respect to AAV2, according to the present nomenclature, an insertion between amino acids 1 and 138 results in an insertion in VP1 only, an insertion between 138 and 203 results in an insertion in VP1 and VP2, and an insertion between 203 and the C-terminus results in an insertion in VP1, VP2, and VP3, as would be expected for insertion site I-587. Thus, the present invention encompasses AAV structural genes with corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0155] Furthermore, due to the high degree of conservation, at least over large stretches and among closely related family members, corresponding insertion sites for AAVs other than those listed can be identified by performing amino acid alignments or by comparing capsid structures. For exemplary alignments of different AAV capsid proteins, see, e.g., Rutledge et al. (1998) J. Virol. 72:309-19 and U.S. Patent No. 9,624,274 (each of which references is incorporated herein by reference in its entirety).

[0156] In some compositions comprising a recombinant viral capsid disclosed herein, the recombinant viral capsid protein is an AAV2 capsid protein VP1 having a heterologous epitope inserted at the I587 site, wherein the heterologous epitope does not comprise an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions comprising a recombinant viral capsid disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein having a heterologous epitope inserted between T448 and N449, wherein the heterologous epitope does not comprise c-myc. In some compositions comprising a recombinant viral capsid disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein having a heterologous epitope inserted at the I-447 site, wherein the heterologous epitope does not comprise L14 or HA.

[0157] In some compositions comprising a recombinant viral capsid, the recombinant viral capsid protein is a VP1 capsid protein having a heterologous epitope inserted at the I587 site, wherein the heterologous epitope comprises an Arg-Gly-Asp (RGD) motif, an NGR motif, or c-myc. In some compositions comprising a recombinant viral capsid disclosed herein, the viral capsid is a VP1 capsid and the heterologous epitope comprises c-myc, wherein the heterologous epitope is inserted between T448 and N449 or between N587 and R588. In some compositions comprising a recombinant viral capsid disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein having a heterologous epitope inserted at the I-447 site, wherein the heterologous epitope comprises L14 or HA. In some compositions comprising recombinant viral capsids disclosed herein, the recombinant viral capsid protein is a VP1 capsid protein with a heterologous epitope inserted between T448 and N449, the heterologous epitope comprising c-myc. U.S. Patent No. 9,624,274 describes I-453 of the AAV capsid protein as a suitable insertion site for a heterologous epitope.

[0158] In some embodiments, the insertion (display) of a heterologous epitope abolishes the natural tropism of the viral vector, e.g., transduction of cells naturally permissive to infection by the wild-type reference viral vector and / or target cells is undetectable in the absence of an appropriate binding molecule. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector, e.g., compared to transduction of cells naturally permissive to infection by the wild-type reference viral vector. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 5%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 10%. In some embodiments, the insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 20%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 30%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 40%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 50%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 60%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 70%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 80%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 90%. In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 95%.In some embodiments, insertion (display) of a heterologous epitope reduces the natural tropism of the viral vector by at least 90%. In those embodiments in which insertion (display) of a heterologous epitope does not eliminate the natural tropism of the recombinant viral capsid, the natural tropism of such recombinant viral capsid may be eliminated by a second, different mutation. For example, in one embodiment, the recombinant viral capsid protein described herein may be derived from an AAV9 capsid gene, may include a heterologous epitope, and may further include a mutation, e.g., a W503A mutation. Other non-limiting examples of second mutations include, e.g., Y445F and V473D for AAV1 or AAV6 capsids.

[0159] This detargeting of the virus from its natural host cells is particularly important when systemic administration of viral vectors is intended, as well as local or locoregional administration, because the effective dose of the viral vector is limited by the uptake of the viral vector by the natural host cells. For AAV2 and AAV6, HSPGs have been reported to be the primary receptors for viral uptake in many cells, especially hepatocytes. For AAV2, HSPG binding activity depends on a group of five basic amino acids: R484, R487, R585, R588, and K532 (Kern et al., (2003) J. Virol. 77(20):11072-81). Recently, it has been reported that the lysine-to-glutamic acid amino acid substitution K531E inhibits the ability of AAV6 to bind to heparin or HSPGs (Wu et al., 2006) J. of Virology 80(22):11393-11397). Thus, preferred point mutations are those that reduce the transduction activity of the viral vector for a given target cell mediated by the native receptor, and, if HSPG is present as the primary receptor, the binding of the viral vector to HSPG, by at least 50%, preferably at least 80%, and particularly at least 95%.

[0160] Therefore, preferred additional mutations for HSPG-binding viral vectors are those that deplete or substitute basic amino acids such as R, K, or H, preferably R or K, involved in HSPG binding of each virus, with non-basic amino acids such as A, D, G, Q, S, and T, preferably A, or with amino acids present at the corresponding positions of a different but highly conserved AAV serotype (which lacks the above-mentioned basic amino acid at this position). Therefore, preferred amino acid substitutions are R484A, R487A, R487G, K532A, K532D, R585A, R585S, R585Q, R585A, or R588T, particularly R585A and / or R588A, for AAV2, and K531A or K531E for AAV6. A particularly preferred embodiment of the present invention is an AAV2 capsid protein mutant that further contains two point mutations, R585A and R588A. These two point mutations are sufficient to largely eliminate HSPG-binding activity, allowing efficient detargeting from HSPG-expressing cells, which in turn improves the specificity of each mutant virus to new target cells for targeting purposes.

[0161] One embodiment of the present invention is a multimeric structure comprising the recombinant viral capsid proteins of the present invention. The multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 recombinant viral capsid proteins comprising the heterologous epitopes described herein. These can form normal viral capsids (empty viral particles) or viral vectors (capsids encapsulating a nucleotide of interest). The formation of a viral vector capable of packaging a viral genome is a highly preferred feature of using the recombinant viral capsids described herein as viral vectors.

[0162] In some embodiments, a targeting ligand can be associated with (e.g., displayed, operably linked, or attached to) a modified AAV capsid protein and the resulting AAV capsid according to an indirect recombinant approach, where the AAV capsid protein is modified to include a first member of a binding pair (e.g., a heterologous scaffold), and optionally the first member of the binding pair is linked (e.g., covalently or non-covalently) to a second cognate member of the binding pair (e.g., an adapter), and further optionally, the second cognate member of the binding pair is fused to the targeting ligand. Non-limiting exemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12:248-265.

[0163] Thus, in some embodiments, the capsid protein modifications described herein generally include modifications resulting from genetic level modifications, e.g., by altering the cap gene, e.g., modifications to insert a first member of a binding pair (e.g., a protein:protein binding pair, a protein:nucleic acid binding pair), a detectable label, etc., for display by the Cap protein.

[0164] In some embodiments, the first member forms a binding pair with an immunoglobulin molecule constant domain. In some embodiments, the first member binds to a metal ion, such as Ni. 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ etc. In some embodiments, the first member is selected from the group consisting of streptavidin, Strep II, HA, L14, 4C-RGD, LH, and protein A.

[0165] In some embodiments, the binding pair comprises an enzyme:nucleic acid binding pair. In some embodiments, the first member comprises an HUH endonuclease or an HUH tag, and the second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises an HUH tag. See, e.g., U.S. Patent Application Publication No. 2021 / 0180082, which is incorporated herein by reference in its entirety.

[0166] In some embodiments, the capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

[0167] In some embodiments, the first and second members of the peptide:peptide binding pair each comprise an intein. See, e.g., Wagner et al., (2021) Adv. Sci. 8: 2004018 (1 of 22); Muik et al. (2017) Biomaterials 144: 84, each of which is incorporated herein by reference in its entirety.

[0168] In some embodiments, the first member is a B-cell epitope, eg, about 1 amino acid to about 35 amino acids in length, that forms a binding pair with an antibody paratope, eg, an immunoglobulin variable domain.

[0169] In some embodiments, the capsid proteins of the invention comprise a first member of a protein:protein binding pair that includes a detectable label, which can also be used for detection and / or isolation of the Cap protein and / or as the first member of the protein:protein binding pair. In some embodiments, the detectable label functions as the first member of a protein:protein binding pair for binding of a targeting ligand, which comprises a multispecific binding protein that can bind both the detectable label and a target expressed by a cell of interest. In some embodiments, the Cap proteins of the invention comprise a first member of a protein:protein binding pair that includes c-myc, FLAG, or HA. The use of a detectable label as the first member of a protein:protein binding pair is described, for example, in WO2019006043.

[0170] In some embodiments, the capsid protein comprises a first member of a protein:protein binding pair, wherein the protein:protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of the protein:protein binding pair is covalently linked via an isopeptide bond to a second cognate member of a peptide:peptide binding pair, and optionally, the second cognate member of the peptide:peptide binding pair is fused to a targeting ligand, wherein the targeting ligand binds to a target expressed by a cell of interest. In some embodiments, the protein:protein binding pair can be selected from SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag003:SpyCatcher003, SpyTag:KTag, Isopeptag:Pilin C, and SnoopTag:SnoopCatcher. In some embodiments, the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion or variant thereof). In some embodiments, the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is KTag (or a biologically active portion or variant thereof). In some embodiments, the first member is KTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion or variant thereof). In some embodiments, the first member is SnoopTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion or variant thereof). In some embodiments, the first member is an isopeptag (or a biologically active portion or variant thereof) and the protein (the second cognate member) is pilin C (or a biologically active portion or variant thereof).In some embodiments, the first member is SpyTag002 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion or variant thereof). In some embodiments, the first member is SpyTag003 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher003 (or a biologically active portion or variant thereof). In some embodiments, the Cap protein of the invention comprises SpyTag or a biologically active portion or variant thereof. The use of first members of protein:protein binding pairs is described in WO2019006046, which is incorporated herein by reference in its entirety.

[0171] In some embodiments, a viral capsid comprising a modified viral capsid protein described herein is a mosaic capsid, e.g., comprising at least two pairs of VP1, VP2, and / or VP3 proteins, each pair encoded by a different cap gene. As used herein, a mosaic capsid generally refers to a mosaic of a first viral capsid protein modified to include a first member of a binding pair and a second corresponding viral capsid protein lacking the first member of the binding pair. In the context of a mosaic capsid, the second viral capsid protein lacking the first member of the binding pair may also be referred to as a reference capsid protein encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP1, VP2, and / or VP3 capsid protein modified with a first member of a protein:protein pair is not a chimeric capsid protein, the VP1, VP2, and / or VP3 reference capsid protein may have an amino acid sequence identical to the viral VP1, VP2, and / or VP3 capsid protein modified with a first member of a binding pair, except for the absence of the first member of the binding pair in the reference capsid protein. In some mosaic capsid embodiments, the VP1, VP2, and / or VP3 reference capsid protein corresponds to the viral VP1, VP2, and / or VP3 capsid protein modified with a first member of a binding pair, except for the absence of the first member of the binding pair in the reference capsid protein. In some embodiments, the VP1 reference capsid protein corresponds to the viral VP1 capsid protein modified with a first member of a binding pair, except for the absence of the first member of the binding pair in the reference capsid protein. In some embodiments, the VP2 reference capsid protein corresponds to a viral VP2 capsid protein modified with a first member of a binding pair, except that the first member of the binding pair is absent from said reference capsid protein. In some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with a first member of a binding pair, except that the first member of the binding pair is absent from said reference capsid protein.In some mosaic capsid embodiments comprising chimeric VP1, VP2, and / or VP3 capsid proteins further modified to comprise a first member of a binding pair, the reference protein may be the corresponding capsid protein, a portion of which forms a portion of the chimeric capsid protein. As a non-limiting example, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP1 capsid protein modified to comprise a first member of a binding pair may comprise as reference capsid proteins: an AAV2 VP1 capsid protein lacking the first member; an AAAV VP1 capsid protein lacking the first member; or a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member. Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP2 capsid protein modified to include a first member of a binding pair may comprise, as reference capsid proteins: an AAV2 VP2 capsid protein lacking the first member; an AAAV VP1 capsid protein lacking the first member; or a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP3 capsid protein modified to include a first member of a binding pair may comprise, as reference capsid proteins: an AAV2 VP2 capsid protein lacking the first member; an AAAV VP1 capsid protein lacking the first member; or a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member. In some mosaic capsid embodiments, the reference capsid protein can be any capsid protein that lacks the first member of the binding pair and is capable of forming a capsid with the first capsid protein modified with the first member of the binding pair.

[0172] Generally, mosaic particles can be produced by transfecting a mixture of modified and reference cap genes into production cells in the indicated ratios. The protein subunit ratio in the particle, e.g., the ratio of modified VP protein to unmodified VP protein, may, but need not, stoichiometrically reflect the ratio of at least two cap genes encoding first capsid proteins modified with first members of binding pairs to one or more reference cap genes, e.g., modified cap genes to reference cap genes, transfected into the packaging cells. In some embodiments, the protein subunit ratio in the particle does not stoichiometrically reflect the ratio of modified cap genes to reference cap genes transfected into the packaging cells.

[0173] In some mosaic virus particle embodiments, the protein subunit ratio is about 1:59 to about 59:1.

[0174] In some non-mosaic viral particle embodiments, the protein subunit ratio may be 1:0, wherein each capsid protein of the non-mosaic viral particle is modified with a first member of a binding pair. In some non-mosaic viral particle embodiments, the protein subunit ratio may be 0:1, wherein each capsid protein of the non-mosaic viral particle is modified with a first member of a binding pair.

[0175] Due to the high degree of conservation, at least for large stretches and among closely related family members, corresponding insertion sites for AAVs other than those listed can be identified by performing amino acid alignments or comparing capsid structures. For exemplary alignments of different AAV capsid proteins, see, e.g., Rutledge et al. (1998) J. Virol. 72:309-19, Mietzsch et al. (2019) Viruses 11, 362, 1-34, and U.S. Pat. No. 9,624,274 (each of which is incorporated herein by reference in its entirety). For example, Mietzsch et al. (2019) provide an overlap of ribbons from different dependoparvoviruses in Figure 7, illustrating variable regions VR I-VR IX. Using such structural and sequence analyses as described in the above documents, one skilled in the art can determine which amino acids within the variable regions correspond to amino acid sequences of AAV that are amenable to insertion of, for example, a targeting ligand, a first member of a binding pair, and / or a detectable label as described herein.

[0176] Generally, the targeting ligand, first member of the binding pair, and / or detectable label can be inserted into a variable region or loop of an AAV capsid protein, the GH loop of an AAV capsid protein, or the like.

[0177] In some embodiments, the first member of the binding pair and / or detectable label is inserted into the VP1 capsid protein of the non-primate AAV after an amino acid position corresponding to an amino acid position selected from the group consisting of G453 of the AAV2 capsid protein VP1, N587 of the AAV2 capsid protein VP1, G453 of the AAV9 capsid protein VP1, and A589 of the AAV9 capsid protein VP1. In some embodiments, the first member of the binding pair and / or detectable label is inserted into the VP1 capsid protein of the non-primate AAV between amino acids corresponding to N587 and R588 of the AAV2 VP1 capsid. Additional suitable insertion sites for non-primate VP1 capsid proteins include those corresponding to I-1, I-34, I-138, I-139, I-161, I-261, I-266, I-381, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, and I-716 of the AAV2 VP1 capsid protein (Wu et al. (2000) J. Virol. 74:8635-8647). The modified viral capsid protein described herein can be a non-primate capsid protein comprising a first member of a binding pair and / or a detectable label inserted at a position corresponding to a position in an AAV2 capsid protein selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. Further suitable insertion sites in non-primate AAVs include sites corresponding to I-587 or I-590 in AAV1, I-589 in AAV1, I-585 in AAV3, I-584 or I-585 in AAV4, and I-575 or I-585 in AAV5.In some embodiments, the modified viral capsid protein described herein may be a non-primate capsid protein comprising a targeting ligand, a first member of a binding pair, and / or a detectable label inserted at a position corresponding to a position selected from the group consisting of I-587(AAV1), I-589(AAV1), I-585(AAV3), I-585(AAV4), I-585(AAV5), and combinations thereof.

[0178] In some embodiments, the first member of the binding pair and / or the detectable label is I444 of avian AAV capsid protein VP1, I580 of avian AAV capsid protein VP1, I573 of bearded dragon AAV capsid protein VP1, I436 of bearded dragon AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, It is inserted into the VP1 capsid protein of a non-primate AAV after an amino acid position corresponding to an amino acid position selected from the group consisting of I432 of the sea lion AAV capsid protein VP1, I433 of the sea lion AAV capsid protein VP1, I434 of the sea lion AAV capsid protein VP1, I436 of the sea lion AAV capsid protein VP1, I437 of the sea lion AAV capsid protein VP1, and I565 of the sea lion AAV capsid protein VP1.

[0179] The nomenclature I-###, I#, etc. refers to the insertion site (I), with ### representing the amino acid number relative to the VP1 protein of the AAV capsid proteins, although such insertions may be located directly N- or C-terminal to one amino acid, preferably C-terminal, in the sequence 5 amino acids N- or C-terminal to a given amino acid, preferably 3, more preferably 2, and especially 1 amino acid N- or C-terminal to a given amino acid. Furthermore, the positions referred to herein are with respect to the VP1 protein encoded by the AAV capsid gene; corresponding positions (and point mutations thereof) with respect to the VP2 and VP3 capsid proteins encoded by the capsid genes can be readily identified by performing sequence alignments of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid genes.

[0180] Thus, insertion of the coding nucleic acid at the corresponding position of one of these sites in the cap gene leads to an insertion in VP1, VP2, and / or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with offset start codons. Thus, for example, with respect to AAV2, according to the present nomenclature, an insertion between amino acids 1 and 138 results in an insertion in VP1 only, an insertion between 138 and 203 results in an insertion in VP1 and VP2, and an insertion between 203 and the C-terminus results in an insertion in VP1, VP2, and VP3, as would be expected for insertion site I-587. Thus, the present invention encompasses AAV structural genes with corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0181] Also provided herein are nucleic acids encoding the VP3 capsid proteins described herein. AAV capsid proteins can be, but are not necessarily, encoded by overlapping reading frames of the same gene with offset start codons. In some embodiments, a nucleic acid encoding a VP3 capsid protein described herein does not also encode a VP2 capsid protein or a VP1 capsid protein of the invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein described herein may also encode a VP2 capsid protein described herein, but not a VP1 capsid of the invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein described herein may also encode a VP2 capsid protein described herein, and a VP1 capsid described herein.

[0182] In some embodiments, viral capsids comprising a modified viral capsid protein comprising a first and second member of a binding pair (e.g., the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) are capable of infecting a particular cell, e.g., have an enhanced ability to target and bind to a particular cell, compared to a control viral capsid comprising, e.g., a control capsid protein identical to the modified viral capsid protein except that one or both of the first and second members of the binding pair are absent. In some embodiments, viral capsids comprising a modified viral capsid protein described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibit a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid.

[0183] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a binding pair (e.g., the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.) is capable of infecting a particular cell, e.g., has an enhanced ability to target and bind to a particular cell, compared to a control viral capsid comprising, e.g., a control capsid protein identical to the modified viral capsid protein except for the absence of one or both of the first and second members of the binding pair. In some embodiments, viral particles of the invention comprising a viral capsid protein that comprises the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, and optionally comprising first and second members of a binding pair (e.g., the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.), exhibit superior ability to evade neutralization by pre-existing antibodies in serum isolated from a human patient compared to a suitable control viral particle (e.g., comprising a viral capsid of an AAV serotype, a portion of which is included in the viral capsid of the invention, e.g., as part of a viral capsid protein that comprises the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof), and the control viral particle also optionally comprises the first and second members of a binding pair (e.g., the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.). In some embodiments, viral particles of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, require at least 2-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to, for example, a suitable control viral particle (e.g., viral particles of the invention have an IC of at least 2-fold higher than that of a control viral particle). 50 value).

[0184] In some embodiments of the invention comprising a detectable label, the targeting ligand comprises a multispecific binding molecule, which comprises (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor that can be attached to the surface of a bead (e.g., for purification) or expressed by a target cell. Thus, the multispecific binding molecule comprises (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor to which the viral particle is targeted. Such "targeting" or "directing" may include scenarios in which wild-type viral particles target multiple cells within a tissue and / or multiple organs within an organism, where broad targeting of tissues or organs is reduced or eliminated by the insertion of a detectable label, and retargeting to specific cells within a tissue or more specific organs within an organism is achieved by the multispecific binding molecule. Such retargeting or redirection may also include scenarios in which wild-type viral particles target a tissue, tissue targeting is reduced or eliminated by the insertion of a detectable label, and retargeting to an entirely different tissue is achieved by the multispecific binding molecule. The antibody paratopes described herein generally comprise at least a complementarity-determining region (CDR), e.g., the CDR3 region of the heavy and / or light chain variable domain, that specifically recognizes a detectable label. In some embodiments, a multispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a detectable label. For example, a multispecific binding molecule may comprise a single-domain heavy chain variable region or a single-domain light chain variable region, which comprises an antibody paratope that specifically binds to a detectable label. In some embodiments, a multispecific binding molecule may comprise an Fv region, e.g., a multispecific binding molecule may comprise an scFV comprising an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecules described herein comprise an antibody paratope that specifically binds to c-myc.

[0185] A further embodiment of the present invention is the use of at least one modified viral capsid protein and / or nucleic acid encoding same, preferably at least one multimeric structure (e.g., a viral particle), for producing a nucleotide of interest and transferring the nucleotide of interest into a target cell.

[0186] In some embodiments, the viral particles described herein comprise components, e.g., capsomeres, glycoproteins, etc., derived from a virus selected from the group consisting of Human Immunodeficiency Virus (HIV), Bovine Immunodeficiency Virus (BIV), Feline Immunodeficiency Virus (FIV), Simian Immunodeficiency Virus (SIV), Equine Infectious Anemia Virus (EIAV), Murine Stem Cell Virus (MSCV), or Murine Leukemia Virus (MLV). In some embodiments, the viral particles described herein comprise an HIV capsomere, multiple HIV capsomeres, and / or HIV capsids, e.g., are HIV viral particles and / or are derived from HIV.

[0187] In some embodiments, the viral particles described herein display a fusogen in addition to a B cell or HSC targeting moiety. In some embodiments, the fusogen is a protein; for example, a viral protein (e.g., a vesiculovirus protein (e.g., vesicular stomatitis virus G glycoprotein (VSVG)), an alphavirus protein (e.g., Sindbis virus glycoprotein), an orthomyxovirus protein (e.g., influenza HA protein), a paramyxovirus protein (e.g., Nipah virus F protein or measles virus F protein)), or a fragment, variant, or derivative thereof. In a specific embodiment, the fusogen is heterologous to the reference wild-type virus from which the particle is derived. In some embodiments, the fusogen is a mutated protein that does not bind to its natural ligand.

[0188] In some embodiments, the targeting moiety and the fusogen are comprised in a single fusion protein.

[0189] In some embodiments described herein, the viral particle comprises a fusogen. Many different protein and non-protein fusogens can be used. In some embodiments, the fusogen is a protein. In a specific embodiment, the fusogen is a viral protein. Non-limiting examples of viral fusogens that can be used include, for example, vesiculovirus fusogens (e.g., vesicular stomatitis virus G glycoprotein (VSVG)), alphavirus fusogens (e.g., Sindbis virus glycoprotein), orthomyxovirus fusogens (e.g., influenza HA protein), paramyxovirus fusogens (e.g., Nipah virus F protein or measles virus F protein), and fusogens derived from dengue virus (DV), Lassa fever virus, tick-borne encephalitis virus, dengue virus, hepatitis B virus, rabies virus, Semliki Forest virus, Ross River virus, Aura virus, Borna disease virus, Hantaan virus, SARS-CoV virus, and various fragments, variants, and derivatives thereof. Other exemplary fusogenic molecules and related methods are described, for example, in US Patent Application Publication Nos. 2005 / 0238626 and 2007 / 0020238.

[0190] In a specific embodiment, the fusogen is heterologous to the virus from which the particle is derived.

[0191] Two classes of viral fusogens have been recognized, both of which can be used as targeting moieties (DS Dimitrov, Nature Rev. Microbio. 2, 109 (2004)). Class I fusogens induce membrane fusion using a helical coiled-coil structure, while class II fusogens induce fusion with a 13-barrel. In some embodiments, class I fusogens are used. In other embodiments, class II fusogens are used. In still other embodiments, both class I and class II fusogens are used. For example, Skehel and Wiley, Annu. Rev. Biochem. 69, 531-569 (2000); Smit, J. et al. J. Virol. 73, 8476-8484 (1999), Morizono et al. J. Virol. 75, 8016-8020 (2005), Mukhopadhyay et al. (2005) Rev. Microbiol. 3, 13-22.

[0192] In some specific embodiments, a form of hemagglutinin (HA) from influenza A / fowl plague virus / Rostock / 34 (FPV), a class I fusogen, is used (Hatziioannou et al., J. Virol. 72, 5313 (1998)). In some specific embodiments, a form of FPV HA is used (Lin et al., Hum. Gene. Ther. 12, 323 (2001)). HA-mediated fusion is generally thought to be independent of receptor binding (Lavillette et al., Cosset, Curr. Opin. Biotech. 12, 461 (2001)).

[0193] In other embodiments, Sindbis virus glycoprotein (class II fusogen) from the Alphavirus genus is used (Wang et al., J. Virol. 66, 4992 (1992); Mukhopadhyay et al., Nature Rev. Microbio. 3, 13 (2005), Morizono et al., Nature Med. 11, 346 (2005)).

[0194] In some embodiments, mutant fusogens are used that retain their membrane fusion ability but have reduced or eliminated binding ability or specificity. The functional properties of mutant fusogens can be tested, for example, in cell culture or by determining their ability to stimulate an immune response in vivo without causing undesirable side effects.

[0195] To select the most effective and non-toxic combination of targeting moiety and fusogen (wild-type or mutant), viral particles carrying these molecules can be tested for their selectivity and / or their ability to promote penetration of the target cell membrane.

[0196] In a specific embodiment, the membrane fusion molecule is Sindbis virus envelope protein (SIN). Sindbis virus imports its RNA into cells through low-pH-mediated membrane fusion. SIN contains five structural proteins: E1, E2, E3, 6K, and capsid. E2 contains a receptor-binding sequence that allows wild-type SIN to bind, while E1 is known to contain the properties necessary for membrane fusion (Konoochik et al., Virology Journal 2011, 8:304). E1, E2, and E3 are encoded by a polyprotein, the amino acid sequences of which are provided, e.g., under accession numbers VHWVB, VHWVB2, and P03316, and the nucleic acid sequences of which are provided, e.g., under accession numbers SVU90536 and V01403 (see also Rice & Strauss, Proc. Nat'l Acad. Sci USA 78:2062-2066 (1981); and Strauss et al., Virology 133:92-110 (1984)).

[0197] In certain embodiments, the Sindbis virus envelope protein is mutated (SINmu). In certain embodiments, this mutation reduces the natural tropism of the Sindbis virus. In certain embodiments, SINmu comprises SIN proteins E1, E2, and E3, wherein at least one of E1, E2, or E3 is mutated compared to the wild-type sequence. For example, one or more of the E1, E2, or E3 proteins may be mutated at one or more amino acid positions. Furthermore, multiple combinations of mutations in E1, E2, and E3, such as E1 and E2, or E2 and E3, or E3 and E1, or mutations in E1, E2, and E3, are encompassed by the fusogens described herein. In certain embodiments, at least E2 is mutated.

[0198] In certain embodiments, SINmu comprises the following envelope protein mutations compared to wild-type Sindbis virus envelope protein: (i) deletion of E3 amino acids 61-64; (ii) E2 KE159-160AA; and (iii) E2 SLKQ68-71AAAA ("SLKQ" and "AAAA" are disclosed as SEQ ID NOs: 10-11, respectively). In further embodiments, SINmu further comprises envelope protein mutation E1 AK226-227SG. Examples of SINmu can be found, for example, in U.S. Pat. No. 9,163,248; WO2011011584; Cronin et al., Curr Gene Ther. 2005 Aug; 5(4): 387-398.

[0199] Other Togaviridae envelopes, such as those from Alphaviruses, such as Semliki Forest virus, Ross River virus, and Equine encephalitis virus, can also be used to pseudotype the vectors described herein. Envelope protein sequences of such alphaviruses are known in the art.

[0200] In certain embodiments, the fusogen is a vesicular stomatitis virus (VSV) envelope protein. In certain embodiments, the fusogen is the VSV G protein (VSV-G; Burns et al., Proc. Natl. Acad. Sci. USA 1993, vol. 90, no. 17, pp. 1833-1837), or a fragment, mutant, derivative, or homolog thereof. VSV-G interacts with phospholipid components of cell (e.g., T cell) membranes to mediate viral entry by membrane fusion (Mastromarino et al., J Gen Virol. 1998, vol. 68, no. 9, pp. 2359-69; Marsh et al., Adv Virus Res. 1989, vol. 107, no. 36, pp. 107-51). Examples of VSV-G can be found, for example, in WO2008058752.

[0201] One embodiment of the present disclosure is a nucleic acid encoding the above-mentioned capsid protein. The nucleic acid is preferably a vector comprising the claimed nucleic acid sequence. The nucleic acid, particularly the vector, is required for the recombinant expression of the capsid protein of the present disclosure.

[0202] A further embodiment of the present disclosure is the use of at least one recombinant viral capsid protein and / or nucleic acid encoding same, preferably at least one multimeric structure (e.g., a viral vector), for the generation of and use as a gene transfer vector.

[0203] Heterologous epitopes Generally, recombinant viral capsid proteins and / or viral vectors comprising recombinant viral capsids comprise a heterologous epitope that allows for retargeting of the viral vector, for example, via a binding molecule (e.g., an antibody). In some embodiments, the heterologous epitope is a B-cell epitope, e.g., about 1 amino acid to about 35 amino acids in length, that forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, the heterologous epitope comprises an affinity tag.

[0204] Numerous tags are known in the art (see, for example, Nilsson et al. (1997) "Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins" Protein Expression and Purification 11: 1-16, Terpe et al. (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60:523-533, and references therein). Affinity tags include, but are not limited to, immobilized divalent cations (e.g., Ni 2+Examples of tags include polyhistidine tags (e.g., His-6, His-8, or His-10 tags) that bind to immobilized avidin (e.g., on an in vivo biotinylated polypeptide sequence), a GST (glutathione S-transferase) sequence that binds to immobilized glutathione, an S tag that binds to immobilized S protein, an antigen that binds to an immobilized antibody or domain or fragment thereof (including, e.g., T7, myc, FLAG, and B tags that bind to the corresponding antibody), a FLASH tag (a high-affinity tag that binds to a specific arsenic-based moiety), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds to immobilized IgG, maltose-binding protein (MBP) that binds to immobilized amylose, an albumin-binding protein that binds to immobilized albumin, a chitin-binding domain that binds to immobilized chitin, a calmodulin-binding peptide that binds to immobilized calmodulin, and a cellulose-binding domain that binds to immobilized cellulose. Another exemplary tag is the SNAP tag, commercially available from Covalys (www.covalys.com). In some embodiments, heterologous epitopes disclosed herein comprise affinity tags that are recognized only by an antibody paratope, hi some embodiments, heterologous epitopes disclosed herein comprise affinity tags that are recognized by an antibody paratope and another specific binding pair.

[0205] In some embodiments, the heterologous epitope and / or affinity tag does not form a binding pair with an immunoglobulin molecule constant domain. In some embodiments, the heterologous epitope and / or affinity tag binds to a metal ion, e.g., Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ etc. In some embodiments, the heterologous epitope is not a polypeptide selected from the group consisting of streptavidin, Strep II, HA, L14, 4C-RGD, LH, and protein A.

[0206] In some embodiments, the affinity tag is selected from the group consisting of FLAG, HA, and c-myc (EQKLISEEDL (SEQ ID NO: 12)). In some embodiments, the heterologous epitope is c-myc.

[0207] In some embodiments, a recombinant viral capsid described herein comprises the amino acid sequence EQKLISEEDL (SEQ ID NO: 12) flanked by and / or operably linked to at least five consecutive amino acids of an AAV VP1 capsid protein. In some embodiments, a recombinant viral capsid described herein comprises the amino acid sequence EQKLISEEDL (SEQ ID NO: 12) flanked by and / or operably linked to at least five consecutive amino acids of an AAV2 VP1 capsid protein. In some embodiments, a recombinant viral capsid described herein comprises EQKLISEEDL (SEQ ID NO: 12) inserted between N587 and R588 of an AAV2 VP1 capsid protein.

[0208] In some embodiments, the heterologous epitope comprises an affinity tag and one or more linkers. In some embodiments, the heterologous epitope comprises an affinity tag sandwiched between linkers, e.g., the heterologous epitope comprises, from N-terminus to C-terminus, a first linker, an affinity tag, and a second linker. In some embodiments, the first linker and the second linker are each independently at least one amino acid in length. In some embodiments, the first linker and the second linker are identical.

[0209] Generally, heterologous epitopes described herein, including, for example, affinity tags alone or in combination with one or more linkers, are about 5 amino acids to about 35 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is at least 5 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 6 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 7 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 8 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 9 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 10 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 11 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 12 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 13 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 14 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 15 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 16 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 17 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 18 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 19 amino acids in length.In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 20 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 21 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 22 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 23 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 24 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 25 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 26 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 27 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 28 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 29 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 30 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 31 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 32 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 33 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 34 amino acids in length. In some embodiments, the heterologous epitope (alone or in combination with one or more linkers) is 35 amino acids in length.

[0210] retargeting part The viral vectors described herein have reduced or eliminated transduction ability in the absence of a binding molecule, particularly a binding molecule that specifically binds to a surface molecule expressed by target cells (e.g., B cells or hematopoietic stem cells). In some embodiments, the binding molecule comprises an antibody (or fragment thereof) comprising an antibody paratope that specifically binds to a heterologous epitope. For example, the binding molecule may comprise a single-domain heavy chain variable region or a single-domain light chain variable region, wherein the single-domain heavy chain variable region or the single-domain light chain variable region comprises an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the binding molecule may comprise an Fv region, e.g., the binding molecule may comprise an scFV comprising an antibody paratope that specifically binds to a heterologous epitope. In some embodiments, the binding molecule described herein comprises an antibody paratope that specifically binds to c-myc.

[0211] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are known in the art and can be used to identify CDRs within the designated HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventional means that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0212] In some embodiments, the binding molecule binds to a protein expressed on the surface of a cell, e.g., a cell surface protein on a hematopoietic cell (HSC), e.g., a B cell or hematopoietic stem cell. There are numerous suitable cell surface proteins, e.g., cell surface receptors, that can be targeted by a retargeting ligand, and for which retargeting ligands, e.g., antibodies or portions thereof, are already available. Such structures include, but are not limited to, B cell receptors and associated proteins (e.g., CD19, CD20, CD22, CD34, CD38, CD40, CD22, CD79, CD180, B cell activating factor (BAFF), ASGR1, CD117, Sca1, etc.), and HSC receptors and associated proteins (e.g., CD34, etc.). The recombinant viral capsids described herein employ binding molecules, including retargeting ligands that bind to differentiated cell surface antigens, as targets for viral vector complexes, thereby enabling specific infection of certain cell types.

[0213] The viral particles described herein may further comprise a second member of a specific binding pair that specifically forms a covalent bond with the first member of the specific binding pair inserted into / displayed by the recombinant viral capsid protein, wherein said second member is fused to the binding molecule.

[0214] In certain exemplary embodiments, the binding molecule is a bispecific antibody. Each antigen-binding domain of a bispecific antibody comprises a heavy chain variable domain (HCVR) and a light chain variable domain (LCVR). In the context of a bispecific antigen-binding molecule (e.g., a bispecific antibody) comprising a first and a second antigen-binding domain, the CDRs of the first antigen-binding domain can be designated with the prefix "A1," and the CDRs of the second antigen-binding domain can be designated with the prefix "A2." Thus, the CDRs of the first antigen-binding domain can be referred to herein as A1-HCDR1, A1-HCDR2, and A1-HCDR3; the CDRs of the second antigen-binding domain can be referred to herein as A2-HCDR1, A2-HCDR2, and A2-HCDR3.

[0215] The first and second antigen-binding domains can be directly or indirectly connected to each other to form a bispecific antigen-binding molecule of the present invention. Alternatively, the first and second antigen-binding domains may each be connected to a separate multimerization domain. Binding of one multimerization domain to another promotes binding between the two antigen-binding domains, thereby forming a bispecific antigen-binding molecule. As used herein, a "multimerizing domain" refers to any macromolecule, protein, polypeptide, peptide, or amino acid capable of binding to a second multimerization domain of the same or similar structure or construct. For example, the multimerizing domain may be a polypeptide containing the CH3 domain of an immunoglobulin molecule. A non-limiting example of a multimerizing component is the Fc portion of an immunoglobulin molecule (containing the CH2-CH3 domain), e.g., the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0216] Bispecific antigen-binding molecules of the invention will typically comprise two multimerization domains, e.g., two Fc domains, each part of a separate antibody heavy chain. The first and second multimerization domains may be of the same IgG isotype, e.g., IgG1 / IgG1, IgG2 / IgG2, IgG4 / IgG4, etc. Alternatively, the first and second multimerization domains may be of different IgG isotypes, e.g., IgG1 / IgG2, IgG1 / IgG4, IgG2 / IgG4, etc.

[0217] In certain embodiments, the multimerization domain is an Fc fragment or an amino acid sequence of 1 to about 200 amino acids in length containing at least one cysteine ​​residue. In other embodiments, the multimerization domain is a cysteine ​​residue or a short cysteine-containing peptide. Other multimerization domains include peptides or polypeptides comprising or consisting of a leucine zipper, a helix loop motif, or a coiled-coil motif.

[0218] Any bispecific antibody format or technology may be used to generate the bispecific antigen-binding molecules of the present invention. For example, a bispecific antigen-binding molecule can be generated by operatively linking (e.g., by chemical bonding, genetic fusion, non-covalent bonding, etc.) an antibody or fragment thereof having a first antigen-binding specificity to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity. Illustrative examples of bispecific formats that can be used in the context of the present invention include, but are not limited to, 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 Mab2 bispecific formats (for reviews of the foregoing formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and references cited therein; see also Brinkmann and Konterman (2017) mAbs 9:182-212, each of which is incorporated by reference in its entirety).

[0219] The present invention also includes bispecific antigen-binding molecules comprising a first CH3 domain and a second Ig CH3 domain, wherein the first and second Ig CH3 domains differ from each other by at least one amino acid, and wherein the at least one amino acid difference reduces binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig CH3 domain binds to protein A and the second Ig CH3 domain comprises a mutation, e.g., an H95R modification (according to IMGT exon numbering; H435R in EU numbering), that reduces or eliminates binding to protein A. The second CH3 may further comprise a Y96F modification (according to IMGT; Y436F in EU). Further modifications that may be found within the second CH3 include: D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (by IMGT; D356E, L358M, N384S, K392N, V397M, and V422I in EU); N44S, K52N, and V82I for IgG2 antibodies (by IMGT; N384S, K392N, and V422I in EU); and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (by IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I in EU). See, for example, WO 2010 / 151792.

[0220] In certain embodiments, the Fc domain may be chimeric, combining Fc sequences from two or more immunoglobulin isotypes. For example, a chimeric Fc domain may comprise part or all of a CH2 sequence from a human IgG1, human IgG2, or human IgG4 CH2 region and part or all of a CH3 sequence from a human IgG1, human IgG2, or human IgG4. A chimeric Fc domain may also comprise a chimeric hinge region. For example, a chimeric hinge may comprise an "upper hinge" sequence from a human IgG1, human IgG2, or human IgG4 hinge region in combination with a "lower hinge" sequence from a human IgG1, human IgG2, or human IgG4 hinge region. A specific example of a chimeric Fc domain that can be included in any of the antigen-binding molecules described herein comprises, from N-terminus 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 described herein comprises, from N-terminus 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 invention are described in WO 2014 / 022540, which is incorporated by reference in its entirety. Chimeric Fc domains with these overall structural arrangements, and variants thereof, can alter Fc receptor binding, which affects Fc effector function.

[0221] Liposomes, lipid nanoparticles, and other carriers In some embodiments, the first component and / or the second component of the systems described herein may be a lipid-based carrier, such as a lipid nanoparticle (LNP), a liposome, a lipidoid, or a lipoplex.

[0222] In some embodiments, the first and / or second components of the systems described herein may comprise liposomes or LNPs. Liposomes and LNPs are vesicles comprising one or more lipid bilayers. In some embodiments, liposomes or LNPs comprise two or more concentric bilayers separated by aqueous compartments. The lipid bilayers can be functionalized and / or crosslinked to each other. The lipid bilayers can comprise one or more proteins, polysaccharides, or other molecules.

[0223] Lipid formulations can protect biomolecules from degradation and improve their cellular uptake. Liposomes or LNPs are particles containing multiple lipid molecules physically bound to each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. Such liposomes or LNPs can be used to encapsulate one or more nucleic acids or proteins for delivery. Formulations containing cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time that nanoparticles can persist in vivo. Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO 2016 / 010840 and WO 2017 / 173054 (each of which is incorporated herein by reference in its entirety for all purposes). Exemplary lipid nanoparticles can include a cationic lipid and one or more other components. In one example, the other components can include a helper lipid such as cholesterol. In another example, the other components can include a helper lipid such as cholesterol and a neutral lipid such as distearoylphosphatidylcholine (DSPC). In another example, the other components can include a helper lipid such as cholesterol, an optional neutral lipid such as DSPC, and a stealth lipid such as S010, S024, S027, S031, or S033.

[0224] Liposomes are amphipathic lipids that can form bilayers in an aqueous environment to encapsulate an aqueous core. Polypeptides (e.g., Cas proteins) or polynucleotides (e.g., guide RNAs) can be incorporated into this aqueous core. These lipids can have anionic, cationic, or zwitterionic hydrophilic head groups. Liposomes can be formed from a single lipid or a mixture of lipids. The mixture can include: (1) a mixture of anionic lipids; (2) a mixture of cationic lipids; (3) a mixture of zwitterionic lipids; (4) a mixture of anionic and cationic lipids; (5) a mixture of anionic and zwitterionic lipids; (6) a mixture of zwitterionic and cationic lipids; or (7) a mixture of anionic, cationic, and zwitterionic lipids. Similarly, the mixture can include both saturated and unsaturated lipids. Exemplary phospholipids include, but are not limited to, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol. Cationic lipids include, but are not limited to, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), dioleoyltrimethylammoniumpropane (DOTAP), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA). Zwitterionic lipids include, but are not limited to, acyl zwitterionic lipids and ether zwitterionic lipids. Examples of useful zwitterionic lipids include dodecylphosphocholine, DPPC, and DOPC.

[0225] Liposomes or LNPs may comprise one or more or all of the following: (i) lipids for encapsulation and endosomal escape; (ii) neutral lipids for stabilization; (iii) helper lipids for stabilization; and (iv) stealth lipids. See, e.g., Finn et al. (2018) Cell Rep. 22(9):2227-2235, and WO 2017 / 173054, each of which is incorporated herein by reference in its entirety for all purposes.

[0226] In some cases, the liposomes or LNPs comprise a cationic lipid, such as (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), or another ionizable lipid. See, e.g., WO 2019 / 067992, WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086 (each of which is incorporated by reference in its entirety for all purposes). In some examples, the molar ratio of cationic lipid amine to RNA phosphate (N:P) of the LNP is about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. In some examples, the terms "cationic" and "ionizable" in the context of LNP lipids are interchangeable (e.g., ionizable lipids are cationic depending on pH).

[0227] The lipid for encapsulation and endosomal escape may be a cationic lipid. The lipid may be a biodegradable lipid, such as a biodegradable ionizable lipid. One example of a suitable lipid is lipid A or LP01, which is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep. 22(9):2227-2235, and WO 2017 / 173054, each of which is incorporated herein by reference in its entirety for all purposes. Another example of a suitable lipid is lipid B, which is ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), also known as ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate). Another example of a suitable lipid is lipid C, which is 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate). Another example of a suitable lipid is lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 3-octylundecanoate. Another suitable lipid is heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (also known as [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butanoate, or Dlin-MC3-DMA (MC3)).

[0228] Additional suitable cationic lipids include, but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethyl Cationic lipids that are positively charged below physiological pH include, but are not limited to, DODAP, DODMA, and DMDMA. In some embodiments, the cationic lipid comprises a C alkyl chain, an ether linkage between the head group and the alkyl chain, and 0 to 3 double bonds. Examples of such lipids include DSDMA, DLinDMA, DLenDMA, and DODMA. The cationic lipid may comprise an ether linkage and a pH-titratable head group. Such lipids include, for example, DODMA. Further cationic lipids are described in U.S. Patent No. 7,745,651; U.S. Patent No. 5,208,036; U.S. Patent No. 5,264,618; U.S. Patent No. 5,279,833; U.S. Patent No. 5,283,185; U.S. Patent No. 5,753,613; and U.S. Patent No. 5,785,992 (incorporated herein by reference).

[0229] In some embodiments, cationic lipids may contain a protonatable tertiary amine head group. Such lipids are referred to herein as ionizable lipids. Ionizable lipids refer to lipid species that contain an ionizable amine head group and typically have a pKa less than about 7. In an acidic pH environment, the ionizable amine head group becomes protonated, allowing the ionizable lipid to preferentially interact with negatively charged molecules (e.g., nucleic acids, such as the recombinant polynucleotides described herein), thereby facilitating liposome or LNP assembly and encapsulation. Thus, in some embodiments, ionizable lipids can increase the loading of nucleic acids within liposomes or LNPs. In environments with a pH greater than about 7 (e.g., at a physiological pH of 7.4), ionizable lipids have a neutral charge. When particles containing ionizable lipids are taken up into the low pH environment of an endosome (e.g., pH < 7), the ionizable lipids again become protonated and associate with the anionic endosomal membrane, facilitating the release of the contents encapsulated by the particle.

[0230] In some embodiments, the liposome or LNP may comprise one or more non-cationic helper lipids. Exemplary helper lipids include (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine) (DLPE), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (D iPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), (1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), ceramide, sphingomyelin, and cholesterol.

[0231] Some of the lipids suitable for use in the liposomes or LNPs described herein are biodegradable in vivo. Examples of biodegradable lipids include, but are not limited to, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate), or another ionizable lipid. See, e.g., WO 2017 / 173054, WO 2015 / 095340, and WO 2014 / 136086. In some embodiments, the terms "cationic" and "ionizable" in the context of liposome or LNP lipids are interchangeable, e.g., ionizable lipids become cationic depending on pH.

[0232] Such lipids may be ionizable depending on the pH of the medium in which they are contained. For example, in a slightly acidic medium, the lipids may be protonated and thus positively charged. Conversely, in a slightly basic medium, such as blood, which has a pH of approximately 7.35, the lipids may not be protonated and may not carry a charge. In some embodiments, the lipids may be protonated at a pH of at least about 9, 9.5, or 10. The ability of such lipids to carry a charge is related to their inherent pKa. For example, the lipids may independently have a pKa ranging from about 5.8 to about 6.2.

[0233] Neutral lipids function to stabilize liposomes or LNPs and improve their processing. Examples of suitable neutral lipids include neutral, uncharged, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), ... , 2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1-myristoyl-2-palmitoylphosphatidylcholine (MPPC), 1-palmitoyl-2-myristoylphosphatidylcholine (PMPC), 1-palmitoyl-2-stearoylphosphatidylcholine (SPPC), distearoylphosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and combinations thereof. For example, the neutral phospholipid can be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE).

[0234] Helper lipids include lipids that enhance transfection. The mechanism by which helper lipids enhance transfection can include enhancing particle stability. In certain cases, helper lipids can enhance membrane fusion. Helper lipids include steroids, sterols, and alkylresorcinols. Examples of suitable helper lipids include cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In some examples, the helper lipid can be cholesterol or cholesterol hemisuccinate.

[0235] Stealth lipids are lipids that change the length of time that nanoparticles can exist in vivo. Stealth lipids can aid in the formulation process, for example, by reducing particle aggregation and controlling particle size. Stealth lipids can adjust the pharmacokinetic properties of liposomes or LNPs. Suitable stealth lipids include lipids with a hydrophilic head group attached to the lipid moiety.

[0236] The hydrophilic head group of the stealth lipid can comprise a polymer moiety selected from, for example, PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyamino acids, and poly(N-(2-hydroxypropyl)methacrylamide)-based polymers. The term "PEG" refers to any polyethylene glycol and other polyalkylene ether polymers. In certain liposome or LNP formulations, the PEG is PEG-2K, also known as PEG2000, with an average molecular weight of approximately 2,000 daltons. See, e.g., International Publication No. WO 2017 / 173054 (incorporated herein by reference in its entirety for all purposes).

[0237] The lipid portion of the stealth lipid can be derived from, for example, diacylglycerol or diacylglycamide, including those containing dialkylglycerol or dialkylglycamide groups having alkyl chain lengths independently containing from about C4 to about C40 saturated or unsaturated carbon atoms, where the chains may contain one or more functional groups, such as, for example, amide or ester. The dialkylglycerol or dialkylglycamide groups can further contain one or more substituted alkyl groups.

[0238] Exemplary stealth lipids include PEG-dilaurylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-distearoylglycamide, PEG-cholesterol (l-[8'-(cholest-5-en-3[β]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[ω]-methyl-poly(ethylene glycol)), PEG-DMB (3,4-ditetradecoxybenzyl-[ω]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl -sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In a particular example, the stealth lipid may be PEG2k-DMG.

[0239] In some embodiments, the liposome or LNP may further comprise one or more PEG-modified lipids comprising poly(ethylene)glycol chains up to 5 kDa in length covalently attached to one or more C6-C20 alkyl-containing lipids. In some embodiments, the liposome or LNP further comprises 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG-amine). In some embodiments, the PEG-modified lipid comprises about 0.1% to about 1% of the total lipid content in the lipid nanoparticle. In some embodiments, the PEG-modified lipid comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0% of the total lipid content in the liposome or lipid nanoparticle.

[0240] In some embodiments, the liposomes or LNPs described herein may contain conjugated lipids that inhibit lipid particle aggregation. Examples of such lipid conjugates include, but are not limited to, PEG-lipid conjugates (see, e.g., U.S. Pat. No. 5,885,613), such as PEG conjugated to dialkyloxypropyl (e.g., PEG-DAA conjugates), PEG conjugated to diacylglycerol (e.g., PEG-DAG conjugates), PEG conjugated to cholesterol, PEG conjugated to phosphatidylethanolamine, and PEG conjugated to ceramide, cationic PEG-lipids, polyoxazoline (POZ) lipid conjugates (e.g., POZ-DAA conjugates), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Further examples of POZ-lipid conjugates are described in International Publication No. WO 2010 / 006282. PEG or POZ can be directly conjugated to the lipid or linked to the lipid via a linker moiety. Any suitable linker moiety for linking PEG or POZ to the lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In certain embodiments, non-ester containing linker moieties such as amides or carbamates are used.

[0241] Liposomes or LNPs can have different molar ratios of component lipids in the formulation. The molar percentage of CCD lipids can be, for example, about 30 mol% to about 60 mol%. The molar percentage of helper lipids can be, for example, about 30 mol% to about 60 mol%. The molar percentage of neutral lipids can be, for example, about 1 mol% to about 20 mol%. The molar percentage of stealth lipids can be, for example, about 1 mol% to about 10 mol%.

[0242] Liposomes or LNPs can have various ratios between the positively charged amine groups (N) of the biodegradable lipids and the negatively charged phosphate groups (P) of the encapsulated nucleic acid. This can be represented mathematically by the formula N / P. For example, the N / P ratio can be from about 0.5 to about 100. The N / P ratio can also be from about 4 to about 6.

[0243] In some embodiments, the liposome or LNP can include a nuclease agent (e.g., a CRISPR / Cas system, a ZFN, or a TALEN), can include a polynucleotide molecule (e.g., a guide RNA), can include a nucleic acid construct encoding a polypeptide of interest (e.g., an antibody or antigen-binding fragment), or can include both a nuclease agent (e.g., a CRISPR / Cas system) and a nucleic acid construct encoding a polypeptide of interest (e.g., a donor template for use in gene editing). With respect to a CRISPR / Cas system, the liposome or LNP can include a Cas protein in any form (e.g., protein, DNA, or mRNA) and / or can include one or more guide RNAs in any form (e.g., DNA or RNA). In one example, the liposome or LNP includes a Cas protein in the form of mRNA (e.g., a modified RNA described herein) and one or more guide RNAs in the form of RNA (e.g., a guide RNA disclosed herein). In another example, the liposome or LNP includes a Cas protein in the form of a protein and one or more guide RNAs in the form of RNA. In some examples, the guide RNA and Cas protein are each introduced in the form of RNA via LNP-mediated delivery in the same LNP. As described in more detail elsewhere herein, one or more of the RNAs can be modified. For example, the guide RNA can be modified to include one or more stabilizing end modifications at the 5' and / or 3' end. Such modifications can include, for example, one or more phosphorothioate linkages at the 5' and / or 3' end and / or one or more 2'-O-methyl modifications at the 5' and / or 3' end. As another example, Cas mRNA modifications can include substitution with pseudouridine (e.g., full substitution with pseudouridine), a 5' cap, and polyadenylation. Other modifications are also contemplated, as disclosed elsewhere herein. Delivery by such methods can result in transient Cas expression and / or transient guide RNA presence, with biodegradable lipids providing improved clearance, increased tolerability, and reduced immunogenicity.

[0244] In certain liposomes or LNPs, the cargo may comprise a guide RNA or a nucleic acid encoding the guide RNA. In certain liposomes or LNPs, the cargo may comprise an mRNA encoding a Cas nuclease, such as Cas9, and a guide RNA or a nucleic acid encoding the guide RNA. In certain liposomes or LNPs, the cargo may comprise a nucleic acid construct encoding a polypeptide of interest (e.g., an antibody or antigen-binding fragment), as described elsewhere herein. In certain liposomes or LNPs, the cargo may comprise an mRNA encoding a Cas nuclease, such as Cas9, a guide RNA or a nucleic acid encoding the guide RNA, and a nucleic acid construct encoding a polypeptide of interest (e.g., an antibody or antigen-binding fragment). In some liposomes or LNPs, the lipid component comprises an amine lipid, such as a biodegradable ionizable lipid. In some examples, the lipid component comprises a biodegradable ionizable lipid, cholesterol, DSPC, and PEG-DMG. For example, Cas9 mRNA and gRNA can be delivered to cells and animals using lipid formulations containing the ionizable lipid ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (also known as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate)), cholesterol, DSPC, and PEG2k-DMG.

[0245] In some liposomes or LNPs, the cargo can include a Cas mRNA (e.g., Cas9 mRNA) and a gRNA. The Cas mRNA and gRNA can be present in various ratios. For example, the ratio of Cas mRNA to gRNA nucleic acid in an LNP formulation can range from about 25:1 to about 1:25. Alternatively, the ratio of Cas mRNA to gRNA nucleic acid in a liposome or LNP formulation can be from about 2:1 to about 1:2. In a particular example, the ratio of Cas mRNA to gRNA nucleic acid can be about 2:1.

[0246] In some liposomes or LNPs, the cargo can include a nucleic acid construct encoding a polypeptide of interest (e.g., an antibody or antigen-binding fragment) and a gRNA. The ratio of the nucleic acid construct encoding the polypeptide of interest (e.g., an antibody or antigen-binding fragment) to the gRNA can be varied. For example, the ratio of the nucleic acid construct to the gRNA nucleic acid in the liposome or LNP formulation can be about 25:1 to about 1:25.

[0247] One specific example of a suitable LNP has a nitrogen to phosphate (N / P) ratio of about 4.5 and comprises a biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of about 45:44:9:2 (about 45:about 44:about 9:about 2). The biodegradable cationic lipid can be (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also referred to as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate. See, e.g., Finn et al. (2018) Cell Rep. 22(9):2227-2235 (incorporated herein by reference in its entirety for all purposes). The weight ratio of Cas9 mRNA to guide RNA can be about 1:1 (about 1:about 1). Another specific example of a suitable LNP comprises Dlin-MC3-DMA (MC3), cholesterol, DSPC, and PEG-DMG in a molar ratio of about 50:38.5:10:1.5 (about 50:about 38.5:about 10:about 1.5). The weight ratio of Cas9 mRNA to guide RNA can be about 1:2 (about 1:about 2). The weight ratio of Cas9 mRNA to guide RNA can be about 1:1 (about 1:about 1). The weight ratio of Cas9 mRNA to guide RNA can be about 2:1 (about 2:about 1).

[0248] Another specific example of a suitable LNP has a nitrogen to phosphate (N / P) ratio of about 6 and comprises a biodegradable cationic lipid, cholesterol, DSPC, and PEG2k-DMG in a molar ratio of about 50:38:9:3 (about 50:about 38:about 9:about 3). The biodegradable cationic lipid can be lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also referred to as 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-octadeca-9,12-dienoate). The weight ratio of Cas9 mRNA to guide RNA can be about 1:2 (about 1:about 2). The weight ratio of Cas9 mRNA to guide RNA can be about 1:1 (about 1:about 1). The weight ratio of Cas9 mRNA to guide RNA can be about 2:1 (about 2:about 1).

[0249] Another specific example of a suitable LNP has a nitrogen-to-phosphate (N / P) ratio of about 3 and includes a cationic lipid, a structural lipid, cholesterol (e.g., cholesterol (ovine) (Avanti 700000)), and PEG2k-DMG (e.g., PEG-DMG 2000 (NOF America - SUNBRIGHT® GM-020 (DMG-PEG)) in a ratio of about 50:10:38.5:1.5 (about 50:about 10:about 38.5:about 1.5), or a ratio of about 47:10:42:1 (about 47:about 10:about 42:about 1). The structural lipid can be, for example, DSPC (e.g., DSPC (Avanti 850365)), SOPC, DOPC, or DOPE. The cationic / ionizable lipid can be, for example, Dlin-MC3-DMA (e.g., Dlin-MC3-DMA (Biofine International)). The weight ratio of mRNA to guide RNA can be about 1:2 (about 1:about 2). The weight ratio of Cas9 mRNA to guide RNA can be about 1:1 (about 1:about 1). The weight ratio of Cas9 mRNA to guide RNA can be about 2:1 (about 2:about 1).

[0250] Another specific example of a suitable LNP comprises Dlin-MC3-DMA, DSPC, cholesterol, and PEG lipid in a ratio of about 45:9:44:2 (about 45:about 9:about 44:about 2). Another specific example of a suitable LNP comprises Dlin-MC3-DMA, DOPE, cholesterol, and PEG lipid or PEG DMG in a ratio of about 50:10:39:1 (about 50:about 10:about 39:about 1). Another specific example of a suitable LNP comprises Dlin-MC3-DMA, DSPC, cholesterol, and PEG2k-DMG in a ratio of about 55:10:32.5:2.5 (about 55:about 10:about 32.5:about 2.5). Another specific example of a suitable LNP comprises Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in a ratio of about 50:10:38.5:1.5 (about 50:about 10:about 38.5:about 1.5). Another specific example of a suitable LNP comprises Dlin-MC3-DMA, DSPC, cholesterol, and PEG-DMG in a ratio of about 50:10:38.5:1.5 (about 50:about 10:about 38.5:about 1.5). The weight ratio of Cas9 mRNA to guide RNA can be about 1:2 (about 1:about 2). The weight ratio of Cas9 mRNA to guide RNA can be about 1:1 (about 1:about 1). The weight ratio of Cas9 mRNA to guide RNA can be about 2:1 (about 2:about 1).

[0251] Other examples of suitable LNPs can be found, for example, in WO 2019 / 067992, WO 2020 / 082042, U.S. Patent Application Publication Nos. 2020 / 0270617, WO 2020 / 082041, U.S. Patent Application Publication Nos. 2020 / 0268906, WO 2020 / 082046 (see, e.g., pages 85-86), and U.S. Patent Application Publication No. 2020 / 0289628, each of which is incorporated herein by reference in its entirety for all purposes.

[0252] Dynamic Light Scattering ("DLS") can be used to characterize the polydispersity index ("PDI") and size of liposomes and LNPs. In some embodiments, the PDI may range from about 0.005 to about 0.75. In some embodiments, the PDI may range from about 0.01 to about 0.5. In some embodiments, the PDI may range from about 0.02 to about 0.4. In some embodiments, the PDI may range from about 0.03 to about 0.35. In some embodiments, the PDI may range from about 0.1 to about 0.35.

[0253] The size of the LNPs disclosed herein can range from about 1 to about 250 nm. In some embodiments, the size of the LNPs can range from about 10 to about 200 nm. In some embodiments, the size of the LNPs can range from about 20 to about 150 nm. In some embodiments, the size of the LNPs can range from about 50 to about 150 nm. In some embodiments, the size of the LNPs can range from about 50 to about 100 nm. In some embodiments, the size of the LNPs can range from about 50 to about 120 nm. In some embodiments, the size of the LNPs can range from about 75 to about 150 nm. In some embodiments, the size of the LNPs can range from about 30 to about 200 nm. In some embodiments, the average size (diameter) of fully formed nanoparticles is measured by dynamic light scattering on a Malvern Zetasizer (e.g., nanoparticle samples can be diluted in phosphate buffered saline (PBS) to achieve a count rate of approximately 200-400 kcals, and the data can be expressed as a weighted average of intensity measurements).

[0254] In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency ranging from about 50% to about 100%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency ranging from about 50% to about 70%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency ranging from about 70% to about 90%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency ranging from about 90% to about 100%. In some embodiments, liposomes or LNPs can be formed with an average encapsulation efficiency ranging from about 75% to about 95%.

[0255] In addition to liposomes and LNPs, the first and / or second components of the systems described herein may be in the form of other carriers for delivery of nucleic acid and / or protein molecules. Examples of other suitable carriers include, but are not limited to, liposomes, lipoids, and lipoplexes, microparticles or polymeric nanoparticles, inorganic nanoparticles, peptide carriers, nanoparticle mimics, nanotubes, conjugates, immune stimulating complexes (ISCOMs), virus-like particles (VLPs), self-assembling proteins, or emulsion delivery systems such as cationic submicron oil-in-water emulsions.

[0256] Polymeric microparticles or nanoparticles can also be used to encapsulate or adsorb polypeptides (e.g., Cas proteins) or polynucleotides (e.g., guide RNA). The particles can be substantially non-toxic and biodegradable. Particles that can be used to deliver polynucleotides (e.g., guide RNA) can have an optimal particle size and zeta potential. For example, the diameter of microparticles can be in the range of 0.02 μm to 8 μm. In examples where the composition is a collection of micro- or nanoparticles with different diameters, at least 80%, 85%, 90%, or 95% of the particles ideally have a diameter in the range of 0.03 to 7 μm. These particles can also have a zeta potential of 40 to 100 mV to provide maximum adsorption of polynucleotides (e.g., guide RNA) to the particles.

[0257] Non-toxic and biodegradable polymers include, but are not limited to, one or more natural polymers such as poly(hydroxy acid), polyhydroxybutyric acid, polylactones (including polycaprolactone), polydioxanone, polyvalerolactone, polyorthoesters, polyanhydrides, polycyanoacrylates, tyrosine-derived polycarbonates, polyvinyl-pyrrolidinone or polyester-amides, polysaccharides (e.g., pullulan, alginic acid, inulin, and chitosan), and combinations thereof. In some embodiments, the particles are formed from poly(hydroxy acid) (e.g., poly(lactide) (PLA)), poly(γ-glutamic acid) (γ-PGA), poly(ethylene glycol) (PEG), polystyrene, copolymers of lactide and glycolide (e.g., poly(D,L-lactide-co-glycolide) (PLG)), and copolymers of D,L-lactide and caprolactone. Useful PLG polymers include those having a lactide / glycolide molar ratio within the range of, for example, 20:80 to 80:20, such as 25:75, 40:60, 45:55, 55:45, 60:40, or 75:25. Useful PLG polymers include those having a molecular weight of, for example, 5,000 to 200,000 Da, such as 10,000 to 100,000, 20,000 to 70,000, or 40,000 to 50,000 Da.

[0258] Polymeric nanoparticles may form hydrogel nanoparticles, i.e., hydrophilic three-dimensional polymer networks with favorable properties including flexible mesh size, large surface area for multivalent conjugation, high water content, and high antigen loading capacity. Poly(L-lactic acid) (PLA), PLGA, PEG, and polysaccharides are suitable for forming hydrogel nanoparticles.

[0259] For example, inorganic nanoparticles can be calcium phosphate nanoparticles, silicon nanoparticles, or gold nanoparticles. Inorganic nanoparticles typically have a rigid structure and include a shell that encapsulates a polypeptide or polynucleotide, or a core to which a polypeptide or polynucleotide can be covalently attached. The core can include one or more atoms such as gold (Au), silver (Ag), copper (Cu), Au / Ag, Au / Cu, Au / Ag / Cu, Au / Pt, Au / Pd, or Au / Ag / Cu / Pd, or calcium phosphate (CaP).

[0260] Other molecules suitable for complexing with the polypeptides or polynucleotides of the disclosure include cationic molecules such as polyamidoamine, dendritic polylysine, polyethyleneiniline or polypropyleneimine, polylysine, chitosan, DNA-gelatin coacervate, DEAE-dextran, dendrimers, or polyethyleneimine (PEI).

[0261] In some embodiments, the polypeptides or polynucleotides of the present disclosure can be complexed with nanoparticles. Nanoparticles that can be used for complexing with the antigens and / or antibodies of the present disclosure include, but are not limited to, chitosan shell nanoparticles, carbon nanotubes, PEGylated liposomes, poly(d,l-lactide-co-glycolide) / montmorillonite (PLGA / MMT) nanoparticles, poly(lactide-co-glycolide) (PLGA) nanoparticles, poly-(malic acid)-based nanoparticles, and other inorganic nanoparticles (e.g., nanoparticles made of magnesium-aluminum layered double hydroxide with disuccinimidyl carbonate (DSC), and TiO nanoparticles).

[0262] Oil-in-water emulsions can be used to deliver polypeptides or polynucleotides (e.g., mRNA) to a subject. Examples of oils that can be used to make the emulsion include animal (e.g., fish) oils or vegetable oils (e.g., nuts, grains, and seeds). The oils can be biodegradable and biocompatible. Exemplary oils include, but are not limited to, tocopherol and squalene, branched shark liver oil, unsaturated terpenoids, and combinations thereof. Terpenoids are branched-chain oils that are biochemically synthesized from five-carbon isoprene units.

[0263] The aqueous component of the emulsion can be water, or water to which additional components have been added. For example, the water can contain a salt, such as a citrate or phosphate salt, such as a sodium salt, to form a buffer. Exemplary buffers include borate buffer, citrate buffer, histidine buffer, phosphate buffer, Tris buffer, or succinate buffer.

[0264] In some embodiments, the oil-in-water emulsion contains one or more cationic molecules. For example, cationic lipids can be included in the emulsion to provide a positively charged droplet surface to which negatively charged polynucleotides (e.g., mRNA) can adhere. Exemplary cationic lipids include, but are not limited to: 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 1,2-dimyristoyl-3-trimethylammoniumpropane (DMTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecylammonium (DDA, e.g., bromide), dipalmitoyl (C16:0) trimethylammoniumpropane (DPTAP), and distearoyltrimethylammoniumpropane (DSTAP). Other useful cationic lipids include benzalkonium hydrochloride (BAK), benzethonium chloride, cholesterol choline ester hemisuccinate, lipopolyamines (e.g., dioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanolamidospermine (DPPES)), cetrimide, cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride (CTAC), cationic derivatives of cholesterol (e.g., cholesteryl-3β-oxysuccinamide ethylenetrimethylammonium salt, cholesteryl-3β-oxysuccinamide ethylene-dimethylamine, cholesteryl-3β-carboxyamido ethylenetrimethylammonium salt, and cholesteryl-3β-carboxyamidoethylenedimethylamine), N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, mixed alkyltrimethylammonium bromides, benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium chloride, benzyltrimethylammonium methoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide (DDAB), methylbenzethonium chloride, decamethonium chloride, mixed methyltrialkylammonium chlorides, methyltrioctylammonium chloride), N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3 tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemethanaminonium chloride (DEBDA), cholesteryl (4'-trimethylammonio)butanoate), N-alkylpyridinium salts (e.g., cetylpyridinium bromide and cetylpyridinium chloride), N-alkylpiperidinium salts, dicationic boraform electrolytes (C12Me6; C12BU6), dialkylglycetylphosphorylcholine, lysolecithin, L-α dioleoylphosphatidylethanolamine, lipopoly-L(or D)-lysine (LPLL, LPDL), poly(L(or D)-lysine) complexed with N-glutarylphosphatidylethanolamine, di Examples include alkyldimethylammonium salts, [1-(2,3-dioleyloxy)-propyl]-N,N,N,trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonio)propane (the acyl group can be dimyristoyl, dipalmitoyl, distearoyl, or dioleoyl), 1,2-diacyl-3(dimethylammonio)propane (the acyl group can be dimyristoyl, dipalmitoyl, distearoyl, or dioleoyl), 1,2-dioleoyl-3-(4'-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester, didodecyl glutamate with a pendant amino group (C GluPhCnN), and ditetradecyl glutamate with a pendant amino group (C GluCnN).

[0265] In some embodiments, in addition to oil and cationic lipid, emulsion can also contain nonionic surfactant and / or zwitterionic surfactant.Examples of useful surfactants include, but are not limited to: polyoxyethylene sorbitan ester surfactants, such as polysorbate 20 and polysorbate 80; copolymers of ethylene oxide, propylene oxide, and / or butylene oxide, linear block copolymers; phospholipids, such as phosphatidylcholine; polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl, and oleyl alcohol; polyoxyethylene-9-lauryl ether; octoxynol; (octylphenoxy) polyethoxyethanol; and sorbitan ester.

[0266] In some embodiments, the polynucleotides described herein may be incorporated into polynucleotide complexes, including, but not limited to, nanoparticles (e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconducting / metallic nanoparticles), gels and hydrogels, polynucleotide complexes with cations and anions, microparticles, and any combination thereof.

[0267] In some embodiments, the polynucleotides described herein can be formulated as self-assembled nanoparticles. As a non-limiting example, polynucleotides can be used to create nanoparticles that can be used in delivery systems for the polynucleotides (see, e.g., WO 2012 / 125987). In some embodiments, polynucleotide self-assembled nanoparticles can include a polynucleotide core disclosed herein and a polymer shell. The polymer shell can be made of any polymer described herein and known in the art. In further embodiments, the polymer shell can be used to protect the core polynucleotide.

[0268] In some embodiments, self-assembled nanoparticles can be microsponges formed from long polymers of polynucleotide hairpins, which form crystalline "pleated" sheets before self-assembling into microsponges. These microsponges are densely packed, sponge-like microparticles that can function as efficient carriers and deliver cargo to cells. Microsponges can range in diameter from 1 μm to 300 nm. Microsponges can be complexed with other agents known in the art to form larger microsponges. As a non-limiting example, microsponges can be complexed with agents such as polycationic polyethyleneimine (PEI) to form an outer layer to promote cellular uptake. This complex can form particles with a diameter of 250 nm that can remain stable at high temperatures (150°C) (Grabow and Jaegar, Nature Materials 2012, 11:269-269). Furthermore, these microsponges can provide exceptional protection against degradation by ribonucleases. In certain embodiments, polymer-based self-assembling nanoparticles, such as but not limited to microsponges, can be fully programmable nanoparticles: By precisely controlling the nanoparticle geometry, size, and stoichiometry, one can create nanoparticles optimized for delivery of cargo, such as but not limited to polynucleotides.

[0269] In some embodiments, the polynucleotides disclosed herein may be incorporated into inorganic nanoparticles (see U.S. Pat. No. 8,257,745). Inorganic nanoparticles include, but are not limited to, water-swellable clay materials. As a non-limiting example, inorganic nanoparticles may include synthetic smectite clays made from simple silicates (see U.S. Pat. Nos. 5,585,108 and 8,257,745).

[0270] In some embodiments, the polynucleotides disclosed herein may be formulated into water-dispersible nanoparticles comprising semiconducting or metallic materials (U.S. Patent Application Publication No. 2012 / 0228565; incorporated herein by reference in its entirety), or may be formed as magnetic nanoparticles (U.S. Patent Application Publication No. 2012 / 0265001 and U.S. Patent Application Publication No. 2012 / 0283503). The water-dispersible nanoparticles may be hydrophobic or hydrophilic.

[0271] In some embodiments, the polynucleotides disclosed herein can be encapsulated in any hydrogel known in the art that can form a gel when injected into a subject. Hydrogels are networks of polymer chains that are hydrophilic and can be viewed as colloidal gels in which water is the dispersion medium. Hydrogels are highly absorbent (capable of containing more than 99% water), natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to that of natural tissue due to their substantial water content. The hydrogels described herein can be used to encapsulate biocompatible, biodegradable, and / or porous lipid nanoparticles.

[0272] As a non-limiting example, the hydrogel can be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel can be programmed to release one or more polynucleotides using polynucleotide hybridization (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413). In some embodiments, the polynucleotides can be encapsulated in lipid nanoparticles, which can then be encapsulated in the hydrogel.

[0273] In some embodiments, the polynucleotides disclosed herein can be encapsulated in fibrin gels, fibrin hydrogels, or fibrin glue. In other embodiments, the polynucleotides may be formulated in lipid nanoparticles or rapidly cleared lipid nanoparticles before encapsulation in fibrin gels, fibrin hydrogels, or fibrin glue. In yet other embodiments, the polynucleotides may be formulated as lipoplexes before encapsulation in fibrin gels, hydrogels, or fibrin glue. Fibrin gels, hydrogels, and glues contain two components: a fibrinogen solution and a calcium-rich thrombin solution (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148: 49-55; Kidd et al. Journal of Controlled Release 2012. 157: 80-85). Varying the concentrations of the components of the fibrin gel, hydrogel, and / or glue can alter the properties, network mesh size, and / or degradation characteristics, including but not limited to, the release characteristics, of the gel, hydrogel, and / or glue (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010, 148: 49-55; Kidd et al., Journal of Controlled Release 2012, 157: 80-85; Catelas et al., Tissue Engineering 2008, 14: 119-128). This characteristic can be advantageous when used to deliver the polynucleotides disclosed herein (see, e.g., Kidd et al., Journal of Controlled Release 2012, 157: 80-85; Catelas et al., Tissue Engineering 2008, 14: 119-128).

[0274] In some embodiments, the polynucleotides disclosed herein may comprise a cation or anion. In one embodiment, the formulation may comprise an anion, such as, but not limited to, Zn 2+ , Ca 2+ , Cu 2+ , Mg 2+ and combinations thereof. As a non-limiting example, the formulation may include a polymer and a polynucleotide complexed with a metal cation (see, e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525).

[0275] In some embodiments, polynucleotides may be formulated into nanoparticles and / or microparticles. These nanoparticles and / or microparticles may be formed to any size, shape, and chemical nature. As an example, nanoparticles and / or microparticles can be made using PRINT® by LIQUIDA TECHNOLOGIES (Morrisville, NC) (see, e.g., WO 2007 / 024323).

[0276] In some embodiments, the polynucleotides disclosed herein may be formulated in NanoJackets and NanoLiposomes manufactured by Keystone Nano (State College, Pennsylvania). NanoJackets are made from compounds naturally occurring in the body, including calcium and phosphate, and may also contain small amounts of silicate. Nanojackets can range in size from 5 to 50 nm and can be used for the delivery of hydrophilic and hydrophobic compounds, including, but not limited to, multiple polynucleotides, primary constructs, and / or single polynucleotides. NanoLiposomes are made from lipids, including, but not limited to, lipids naturally occurring in the body. NanoLiposomes can range in size from 60 to 80 nm and can be used for the delivery of hydrophilic and hydrophobic compounds, including, but not limited to, multiple polynucleotides, primary constructs, and / or single polynucleotides. In one aspect, the polynucleotides disclosed herein are formulated in NanoLiposomes, including, but not limited to, Ceramide NanoLiposomes.

[0277] Gene editing system In one aspect, the disclosed system or composition can introduce a gene editing system (e.g., a CRISPR / Cas system) into a target cell (e.g., a B cell or HSC). The system includes, as one component, a gene editing molecule or a polynucleotide molecule comprising a sequence encoding the gene editing molecule.

[0278] In some embodiments, at least one component of the systems described herein may further comprise a guide RNA (gRNA) molecule or a sequence encoding said gRNA molecule.

[0279] In one embodiment, a system or composition of the present disclosure includes a recombinant viral particle comprising a gene-editing molecule and a second recombinant viral particle comprising a guide RNA (gRNA) and a sequence encoding an antibody or a fragment thereof. In certain embodiments, the gene-editing molecule is a functional fragment or derivative thereof.

[0280] A "gene-editing molecule" is a molecule (e.g., a protein or a polynucleotide molecule (e.g., mRNA) encoding such a protein) used to modify a genomic locus of interest (i.e., a target) in a cell (e.g., a eukaryotic cell, a mammalian cell, a human cell, or a non-human cell). Such modifications include, but are not limited to, disrupting, deleting, repairing, mutating, adding, altering, or correcting a gene sequence at a target locus within a gene. Examples of gene-editing molecules include, but are not limited to, endonucleases. Endonucleases are enzymes that cleave phosphodiester bonds within a polynucleotide chain, but only cleave internal phosphodiester bonds. Examples of gene editing endonucleases that can be used in the compositions and methods of the present disclosure include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, restriction endonucleases, recombinases, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) proteins.

[0281] A.Cas fusion molecule The methods and compositions disclosed herein can utilize a clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system, or components of such a system, to modify the genome in a cell. A CRISPR / Cas system includes transcripts and other elements involved in the expression of or directing the activity of Cas genes. A CRISPR / Cas system can be, for example, a Type I, Type II, or Type III system. Alternatively, the CRISPR / Cas system can be a Type V system (e.g., subtype VA or subtype VB). The methods and compositions disclosed herein can employ a CRISPR / Cas system by utilizing a CRISPR complex (including a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of target genes.

[0282] The CRISPR / Cas systems used in the methods and compositions disclosed herein may not be naturally occurring. A "non-naturally occurring" system includes any that exhibit human intervention, such as one in which one or more components of the system are altered or mutated from their naturally occurring state, or are at least substantially absent from at least one other component with which they are naturally associated in nature, or are associated with at least one other component with which they are not naturally associated. For example, some CRISPR / Cas systems use non-naturally occurring CRISPR complexes that include gRNAs and Cas proteins that do not occur together in nature, use non-naturally occurring Cas proteins, or use non-naturally occurring gRNAs.

[0283] (i)Cas molecule A "Cas molecule," "Cas protein," or "Cas nuclease" that can be used in the compositions and methods of the present invention generally comprises at least one RNA recognition or binding domain capable of interacting with a guide RNA (gRNA, described in more detail below). Cas proteins can also comprise a nuclease domain (e.g., a DNase or RNase domain), a DNA-binding domain, a helicase domain, a protein-protein interaction domain, a dimerization domain, and other domains. The nuclease domain possesses catalytic activity for nucleic acid cleavage, including cleavage of covalent bonds in nucleic acid molecules. Cleavage can generate blunt or staggered ends, which can be single-stranded or double-stranded. For example, wild-type Cas9 proteins will typically generate blunt cleavage products. Alternatively, a wild-type Cpf1 protein (e.g., FnCpf1) can result in a cleavage product with a 5-nucleotide 5' overhang, with cleavage occurring after the 18th base pair of the PAM sequence on the non-target strand and after the 23rd base pair on the target strand. The Cas protein can have full cleavage activity, creating a double-stranded break (e.g., a blunt-ended double-stranded break) at the target genomic locus, or it can be a nickase, creating a single-stranded break at the target genomic locus.

[0284] Examples of Cas proteins that can be used in the compositions and methods of the invention include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Casl0d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Css1 (CasA), Css2 (CasB), Css1 (CasB), Css2 (CasC), Css1 (CasC), Css1 (CasC), Css1 (CasD ... These include se3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs and modified versions thereof.

[0285] An exemplary Cas protein is the Cas9 protein from a type II CRISPR / Cas system, or a protein derived from Cas9. Cas9 proteins are from type II CRISPR / Cas systems and typically share four major motifs with a conserved architecture. Motifs 1, 2, and 4 are RuvC-like motifs, and motif 3 is an HNH motif. Exemplary Cas9 proteins are found in Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp.), Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldas caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp.), Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp.Cas9 proteins are derived from Bacillus subtilis, Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Neisseria meningitidis, or Campylobacter jejuni. Further examples of Cas9 family members are described in WO 2014 / 131833, which is incorporated herein by reference in its entirety for all purposes. Cas9 from Streptococcus pyogenes (SpCas9) (assigned SwissProt accession number: Q99ZW2) is an exemplary Cas9 protein. Cas9 from Staphylococcus aureus (SaCas9) (assigned UniProt accession number: J7RUA5) is another exemplary Cas9 protein. Cas9 from Campylobacter jejuni (CjCas9) (assigned UniProt accession number: Q0P897) is another exemplary Cas9 protein. See, e.g., Kim et al. (2017) Nat. Comm. 8:14500 (incorporated by reference in its entirety for all purposes). SaCas9 is smaller than SpCas9, and CjCas9 is smaller than both SaCas9 and SpCas9.

[0286] Another example of a Cas protein is the Cpf1 (CRISPR from Prevotella and Francisella 1) protein. Cpf1 is a large protein (approximately 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain in Cas9 and a counterpart of Cas9's characteristic arginine-rich cluster. However, Cpf1 lacks the HNH nuclease domain present in the Cas9 protein, and the RuvC-like domain is contiguous with the Cpf1 sequence, in contrast to Cas9, which contains a long insert containing the HNH domain. See, e.g., Zetsche et al. (2015) Cell 163(3):759-771, incorporated herein by reference in its entirety for all purposes.Exemplary Cpf1 proteins are found in Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, and the like. GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3), Prevotella disiens, and Porphyromonas macacae. Cpf1 from Francisella novicida U112 (FnCpf1; assigned UniProt accession number: A0Q7Q2) is an exemplary Cpf1 protein.

[0287] The Cas protein can be a wild-type protein (i.e., naturally occurring), a modified Cas protein (i.e., a variant of a Cas protein), or a fragment of a wild-type or Cas protein. The Cas protein can also be a variant or fragment that is active with respect to the catalytic activity of a wild-type or modified Cas protein. A catalytically active variant or fragment can have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even higher sequence identity to a wild-type or modified Cas protein or portion thereof, and the active variant retains the ability to cleave at the desired cleavage site and thus retains nick-inducing or double-strand break-inducing activity. Assays for nick-inducing or double-strand break-inducing activity are known and generally measure the overall activity and specificity of a Cas protein on a DNA substrate containing the cleavage site.

[0288] One example of a modified Cas protein is the modified SpCas9-HF1 protein, which is a high-fidelity variant of Streptococcus pyogenes Cas9 with modifications (N497A / R661A / Q695A / Q926A) designed to reduce nonspecific DNA contacts. See, e.g., Kleinstiver et al. (2016) Nature 529(7587):490-495 (incorporated herein by reference in its entirety for all purposes). Another example of a modified Cas protein is the modified eSpCas9 variant (K848A / K1003A / R1060A) designed to reduce off-target effects. See, e.g., Slaymaker et al. (2016) Science 351(6268):84-88 (incorporated herein by reference in its entirety for all purposes). Other SpCas9 variants include K855A and K810A / K1003A / R1060A.

[0289] Cas proteins can be modified to increase or decrease one or more of nucleic acid binding affinity, nucleic acid binding specificity, and enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of a Cas protein can be modified, deleted, or inactivated, or the Cas protein can be truncated to remove domains that are not essential for protein function or to optimize (e.g., enhance or reduce) the activity of the Cas protein.

[0290] Cas proteins can include at least one nuclease domain, such as a DNase domain. For example, wild-type Cpfl proteins generally include a RuvC-like domain, which may be in a dimeric configuration and cleaves both strands of target DNA. Cas proteins can also include at least two nuclease domains, such as a DNase domain. For example, wild-type Cas9 proteins generally include a RuvC-like nuclease domain and an HNH-like nuclease domain. The RuvC domain and the HNH domain can each cleave different strands of double-stranded DNA, resulting in a double-stranded break in DNA. See, e.g., Jinek et al. (2012) Science 337:816-821 (incorporated herein by reference in its entirety for all purposes).

[0291] Deleting or mutating one or more of the nuclease domains can eliminate function or reduce nuclease activity. For example, if one or more of the nuclease domains in a Cas9 protein are deleted or mutated, the resulting Cas9 protein can be called a nickase and can generate single-stranded breaks at the guide RNA recognition sequence in double-stranded DNA but cannot generate double-stranded breaks (i.e., it can cleave either the complementary strand or the non-complementary strand, but not both). If both nuclease domains are deleted or mutated, the resulting Cas protein (e.g., Cas9) will have a reduced ability to cleave both strands of double-stranded DNA (e.g., a nuclease-null or nuclease-inactive Cas protein, or a catalytically inactive Cas protein (dCas)). One example of a mutation that converts Cas9 into a nickase is the D10A (aspartic acid at position 10 of Cas9 becomes alanine) mutation in the RuvC domain of Cas9 from Streptococcus pyogenes. Similarly, Cas9 from Streptococcus pyogenes can be converted into a nickase by H939A (histidine at amino acid position 839 is replaced by alanine), H840A (histidine at amino acid position 840 is replaced by alanine), or N863A (asparagine at amino acid position N863 is replaced by alanine) in the HNH domain. Other examples of mutations that convert Cas9 into a nickase include the corresponding mutations in Cas9 from Streptococcus thermophilus. See, e.g., Sapranauskas et al. (2011) Nucleic Acids Research 39:9275-9282 and WO 2013 / 141680 (each of which is incorporated by reference in its entirety for all purposes). Such mutations can be generated using methods such as site-directed mutagenesis, PCR-mediated mutagenesis, or total gene synthesis. Other examples of nickase-generating mutations can be found, for example, in WO 2013 / 176772 and WO 2013 / 142578, each of which is incorporated herein by reference in its entirety for all purposes.When all nuclease domains in a Cas protein are deleted or mutated (e.g., both nuclease domains in a Cas9 protein are deleted or mutated), the resulting Cas protein (e.g., Cas9) has a reduced ability to cleave both strands of double-stranded DNA (e.g., a nuclease-null or nuclease-inactive Cas protein). One specific example is the D10A / H840A S. pyogenes Cas9 double mutant, or the corresponding double mutant of a Cas9 from another species when optimally aligned with S. pyogenes Cas9. Another specific example is the D10A / N863A S. pyogenes Cas9 double mutant, or the corresponding double mutant of a Cas9 from another species when optimally aligned with S. pyogenes Cas9.

[0292] Examples of inactivating mutations in the catalytic domain of the Staphylococcus aureus Cas9 protein are also known. For example, the Staphylococcus aureus Cas9 enzyme (SaCas9) can contain substitutions at positions N580 (e.g., an N580A substitution) and D10 (e.g., a D10A substitution) to generate a nuclease-inactive Cas protein. See, e.g., WO 2016 / 106236 (incorporated herein by reference in its entirety for all purposes).

[0293] Examples of inactivating mutations in the catalytic domain of Cpf1 proteins are also known. For example, in the Cpf1 proteins from Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), and Moraxella boehmich237 (MbCpf1 Cpf1), such mutations include mutations at positions 908, 993, or 1263 of AsCpf1 or corresponding positions in Cpf1 orthologs, or at positions 832, 925, 947, or 1180 of LbCpf1 or corresponding positions in Cpf1 orthologs. Such mutations can include, for example, one or more of the mutations D908A, E993A, and D1263A in AsCpf1, or corresponding mutations in Cpf1 orthologs, or the mutations D832A, E925A, D947A, and D1180A in LbCpf1, or corresponding mutations in Cpf1 orthologs. See, e.g., U.S. Patent Application Publication No. 2016 / 0208243, which is incorporated herein by reference in its entirety for all purposes.

[0294] The Cas fusion protein can also be tethered to a labeled nucleic acid. Such tethering (i.e., physical linkage) can be achieved by covalent or non-covalent interactions, and can be direct (e.g., by direct fusion or chemical conjugation, which can be achieved by modifying cysteine ​​or lysine residues on the protein or by intein modification) or via one or more intervening linker or adapter molecules, such as streptavidin or aptamers. See, e.g., Pierce et al. (2005) Mini Rev. Med. Chem. 5(1):41-55; Duckworth et al. (2007) Angew. Chem. Int. Ed. Engl. 46(46):8819-8822; Schaeffer and Dixon (2009) Australian J. Chem. 62(10):1328-1332; Goodman et al. (2009) Chembiochem. 10(9):1551-1557; and Khatwani et al. (2012) Bioorg. Med. Chem. 20(14):4532-4539, each of which is incorporated by reference in its entirety for all purposes. Non-covalent strategies for the synthesis of protein-nucleic acid conjugates include the biotin-streptavidin method and the nickel-histidine method. Covalent protein-nucleic acid conjugates can be synthesized by connecting appropriately functionalized nucleic acids and proteins using a wide variety of chemical reactions. Some of these chemical reactions involve the direct attachment of oligonucleotides to amino acid residues on the protein surface (e.g., lysine amines or cysteine ​​thiols), while other, more complex schemes require post-translational modifications of the protein or the involvement of catalytic or reactive protein domains. Methods for covalently attaching proteins to nucleic acids include, for example, chemical cross-linking of oligonucleotides to lysine or cysteine ​​residues of the protein, ligation of expressed proteins, chemoenzymatic methods, and the use of photoaptamers. Labeled nucleic acids can be tethered to the C-terminus, N-terminus, or internal regions of the Cas protein.Preferably, the labeled nucleic acid is tethered to the C-terminus or N-terminus of the Cas protein. Similarly, the Cas protein can be tethered to the 5'-terminus, 3'-terminus, or an internal region of the labeled nucleic acid. That is, the labeled nucleic acid can be tethered in any orientation and polarity. Preferably, the Cas protein is tethered to the 5'-terminus or 3'-terminus of the labeled nucleic acid.

[0295] In some embodiments, nucleic acids encoding the Cas proteins of the invention, or functional fragments or derivatives thereof, can be codon-optimized for efficient translation into proteins in a particular cell or organism. For example, nucleic acids encoding the Cas proteins, or functional fragments or derivatives thereof, can be modified to replace codons that are frequently used in bacterial, yeast, human, non-human, mammalian, rodent, mouse, rat, or any other host (e.g., packaging) and / or target cell of interest. Introducing a fusion RNA encoding a Cas protein, or functional fragment or derivative thereof, into a cell allows for transient or conditional expression of the Cas protein, or functional fragment or derivative thereof, in the cell.

[0296] In certain embodiments, the Cas molecule is a Cas9 molecule, or a functional fragment or derivative thereof. In certain embodiments, the Cas9 can be wild-type Cas9, Cas9 nickase, inactive Cas9 (dCas9), split-Cas9, and Cas9 fusion proteins. In certain embodiments, the Cas9 is Streptococcus pyogenes or Staphylococcus aureus Cas9. In certain embodiments, the sequence of the Cas9 mRNA is codon-optimized for expression in eukaryotic cells.

[0297] Optionally, the Cas mRNA can be codon-optimized for efficient translation into a Cas protein, or a functional fragment or derivative thereof, within a particular cell or organism. For example, the nucleic acid sequence encoding the Cas protein, or a functional fragment or derivative thereof, can be modified to replace codons that are frequently used in bacterial, yeast, human, non-human, mammalian, rodent, mouse, rat, hamster, or any other host (e.g., packaging) and / or target cell of interest.

[0298] In certain embodiments, the Cas protein is Cas9, or a functional fragment or derivative thereof. In certain embodiments, the Cas9 is selected from the group consisting of wild-type Cas9, Cas9 nickase, inactive Cas9 (dCas9), split-Cas9, inducible Cas9, and Cas9 fusion proteins. In certain embodiments, the Cas9 is Streptococcus pyogenes or Staphylococcus aureus Cas9. In certain embodiments, the sequence of the Cas9 mRNA is codon-optimized for expression in eukaryotic cells.

[0299] The Cas protein, or a functional fragment or derivative thereof, can also be operably linked to another heterologous polypeptide as a fusion protein. For example, the Cas protein, or a functional fragment or derivative thereof, can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. See WO 2014 / 089290 (incorporated herein by reference in its entirety for all purposes). Examples of transcriptional activation domains include the herpes simplex virus VP16 activation domain, VP64 (a tetrameric derivative of VP16), the NFκB p65 activation domain, p53 activation domains 1 and 2, the CREB (cAMP response element binding protein) activation domain, the E2A activation domain, and the NFAT (nuclear factor of activated T-cells) activation domain. Other examples include activation domains from Oct1, Oct-2A, SP1, AP-2, CTF1, P300, CBP, PCAF, SRC1, PvALF, ERF-2, OsGAI, HALF-1, C1, AP1, ARF-5, ARF-6, ARF-7, ARF-8, CPRF1, CPRF4, MYC-RP / GP, TRAB1PC4, and HSF1. See, e.g., U.S. Patent Application Publication No. 2016 / 0237456, European Patent Application No. 3045537, and International Publication No. WO 2011 / 145121 (each of which is incorporated by reference in its entirety for all purposes). In some cases, a transcription activation system comprising a dCas9-VP64 fusion protein paired with MS2-p65-HSF1 can be used. Guide RNAs for such systems can be designed by appending aptamer sequences to the sgRNA tetraloop and stem-loop 2, which are designed to bind to dimerized MS2 bacteriophage coat protein. See, e.g., Konermann et al. (2015) Nature 517(7536):583-588, which is incorporated herein by reference in its entirety for all purposes.Examples of transcriptional repressor domains include the inducible cAMP early repressor (ICER) domain, the Kruppel-associated box A (KRAB-A) repressor domain, the YY1 glycine-rich repressor domain, Sp1-like repressor, the E(spl) repressor, the IκB repressor, and MeCP2. Other examples include transcriptional repressor domains from A / B, KOX, TGF-β-inducible early genes (TIEG), v-erbA, SID, SID4X, MBD2, MBD3, DNMT1, DNMG3A, DNMT3B, Rb, and ROM2. See, e.g., EP 3045537 and WO 2011 / 145121 (each of which is incorporated by reference in its entirety for all purposes). Cas proteins can also be fused to heterologous polypeptides that provide increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or internally of the Cas protein or a functional fragment or derivative thereof.

[0300] For example, a Cas protein or a functional fragment or derivative thereof can be fused to one or more heterologous polypeptides that provide subcellular localization. Such heterologous polypeptides can include one or more nuclear localization signals (NLSs), such as the SV40 NLS and / or the α-importin NLS, for targeting to the nucleus, a mitochondrial localization signal for targeting to mitochondria, an ER retention signal, etc. See, e.g., Lange et al. (2007) J. Biol. Chem. 282:5101-5105, incorporated herein by reference in its entirety for all purposes. Such subcellular localization signals can be located at the N-terminus, C-terminus, or anywhere within the Cas protein or its functional fragment or derivative. The NLS can comprise a stretch of basic amino acids and can be a mono- or bi-karyotic sequence. Optionally, the Cas protein, or functional fragment or derivative thereof, comprises two or more NLSs, including an N-terminal NLS (e.g., an alpha importin NLS) and / or a C-terminal NLS (e.g., an SV40 NLS).

[0301] The Cas protein or a functional fragment or derivative thereof can also be operably linked to a heterologous polypeptide, such as a fluorescent protein, purification tag, or epitope tag, to facilitate tracking or purification. Examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, eGFP, Emerald, Azami Green, monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (mKate, mK), and the like. ate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred), orange fluorescent protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato), and any other suitable fluorescent protein.Examples of tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, hemagglutinin (HA), nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, histidine (His), biotin carboxyl carrier protein (BCCP), and calmodulin.

[0302] Cas fusion proteins can be generated using conventional molecular biology techniques, such as those described above or in He et al., supra. Alternatively, Cas fusion proteins can be prepared in a variety of other ways.

[0303] In certain embodiments, the nucleic acid encoding the Cas fusion protein comprises a regulatory element, such as a promoter, enhancer, or transcriptional repressor binding element. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, (1990) Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (incorporated by reference in its entirety for all purposes).

[0304] B. Transcription Activator-Like Effector Nucleases, Zinc Finger Nucleases, Meganucleases, and Restriction Endonucleases In certain embodiments, the gene editing molecules can be zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and / or restriction endonucleases. Fusion RNA and fusion protein molecules using these gene editing molecules, or functional fragments or derivatives thereof, for use in the compositions and methods of the invention can be made in the same manner and configuration as disclosed above for Cas molecules, or functional fragments or derivatives thereof.

[0305] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave target sequences in DNA. They are created by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease that cleaves DNA strands). TAL effector nucleases are a class of sequence-specific nucleases that can be used to create double-strand breaks at specific target sequences within the genomes of prokaryotes or eukaryotes. TAL effector nucleases are created by fusing a native or engineered transcription activator-like (TAL) effector, or a functional portion thereof, to the catalytic domain of an endonuclease, such as FokI. The unique modularity of the TAL effector DNA-binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA-binding domain of a TAL effector nuclease can be engineered to recognize a specific DNA target site and can be used to create a double-strand break at a desired target sequence. See WO 2010 / 079430; Morbitzer et al. (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. doi: 10.1093 / nar / gkq704; and Miller et al. (2011) Nature Biotechnology 29:143-148, all of which are incorporated by reference in their entirety for all purposes.

[0306] Examples of suitable TAL nucleases and methods for preparing suitable TAL nucleases are disclosed, for example, in U.S. Patent Application Publication No. 2011 / 0239315, U.S. Patent Application Publication No. 2011 / 0269234, U.S. Patent Application Publication No. 2011 / 0145940, U.S. Patent Application Publication No. 2003 / 0232410, U.S. Patent Application Publication No. 2005 / 0208489, U.S. Patent Application Publication No. 2005 / 0026157, U.S. Patent Application Publication No. 2005 / 0064474, U.S. Patent Application Publication No. 2006 / 0188987, and U.S. Patent Application Publication No. 2006 / 0063231, each of which is incorporated by reference in its entirety for all purposes. In various embodiments, the TAL effector nuclease is engineered to cleave at or near a target nucleic acid sequence, e.g., within a genomic locus of interest, where the target nucleic acid sequence is at or near a sequence modified by a targeting vector. TAL nucleases suitable for use with the various methods and compositions provided herein include those specifically designed to bind to or near a sequence modified by a targeting vector.

[0307] In one embodiment, each TALEN monomer contains 12-25 TAL repeats, each binding to a 1-bp subsite. In a specific embodiment, the gene editing molecule is a chimeric protein comprising a TAL repeat-based DNA-binding domain operably linked to an independent nuclease. In a specific embodiment, the independent nuclease is a FokI endonuclease. In one embodiment, the gene editing molecule comprises a first TAL repeat-based DNA-binding domain and a second TAL repeat-based DNA-binding domain, each operably linked to a FokI nuclease, the first and second TAL repeat-based DNA-binding domains recognizing two consecutive target DNA sequences in each strand of the target DNA sequence, separated by a cleavage site of about 6 bp to about 40 bp, and the FokI nuclease dimerizes to make a double-stranded cleavage in the target sequence.

[0308] In certain embodiments, the gene editing molecule comprises a first TAL repeat-based DNA-binding domain and a second TAL repeat-based DNA-binding domain, wherein the first and second TAL repeat-based DNA-binding domains are each operably linked to a FokI nuclease, wherein the first and second TAL repeat-based DNA-binding domains recognize two consecutive target DNA sequences in each strand of the target DNA sequence separated by a 5 bp or 6 bp cut site, and wherein the FokI nuclease dimerizes and makes a double-stranded break.

[0309] The gene editing molecules used in the various methods and compositions disclosed herein can further include zinc finger nucleases (ZFNs). Zinc finger nucleases (ZFNs) are a class of engineered DNA-binding proteins that assist in targeted genome editing by generating double strand breaks (DSBs) in DNA at targeted sites. ZFNs contain two functional domains: i) a DNA-binding domain (multiple two-finger modules are stitched together to form zinc finger proteins, each with a specificity of 24 bp or more), which contains a chain of two-finger modules (each recognizing a unique hexamer (6 bp) sequence in DNA), and ii) a DNA-cleavage domain, which contains a nuclease domain of FokI. When the DNA-binding domain and the DNA-cleavage domain are fused together, a pair of "genomic scissors" with high specificity is generated.

[0310] In certain embodiments, each monomer of the ZFN comprises three or more zinc finger-based DNA-binding domains, each of which binds to a 3-bp subsite. In other embodiments, the ZFN is a chimeric protein comprising a zinc finger-based DNA-binding domain operably linked to an independent nuclease. In certain embodiments, the independent endonuclease is a FokI endonuclease. In certain embodiments, the gene-editing molecule comprises a first ZFN and a second ZFN, each of which is operably linked to a FokI nuclease, wherein the first and second ZFNs recognize two consecutive target DNA sequences within each strand of the target DNA sequence, separated by a cleavage site of about 6 bp to about 40 bp, or a cleavage site of about 5 bp to about 6 bp, and the FokI nuclease dimerizes to make a double-stranded cleavage. See, e.g., U.S. Patent Application Publication No. 20060246567; U.S. Patent Application Publication No. 20080182332; U.S. Patent Application Publication No. 20020081614; U.S. Patent Application Publication No. 20030021776; WO 2002 / 057308; U.S. Patent Application Publication No. 20130123484; U.S. Patent Application Publication No. 20100291048; and WO 2011 / 017293 (each of which is incorporated by reference in its entirety into this application for all purposes).

[0311] In certain embodiments of the compositions and methods provided herein, the gene editing molecule comprises: (a) a chimeric protein comprising a zinc finger-based DNA binding domain fused to a FokI endonuclease; or (b) a chimeric protein comprising a transcription activator-like effector nuclease (TALEN) fused to a FokI endonuclease.

[0312] In yet another embodiment, the gene editing molecule is a meganuclease. Meganucleases are classified into four families based on conserved sequence motifs: LAGLIDADG family, GIY-YIG family, HNH family, and His-Cys box family. These motifs are involved in metal ion coordination and phosphodiester bond hydrolysis. The notable features of HEases are their long recognition sites and their tolerance of some sequence polymorphism of DNA substrates. Meganuclease domains, structures, and functions are known, see, e.g., Guhan and Muniyappa (2003) Crit Rev Biochem Mol Biol 38:199-248; Lucas et al., (2001) Nucleic Acids Res 29:960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55:1304-26; Stoddard, (2006) Q Rev Biophys 38:49-95; and Moure et al., (2002) Nat Struct Biol 9:764. In some instances, naturally occurring variant and / or engineered derivative meganucleases are used.Methods for modifying kinetics, cofactor interactions, expression, optimal conditions, and / or recognition site specificity, as well as screening for activity, are known, see, for example, Epinat et al., (2003) Nucleic Acids Res 31:2952-62; Chevalier et al., (2002) Mol Cell 10:895-905; Gimble et al., (2003) Mol Biol 334:993-1008; Seligman et al., (2002) Nucleic Acids Res 30:3870-9; Sussman et al., (2004) J Mol Biol 342:31-41; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; Chames et al., (2005) Nucleic Acids Res 33:e178; Smith et al., (2006) Nucleic Acids Res 34:e149; Gruen et al., (2002) Nucleic Acids Res 30:e29; Chen and Zhao, (2005) Nucleic Acids Res 33:e154; WO2005105989; WO2003078619; WO2006097854; WO2006097853; WO2006097784; and WO2004031346.

[0313] In this specification, any meganuclease can be used, including but not limited to the following: I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-Aural, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nspx236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpβIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpβIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, PI-TliII, or any active variant or fragment thereof.

[0314] In one embodiment, the meganuclease recognizes a double-stranded DNA sequence of 12 to 40 base pairs. In one embodiment, the meganuclease recognizes a single perfect match target sequence within a genome. In one embodiment, the meganuclease is a homing nuclease. In one embodiment, the homing nuclease is a member of the LAGLIDADG family of homing nucleases. In one embodiment, the LAGLIDADG family of homing nucleases is selected from I-SceI, I-CreI, and I-Dmol.

[0315] The gene editing molecule can further comprise a restriction endonuclease, including type I, type II, type III, and type IV endonucleases. Type I and type III restriction endonucleases recognize specific recognition sites but typically cleave at a variable location from the nuclease binding site, which can be hundreds of base pairs away from the cleavage site (recognition site). In type II systems, restriction activity is independent of methylase activity, and cleavage typically occurs at a specific site within or near the binding site. Most type II enzymes cleave palindromic sequences, while type IIa enzymes recognize non-palindromic recognition sites and cleave outside the recognition site, type IIb enzymes cleave the sequence twice, with these two sites outside the recognition site, and type IIs enzymes recognize asymmetric recognition sites and cleave on one side at a predetermined distance of approximately 1 to 20 nucleotides from the recognition site. Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, for example, in the REBASE database (webpage rebase.neb.com; Roberts et al., (2003) Nucleic Acids Res 31:418-0), Roberts et al., (2003) Nucleic Acids Res 31:1805-12, Belfort et al., (2002) in Mobile DNA II, pp. 761-783, Eds. Craigie et al. (ASM Press, Washington, DC).

[0316] ZFN and TALEN introduce DSB into target genomic sequence, activating non-homologous end-joining (NHEJ)-mediated DNA repair, thereby generating mutant alleles containing insertion or deletion of nucleic acid sequences at the genomic locus of interest, thus disrupting the genomic locus of interest in cells. DSB also stimulates homology-directed repair (HDR) through homologous recombination when a repair template is provided. HDR can result in perfect repair, restoring the original sequence at the broken site, or can be used to induce designed modifications, such as deletion, insertion, or substitution of sequences at the site of double-strand break.

[0317] C. Guide RNA In one aspect, the systems described herein include a guide RNA (gRNA).

[0318] A "guide RNA" or "gRNA" is an RNA molecule that binds to a Cas protein (e.g., a Cas9 protein) or a functional fragment or derivative thereof, targeting the Cas protein to a specific location within a target DNA. A guide RNA can comprise two segments: a "DNA-targeting segment" and a "protein-binding segment." A "segment" includes a section or region of a molecule, such as a series of consecutive nucleotides within an RNA. Some gRNAs, such as the gRNA for Cas9, can comprise two separate RNA molecules: an "activator RNA" (e.g., a tracrRNA) and a "targeter RNA" (e.g., a CRISPR RNA or crRNA). Other gRNAs are single RNA molecules (single RNA polynucleotides), sometimes referred to as "single-molecule gRNAs," "single-guide RNAs," or "sgRNAs." See, e.g., WO 2013 / 176772, WO 2014 / 065596, WO 2014 / 089290, WO 2014 / 093622, WO 2014 / 099750, WO 2013 / 142578, and WO 2014 / 131833 (each of which is incorporated by reference in its entirety for all purposes). For example, with respect to Cas9, a single guide RNA can comprise a crRNA fused to a tracrRNA (e.g., via a linker). For example, with respect to Cpfl, only one crRNA is required to achieve binding to the target sequence. Both the terms "guide RNA" and "gRNA" include both two-molecule (i.e., modular) gRNAs and one-molecule gRNAs.

[0319] An exemplary bimolecular gRNA comprises a crRNA-like ("CRISPR RNA," or "targeter RNA," or "crRNA," or "crRNA repeat") molecule and a corresponding tracrRNA-like ("trans-acting CRISPR RNA," or "activator RNA," or "tracrRNA") molecule. The crRNA comprises both the DNA-targeting segment (single strand) of the gRNA and a series of nucleotides that form one half of the dsRNA duplex of the protein-binding segment of the gRNA.

[0320] The corresponding tracrRNA (activator RNA) contains a sequence of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the gRNA. The sequence of nucleotides in the crRNA is complementary to the sequence of nucleotides in the tracrRNA, which hybridizes to form the dsRNA duplex of the protein-binding segment of the gRNA. Thus, each crRNA can be said to have a corresponding tracrRNA.

[0321] In systems requiring both a crRNA and a tracrRNA, the crRNA and the corresponding tracrRNA hybridize to form a gRNA. In systems requiring only a crRNA, the crRNA can serve as the gRNA. The crRNA also provides a single-stranded DNA targeting segment that hybridizes to the guide RNA recognition sequence. When used for intracellular modification, the exact sequence of a given crRNA or tracrRNA molecule can be designed to be specific for the species in which the RNA molecule is used. See, e.g., Mali et al. (2013) Science 339:823-826; Jinek et al. (2012) Science 337:816-821; Hwang et al. (2013) Nat. Biotechnol. 31:227-229; Jiang et al. (2013) Nat. Biotechnol. 31:233-239; and Cong et al. (2013) Science 339:819-823 (each of which is incorporated by reference in its entirety for all purposes).

[0322] The DNA-targeting segment (crRNA) of a given gRNA contains a nucleotide sequence complementary to a sequence within the target DNA (i.e., the guide RNA recognition sequence). The DNA-targeting segment of a gRNA interacts with the target DNA in a sequence-specific manner through hybridization (i.e., base pairing). Thus, the nucleotide sequence of the DNA-targeting segment can be varied and determines the location within the target DNA where the gRNA and target DNA interact. The DNA-targeting segment of a given gRNA can be engineered to hybridize with any desired sequence within the target DNA. Naturally occurring crRNAs vary depending on the CRISPR / Cas system and organism, but often contain a targeting segment 21 to 72 nucleotides in length flanked by two direct repeats (DRs) 21 to 46 nucleotides in length (see, e.g., WO 2014 / 131833, incorporated herein by reference in its entirety for all purposes). In Streptococcus pyogenes, the DRs are 36 nucleotides in length, and the targeting segment is 30 nucleotides in length. The 3'-located DR is complementary to and hybridizes with the corresponding tracrRNA, which then binds to the Cas protein.

[0323] The length of a DNA-targeting segment can be at least about 12 nucleotides, at least about 15 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, or at least about 40 nucleotides. The length of such a DNA-targeting segment can be about 12 nucleotides to about 100 nucleotides, about 12 nucleotides to about 80 nucleotides, about 12 nucleotides to about 50 nucleotides, about 12 nucleotides to about 40 nucleotides, about 12 nucleotides to about 30 nucleotides, about 12 nucleotides to about 25 nucleotides, or about 12 nucleotides to about 20 nucleotides. For example, a DNA-targeting segment can be about 15 nucleotides to about 25 nucleotides (e.g., about 17 nucleotides to about 20 nucleotides, or about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, or about 20 nucleotides). See, e.g., U.S. Patent Application Publication No. 2016 / 0024523 (incorporated herein by reference in its entirety for all purposes). For Cas9 from Streptococcus pyogenes, the length of the DNA-targeting segment is typically 16-20 nucleotides or 17-20 nucleotides. For Cas9 from Staphylococcus aureus, the length of the DNA-targeting segment is typically 21-23 nucleotides. For Cpf1, the length of the DNA-targeting segment is typically at least 16 nucleotides or at least 18 nucleotides.

[0324] The tracrRNA can be in any form (e.g., full-length or active portion) and of various lengths. These can include primary transcripts or processed forms. For example, the tracrRNA (as part of a single guide RNA or as a separate molecule as part of a bimolecular gRNA) can comprise or consist of all or a portion of the wild-type tracrRNA sequence (e.g., about 20 or more, 26 or more, 32 or more, 45 or more, 48 or more, 54 or more, 63 or more, 67 or more, 85 or more, or even more nucleotides of the wild-type tracrRNA sequence). Examples of wild-type tracrRNA sequences from Streptococcus pyogenes include the 171-nucleotide version, the 89-nucleotide version, the 75-nucleotide version, and the 65-nucleotide version. See, e.g., Deltcheva et al. (2011) Nature 471:602-607; WO 2014 / 093661 (each of which is incorporated herein by reference in its entirety for all purposes). Examples of tracrRNAs within single guide RNAs (sgRNAs) include tracrRNA segments found within +48, ​​+54, +67, and +85 versions of sgRNAs, where "+n" indicates that the sgRNA contains up to +n nucleotides of the wild-type tracrRNA. See U.S. Patent No. 8,697,359 (incorporated herein by reference in its entirety for all purposes).

[0325] The percent complementarity between the DNA-targeting sequence and the guide RNA recognition sequence in the target DNA may be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%). The percent complementarity between the DNA-targeting sequence and the guide RNA recognition sequence in the target DNA may be at least 60% over about 20 contiguous nucleotides. As an example, the percent complementarity between the DNA-targeting sequence and the guide RNA recognition sequence in the target DNA may be 100% over 14 contiguous nucleotides at the 5' end of the guide RNA recognition sequence in the complementary strand of the target DNA, and as low as 0% for the remainder. In such cases, the length of the DNA-targeting sequence may be considered to be 14 nucleotides. As another example, the percent complementarity between a DNA-targeting sequence and a guide RNA recognition sequence in the target DNA may be 100% over the 7 contiguous nucleotides at the 5' end of the guide RNA recognition sequence in the complementary strand of the target DNA, and as low as 0% for the remainder. In such cases, the length of the DNA-targeting sequence may be considered 7 nucleotides. For some guide RNAs, at least 17 nucleotides in the DNA target sequence are complementary to the target DNA. For example, the DNA-targeting sequence may be 20 nucleotides in length and may contain one, two, or three mismatches with the target DNA (guide RNA recognition sequence). Preferably, these mismatches are not adjacent to a protospacer adjacent motif (PAM) sequence (e.g., the mismatch is at the 5' end of the DNA-targeting sequence, or the mismatch is separated from the PAM sequence by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 base pairs).

[0326] The protein-binding segment of a gRNA can comprise two sequences of nucleotides that are complementary to each other. These complementary nucleotides of the protein-binding segment hybridize to form a double-stranded RNA duplex (dsRNA). The protein-binding segment of a subject gRNA interacts with a Cas protein or a functional fragment or derivative thereof, and the gRNA guides the bound Cas protein or a functional fragment or derivative thereof to a specific nucleic acid sequence within the target DNA via the DNA-targeting segment.

[0327] Guide RNAs can include modifications or sequences that provide additional desirable characteristics (e.g., modified or regulated stability; intracellular targeting; tracking using fluorescent labels; binding sites for proteins or protein complexes, etc.). Examples of such modifications include, for example, a 5' cap (e.g., a 7-methylguanylate cap (m7G)); a 3' polyadenylation tail (i.e., a 3' poly(A) tail); a riboswitch sequence (e.g., to allow for regulated stability and / or regulated accessibility by proteins and / or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (i.e., a hairpin); a modification or sequence that targets the RNA to a certain intracellular location (e.g., the nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides tracking (e.g., direct conjugation with a fluorescent molecule, conjugation with a moiety that facilitates fluorescent detection, a sequence that allows fluorescent detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including a transcriptional activator, a transcriptional repressor, a DNA methyltransferase, a DNA demethylase, a histone acetyltransferase, a histone demethylase, etc.); and combinations thereof. Other examples of modifications include an engineered stem-loop duplex structure, an engineered bulge region, an engineered hairpin 3' of a stem-loop duplex structure, or any combination thereof. See, e.g., U.S. Patent Application Publication No. 2015 / 0376586, incorporated herein by reference in its entirety for all purposes. A bulge can be an unpaired region of nucleic acid within a duplex composed of a crRNA-like region and a minimal tracrRNA-like region. A bulge can include an unpaired 5'-XXXY-3' (where X is any purine and Y can be a nucleotide that can form a wobble pair with a nucleotide on the opposite strand) on one side of the duplex and an unpaired nucleotide region on the other side of the duplex.

[0328] In some cases, a transcription activation system comprising a dCas9-VP64 fusion protein paired with MS2-p65-HSF1 can be used. The guide RNA for such a system can be designed by appending an aptamer sequence to the sgRNA tetraloop and stem-loop 2, which are designed to bind to the dimerized MS2 bacteriophage coat protein. See, e.g., Konermann et al. (2015) Nature 517(7536):583-588 (incorporated herein by reference in its entirety for all purposes).

[0329] Guide RNAs can be provided in any form. For example, gRNAs can be provided in the form of RNA, either as two molecules (separate crRNA and tracrRNA) or as a single molecule (sgRNA). gRNAs can also be provided in the form of DNA encoding the gRNA. The DNA encoding the gRNA can encode a single RNA molecule (sgRNA) or multiple separate RNA molecules (e.g., separate crRNA and tracrRNA). In the latter case, the DNA encoding the gRNA can be provided as a single DNA molecule or as separate DNA molecules encoding the crRNA and tracrRNA, respectively.

[0330] When the gRNA is provided in the form of DNA, the gRNA can be expressed transiently, conditionally, or constitutively in the cell. The DNA encoding the gRNA can be stably integrated into the genome of the cell and operably linked to a promoter active in the cell. Alternatively, the DNA encoding the gRNA can be operably linked to a promoter in an expression construct. For example, the DNA encoding the gRNA can be in a vector containing a heterologous nucleic acid. Promoters that can be used in such expression constructs include promoters active in one or more of eukaryotic cells, human cells, non-human cells, mammalian cells, non-human mammalian cells, rodent cells, mouse cells, rat cells, hamster cells, rabbit cells, pluripotent cells, embryonic stem (ES) cells, adult stem cells, developmentally restricted progenitor cells, induced pluripotent stem (iPS) cells, or one-cell embryos. Such promoters can be, for example, conditional, inducible, constitutive, or tissue-specific promoters. Such promoters can also be, for example, bidirectional promoters. In certain embodiments, an RNA Pol III promoter can be operably linked to a gRNA sequence (if included in a lentiviral vector) to control the expression of such a sequence. RNA Pol III promoters are frequently used to express small interfering RNA (siRNA) / short hairpin RNA (shRNA) and guide RNA sequences used in the CRISPR-Cas9 system. Examples of RNA Pol III promoters that can be used in the present invention include, but are not limited to, human U6 promoter, rat U6 polymerase III promoter, or mouse U6 polymerase III promoter, and H1 promoter, as described, for example, in Goomer and Kunkel, Nucl. Acids Res., 20 (18): 4903-4912 (1992), and Myslinski et al., Nucleic Acids Res., 29 (12): 2502-9 (2001).

[0331] D. Guide RNA Recognition Sequence The term "guide RNA recognition sequence" includes a nucleic acid sequence present in target DNA to which the DNA-targeting segment of a gRNA binds when sufficient conditions for binding exist. For example, the gRNA recognition sequence includes a sequence to which the gRNA is designed to have complementarity, and hybridization between the guide RNA recognition sequence and the DNA-targeting sequence promotes the formation of a CRISPR complex. Perfect complementarity is not necessary, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. The guide RNA recognition sequence also includes a cleavage site for a Cas protein, which is described in more detail below. The gRNA recognition sequence can include any polynucleotide that can be located, for example, in the nucleus or cytoplasm of a cell, or within a cellular organelle, such as a mitochondria or chloroplast.

[0332] The gRNA recognition sequence in the target DNA can be targeted by (i.e., can bind to, hybridize with, or be complementary to) a Cas protein or gRNA. Suitable DNA / RNA binding conditions include physiological conditions normally present in a cell. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001), incorporated herein by reference in its entirety for all purposes). The strand of the target DNA that is complementary to or hybridized with a Cas protein or gRNA is referred to as the "complementary strand," and the strand of the target DNA that is complementary to (and therefore not complementary to) a "complementary strand" is referred to as the "noncomplementary strand" or "template strand."

[0333] Cas proteins can cleave nucleic acids at sites within or outside of the nucleic acid sequence present in the target DNA to which the DNA-targeting segment of the gRNA binds. A "cleavage site" includes the location in a nucleic acid at which a Cas protein generates a single-stranded or double-stranded break. For example, formation of a CRISPR complex (including a gRNA hybridized to a guide RNA recognition sequence and complexed with a Cas protein) can cleave one or both strands within or near (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the target DNA to which the DNA-targeting segment of the gRNA binds. If the cleavage site is outside of the nucleic acid sequence to which the DNA-targeting segment of the gRNA binds, the cleavage site is still considered to be within the "guide RNA recognition sequence." The cleavage site may be present in only one strand of the nucleic acid, or in both strands. The cleavage sites can be located at the same position on both strands of the nucleic acid (generating a blunt end) or at different sites on the strands (generating a sticky end (i.e., an overhang)). Sticky ends can be generated, for example, by generating double-stranded breaks using two Cas proteins, each generating a single-stranded break at a different cleavage site on a different strand. For example, a first nickase can generate a single-stranded break in a first strand of double-stranded DNA (dsDNA), and a second nickase can generate a single-stranded break in a second strand of dsDNA, thereby generating an overhanging sequence. In some cases, the guide RNA recognition sequence of the nickase on the first strand is separated from the guide RNA recognition sequence of the nickase on the second strand by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, or 1,000 base pairs.

[0334] Site-specific binding and cleavage of target DNA by a Cas protein can occur at a location determined by both (i) base-pairing complementarity between the gRNA and the target DNA and (ii) a short motif within the target DNA, called a protospacer adjacent motif (PAM). The PAMs can flank the guide RNA recognition sequence. Optionally, the guide RNA recognition sequence can be flanked by PAMs at its 3' end. Alternatively, the guide RNA recognition sequence can be flanked by PAMs at its 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10 or about 2 to about 5 base pairs (e.g., 3 base pairs) upstream or downstream of the PAM sequence. In some cases (e.g., when using Cas9 from Streptococcus pyogenes or a closely related Cas9), the PAM sequence on the non-complementary strand can be 5'-N1GG-3', where N1 is any DNA nucleotide, and is adjacent to the 3' end of the guide RNA recognition sequence on the non-complementary strand of the target DNA. Thus, the PAM sequence on the complementary strand is 5'-CCN2-3', where N2 is any DNA nucleotide adjacent to the 5' end of the guide RNA recognition sequence on the complementary strand of the target DNA. In some such cases, N1 and N2 can be complementary, and the N1-N2 base pair can be any base pair (e.g., N1 = C and N2 = G; N1 = G and N2 = C; N1 = A and N2 = T; or N1 = T and N2 = A). In the case of Cas9 derived from Staphylococcus aureus, the PAM can be NNGRRT or NNGRR, where N can be A, G, C, or T, and R can be G or A. In the case of Cas9 derived from Campylobacter jejuni, the PAM can be, for example, NNNNACAC or NNNNRYAC, where N can be A, G, C, or T, and R can be G or A. In some cases (eg, in the case of FnCpf1), the PAM sequence can be upstream of the 5' end and can have the sequence 5'-TTN-3'.

[0335] Examples of gRNA recognition sequences include DNA sequences complementary to the DNA targeting segment of the gRNA, or PAM sequences plus such DNA sequences. For example, the target motif is GN 19 NGG or N 20The guide RNA recognition sequence may be a 20-nucleotide DNA sequence immediately preceding the NGG motif recognized by the Cas9 protein, such as NGG. See, for example, International Publication No. WO 2014 / 165825 (incorporated herein by reference in its entirety for all purposes). A guanine at the 5' end can promote transcription by RNA polymerase in cells. Another example of a guide RNA recognition sequence is a 5' end with two guanine nucleotides (e.g., GGN ) at the 5' end to promote efficient in vitro transcription by T7 polymerase. 20 Other guide RNA recognition sequences can be 4-22 nucleotides in length, including a 5' G or GG and a 3' GG or NGG. Still other guide RNA recognition sequences can be 14-20 nucleotides in length.

[0336] In various embodiments, the gRNA is complementary to a sequence at the IgH locus, J chain locus, or Igκ locus in a target cell (e.g., a B cell or HSC). In some embodiments, the gRNA is complementary to a sequence at the J chain locus. In one embodiment, the gRNA is complementary to a sequence within the fourth exon of the J chain locus. In one embodiment, the gRNA is complement...

Claims

1. A system for producing antibodies or antigen-binding fragments thereof within the body of a target, a) A first component comprising a polynucleotide molecule containing a sequence encoding the antibody or its antigen-binding fragment; and b) A second component comprising a gene editing molecule, or a polynucleotide molecule containing a sequence encoding the gene editing molecule. A system that includes this.

2. The system according to claim 1, wherein the antibody or its antigen-binding fragment binds to an antigen associated with a disease or disorder.

3. The system according to claim 1, wherein the gene editing molecule is a Cas nuclease.

4. The system according to claim 3, wherein the Cas nuclease is Cas9 nuclease.

5. The system according to claim 1, wherein the first component or the second component further comprises a guide RNA (gRNA) molecule or a sequence encoding a gRNA molecule.

6. a) The first component comprises a sequence encoding the antibody or its antigen-binding fragment and a sequence encoding a gRNA molecule, or b) The first component comprises (i) a first polynucleotide molecule containing a sequence encoding the antibody or its antigen-binding fragment, and (ii) a second polynucleotide molecule containing a sequence encoding a gRNA molecule, or c) The first component comprises (i) a first polynucleotide molecule containing a sequence encoding the antibody or its antigen-binding fragment, and (ii) a gRNA molecule, or d) The system according to claim 5, wherein the second component comprises a gRNA molecule or a sequence encoding a gRNA molecule.

7. The system according to claim 5, wherein the gRNA molecule is complementary to the sequence at the IgH locus, the J chain locus, or the Igκ locus.

8. The system according to claim 7, wherein the gRNA molecule is complementary to the sequence at the J-chain locus.

9. a) The gRNA molecule is complementary to the sequence in the fourth exon of the J-chain locus, or b) The system according to claim 8, wherein the gRNA molecule is complementary to the sequence in the first intron of the J-chain locus.

10. a) The sequence encoding the antibody or its antigen-binding fragment is incorporated into the IgH locus in the genomic region downstream of the last J gene and upstream of the Eμ enhancer, or b) The system according to claim 1, wherein when the sequence encoding the antibody or its antigen-binding fragment is incorporated into the DNA of a B cell or hematopoietic stem cell (HSC), an interruption of the κ light chain constant region is caused.

11. a) A polynucleotide molecule comprising a sequence encoding the antibody or its antigen-binding fragment comprises, from 5' to 3', a 5'IgH homology region, a splice acceptor, a 2A sequence having a 5'Furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence having a 5'Furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a splice donor sequence, and a 3'IgH homology region, wherein the heavy chain sequence and the light chain sequence may be arranged in any order, or b) The system according to claim 1, wherein the polynucleotide molecule comprising a sequence encoding the antibody or its antigen-binding fragment comprises, from 5' to 3', a 5' J chain exon 4 homology region, a 2A sequence having a 5' Furin cleavage sequence, a sequence encoding the light chain variable region of the antibody, a sequence encoding the light chain constant region of the antibody, a 2A sequence having a 5' Furin cleavage sequence, a sequence encoding the heavy chain variable region of the antibody, a sequence encoding the heavy chain constant region of the antibody, and a 3' J chain exon 4 homology region, wherein the heavy chain sequence and the light chain sequence may be arranged in any order.

12. The system according to claim 1, wherein the sequence encoding the antibody or its antigen-binding fragment does not include a promoter sequence.

13. a) When the sequence encoding the antibody or its antigen-binding fragment is incorporated into the DNA of a B cell or hematopoietic stem cell (HSC), the sequence is subject to transcriptional regulation by an endogenous heavy chain promoter in the B cell or HSC, or b) The system according to claim 12, wherein when the sequence encoding the antibody or its antigen-binding fragment is incorporated into the DNA of a B cell or HSC, the sequence is subject to transcriptional control by an endogenous J chain promoter in the B cell or HSC.

14. The system according to claim 1, wherein the polynucleotide molecule containing a sequence encoding the antibody or its antigen-binding fragment includes a promoter sequence, and the sequence encoding the antibody or its antigen-binding fragment is under the transcriptional control of the promoter.

15. a) The promoter is a B cell-specific promoter or a hematopoietic stem cell (HSC)-specific promoter, b) The promoter is an Hg38-mCP promoter, or c) The system according to claim 14, wherein the promoter is a splenic focal virus (SFFV) promoter or a fragment thereof.

16. (i) Both the first component and the second component are viral vectors, or (ii) The system according to claim 1, wherein the first component and / or the second component is a viral vector further comprising a targeting portion.

17. (i) The first component and the second component are viral vectors derived from the same virus species, or (ii) The system according to claim 16, wherein the first component and the second component are viral vectors derived from different virus species.

18. The system according to claim 16, wherein the viral vector is an adeno-associated virus (AAV) vector.

19. The system according to claim 18, wherein the AAV vector is derived from AAV1, AAV2, AAV6, AAV9, or AAV9.PHP.

20. The system according to claim 18, wherein the capsid of the AAV vector contains one or more mutations, the one or more mutations cause the innate directivity of the AAV vector to disappear.

21. The system according to claim 18, wherein the AAV vector further comprises a targeting portion, the targeting portion being inserted into a protein forming a viral capsid, or being covalently or noncovalently attached to a protein forming a viral capsid.

22. The system according to claim 21, wherein the targeted portion attaches to the viral capsid via a first member and a second member of a binding pair, and the first member and the second member form an isopeptide bond.

23. The system according to claim 16, wherein the viral vector is a retroviral vector.

24. The system according to claim 23, wherein the retroviral vector is a lentiviral vector.

25. The system according to claim 24, wherein the lentiviral vector further comprises a targeting portion, the targeting portion being covalently or noncovalently attached to the fusogen.

26. The system according to claim 16, wherein the targeted portion comprises a targeted antibody or an antigen-binding fragment thereof.

27. A pharmaceutical composition comprising the system described in any one of claims 1 to 26 and a pharmaceutically acceptable carrier or excipient.

28. (i) A kit comprising the system according to any one of claims 1 to 26, and optionally (ii) a container and / or instructions for use.

29. A method for ex vivo production of modified B cells or modified hematopoietic stem cells (HSCs) that produce antibodies or antigen-binding fragments thereof, comprising ex vivo transduction of an effective amount of the system described in any one of claims 1 to 26 into B cells or HSCs, wherein the first and second components of the system are administered to the cells simultaneously or sequentially in any order, and the administration of the first and second components incorporates a sequence encoding the antibody or antigen-binding fragment thereof into the DNA of the cells, thereby making the cells modified cells.

30. a) The B cells or HSCs are cultured under stimulating conditions before and / or after transduction, and / or b) The method according to claim 29, further comprising culturing the prepared modified B cells or modified HSCs under differentiation conditions to promote the differentiation of the modified B cells or modified HSCs into modified plasma cells.

31. A composition for producing an antibody or an antigen-binding fragment thereof in the body of a subject requiring it, comprising a modified B cell or modified HSC prepared by the method of Claim 29, wherein the modified B cell or modified HSC is introduced into the body of the subject.

32. The composition according to claim 31, wherein the subject is CD20+ cells that are depleted before the introduction of the modified cells.

33. A system for use in a method for producing an antibody or an antigen-binding fragment thereof in vivo in the body of a subject requiring such production, the method comprising administering an effective amount of the system to the subject, wherein the first and second components of the system are administered simultaneously or sequentially in any order, and by administering the first and second components to the subject, a sequence encoding the antibody or the antigen-binding fragment thereof is incorporated into the DNA of the subject's B cells and / or hematopoietic stem cells (HSCs), thereby resulting in the production of the antibody or the antigen-binding fragment in the subject's body, according to any one of claims 1 to 26.

34. The system according to claim 33, further comprising administering an effective amount of a CD40 agonist and / or a CD180 agonist to the subject before administering the system to the subject.

35. The method includes administering an effective amount of a first antigen to the subject, and administering an effective amount of a second antigen to the subject. The first antigen has low affinity for the antibody or its antigen-binding fragment, and the first antigen is administered before the administration of the first and second components of the system. The system according to claim 33, wherein the second antigen has a high affinity for the antibody or its antigen-binding fragment, and the second antigen is administered after the administration of the first and second components of the system.

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