Ex VIVO delivery of lipid nanoparticles for delivery of gene modifying systems to t cells

Abstract

The disclosure provides methods to administer therapeutic composition generated by contacting blood with lipid nanoparticles (LNPs) encapsulating a gene editing system targeting immune cells to a patient. The provided methods provide ex vivo gene editing by LNPs in less than 8 hours.

Description

EX VIVO DELIVERY OF LIPID NANOPARTICLES FOR DELIVERY OF GENE MODIFYING SYSTEMS TO T CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application 63 / 549,324, filed on February 2, 2024 and U.S. Provisional Application 63 / 641,851, filed on May 2, 2024, the contents of each of which are hereby incorporated herein by reference in their entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The content of the electronic sequence listing (252052002840seqlist.xml; Size: 565,294 bytes; and Date of Creation: January 31, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0003] Autologous ex vivo chimeric antigen receptor (CAR) T-cell therapies have demonstratable efficacy against B-cell driven hematological malignancies and represent an effective cure for many patients.

[0004] Despite these successes, CAR-T therapies are still plagued with significant manufacturing challenges, such as long needle-to-needle times, high costs, and supply chain bottlenecks from both the vector and drug product manufacturing sides. These barriers to patient therapy are beginning to be addressed through shortening CAR-T manufacturing times and reducing dose through improvements in potency. However, a true “same-day” bedside manufacturing paradigm or other in vivo-based CAR-T therapies remain elusive for traditional CAR-T therapies. Accordingly, there exists a need to create safe and effective “same-day” CAR- T therapies.SUMMARY OF THE INVENTION

[0005] The disclosure provides an ex vivo gene editing method for editing patient cells and reinfusing the edited cells into the patient within 10 hours of collecting the patient cells. In some aspects, the disclosure provides an ex vivo gene editing method for integrating a heterologous sequence into the genomes of patient cells wherein edited cells are reinfused into the patient within 10 hours of collecting the patient cells. In some embodiments, the method comprises contacting patient cells with lipid nanoparticles (LNPs) or conjugates comprising a lipid nanoparticle (LNP) encapsulating a gene modifying system. In some embodiments, the edited1cells are generated ex vivo. In some embodiments, the population of edited cells expands in vivo once they have been reinfused into the patient.

[0006] In some aspects, the disclosure provides an ex vivo gene editing method for introducing a CAR into T-cells wherein edited cells are reinfused into the patient within 10 hours of collecting the patient cells. In some embodiments, the method comprises contacting patient immune cells, such as T cells or a population of immune cells comprising T cells, with lipid nanoparticles (LNPs) or conjugates comprising a lipid nanoparticle (LNP) encapsulating a gene modifying system. In some aspects, CAR-T cells are generated ex vivo. In some embodiments, the population of CAR-T cells expand in vivo once reinfused into the patient.

[0007] In some aspects, the disclosure provides a method for administering a therapeutic composition to a patient, comprising: (a) collecting a blood fraction comprising lymphocytes from the patient; (b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a gene modifying system to create a blood-LNP composition, wherein the gene modifying system comprises: (i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and (ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequence into the genome of at least one lymphocyte to produce at least one edited lymphocyte; (c) optionally, removing residual LNP from the blood-LNP composition to create a therapeutic composition comprising the at least one edited lymphocyte; and (d) reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction.

[0008] In some aspects, the disclosure provides a method for ex vivo gene editing of patient lymphocytes, comprising: (a) collecting a blood fraction comprising lymphocytes from a patient; (b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a gene modifying system to create a blood-LNP composition, wherein the gene modifying system comprises: (i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and (ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequence into the genome of at least one lymphocyte to produce at least one edited lymphocyte; wherein following the contacting for at least about one hour, at least 1% of the lymphocytes in the blood-LNP composition are edited.2

[0009] In some aspects, the disclosure provides a method for treating cancer in a patient comprising: (a) collecting a blood fraction comprising lymphocytes from the patient; (b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a gene modifying system to create a blood-LNP composition, wherein the gene modifying system comprises: (i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and (ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequence into the genome of at least one lymphocyte to produce at least one edited lymphocyte; (c) optionally, removing residual LNP from the blood-LNP composition to create a therapeutic composition comprising the at least one edited lymphocytes; (d) reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction, wherein the edited lymphocytes target cancer cells.

[0010] In some aspects, the LNPs comprise an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from lipids in Table LI and Table L3.

[0011] In some aspects, the ionizable lipid has one of the following structures: ox34

[0012] In some aspects, the ionizable lipid has the structure:

[0013] In some aspects, the ionizable lipid has the structure:

[0014] In some aspects, the ionizable lipid has the structure:5

[0015] In some aspects, the ionizable lipid has the structure:

[0016] In some aspects, the ionizable lipid has the structure:

[0017] In some aspects, the blood fraction is collected using leukapheresis. In some aspects, the blood fraction comprises peripheral blood mononuclear cells (PBMCs). In some aspects, the methods further comprise performing a wash to remove platelets from the blood fraction. In some aspects, the methods further comprise a spinning membrane separation remove the platelets. In some aspects, the method further comprise using a device comprising a centrifugation camber to remove the platelets.

[0018] In some aspects, the blood fraction comprises a lymphocyte concentration of about 20x106cells / mL to about 10Ox106cells / mL. In some aspects, wherein the blood fraction comprises a cell density of about 20x106cells / mL to about 100x106cells / mL.

[0019] In some aspects, the LNP is contacted with the blood fraction ex vivo. In some aspects, the LNP is contacted with the blood fraction ex vivo using an extra-corporeal delivery device.

[0020] In some aspects, the gene modifying system comprises the gene modifying polypeptide. In some aspects, the gene modifying polypeptide comprises a nickase domain, a DNA binding domain, a RNA binding domain, and a reverse transcriptase domain. In some aspects, the gene modifying polypeptide comprises an amino acid sequence set forth Table R2, Table E3, or Table6E6. In some aspects, the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl. In some aspects, the gene modifying polypeptide comprises a Cas domain and a reverse transcriptase domain.

[0021] In some aspects, the gene modifying system comprises the nucleic acid encoding the gene modifying polypeptide. In some aspects, the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule. In some aspects, the nucleic acid encoding the gene modifying polypeptide is a DNA molecule. In some aspects, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth Table E3 or Table E6. In some aspects, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

[0022] In some aspects, the blood fraction is further contacted with a LNP comprising a heterologous gene modifying system.

[0023] In some aspects, the disclosed methods further comprise stimulating the lymphocytes in the blood fraction with a T-cell stimulating reagent. In some aspects, the stimulating takes place before the contacting the LNP with the blood fraction. In some aspects, the stimulating takes place concurrently with the contacting the LNP with the blood fraction. In some aspects, the T- cell stimulating reagent comprises a CD3 agonist and / or a CD28 agonist. In some aspects, the T- cell stimulating reagent comprises a colloidal polymeric nanomatrix conjugated to a CD3 agonist and a CD28 agonist. In some aspects, the lymphocytes are stimulated for about 30 minutes to about 4 hours. In some aspects, the LNP is contacted with the blood fraction for about 30 minutes to 4 about hours.

[0024] In some aspects, the disclosed methods do not comprise stimulating the lymphocytes in the blood fraction with a T-cell stimulating reagent. For instance, in some embodiments, the LNPs composition is contacted with the blood fraction without any prior or concurrent stimulation. In some embodiments, the LNP composition itself is capable of stimulating T cells in the blood fraction without ant prior or concurrent stimulation.

[0025] In some aspects, the blood-LNP composition comprises about 0.1 μg of the LNP per lx 106cells to about 5 μg of the LNP per 1x106cells. In some aspects, the blood-LNP composition comprises about 20 cells / mL to about 100 x 106cells / mL and about 54μL / mL to about 6.7μL / mL of T cell stimulating reagent.7

[0026] In some aspects, the blood-LNP composition comprises the LNPs encapsulating the gene modifying system. In some embodiments, the gene modifying system comprises RNA. In some embodiments, the blood-LNP composition comprises about 0.1 μg of the RNA per lx 106cells to about 10 μg of the RNA per 1x106cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg of the RNA per lx 106cells to about 5 μg of the RNA per 1x106cells. In some embodiments, the blood-LNP composition comprises about 1 μg of the RNA per lx 106cells to about 5 μg of the RNA per 1x106cells. In some embodiments, the blood-LNP composition comprises about 2 μg of the RNA per lx 106cells to about 5 μg of the RNA per 1x106cells.

[0027] In some aspects, the heterologous object sequence, encodes a chimeric antigen receptor (CAR). In some aspects, the edited lymphocytes comprise the CAR integrated at a genomic locus. In some aspects, the edited lymphocytes express a CAR. In some aspects, about 1% to about 30% of lymphocytes in the therapeutic composition are edited lymphocytes.

[0028] In some aspects, the therapeutic composition further comprises a pharmaceutically acceptable buffer. In some aspects, the methods further comprises performing sterility testing before reinfusion. In some aspects, the methods, further comprise assaying the therapeutic composition to determine the number or percentage of edited lymphocytes. In some aspects, the therapeutic composition does not comprise microbial contaminants.

[0029] In some aspects, the therapeutic composition is reinfused into the patient within about 1 hour to about 9 hours.

[0030] In some aspects, the edited lymphocytes expand in-vivo after the therapeutic composition is reinfused into the patient. In some aspects, about 7 days after reinfusion, about 0% - about 20% of the patient’s lymphocytes are edited lymphocytes. In some aspects, about 7 days after reinfusion, the patient T cells comprise between about 30 million and about 1 billion CAR-T cells.

[0031] In some aspects, the method is carried out in a single in-line procedure to maintain a closed or functionally closed fluid circuit.

[0032] In some aspects, (i) the gene modifying polypeptide, or the nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence, are encapsulated in separate LNPs. In some aspects, the blood fraction is contacted with the LNPs8encapsulating (i) the gene modifying polypeptide, or the nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence at a ratio of between about 1:2 to about 1:25.

[0033] In some aspects, the (i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence, are encapsulate in the same LNP. In some aspects, the blood fraction is contacted with the LNP encapsulating (i) the gene modifying polypeptide, or the nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence at a ratio of between about 1 :2 to about 1 :25. In some aspects, the template nucleic acid is a RNA molecule. In some aspects, the template nucleic acid comprises the sequence set forth in SEQ ID NO: 575.

[0034] In some aspects, the LNPs comprise a targeting moiety. In some aspects, the targeting moiety is conjugated to the LNPs through a linker, and wherein the linker comprises an enzyme recognition sequence and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNPs. In some aspects, the Click reaction is an inverse electron demand Diels-Adler reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the first or second Click handle. In some aspects, the targeting moiety binds to a surface protein on T cells. In some aspects, the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7. In some aspects, the targeting moiety comprises an anti-CD3 moiety. In some aspects, the anti-CD3 moiety comprises any of the sequences set forth in Table E7.

[0035] In some aspects, the CAR comprises an antigen-binding domain, a transmembrane domain, a first intracellular signaling domain, and a second intracellular signaling domain. In some aspects, the CAR comprises an antigen-binding domain that binds to one or more antigens of a blood cancer. In some aspects, the blood cancer is leukemia, lymphoma, or multiple myeloma. In some aspects, the one or more antigens is a B cell antigen. In some aspects, the antigen binding domain binds to one or more antigens of a solid tumor. In some aspects, the antigen binding domain comprises an amino acid sequence or an antigen binding domain set forth in Table 4. In some aspects, the antigen binding domain comprises an scFv. In some aspects, the CAR comprises a linker domain comprising an amino acid sequence of a linker domain set forth in Table Linker 1. In some aspects, the CAR comprises a hinge domain. In9some aspects, the first intracellular signaling domain comprises an amino acid sequence of an intracellular signaling domain set forth in Table 5 or Table 6. In some aspects, the second intracellular signaling domain comprises an amino acid sequence of an intracellular signaling domain set forth in Table 5 or Table 6. In some aspects, the CAR comprises a costimulatory domain comprising an amino acid sequence of a costimulatory domain set forth in Table 5 or Table 6.

[0036] In some aspects, disclosed herein is a system for administering a therapeutic composition to a patient the system comprising: (a) an incoming processing unit for collecting a blood fraction from the subject; (b) a chamber for contacting lipid nanoparticles (LNPs) encapsulating components of a gene modifying system with the blood fraction to create a blood-LNP composition, wherein the gene modifying system comprises: (i) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, and (ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence; wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, (c) optionally, a processing unit for removing residual LNPs from the blood-LNP composition to create a therapeutic composition; and (d) a transfer container for reinfusing the therapeutic composition into the same subject within 10 hours of removing the blood fraction.

[0037] In some aspects, the incoming processing unit is a leukapheresis device.

[0038] In some aspects, the gene modifying system comprises the gene modifying polypeptide. In some aspects, the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl.

[0039] In some aspects, the blood fraction is further contacted with a LNP comprising a heterologous gene modifying system.

[0040] In some aspects, the gene modifying polypeptide comprises an amino acid sequence set for in Table R2, Table E3 or Table E6. In some aspects, the gene modifying system comprises the nucleic acid encoding the gene modifying polypeptide. In some aspects, the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule. In some aspects, the nucleic acid encoding the gene modifying polypeptide is a DNA molecule. In some aspects, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth in Table Rl, Table E3, or Table E6. In some aspects, the nucleic acid encoding the gene modifying10polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

[0041] In some aspects, described herein is a blood-LNP composition comprising: (a) lymphocytes; wherein the concentration of lymphocytes is around 20x106cells / mL to about 100x106cells / mL (b) lipid nanoparticles (LNPs) encapsulating a gene modifying system with the blood fraction to create a blood-LNP composition, wherein the gene modifying system comprises: (i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and (ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence; and wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the concentration of the LNPs is around 0.1 μg LNP per lx 106cells - 5 μg LNP per 1x106cells; and (c) optionally, a T-cell stimulating reagent.

[0042] In some aspects, described herein is a blood-LNP composition comprising: (a) lymphocytes; wherein the concentration of lymphocytes is around 20x106cells / mL to about 200x106cells / mL or wherein the concentration of lymphocytes is around 100x106cells / mL to about 200x106cells / mL (b) lipid nanoparticles (LNPs) encapsulating a gene modifying system with the blood fraction to create a blood-LNP composition, wherein the gene modifying system comprises: (i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and (ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence; and wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the concentration of the LNPs is around 0.1 μg LNP per lx 106cells - 5 μg LNP per 1x106; and (c) optionally, a T-cell stimulating reagent.

[0043] In some aspects, the gene modifying system comprises the gene modifying polypeptide. In some aspects, the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl. In some aspects, the LNPs encapsulate a heterologous gene modifying system. In some aspects, the gene modifying polypeptide comprises an amino acid sequence set forth in Table R2 or Table E3. In some aspects, the gene modifying system comprises the nucleic acid encoding the gene modifying polypeptide. In some aspects, the nucleic acid encoding the gene modifying polypeptide is a DNA molecule. In some aspects, the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule. In some aspects, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth in Table Rl or Table E3. In11some aspects, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

[0044] In some aspects, the LNP comprises a targeting moiety. In some aspects, the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7. In some aspects, the targeting moiety comprises an anti-CD3 moiety. In some aspects, the anti-CD3 moiety comprises any of the sequences set forth in Table E7.

[0045] In some embodiments, disclosed is a method for administering a therapeutic composition to a patient, comprising: (a) collecting a blood fraction comprising lymphocytes from the patient; (b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating heterologous gene modifying system to create a blood-LNP composition, wherein the heterologous gene modifying system comprises: (i) a heterologous gene modifying polypeptide, or a nucleic acid encoding the heterologous gene modifying polypeptide comprising (1) a Cas domain (e.g., a Case nickase domain, e.g. a Cas9 nickase domain) and (2) a reverse transcriptase domain, and (ii) a template nucleic acid as described herein (e.g., a template RNA), wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the heterologous gene modifying system edits the genome of the at least one lymphocyte to produce edited lymphocytes; (c) optionally removing residual LNP from the blood-LNP composition to create a therapeutic composition comprising the edited lymphocytes; and(d) reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction.

[0046] In some embodiments, disclosed is a method for ex vivo gene editing of patient lymphocytes, comprising: (a) collecting a blood fraction comprising lymphocytes from a patient; (b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a heterologous gene modifying system with the blood fraction to create a blood-LNP composition, wherein the heterologous gene modifying system comprises: (i) a heterologous gene modifying polypeptide, or a nucleic acid encoding the heterologous gene modifying polypeptide, comprising (1) a Cas domain (e.g., a Cas nickase domain, e.g. a Cas9 nickase domain) and (2) a reverse transcriptase domain, and (ii) a template nucleic acid as described herein (e.g., a template RNA), wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the heterologous gene modifying polypeptide system edits the genome of at least one lymphocyte to produce edited lymphocytes; wherein following the contacting for at least about one hour, at least 1% of the lymphocytes in the blood-LNP composition are edited.12

[0047] In some embodiments, provided herein is a method for treating cancer in a patient comprising: (a) collecting a blood fraction comprising lymphocytes from the patient; (b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a heterologous gene modifying system with the blood fraction to create a blood-LNP composition, wherein the heterologous gene modifying system comprises: (i) a heterologous gene modifying polypeptide, or a nucleic acid encoding the heterologous gene modifying polypeptide, comprising (1) a Cas domain (e.g., a Cas nickase domain, e.g. a Cas9 nickase domain) and (2) a reverse transcriptase domain, and (ii) a template nucleic acid as described herein (e.g., a template RNA), wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the heterologous gene modifying system edits the genome of at least one lymphocyte to produce edited lymphocytes; (c) optionally, removing residual LNPs from the blood-LNP composition to create a therapeutic composition comprising the edited lymphocytes; (d) reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction, wherein the edited lymphocytes target cancer cells.

[0048] In some aspects, the methods described herein can be used to treat autoimmune diseases. In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, diabetes type I, aplastic anemia, Grave’s disease, coeliac disease, Crohn’s disease, lupus, arthritis, osteoarthritis, autoimmune uveitis and myasthenia gravis.BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 shows that higher levels of transfection of targeted LNPs in activated T cells are achieved in the absence of serum.

[0050] FIGs. 2A and 2D, when FBS was present, the transfection efficiency in activated T cells appeared more normalized across the anti-CD3 targeting moieties. FIGs. 2B and 2C show that in the absence of serum, all anti-CD3 targeting moieties screened improved transfection of activated T cells when conjugated to an LNP relative to the base LNP (non-conjugated to an anti-CD3 targeting moiety).

[0051] FIGs. 3A-3D show that in rested T cells, all tLNPs conjugated to the anti-CD3 targeting moieties that were screened enhanced transfection efficiency above that of non-targeted base LNPs, both in the presence and absence of serum. Serum had less of a normalization effect in rested cells (compared to activated cells), as rested cells transfected with anti-CD3-8 ttLNPs showed over a 120-fold increase in MFI relative to cells transfected with non-targeted base LNPs13at the highest dose tested (FIG. 3D). In the absence of serum, GFP expression is over 370-fold higher in cells transfected by anti-CD8-8 tLNPs compared to non-targeted base LNPs (FIG. 3C).

[0052] FIG. 4A shows that the anti-CD3 targeting moieties induced variable expression of the T cell activation marker CD25.

[0053] FIG. 5A shows that close to 100% of living activated T cells transfected with LNPs comprising Lipid092 or Lipidl54 expressed GFP, starting at the lowest dose. Fewer T cells were transfected with the other LNPs tested, including the baseline control LNPs, across all dose levels. FIG. 5B shows that transfection with Lipidl54 LNPs resulted in the highest GFP expression levels (MFI) in the cells, followed by LNPs comprising Lipid092. FIG. 5C shows that Lipid092 and Lipidl54 LNPs transfected the largest numbers of cells at the 10Ong to 400 ng doses (per 2x105cells), but then the percentage of GFP+ cells fell at higher doses of the Lipidl54 LNP. The LNPs formulated with the V003 ionizable lipid transfected smaller numbers of rested T cells at all doses tested. FIG. 5D shows that transfection of LNPs with Lipid092 GFP expression resulted in the highest levels of GFP expression at most doses, followed by LNPs with Lipidl54.

[0054] FIG. 6A shows that at 4 days following transfection, substantially more activated T cells expressed GFP at all doses when Lipid092 LNPs or Lipidl54 LNPs delivered the gene modifying system compared to activated T cells that were contacted with the V003 LNPs, with the Lipidl54 LNPs showing the highest levels of delivery. FIG. 6B shows that activated T cells transduced with the Lipid092 or Lipidl54 expressed GFP at higher levels (higher MFI) relative to activated T cells transduced with LNPs comprising the V003 ionizable lipid. FIG. 6B shows that activated T cells transduced with the Lipid092 or Lipidl54 expressed GFP at higher levels (higher MFI) relative to activated T cells transduced with LNPs comprising the V003 ionizable lipid.

[0055] FIGs. 7A-D show that delivery of a gene modifying system payload to activated cells using targeted LNPs formulated with Lipidl54 and 22% DSPC generated more cells that expressed GFP (%GFP+) and at higher levels (MFI) relative to the baseline control tLNP that was the identical except that it was formulated with 8% DSPC. At four days (FIGs. 7A and B) and at 7 days (FIGs. 7C and D) following transfection of tLNPs comprising 22% DSPC at all doses tested, more activated T cells expressed GFP at higher levels relative to the cells transfected with tLNPs comprising 8% DSPC.14

[0056] FIGs. 8A and 8B shows that through day 7, cell culture viabilities remain high and population doubling levels increase for ex vivo culture of edited cells.

[0057] FIG. 9 shows that the frequency of cells expressing CAR in ex vivo cultures increase between day 4 and day 7.

[0058] FIGs. 10A-10C shows BCMA CAR-T cells effectively clear individual animal RPMI- 8226 tumors by day 31 whereas individual animal RPMI-8226 tumor growth increases for animals treated with vehicle T cells in FIG. 10A and untransfected T cells in FIG 10B. (vehicle treated in FIG. 10A, untransfected T cells treated in FIG. 10B, and BCMA CAR-T cells treated in FIG. IOC).

[0059] FIG. 11 shows that CAR expression was visible in treated T cells with as little as 1 hour of treatment.

[0060] FIG. 12 shows quantification of the percentage of edited cells expressing the CAR in mock treated PBMCs (left) or PBMCs treated with the RNA Gene Writer system (right).

[0061] FIG. 13 shows quantification of the percentage of BCMA tumor cells killed with CAR-T cells generated by the mock system or the RNA Gene Writer system.

[0062] FIG. 14 shows quantification of the IFN-y cytokine in that did not receive the anti-CD3 tLNPs formulated with the gene modifying system (left) and cells that did receive the anti-CD3 tLNPs formulated with a gene modifying system (right).

[0063] FIG. 15 shows quantification of the percentage of edited cells expressing the CAR in cells that received tLNPs conjugated to four different anti-CD3 targeting moieties (fab fragments) at two doses.

[0064] FIG. 16 shows quantification of CAR expression levels (MFI) in the CAR-T cells generated by with four different anti-CD3 tLNPs at two doses.

[0065] FIG. 17 shows quantification of the percentage of BCMA tumor cells killed by CAR-T cells generated using four anti-CD3 tLNPs comprising an exemplary gene modifying system.

[0066] FIG. 18 shows quantification of the percentage of edited cells expressing the CAR in mock treated PBMCs (left), PBMCs treated anti-CD3 tLNPs formulated with an exemplary gene modifying system (middle), and PBMCs with CAR transgenes introduced via lentivirus transduction (right).15

[0067] FIG. 19 shows quantification of the percentage of BCMA tumor cells killed with edited cells expressing the CAR in mock treated PBMCs, PBMCs treated with anti-CD3 tLNPs formulated with an exemplary gene modifying system, and PBMCs with CAR transgenes introduced via lentivirus transduction.

[0068] FIG 20A- 20B shows a systematic representation of some of the embodiments described herein. FIG 20A shows a systematic representation of a method of administering a therapeutic composition to a patient according to some embodiments without use of a T-cell stimulating agent. FIG 20B shows a systematic representation of a method of administering a therapeutic composition to a patient according to some embodiments including the use of a T-cell stimulating agent.DETAILED DESCRIPTION

[0069] The disclosed method removes traditional CAR-T clinical roadblocks by leveraging a lipid nanoparticle (LNP) platform that delivers a gene modifying system to primary human T cells from a patient to generate CAR-T cells in a same-day manufacturing process, wherein CAR-T cells expand in vivo post reinfusion. The disclosed gene modifying systems leverage target-primed reverse transcription (TPRT) biochemistry evolved from non-LTR retrotransposon mobile genetic elements to modify the genome without generating double-stranded DNA breaks. Moreover, the gene modifying system can be engineered to catalyze a variety of editing reactions such as substitutions, deletions, and insertions of transgenes from an RNA template. These edits can be achieved with all-RNA delivery in primary cells, eliminating the need for viral vectors and DNA template-based gene editing.

[0070] Disclosed herein are methods for administering a therapeutic composition to a patient, comprising collecting a blood fraction comprising lymphocytes from the patient, contacting any of the lipids, LNPs, or conjugates encapsulating a gene modifying system, described herein, with the blood fraction to create a blood-LNP composition. In some embodiments the gene modifying system comprises a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide and a template nucleic acid comprising a sequence that binds to the gene modifying polypeptide and a heterologous object sequence, wherein the gene modifying polypeptide integrates the heterologous object sequences into the genome of the lymphocyte to produce edited lymphocytes. The method may further comprise removing residual lipids, LNPs, or conjugates from the blood-LNP composition to create a therapeutic composition comprising edited lymphocytes. In some embodiments, the method further comprises reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction.16

[0071] Disclosed herein are methods for ex vivo gene editing of patient lymphocytes comprising collecting a blood fraction comprising lymphocytes from the patient, and contacting any of the lipid, LNPs, or conjugates encapsulating a gene modifying system, described herein, with the blood fraction to create a blood-LNP composition. In some embodiments the gene modifying system comprises a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide and a template nucleic acid comprising a sequence that binds to the gene modifying polypeptide and a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequences into the genome of the lymphocyte to produce edited lymphocytes. Following at least about a half an hour of contacting, at least 1% of the lymphocytes in the blood-LNP composition are edited.

[0072] In some embodiments, described herein are methods for treating cancer in a patient. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the methods comprise collecting a blood fraction comprising lymphocytes from the patient, contacting any of the lipid, LNPs, or conjugates encapsulating a gene modifying system, described herein, with the blood fraction to create a blood-LNP composition. In some embodiments the gene modifying system comprises a gene modifying peptide, or a nucleic acid encoding a gene modifying polypeptide and a template nucleic acid comprising a sequence that binds to the gene modifying polypeptide and a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequences into the genome of the lymphocyte to produce edited lymphocytes. The method may further comprise removing residual lipids, LNPs, or conjugates from the blood-LNP composition to create a therapeutic composition comprising edited lymphocytes. In some embodiments, the method further comprises reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction. In some embodiments the edited lymphocytes target cancer cells.

[0073] In some embodiments, the methods described herein can be used to treat autoimmune diseases. In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, diabetes type I, aplastic anemia, Grave’s disease, coeliac disease, Crohn’s disease, lupus, arthritis, osteoarthritis, autoimmune uveitis and myasthenia gravis.

[0074] FIG. 20A - 20B provide a systematic representation of the methods described herein for administering a therapeutic composition to a patient and for treating a cancer or autoimmune disease in the patient. In FIG 20A, a blood fraction comprising lymphocytes is collected from a17patient using a leukapheresis device. The blood fraction comprising lymphocytes is washed to remove platelets from the blood fraction. The blood fraction is contacted with LNPs encapsulating a gene modifying system to create a blood-LNP composition. A therapeutic composition is created by optionally, removing residual LNPs from the blood-LNP composition and formulating a therapeutic composition with a clinical buffer. The therapeutic composition is reinfused into the patient within about 10 hours of collecting the blood fraction. After reinfusion of the therapeutic composition CAR-T cells are generated in vivo. In FIG 20B the methods are similar to the methods outlined in FIG 20A, but the methods in FIG 20B comprise contacting the blood fraction comprising lymphocytes with both the LNPs encapsulating a gene modifying system and a T-cell stimulating agent (e.g.TransACT).I. DEFINITIONS

[0075] Antigen binding domain: The term “antigen binding domain” as used herein refers to that portion of antibody or a chimeric antigen receptor which binds an antigen. In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments an antigen binding domain binds an antigen characteristic of a cancer, e.g., a tumor associated antigen in a neoplastic cell. In some embodiments, an antigen binding domain binds an antigen characteristic of an infectious disease, e.g. a virus associated antigen in a virus infected cell. In some embodiments, an antigen binding domain binds an antigen characteristic of a cell targeted by a subject’s immune system in an autoimmune disease, e.g., a 0-antigen. In some embodiments, an antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, an antigen binding domain is or comprises an scFv or Fab.

[0076] Expression cassette: The term “expression cassette,” as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention.

[0077] gRNA spacer: A “gRNA spacer”, as used herein, refers to a portion of a nucleic acid that has complementarity to a target nucleic acid and can, together with a gRNA scaffold, target a Cas protein to the target nucleic acid.

[0078] gRNA scaffold: A “gRNA scaffold”, as used herein, refers to a portion of a nucleic acid that can bind a Cas protein and can, together with a gRNA spacer, target the Cas protein to the target nucleic acid. In some embodiments, the gRNA scaffold comprises a crRNA sequence, tetraloop, and tracrRNA sequence.18

[0079] Gene modifying polypeptide: A “gene modifying polypeptide”, as used herein, refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, the gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, the gene modifying polypeptide integrates a sequence into a random position in a genome, and in some embodiments, the gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, a gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the template nucleic acid into the target DNA. Gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence. Gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. Exemplary gene modifying polypeptides, and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a gene modifying polypeptide integrates a sequence into a sequence outside of a gene.

[0080] Gene modifying system: A “gene modifying system,” as used herein, refers to a system comprising a gene modifying polypeptide, or a nucleic acid (e.g., an mRNA) encoding the gene modifying polypeptide, and a template nucleic acid.

[0081] Domain: The term “domain” as used herein refers to a structure of a biomolecule that contributes to a specified function of the biomolecule. A domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, an19endonuclease domain, a DNA binding domain, a reverse transcriptase domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain.

[0082] Exogenous: As used herein, the term “exogenous,” when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell, or organism by the hand of man. For example, a nucleic acid that is as added into an existing genome, cell, tissue, or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.

[0083] Heterologous: The term “heterologous”, when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector). In some embodiments, a domain is heterologous relative to another domain, if the first domain is not naturally comprised in the same polypeptide as the other domain (e.g., a fusion between two domains of different proteins from the same organism).20

[0084] Heterologous gene modifying polypeptide: As used herein, the term “heterologous gene modifying polypeptide” refers to a polypeptide comprising a retroviral reverse transcriptase, or a polypeptide comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a retroviral reverse transcriptase, which is capable of integrating a nucleic acid sequence (e.g., a sequence provided on a template nucleic acid) into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell). In some embodiments, the heterologous gene modifying polypeptide is capable of integrating the sequence substantially without relying on host machinery. In some embodiments, the heterologous gene modifying polypeptide integrates a sequence into a random position in a genome, and in some embodiments, the heterologous gene modifying polypeptide integrates a sequence into a specific target site. In some embodiments, the sequence that is integrated comprises a deletion, substitution, or insertion relative to the target DNA molecule. In some embodiments, a heterologous gene modifying polypeptide includes one or more domains that, collectively, facilitate 1) binding the template nucleic acid, 2) binding the target DNA molecule, and 3) facilitate integration of the at least a portion of the template nucleic acid into the target DNA. Heterologous gene modifying polypeptides include both naturally occurring polypeptides as well as engineered variants of the foregoing, e.g., having one or more amino acid substitutions to the naturally occurring sequence. Heterologous gene modifying polypeptides also include heterologous constructs, e.g., where one or more of the domains recited above are heterologous to each other, whether through a heterologous fusion (or other conjugate) of otherwise wild-type domains, as well as fusions of modified domains, e.g., by way of replacement or fusion of a heterologous sub-domain or other substituted domain. Exemplary heterologous gene modifying polypeptides, and systems comprising them and methods of using them, that can be used in the methods provided herein are described, e.g., in PCT / US2021 / 020948, which is incorporated herein by reference with respect to heterologous gene modifying polypeptides that comprise a retroviral reverse transcriptase domain. In some embodiments, a heterologous gene modifying polypeptide integrates a sequence into a gene. In some embodiments, a heterologous gene modifying polypeptide integrates a sequence into a sequence outside of a gene. A “heterologous gene modifying system,” as used herein, refers to a system comprising a heterologous gene modifying polypeptide and a template nucleic acid.

[0085] Mutation or Mutated: The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a21point mutation) or multiple nucleotides may be inserted, deleted, or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art. In some embodiments a mutation occurs naturally. In some embodiments a desired mutation can be produced by a system described herein.

[0086] Nucleic acid molecule: “Nucleic acid molecule” refers to both RNA and DNA molecules including, without limitation, complementary DNA (“cDNA”), genomic DNA (“gDNA”), and messenger RNA (“mRNA”), and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. The nucleic acid molecule can be double-stranded or single-stranded, circular, or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:,” or “nucleic acid comprising SEQ ID NO: 1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ ID NO: 1, or (ii) a sequence complimentary to SEQ ID NO: 1. The choice between the two is dictated by the context in which SEQ ID NO: 1 is used. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). Also included are chemically modified bases, backbone, and modified caps. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule, e.g., peptide nucleic acids (PNAs). Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids (LNAs). In various embodiments, the nucleic acids are in operative association with additional genetic elements, such as tissue-specific22expression-control sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats / direct repeats, homology regions (segments with various degrees of homology to a target DNA), untranslated regions (UTRs) (5', 3', or both 5' and 3' UTRs), and various combinations of the foregoing. The nucleic acid elements of the systems provided by the invention can be provided in a variety of topologies, including single-stranded, double-stranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), closed-ended DNA (ceDNA).

[0087] Primer Binding Sequence: The term “primer binding site sequence” or “PBS sequence,” as used herein, refers to a portion of a template RNA capable of binding to a region comprised in a target nucleic acid sequence. In some instances, a PBS sequence is a nucleic acid sequence comprising at least 3, 4, 5, 6, 7, or 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. In some embodiments the primer region comprises at least 5, 6, 7, 8 bases with 100% identity to the region comprised in the target nucleic acid sequence. Without wishing to be bound by theory, in some embodiments when a template RNA comprises a PBS sequence and a heterologous object sequence, the PBS sequence binds to a region comprised in a target nucleic acid sequence, allowing a reverse transcriptase domain to use that region as a primer for reverse transcription, and to use the heterologous object sequence as a template for reverse transcription.

[0088] It is understood that aspects and embodiments described herein as “comprising” include “consisting of’ and “consisting essentially of’ embodiments. n. LIPID NANOPARTICLES CONJUGATES

[0089] In one aspect, the disclosure provides an LNP (conjugate) comprising an ionizable lipid as described herein (e.g., in Table LI), wherein the LNP can deliver a payload, such as a therapeutic agent (e.g., a gene modifying system, such as a retrotransposon gene modifying system and / or a heterologous gene modifying system, as described herein) to an immune cell (e.g., a T cell). In another aspect, the LNP (conjugate) comprises a targeting moiety that binds to a protein (e.g., a protein receptor) on an immune cell (e.g., a T cell), as described herein. In some embodiments, an LNP (conjugate) comprises both an ionizable lipid and a targeting moiety. In some embodiments, the LNP (conjugate) delivers greater than 90% of the pay load to T cells. In some embodiments, the LNP (conjugate) delivers from about 90% to about 100% of the pay load to T cells.23

[0090] In one aspect, the disclosure provides targeted LNPs (conjugates) comprising a targeting moiety and a lipid nanoparticle (LNP) encapsulating a payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system), wherein the targeting moiety binds to a protein (e.g., protein receptor) on an immune cell (e.g., T cell). In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In some instances, the targeting moiety is an antibody, a Fab fragment, a scFv, a D ARPIN, a VHH domain antibody, a FN3 domain, a nanobody, a single domain antibody or a Centyrin. In other embodiments, the targeting moiety is a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin or a N-Acetylgalactosamine (GalNac). In some embodiments, the payload (e.g., a therapeutic agent, as described herein, such as a gene modifying polypeptide or a gene modifying system) is capable of modifying one or more genes of the target immune cell (e.g., T cell).

[0091] The conjugates described herein may be used to target and modify immune cells. In some embodiments, the conjugates may be used to modify T cells. In some embodiments, T-cells may include any subpopulation of T-cells, e.g., CD4+, CD8+, gamma-delta, naive T cells, stem cell memory T cells, central memory T cells, or a mixture of subpopulations. In some embodiments, the conjugates may be used to deliver or modify a sequence encoding a T-cell receptor (TCR) in a T cell. In some embodiments, the conjugates may be used to deliver at least one sequence encoding a chimeric antigen receptor (CAR) to T-cells. For instance, in specific embodiments, the conjugates can be used to deliver an RNA encoding a CAR to T-cells.A. Targeting moieties

[0092] In some embodiments, the LNP comprises a targeting moiety. In some embodiments, the targeting moiety is a T-cell targeting moiety, for example, an antibody, Fab fragment or ScFv that binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CDS, CD28, CD137, CD45, T-cell receptor (TCR)P,TCR-a, TCR-a / p, TCR-y / 5, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CDlla, TLR2, TLR4, TLR5, IL-7 receptor, or IL-15 receptor.

[0093] In certain embodiments, the targeting moiety is a T-cell targeting moiety, for example, an antibody, Fab fragment or ScFv that binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CDS, CD28, CD137, CD45, T-cell receptor (TCR)P,TCR-o, TCR-a / p, TCR-y / 5, PD1, CTLA4, TIM3, LAG3, GDIS, IL-2 receptor, CDlla, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor. In some embodiments the CD80 targeting moiety is a CD80 extracellular domain (ECD).24

[0094] In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that targets a receptor on the surface of the T cell selected from CD2, CD3, CD4, CD5, CD6, CD7, and CDS. In some embodiments, the targeting moiety targets a CD3 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD7 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD5 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CD2 receptor on the surface of the T cell. In some embodiments, the targeting moiety targets a CDS receptor on the surface of the T cell.

[0095] In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that targets CD3 on the surface of the T cell, wherein the targeting moiety is an antibody, Fab fragment or ScFv selected from SP34, teclistamab, mosunetuzumab, odronextamab, tebentafusp, tepilizumab, muromonab and visilizumabm, or an antigen-binding portion thereof. In certain embodiments, the targeting moiety is SP34 or an antigen-binding portion thereof In other embodiments, the targeting moiety is teclistamab or an antigen-binding portion thereof In other embodiments, the targeting moiety is mosunetuzumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is odronextamab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is tebentafusp or an antigen-binding portion thereof. In other embodiments, the targeting moiety is muromonab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is visilizumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is tepilizumab or an antigen-binding portion thereof. . In other embodiments, the targeting moiety is Plamotamab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is HPN536 or an antigen-binding portion thereof. In other embodiments, the targeting moiety is Pasotuxizumab or an antigen-binding portion thereof. In other embodiments, the targeting moiety is Flotetuzumab or an antigenbinding portion thereof.

[0096] In some embodiments, the targeted LNP (conjugate) comprises a plurality of targeting moieties conjugated to the LNP, wherein the plurality of targeting moieties bind to at least one targeting moiety on a T cell.

[0097] In some embodiments, the plurality of targeting moieties bind to two or more T-cell antigens selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CDS, CD28, CD80, CD137, CD45, T-cell receptor (TCR)-|3,TCR-a, TCR-a / p, TCR-y / 5, PD1, CTLA4, TIM3, LAG3, CD18, IL-2 receptor, CDlla, TLR2, TLR4, TLR5, IL-7 receptor, and IL-15 receptor. In some embodiments, the targeted LNP (conjugate) comprises a targeting moiety that targets a25receptor on the surface of the T cell selected from CD2, CD3, CD4, CD5, CD6, CD7, and CD28. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD7. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD28. In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD3. In some embodiments, the one targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the other targeting moiety that binds to CD3.

[0098] In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD5. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD7. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD5 and the other targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein one targeting moiety binds to CD7 and the other targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD). In some embodiments, a targeted LNP comprises two targeting moieties, wherein one targeting moiety binds to CD3 and the other targeting moiety binds to CD3. In some embodiments, the one targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the other targeting moiety that binds to CD3.

[0099] In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein each targeting moiety binds to the same target (e.g., receptor) on the T cell. For instance, in some embodiments, both targeting moieties of the conjugate bind to CD3. In some such embodiments, one of the targets is SP34 or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof. In other such embodiments, one of the targets is SP34 or an antigen-binding portion thereof and the other is visilizumab or an antigenbinding portion thereof. In other such embodiments, one of the targets is SP34 or an antigenbinding portion thereof and the other is tepilizumab or an antigen-binding portion thereof. In other such embodiments, one of the targets is visilizumab or an antigen-binding portion thereof and the other is tepilizumab or an antigen-binding portion thereof. In other such embodiments, 26one of the targets is visilizumab or an antigen-binding portion thereof and the other is teclistamab or an antigen-binding portion thereof. In some embodiments, both targeting moieties of the conjugate bind to CD3. In some embodiments, both targeting moieties of the conjugate bind to CD7.

[0100] In certain embodiments, the targeting moiety binds to a CD4+ and / or CD8+ T cell. In other embodiments, the targeting moiety binds to a natural killer (NK) cell. In other embodiments, the targeting moiety binds to a hematopoietic stem cell. In other embodiments, the targeting moiety binds to a lymphoid progenitor cell. In other embodiments, the targeting moiety binds to a myeloid cell. In other embodiments, the targeting moiety binds to a macrophage.CD2 Targeting Moieties

[0101] In some embodiments, the target molecule is CD2. In some embodiments, the target cell is CD2+. The glycoprotein CD2 is a costimulatory receptor expressed mainly on T cells, NK cells, thymocytes, and dendritic cells that binds to lymphocyte-associated antigen 3 (LF A3; also known as CD58) which is expressed on the surface of B cells, T cells, monocytes, granulocytes, thymic epithelial cells. CD2 also binds to CD48, albeit with a relatively lower affinity. CD2 has an important role in the formation and organization of the immunological synapse that is formed between T cells and antigen-presenting cells upon cell-cell conjugation and associated intracellular signaling. CD2 expression is upregulated on memory T cells as well as activated T cells and plays an important role in activation of memory T cells. See, e.g., Binder et al. (2020) Front. Immunol. 11:1090, hereby incorporated by reference in its entirety.

[0102] In some embodiments, the CD2 targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD2. In some embodiments, the CD2 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’)2, Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin). In other embodiments, the CD2 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine. In some embodiments, the CD2 targeting moiety is an anti-CD2 antibody or antigen binding fragment thereof. In some embodiments, the CD2 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD2 targeting moiety is a bispecific or multispecific antibody or fragment thereof. In some embodiments, the CD2 targeting moiety is a humanized antibody or antigen-binding fragment thereof.27

[0103] Exemplary anti-CD2 binders, antibodies, or antigen-binding fragments thereof include Siplizumab (i.e., MEDI-507 or TCD601, ITB-Med LLC), BTI-322 (Lo-CD2a), Alefacept (i.e., a chimeric fusion protein consisting of the CD2-binding portion of human LFA3-Fc, Biogen) CB.219 (e.g., BioXCell), UMCD2 (e.g., Santa Cruz Biotechnology), TS1 / 8, RPA-2.10, TS1 / 18, TS1 / 18.1.1, TS2 / 18, AB75, and ZR100, as well as anti-CD2 antibodies or antigen-binding fragments thereof disclosed in any of: US 5,730,979; US 5,928,643; US 5,951,983; US 6,764,681; US 7,858,095; US 6,162,432; US 11,732,042; US 12,037,378; US20210032308;US20030068320; US20230365687; WO1999058147; WO2014025198; WO2024180185; WO2023126445; W02024079046; W02024079046; etc., each hereby incorporated by reference in its entirety.

[0104] In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:269 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:270. In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 280 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:281. In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:291 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:292. In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 302 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:292. SEQ ID NOs:269, 270, 280, 281, 291, 292, and 302 are shown in Table 2N.

[0105] In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:269, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:270. In some embodiments, the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence SYWVN (SEQ ID NO:271), a CDR-H2 comprising an amino acid sequence RIDPYDSETHYNQKFTD (SEQ ID NO:272), a CDR-H3 comprising an amino acid sequence SPRDSSTNLAD (SEQ ID NO:273), a CDR-L1 comprising an amino acid sequence RASQSISDYLH (SEQ ID NO:274), a CDR-L2 comprising an amino acid sequence YASQSIS (SEQ ID NO:275), and a CDR-L3 comprising an amino acid sequence QNGHSFPLT (SEQ ID NO:276). In some embodiments, the CD2 targeting moiety is a Fab 28fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:266 or 267, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:268.

[0106] In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:280, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:281. In some embodiments, the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RYWIH (SEQ ID NO:282), a CDR-H2 comprising an amino acid sequence NIDPSDSETHYNQKFKD (SEQ ID NO:283), a CDR-H3 comprising an amino acid sequence EDLYYAMEY (SEQ ID NO:284), a CDR-L1 comprising an amino acid sequence KSSQSVLYSSNQKNYLA (SEQ ID NO:285), a CDR-L2 comprising an amino acid sequence WASTRES (SEQ ID NO: 147), and a CDR-L3 comprising an amino acid sequence HQYLSSHT (SEQ ID NO:287). In some embodiments, the CD2 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:277 or 278, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:279.

[0107] In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:291, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:292. In some embodiments, the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence EYYMY (SEQ ID NO:293), a CDR-H2 comprising an amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO: 294), a CDR-H3 29comprising an amino acid sequence GKFNYRFAY (SEQ ID NO:295), a CDR-L1 comprising an amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO:296), a CDR-L2 comprising an amino acid sequence LVSKLES (SEQ ID NO:297), and a CDR-L3 comprising an amino acid sequence MQFTHYPYT (SEQ ID NO:298). In some embodiments, the CD2 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:288 or 289, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:290.

[0108] In some embodiments, the CD2 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:302, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:292. In some embodiments, the CD2 targeting moiety comprises a CDR-H1 comprising an amino acid sequence EYYMY (SEQ ID NO:293), a CDR-H2 comprising an amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO: 294), a CDR-H3 comprising an amino acid sequence GKFNYRFAY (SEQ ID NO:295), a CDR-L1 comprising an amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO:296), a CDR-L2 comprising an amino acid sequence LVSKLES (SEQ ID NO:297), and a CDR-L3 comprising an amino acid sequence MQFTHYPYT (SEQ ID NO:298). In some embodiments, the CD2 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:299 or 300, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:290.CD 3 Targeting Moieties

[0109] In some embodiments, the target molecule is CD3. In some embodiments, the target cell is CD3+. CD3 is a multimeric protein complex made up of four polypeptide chains (CD3-epsilon (E), CD3-gamma (y), CD3-delta (5), and CD3-zeta (Q) to form a CD3ye-CD35e-CD3^30signaling hexamer that associates with the T cell receptor (TCR). The CD3 / TCR complex is critical for T cells to recognize foreign antigens and activate T-cell adaptive immunity. CD3 is expressed by all T cells and is a defining marker of the T lymphocyte lineage. See, e.g., Dong et al. (2019) Nature 573:546-552, hereby incorporated by reference in its entirety.

[0110] In some embodiments, the CD3 targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD3. In some embodiments, the CD3 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’)2, Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin). In other embodiments, the CD3 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine. In some embodiments, the CD3 targeting moiety is an anti-CD3 antibody or antigen binding fragment thereof. In some embodiments, the CD3 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD3 targeting moiety is a bispecific or multispecific antibody or fragment thereof. In some embodiments, the CD3 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0111] Exemplary anti-CD3 binders, antibodies, or antigen-binding fragments thereof include SP34 mouse monoclonal antibody (see, for example, Pressano, S. The EMBO J. 4:337-344, 1985; Alarcon, B. EMBO J. 10:903-912, 1991; Salmeron A. etal., J. Immunol. 147:3047-52, 1991; Yoshino N. etal., Exp. Anim 49:97-110, 2000; Conrad M L. etal., Cytometry 71A:925- 33, 2007; Yang etal., J. Immunol. 137:1097-1100: 1986; US 8,846,042; US 11,013,800; and US 10,870,701), Cris-7 monoclonal antibody (Reinherz, E. L. etal. (eds.), Leukocyte typing II, Springer Verlag, New York, (1986)), BC3 monoclonal antibody (Anasetti etal. (1990) J. Exp. Med. 172:1691), OKT3 (Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med. 313:337) and derivatives thereof such as OKT3 ala-ala (Herold et al. (2003) J. Clin. Invest. 11:409), visilizumab (Carpenter etal. (2002) Blood 99:2712), mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, muromonab, plamotamab, HPN536, pasotuxizumab, flotetuzumab, and 145-2C11 monoclonal antibody (Hirsch etal. (1988) J. Immunol. 140: 3766). Further CD3 binding molecules contemplated herein include UCHT-1 (Beverley, P C and Callard, R. E. (1981) Eur. J. Immunol. 11: 329-334) and CD3 binding molecules described in W02004 / 106380; W02010 / 037838; W02008 / 119567; W02007 / 042261; W02010 / 0150918; the contents of each of which are incorporated herein by reference in their entirety.

[0112] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141 and a light chain variable region31comprising the amino acid sequence of SEQ ID NO: 142. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 153. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 163 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 164. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 174 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 175. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 185 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 186. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 196 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 197. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:207 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:208. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:218 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:219. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:228 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:229. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 238 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:239. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:248 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:249. In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 258 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:259. SEQ ID NOs: 141-142, 152-153, 163-163, 174-175, 185-186, 196-197, 207-208, 218-219, 228-229, 238-239, 248-249, and 258-259 are shown in tables below.

[0113] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 141, and / or a light chain variable region comprising an32amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 142. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NYYIH (SEQ ID NO: 143), a CDR-H2 comprising an amino acid sequence WIYPGDGNTKYNEKFKG (SEQ ID NO: 144), a CDR-H3 comprising an amino acid sequence DSYSNYYFDY (SEQ ID NO: 145), a CDR-L1 comprising an amino acid sequence KSSQSLLNSRTRKNYLA (SEQ ID NO: 146), a CDR-L2 comprising an amino acid sequence WASTRES (SEQ ID NO: 147), and a CDR-L3 comprising an amino acid sequence TQSFILRT (SEQ ID NO: 148). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 138 or 139, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 140. In some embodiments, the CD3 targeting moiety is mosunetuzumab or an antigen-binding fragment thereof.

[0114] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 152, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 153. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence DYTMH (SEQ ID NO: 154), a CDR-H2 comprising an amino acid sequence GISWNSGSIGY ADSVKG (SEQ ID NO: 155), a CDR-H3 comprising an amino acid sequence DNSGYGHYYYGMDV (SEQ ID NO:156), a CDR-L1 comprising an amino acid sequence RASQSVSSNLA (SEQ ID NO:157), a CDR-L2 comprising an amino acid sequence GASTRAT (SEQ ID NO: 158), and a CDR-L3 comprising an amino acid sequence QHYINWPLT (SEQ ID NO: 159). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 149 or 150, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at33least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 151. In some embodiments, the CD3 targeting moiety is odronextamab or an antigen-binding fragment thereof.

[0115] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 163, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 164. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence GYTMN (SEQ ID NO: 165), a CDR-H2 comprising an amino acid sequence LINPYKGVSTYNQKFKD (SEQ ID NO: 166), a CDR-H3 comprising an amino acid sequence SGYYGDSDWYFDV (SEQ ID NO: 167), a CDR-L1 comprising an amino acid sequence RASQDIRNYLN (SEQ ID NO: 168), a CDR-L2 comprising an amino acid sequence YTSRLES (SEQ ID NO: 169), and a CDR-L3 comprising an amino acid sequence QQGNTLPWT (SEQ ID NO: 170). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 160 or 161, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 162. In some embodiments, the CD3 targeting moiety is tebentafusp or an antigen-binding fragment thereof.

[0116] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 174, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 175. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RYTMH (SEQ ID NO: 176), a CDR-H2 comprising an amino acid sequence YINPSRGYTNYNQKVKD (SEQ ID NO: 177), a CDR-H3 comprising an amino acid sequence YYDDHYCLDY (SEQ ID NO: 178), a CDR-L1 comprising 34an amino acid sequence SASSSVSYMN (SEQ ID NO: 179), a CDR-L2 comprising an amino acid sequence DTSKLAS (SEQ ID NO: 180), and a CDR-L3 comprising an amino acid sequence QQWSSNPFT (SEQ ID NO: 181). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:171 or 172, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 173. In some embodiments, the CD3 targeting moiety is teplizumab or an antigen-binding fragment thereof.

[0117] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 185, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 186. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NTYAMN (SEQ ID NO: 187), a CDR-H2 comprising an amino acid sequence RIRSKYNNYATYYAASVKG (SEQ ID NO: 188), a CDR- H3 comprising an amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 189), a CDR-L1 comprising an amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 190), a CDR-L2 comprising an amino acid sequence GTNKRAP (SEQ ID NO: 191), and a CDR-L3 comprising an amino acid sequence ALWYSNLWV (SEQ ID NO: 192). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 182 or 183, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 184. In some embodiments, the CD3 targeting moiety is teclistamab or an antigen-binding fragment thereof.

[0118] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to 35the amino acid sequence of SEQ ID NO: 196, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 197. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence SYTMH (SEQ ID NO: 198), a CDR-H2 comprising an amino acid sequence YINPRSGYTHYNQKLKD (SEQ ID NO: 199), a CDR-H3 comprising an amino acid sequence SAYYDYDGFAY (SEQ ID N0:200), a CDR-L1 comprising an amino acid sequence SASSSVSYMN (SEQ ID NO: 179), a CDR-L2 comprising an amino acid sequence DTSKLAS (SEQ ID NO: 180), and a CDR-L3 comprising an amino acid sequence QQWSSNPPT (SEQ ID NO:203). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 193 or 194, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 195. In some embodiments, the CD3 targeting moiety is visilizumab or an antigen-binding fragment thereof.

[0119] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:207, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:208. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RYTMH (SEQ ID NO: 176), a CDR-H2 comprising an amino acid sequence YINPSRGYTNYNQKFKD (SEQ ID NO:210), a CDR-H3 comprising an amino acid sequence YYDDHYCLDY (SEQ ID NO: 178), a CDR-L1 comprising an amino acid sequence SASSSVSYMN (SEQ ID NO: 179), a CDR-L2 comprising an amino acid sequence DTSKLAS (SEQ ID NO: 180), and a CDR-L3 comprising an amino acid sequence QQWSSNPFT (SEQ ID NO: 181). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:204 or 205, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least3692%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:206. In some embodiments, the CD3 targeting moiety is muromonab or an antigen-binding fragment thereof.

[0120] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:218, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:219. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence KYAMN (SEQ ID NO:220), a CDR-H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR- H3 comprising an amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:222), a CDR-L1 comprising an amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:223), a CDR-L2 comprising an amino acid sequence GTKFLAP (SEQ ID NO:224), and a CDR-L3 comprising an amino acid sequence VLWYSNRWV (SEQ ID NO:225). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:215 or 216, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:217. In some embodiments, the CD3 targeting moiety is SP34 or an antigen-binding fragment thereof.

[0121] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:228, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:229. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence TYAMN (SEQ ID NO:230), a CDR-H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKG (SEQ ID NO:231), a CDR- H3 comprising an amino acid sequence HGNFGDSYVSWFAY (SEQ ID NO:232), a CDR-L1 comprising an amino acid sequence GSSTGAVTTSNYAN (SEQ ID NO:233), a CDR-L2 37comprising an amino acid sequence GTNKRAP (SEQ ID NO: 191), and a CDR-L3 comprising an amino acid sequence ALWYSNHWV (SEQ ID NO:235). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 226, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:227. In some embodiments, the CD3 targeting moiety is plamotamab or an antigen-binding fragment thereof.

[0122] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:238, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:239. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence KYAIN (SEQ ID NO:240), a CDR-H2 comprising an amino acid sequence RIRSKYNNYATYYADQVKD (SEQ ID NO:241), a CDR-H3 comprising an amino acid sequence HANFGNSYISYWAY (SEQ ID NO:242), a CDR-L1 comprising an amino acid sequence ASSTGAVTSGNYPN (SEQ ID NO:243), a CDR-L2 comprising an amino acid sequence GTKFLVP (SEQ ID NO:244), and a CDR-L3 comprising an amino acid sequence TLWYSNRWV (SEQ ID NO:245). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:236, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:237. In some embodiments, the CD3 targeting moiety is HPN536 or an antigen-binding fragment thereof.

[0123] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to 38the amino acid sequence of SEQ ID NO:218, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:249. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence KYAMN (SEQ ID NO:220), a CDR-H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR- H3 comprising an amino acid sequence HGNFGNSYISYWAY (SEQ ID NO:222), a CDR-L1 comprising an amino acid sequence GSSTGAVTSGNYPN (SEQ ID NO:223), a CDR-L2 comprising an amino acid sequence GTKFLAP (SEQ ID NO:224), and a CDR-L3 comprising an amino acid sequence VLWYSNRWV (SEQ ID NO:225). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 246, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:247. In some embodiments, the CD3 targeting moiety is pasotuxizumab or an antigen-binding fragment thereof.

[0124] In some embodiments, the CD3 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:258, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:259. In some embodiments, the CD3 targeting moiety comprises a CDR-H1 comprising an amino acid sequence TYAMN (SEQ ID NO:230), a CDR-H2 comprising an amino acid sequence RIRSKYNNYATYYADSVKD (SEQ ID NO:221), a CDR- H3 comprising an amino acid sequence HGNFGNSYVSWFAY (SEQ ID NO: 189), a CDR-L1 comprising an amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 190), a CDR-L2 comprising an amino acid sequence GTNKRAP (SEQ ID NO: 191), and a CDR-L3 comprising an amino acid sequence ALWYSNLWV (SEQ ID NO: 192). In some embodiments, the CD3 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of39SEQ ID NO:256, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:257. In some embodiments, the CD3 targeting moiety is flotetuzumab or an antigen-binding fragment thereof.CDS Targeting Moieties

[0125] In some embodiments, the target molecule is CD5. In some embodiments, the target cell is CD5+. CD5 is a type-I transmembrane glycoprotein with an extracellular region composed of three scavenger receptor cysteine-rich (SRCR) domains. Several CD5 ligands have been reported such as CD72, the IgV(H) frame-work region and several polypeptides (gp40-80, gp!50) whose identity remains undetermined. CD5 regulates T cell functions and development, including negative regulation of TCR signaling. CD 5 is an activation marker of T cells, wherein the expression of CD5 increases according to the magnitude of the signal delivered by the TCR. Consequently, CD5 expression reflects the heterogeneity of the signal strength associated with each individual TCR within a polyclonal T cell population. See, e.g., Voisinne et al. (2018) Front. Immunol. 9:2900, hereby incorporated by reference in its entirety.

[0126] In some embodiments, the CD5 targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD5. In some embodiments, the CD5 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’)2, Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin). In other embodiments, the CD5 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine. In some embodiments, the CD5 targeting moiety is an anti-CD5 antibody or antigen binding fragment thereof. In some embodiments, the CD5 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD5 targeting moiety is a bispecific or multispecific antibody or fragment thereof. In some embodiments, the CD5 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0127] Exemplary anti-CD5 binders, antibodies, or antigen-binding fragments thereof include AFM 16 (e.g., Affimed Therapeutics); AFM 17 (e.g., Affimed Therapeutics), RM354, L17F12, CRIS-1, UCHT2, RM314, SP19, and CD5-5D7, as well as anti-CD5 antibodies or antigenbinding fragments thereof disclosed in any of: US 10,786,549; US20110250203; Dai et al. (2021) Mol Then 29(9)2707-2722; etc., each hereby incorporated by reference in its entirety.40

[0128] In some embodiments, the CD5 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:357 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:358. SEQ ID NOs:357 and 358 are shown in Table 20, with complementary determining regions (CDRs) marked in bold.

[0129] In some embodiments, the CD5 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:357, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:358. In some embodiments, the CD5 targeting moiety comprises a CDR-H1 comprising an amino acid sequence TSGMGVG (SEQ ID NO:359), a CDR-H2 comprising an amino acid sequence HIWWDDDVYYNPSLKS (SEQ ID NO:360), a CDR-H3 comprising an amino acid sequence RRATGTGFDY (SEQ ID NO:361), a CDR-L1 comprising an amino acid sequence QASQDVGTAVA (SEQ ID NO: 362), a CDR-L2 comprising an amino acid sequence WTSTRHT (SEQ ID NO:363), and a CDR-L3 comprising an amino acid sequence HQYNSYNT (SEQ ID NO:364). In some embodiments, the CD5 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 354 or 355, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:356.CD 7 Targeting Moieties

[0130] In some embodiments, the target molecule is CD7. In some embodiments, the target cell is CD7+. CD7 (also known as GP40, LEU-9, Tp40, and TP41) is a transmembrane glycoprotein expressed by T cells, NK cells, and their precursors. It is present in >95% of lymphoblastic T- cell leukemias and lymphomas and a subset of PTCLs. CD7 has a costimulatory role in T-cell activation and cytokine production (e.g., IL-2) upon binding to its ligand, K12 / SECTM1.

[0131] In some embodiments, the CD7 targeting moiety includes an antibody or antigen-binding fragment thereof that binds to CD7. In some embodiments, the CD7 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’)2, Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin). In other 41embodiments, the CD7 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine. In some embodiments, the CD7 targeting moiety is an anti-CD7 antibody or antigen binding fragment thereof. In some embodiments, the CD7 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD7 targeting moiety is a bispecific or multispecific antibody or fragment thereof. In some embodiments, the CD7 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0132] Exemplary anti-CD7 binders, antibodies, or antigen-binding fragments thereof include SP94 (e.g., Roche Diagnostics), A20153E (e.g., BioLegend), 4H9 / CD7 (e.g., BioLegend), 124- 1D1, CD7-6B7, B-F12, 4H9, 3A1E, LT7, MEM-186, and MG34, as well as anti-CD7 antibodies or antigen-binding fragments thereof disclosed in any of: US 11,440,958; US 11,390,658; US20240075143; US20230128800; US20230399398; US20230159636; W02003051926 WO2023185256; Wang et al. (2024) Biomolecules 14(l):106; etc., each hereby incorporated by reference in its entirety.

[0133] In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:313 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:314. In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 324 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:325. In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:335 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:336. In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 346 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:347. SEQ ID NOs:313-314, 324-325, 335-336, and 346-347 are shown in tables below.

[0134] In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:313, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:314. In some embodiments, the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NYGMN (SEQ ID NO:315), a CDR-H242comprising an amino acid sequence WINTYTGEPTYADDFKG (SEQ ID NO:316), a CDR-H3 comprising an amino acid sequence WAYFYGSSPYFFDY (SEQ ID NO:317), a CDR-L1 comprising an amino acid sequence RSSTGAVTTSNYAN (SEQ ID NO: 190), a CDR-L2 comprising an amino acid sequence GTNNRAP (SEQ ID NO:319), and a CDR-L3 comprising an amino acid sequence ALWCSNHLV (SEQ ID NO:320). In some embodiments, the CD7 targeting moiety i5s a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:310 or 311, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:312. In some embodiments, the CD7 targeting moiety is grisnilimab or an antigen-binding fragment thereof.

[0135] In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:324, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 325. In some embodiments, the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NY AMS (SEQ ID NO:326), a CDR-H2 comprising an amino acid sequence TISGSGGSTYYADSAK (SEQ ID NO:327), a CDR-H3 comprising an amino acid sequence GGLLYFGEFHFDY (SEQ ID NO:328), a CDR-L1 comprising an amino acid sequence RASQGISNYLA (SEQ ID NO:329), a CDR-L2 comprising an amino acid sequence AASSLQS (SEQ ID NO:330), and a CDR-L3 comprising an amino acid sequence QHYNSYPLT (SEQ ID NO:331). In some embodiments, the CD7 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 321 or 322, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 323.

[0136] In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 4394%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:335, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 336. In some embodiments, the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence NAWMS (SEQ ID NO:337), a CDR-H2 comprising an amino acid sequence RIKSKTDGGTTDYAAPVKG (SEQ ID NO:338), a CDR- H3 comprising an amino acid sequence TIEAVAGHFDY (SEQ ID NO:339), a CDR-L1 comprising an amino acid sequence RASQSISSWLA (SEQ ID NO:340), a CDR-L2 comprising an amino acid sequence KASSLES (SEQ ID NO:341), and a CDR-L3 comprising an amino acid sequence QQYNNYSPT (SEQ ID NO:342). In some embodiments, the CD7 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 332 or 333, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:334.

[0137] In some embodiments, the CD7 targeting moiety comprises a heavy chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO:346, and / or a light chain variable region comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 347. In some embodiments, the CD7 targeting moiety comprises a CDR-H1 comprising an amino acid sequence RY AMS (SEQ ID NO:348), a CDR-H2 comprising an amino acid sequence SISASGATTFYADPVKG (SEQ ID NO:349), a CDR-H3 comprising an amino acid sequence DQDFDILTGYLNWFDP (SEQ ID NO:350), a CDR-L1 comprising an amino acid sequence RVSQSVSSYLA (SEQ ID NO:351), a CDR-L2 comprising an amino acid sequence DTSNRAT (SEQ ID NO: 352), and a CDR-L3 comprising an amino acid sequence QQRRNWPLT (SEQ ID NO:353). In some embodiments, the CD7 targeting moiety is a Fab fragment comprising a first polypeptide comprising an amino acid sequence having at least 90% (e.g., at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 343 or 344, and a second polypeptide comprising an amino acid sequence having at least 90% (e.g., at44least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity to the amino acid sequence of SEQ ID NO: 345.CD28 Targeting Moieties

[0138] In some embodiments, the target molecule is CD28. In some embodiments, the target cell is CD28+. CD28 is a T-cell costimulatory molecule. It is a homodimeric glycoprotein member of the Ig gene superfamily and has a single IgV domain. It is expressed on T cells where it is activated upon binding to its ligands B7-1 or B7-2 (CD80 or CD86), which are expressed on professional antigen-presenting cells. CD28 does not affect T cell activation unless the T-cell receptor is first engaged by cognate antigen. Upon antigen recognition, CD28 signaling strongly amplifies T-cell receptor signaling to activate T cells, and CD28 co-stimulation of T cells increases glucose uptake and glycolysis during an immune response.

[0139] In some embodiments, the CD28 targeting moiety includes an antibody or antigenbinding fragment thereof that binds to CD28. In some embodiments, the CD28 targeting moiety is an antibody or antigen-binding fragment thereof (e.g., a Fab, Fab’, F(ab’)2, Fv fragment, scFv, DARPIN, VHH domain, FN3 domain, nanobody, single domain antibody, or Centyrin). In other embodiments, the CD28 targeting moiety includes a ligand, a folate moiety, an antibiotic mimetic, a polynucleotide (such as a DNA or RNA apatamer), a carbohydrate, a vitamin, a cytokine, or a chemokine. In some embodiments, the CD28 targeting moiety is an anti-CD28 antibody or antigen binding fragment thereof In some embodiments, the CD28 targeting moiety is an IgA, IgG, IgE, or IgM antibody. In some embodiments, the CD28 targeting moiety is a bispecific or multi-specific antibody or fragment thereof. In some embodiments, the CD28 targeting moiety is a humanized antibody or antigen-binding fragment thereof.

[0140] Exemplary anti-CD28 binders, antibodies, or antigen-binding fragments thereof include Theralizumab (i.e., TGN1412, TAB08, or CD28-SuperMAB, e.g., TeGenero), davoceticept (i.e., ALPN-202, e.g., Alpine Immune Sciences, Inc.), FPT155 (Five Prime Therapeutics, Inc.), 10F3, RM404, 15E8, CD28.3, Leu-2, 9.3, EX5.3D10, YTH913.12, S20013F, S20013B, and QA17A12, as well as anti-CD28 antibodies or antigen-binding fragments thereof disclosed in any of: US 7,175,843; US 8,168,759; US 8,785,138; US 8,785,604; US 10,273,281; US 11,117,949; US20180112000; US20230227530; US20230348600; US20230382972;WO1994029436; W02002051871; Tan et al. (2002) J Immunol 169:1119-1125; Elsyed et al. (2023) Mabs 15(l):2220839; etc., each hereby incorporated by reference in its entirety.45

[0141] In some embodiments, the CD28 targeting moiety is a CD28 receptor ligand. In some embodiments, the CD28 receptor ligand is CD80. Accordingly, in some embodiments, the CD28 targeting moiety is CD80. CD80 is a costimulatory molecule known for its role in T-cell activation and also in regulating the activity of normal and malignant B cells. Surface CD80 is expressed transiently on activated B cells, macrophages, and DCs. In certain embodiments, the CD28 targeting moiety is a CD80 extracellular domain (ECD), for example a CD80 ECD comprising an amino acid sequence of SEQ ID NO:365 or comprising at least about 80% (such as about any of 81%, 82%, 83%, 84%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO:365.Exemplary Dual Targeting Moieties

[0142] In some embodiments, the targeted LNP (conjugate) comprises two or more targeting moieties, wherein each targeting moiety independently binds to a T-cell antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CDS, CD28, CD80, CD137, CD45, T-cell receptor (TCR)-β ,TCR- α TCR-α / β, TCR-Υ / δ, PD1, CTLA4, TIM3, LAG3, CD 18, IL-2 receptor, CD 11 a, TLR2, TLR4, TLR5, IL- 7 receptor, and IL- 15 receptor. In some embodiments, the targeted LNP (conjugate) comprises two or more targeting moieties, wherein each targeting moiety independently targets a receptor on the surface of the T cell selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, and CD28. In some embodiments, the targeted LNP (conjugate) comprises two or more targeting moieties, wherein at least a first targeting moiety targets CD3, and wherein at least a second targeting moiety targets a receptor on the surface of the T cell is selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD7, and CD28. In some embodiments, the first targeting moiety comprises a CD3-binding domain comprising a VH comprising a CDR-H1, a CDR-H2, a CDR-H3 of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab, and a VL comprising a CDR-L1, a CDR-L2, and a CDR-L3 of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab. In some embodiments, the first targeting moiety comprises a CD3-binding domain comprising the VH and the VL of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab. Exemplary sequences for the first targeting moiety and / or the second targeting moiety can be found in tables provided below.46

[0143] In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD5. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD7. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD5 and a second targeting moiety binds to CD28. In some embodiments, the targeted LNP (conjugate) comprises two targeting moieties, wherein a first targeting moiety binds to CD7, and a second targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD). In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD3. In some embodiments, the first targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the second targeting moiety that binds to CD3.

[0144] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD5. In some embodiments, the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, and the second targeting moiety that binds to CD5 comprises a VH comprising the amino acid of SEQ ID NO:357, and a VL comprising the amino acid sequence of SEQ ID NO:358.

[0145] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD2. In some embodiments, the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, and the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO:269, and a VL comprising the amino acid sequence of SEQ ID NO:270. In some embodiments, the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the 47group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, and the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO: 280, and a VL comprising the amino acid sequence of SEQ ID NO: 281. In some embodiments, the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, and the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO:291, and a VL comprising the amino acid sequence of SEQ ID NO:292. In some embodiments, the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, and the second targeting moiety that binds to CD2 comprises a VH comprising the amino acid of SEQ ID NO: 301, and a VL comprising the amino acid sequence of SEQ ID NO: 292.

[0146] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD7. In some embodiments, the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, and the second targeting moiety that binds to CD7 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 313, 324, 335, and 346, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 314, 325, 336, and 347. In some embodiments, the first targeting moiety comprises a CD3-binding domain comprising the VH and the VL of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab, and the second targeting moiety comprises a CD7 -binding domain comprising the VH and the VL of grisnilimab.

[0147] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD28. In some embodiments the CD28 targeting moiety is a CD80 extracellular domain (ECD). In some embodiments, the first targeting moiety that binds to CD3 comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141, 152, 163, 174, 185, 196, 48207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, and the second targeting moiety that binds to CD28 comprises the amino acid sequence of SEQ ID NO:365. In some embodiments, the first targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO:218 and a VL comprising the amino acid sequence of SEQ ID NO:219, and the second targeting moiety that binds to CD28 comprises the amino acid sequence of SEQ ID NO: 365.

[0148] In some embodiments, a targeted LNP comprises two targeting moieties, wherein a first targeting moiety binds to CD3, and a second targeting moiety binds to CD3. In some embodiments, the first targeting moiety that binds to CD3 comprises a different anti-CD3 antibody, Fab fragment, or scFv than the second targeting moiety that binds to CD3. In some embodiments, the first targeting moiety and the second targeting moiety each independently comprises a CD3-binding domain comprising the VH and the VL of any one of mosunetuzumab, odronextamab, tebentafusp, teplizumab, teclistamab, visilizumab, muromonab, SP34, plamotamab, HPN536, pasotuxizumab, and flotetuzumab, wherein the first targeting moiety comprises a different a CD3-binding domain than the second targeting moiety. In some embodiments, the first targeting moiety and the second targeting moiety each independently comprises a CD3-binding domain comprising a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:141, 152, 163, 174, 185, 196, 207, 218, 228, 238, and 258, and a VL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 142, 153, 164, 175, 186, 197, 208, 219, 229, 239, 249, and 259, wherein the first targeting moiety comprises a different a CD3-binding domain than the second targeting moiety. In some embodiments, the first targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 196 and a VL comprising the amino acid sequence of SEQ ID NO:197, and the second targeting moiety comprises a VH comprising the amino acid sequence of SEQ ID NO:218 and a VL comprising the amino acid sequence of SEQ ID NO:219.B. Methods of Making Targeted LNPs (conjugates)

[0149] Different approaches can be used to introduce a targeting moiety onto the surface of an LNP. For example, one approach relies on functionalizing a preformed LNP with a targeting moiety. The LNP generally includes a lipid that has polyethylene glycol (PEG) spacer functionalized with a reactive moiety such as a thiol, amine, maleimide or carboxylic acid group.49The functionalized lipid of the LNP reacts with a complementary group that is covalently bonded to a targeting moiety, hence generating a conjugate of the LNP and the targeting moiety.

[0150] In some embodiments, the targeting moiety is conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some such embodiments, the targeting moiety is an antibody or antigen binding fragment thereof. In other such embodiments, the targeting moiety is a ScFv. In some embodiments, the targeting moiety is a Fab fragment.

[0151] In one embodiment, the Click product can be formed using a copper-catalyzed Click reaction. One such copper-catalyzed Click reaction is a Huisgen 1,3-dipolar cycloaddition (CuAAC) between an azide and an alkyne. In some embodiments, the first or second Click handle comprises a cyclic derivative of the alkynyl group. In some embodiments, the cyclic derivative of the alkynyl group is selected from dibenzocyclooctyne, cyclooctyne, and difluorinated cyclooctyne. In some embodiments, the click chemistry involves strain promoted cycloaddition of azides. In some embodiments, the click chemistry is based upon reaction of strained alkenes.

[0152] In another embodiment, the Click product can be formed using copper-free Click chemistry. For example, the Click product can be formed between an azide and dibenzocyclooctene (DBCO). Alternatively, the Click product can be formed using a Staudinger reaction between an azide and a phosphine, hence producing an aza-ylide.

[0153] In some embodiments, the Click product can be formed from an inverse electron demand Diels-Alder reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the first or second Click handle. In some embodiments, the first Click handle comprises a tetrazine (Tz) ring and the second Click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is unsubstituted. In some such embodiments, the tetrazine rung is methyltetrazine. In some embodiments, the tetrazine ring is a 6-methyl substituted tetrazine.

[0154] In another embodiment, the targeting moiety (e.g., antibody, Fab fragment or ScFv) is first selectively modified with an enzyme recognition sequence. An enzyme recognizing the enzyme recognition sequence can site-specifically introduce the first Click handle onto the targeting moiety through covalent attachment. The first Click handle can next react with the second Click handle on the LNP to produce the targeted LNP. Hence, in one embodiment, an 50antibody, Fab fragment or single chain variable fragment (ScFv) that is covalently linked to a first Click handle through a linker comprising an enzyme recognition sequence is reacted with an LNP comprising a second Click handle, thereby forming a Click reaction product that conjugates the antibody, Fab fragment or ScFv to the LNP. In some embodiments, the antibody Fab fragment or ScFv is directly bonded to the enzyme recognition sequence. In some embodiments, the targeting moiety (e.g., antibody Fab fragment or ScFv) is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C-terminus of the targeting moiety (e.g., antibody, Fab fragment or ScFv) include, but are not limited to (GGGGS)V(SEQ ID NO: 1), (G)v(SEQ ID NO: 5), (EAAAK)v(SEQ ID NO: 3), (PAPAP)v(SEQ ID NO: 4), (AP)v(SEQ ID NO: 6) and A(EAAAK)UALEA(EAAAK)vA(SEQ ID NO: 2), wherein u is 1-10 and v is 1-10.

[0155] In some embodiments, the enzyme recognition sequence is a sortase recognition motif or an LplA acceptor peptide. In some embodiments where the LNP is conjugated to an antibody, the C-terminus of one or more of the heavy or light chains of the antibody is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide) either directly or through a linker comprising one or more amino acid residues, a set forth herein. In some embodiments where the LNP is conjugated to a Fab fragment, the C-terminus of the heavy or light chain of the Fab fragment is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some embodiments where the LNP is conjugated to s ScFv, the C-terminus of the ScFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some of the foregoing embodiments, the first Click handle comprises a tetrazine ring or TCO moiety and the second Click handle comprises a tetrazine ring or TCO moiety. In some such embodiments, the first Click handle comprises a tetrazine ring and the second Click handle comprises a TCO moiety. In some embodiments, the tetrazine ring is a methyltetrazine. In some embodiments, the conjugation efficiency achieved by the disclosed method is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the conjugation efficiency achieved by the disclosed method is from about 60% to about 95%. In some embodiments, the conjugation efficiency achieved by the disclosed methods are from about 70% to about 85%.

[0156] In some embodiments, the linker further comprises a spacer between the targeting moiety and the Click product. The spacer can include additional functional groups that indirectly link the targeting moiety to the Tz group. The spacer may also include additional amino acid residues that indirectly links the targeting moiety to the Tz group.51

[0157] In some embodiments, the spacer that links the targeting moiety to the Tz ring is an enzyme recognition sequence. Accordingly, the disclosure provides methods of conjugating an LNP to a targeting moiety that has been modified with an enzyme recognition sequence, wherein said conjugating is accomplished via a Click reaction between a Tz ring covalently bound to the targeting moiety and a TCO moiety bound to the LNP. For instance, the disclosure provides methods of conjugating an LNP to an antibody, Fab fragment or single chain variable fragment (ScFv), wherein the antibody, Fab fragment or ScFv is covalently linked to a first Click handle (Tz ring) through a linker comprising an enzyme recognition sequence and the LNP is covalently linked to a second Click handle (TCO moiety), said method comprising contacting the LNP with an antibody, Fab fragment or ScFv such that first Click handle reacts with the second Click handle to form a Click reaction product (dihydropyridazine) that conjugates the antibody, Fab fragment or ScFv to the LNP. In some embodiments, the antibody, Fab fragment or ScFv is directly bonded to the enzyme recognition sequence. In some embodiments, the antibody Fab fragment or ScFv is bonded to the enzyme recognition sequence via one or more amino acid residues. Particular amino acid residues added that can be covalently attached to the C-terminus of the antibody, Fab fragment or ScFv include, but are not limited to (GGGGS)V(SEQ ID NO: 1), (G)v (SEQ ID NO: 5), (EAAAK)v (SEQ ID NO: 3), (PAPAP)v (SEQ ID NO: 4), (AP)v (SEQ ID NO: 6) and A(EAAAK)UALEA(EAAAK)vA (SEQ ID NO: 2), wherein u is 1-10 and v is 1- 10.

[0158] In some embodiments, the enzyme recognition sequence is a sortase recognition motif or a LplA acceptor peptide. In some embodiments where the LNP is conjugated to an antibody, the C-terminus of one or more of the heavy or light chains of the antibody is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide) either directly or through a linker comprising one or more amino acid residues, a set forth herein. In some embodiments where the LNP is conjugated to a Fab fragment, the C-terminus of the heavy or light chain of the Fab fragment is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some embodiments where the LNP is conjugated to s ScFv, the C-terminus of the ScFv is covalently bonded to the enzyme recognition sequence (e.g., sortase recognition motif or LplA acceptor peptide). In some embodiments, the conjugation efficiency achieved by the disclosed method is greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the conjugation efficiency achieved by the disclosed method is from about 60% to about 95%. In some embodiments, the conjugation efficiency achieved by the disclosed methods are from about 70% to about 85%.52

[0159] In some embodiments, the conjugate produced by methods disclosed herein comprises a protein targeting moiety as set forth above (e.g., antibody, Fab fragment or ScFv), conjugated to the lipid nanoparticle through a linker, and wherein the linker comprises a lipoic acid ligase (LplA) acceptor peptide and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNP. In some embodiments, the linker further comprises one or more additional amino acid residues between the protein targeting moiety (e.g., antibody, Fab fragment or ScFv) and the LplA acceptor peptide. In some embodiments, the LplA acceptor peptide has the sequence GFEDKVWYDLDA (SEQ ID NO: 577). In some embodiments, the conjugate comprises an antibody, wherein the C-terminus of one or more of the heavy or light chains of the antibody is bonded to the linker. In some embodiments, the conjugate comprises a Fab fragment, the C-terminus of the heavy or light chain of the Fab fragment is bonded to the linker. In some embodiments, the conjugate comprises a ScFv, wherein the C-terminus of the ScFv is bonded to the linker.

[0160] In some embodiments, the linker comprises additional amino acid residues between the targeting moiety (e.g., antibody, Fab fragment or ScFv) and the LplA acceptor peptide. In such embodiments, a C -terminus of the targeting moiety (e.g., antibody, Fab Fragment or ScFv) can be covalently modified with one or more amino acid residues prior to covalently linking the LplA acceptor peptide. For instance, in particular embodiments, the conjugates have the structure targeting moiety -Z-LplA acceptor peptide-Click product-LNP (e.g., Antibody -Z- LplA acceptor peptide -Click product-LNP, Fab fragment-Z- LplA acceptor peptide -Click product- LNP, or ScFv-Z- LplA acceptor peptide -Click product-LNP), wherein Z is a linker between the antibody (or Fab fragment or ScFv) and the glycine residue of the LplA acceptor peptide. In some embodiments, Z comprises one or more amino acid residues. In some embodiments Z is (GGGGS)V(SEQ ID NO: 1), (G)v(SEQ ID NO: 5), (EAAAK)v (SEQ ID NO: 3), (PAPAP)V(SEQ ID NO: 4), (AP)v (SEQ ID NO: 6) and A(EAAAK)UALEA(EAAAK)vA (SEQ ID NO: 2), wherein u is 1-10 and v is 1-10. In some embodiments, Z is GG, GGG, GGGG (SEQ ID NO: 114), GGGGG (SEQ ID NO: 115), GGGGGG (SEQ ID NO: 116), and GGGGGGG (SEQ ID NO: 117) or GGGGGS (SEQ ID NO: 138).

[0161] It will be understood that in the forgoing embodiments, the lysine (K) residue of the LplA acceptor peptide is covalently linked to Click product, which is covalently linked to the LNP. Specifically, to generate conjugates, the side chain lysyl group reacts with the carboxylic acid compound that includes the first Click handle. The resultant modified targeting moieties (antibodies or Fab fragments or ScFvs) are reacted with an LNP that has been modified with a second Click handle, as disclosed herein, thereby generating a Click product. An LNP surface 53modified with a Fab fragment is depicted below, where R is a lipid group (e.g., C2-C30 alkyl group).

[0162] In other embodiments, the enzyme recognition sequence is a transglutaminase enzyme recognition sequence (LLQG). The transglutaminase enzyme recognition sequence (LLQG) is also referred to as Q-tag. The Q-tag may be present on or can be inserted at one or more locations of targeting moiety, (e.g., antibody, Fab fragment or ScFv), for instance at a C- terminus. The transglutamine enzyme catalyzes the reaction between a side-chain amide group on the Q-tag and an alkyl-primary amine on a component of the LNP (e.g., a lipid), thus linking the antibody to the LNP through an amide bond.

[0163] In other embodiments, the enzyme recognition sequence is a sequence recognized by formylglycine generating enzyme, specifically CXPXR, wherein each X is any amino acid. In such embodiments, the CXPXR sequence can be inserted at one or more locations of the targeting moiety (e.g., antibody, Fab fragment or ScFv), for instance at a C -terminus. The formylglycine generating enzyme converts the cysteine thiol of CXPXR into an aldehyde group, which can be reacted with an aminooxy or hydrazine group covalently bonded to a component of the LNP.

[0164] In some embodiments, the Click product can be formed using any suitable photo-induced Click chemistry reaction. In some embodiments, the Click product can be formed using photoinducible 1,3-dipolar cycloaddition reaction between a tetrazole and an alkene (see, e.g., Song et ak.,Angew. Chem., Int. Ed. 2008, 47 (15), 2832-2835).

[0165] In some embodiments, the Click product can be formed using oxime and hydrazone ligations. In some embodiments, a ketone or aldehyde can react with a effect amine, such as hydroxylamine, hydrazine and hydrazide (see, e.g., Agten et al., ChemBioChem 2013, 14 (18), 2431-2434 and Dirksen et al., J. Am. Chem. Soc. 2006, 128 (49), 15602-15603).

[0166] In some embodiments, the conjugate can comprise more than 10 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise more than 20 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise more than 30 targeting moieties (e.g., antibodies, Fab fragments or ScFvs). In some embodiments, the conjugate can comprise more than 50 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise more than 75 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise more than 100 54targeting moieties (e.g., antibodies, Fab fragments or ScFvs). In some embodiments, the conjugate can comprise from about 50 to about 200 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 targeting moieties (e.g., antibodies, Fab fragments or ScFvs) per LNP.

[0167] In some embodiments, the weight ratio between a targeting moiety on the surface of the LNP and the payload (e.g., RNA) encapsulated in the LNP can be about 1:20, about 1:15, about 1:10, about 1:9, about 1:8, about 1:7. about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1.Interchain CL.-CHI disulfide reduction followed by conjugation

[0168] IgG antibodies consist of four polypeptide chains linked by disulfide bonds. The two polypeptide chains of low molecular weight are call light chains (L). The light chains consist of a variable light chain domain (VL) and a constant light chain domain (CL). The heavy chains consist of a variable heavy light domain (VH) and three constant heavy chain domains (CHI, CH2, and CH3). The Fab region of the antibody includes the VL, CL, VH, and CHI domains. The Fc region includes the constant heavy chain domains CH2, and CH3. A hinge region of the IgG antibody covalently links the CHI domain to the CH2 domain. The two heavy chains of IgG antibodies are connected in the hinge region by a variable number of disulfide bonds depending on the IgG subclass. Different subclasses of IgG antibodies have varying numbers of interchain disulfide bonds. Additionally, the light chain is covalently linked to the heavy chain via a disulfide bond between the light chain and the heavy chain. Using standard IgG nomenclature, this natural interchain disulfide bond is also referred to as the CL-CH1 disulfide bond to distinguish it from disulfide bonds present in the hinge region. Therapeutic antibodies of type IgGl possess an intermolecular disulfide bond between Cys233 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain. Therapeutic antibodies of type IgG4 possess an intermolecular disulfide bond between Cysl27 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain. Therapeutic antibodies of55type IgG2 possess an intermolecular disulfide bond between Cysl35 (Kabat numbering) of the heavy domain and Cys214 (Kabat numbering) of the light domain.

[0169] Proteolytic cleavage of an IgG antibody results in the formation of a Fab fragment known as a F(ab’)2 fragment. The F(ab’)2 fragment does not include the CH2 domain or the CH3 domain. However, the hinge region of the antibody is retained in a F(ab’)2 fragment. The F(ab’)2 fragment includes disulfide bonds that covalently link two Fab fragments. Reduction of the disulfide bond in the F(ab’)2 generates two F(ab’) fragments. The sulfhydryl (thiol) groups of the F(ab’) could potentially react with a thiol-reactive group on the surface of an LNP, hence generating a conjugate. However, owing to the presence of multiple sulfhydryl groups in the hinge region of the F(ab’) fragment, site-specific conjugation is challenging. Moreover, the reduction of the F(ab’)2 to the F(ab’) fragments could also disrupt the natural interchain disulfide bonds between the CL and CHI regions of the Fab fragments, hence further compromising sitespecific conjugation.

[0170] As set forth herein, Fab fragment can be site-selectively conjugated to the surface of a precursor lipid nanoparticle (LNP) through the natural interchain disulfide bond between the heavy chain and the light chain (i.e., the CL-CH1 disulfide bond) of the Fab fragment to make a targeted LNP (conjugate).

[0171] The term “precursor LNP” or “base LNP” refers to an LNP that has been functionalized with a reactive moiety (e.g., thiol-reactive group or polyglycine) prior to reacting with the Fab fragment. The process for conjugating a targeting moiety, as disclosed herein, involves reducing the natural interchain disulfide bond between the CL and CHI domains of a Fab fragment, and reacting the reduced Fab fragment with a thiol-reactive group (e.g., a maleimide or DBM group) covalently bonded to the surface of a precursor LNP, thus forming a conjugate. Alternatively, the reduced Fab fragment can be reacted with a lipid that has been chemically modified (functionalized) with a thiol-reactive group (e.g., maleimide or DBM group). The resultant lipid can then be inserted into a preexisting LNP, thus generating a conjugate. As described herein, despite the removal of the natural interchain disulfide bond linking the heavy and light chains of the Fab fragment, the resulting conjugates are able to effectively target specific cell types depending on the nature of the Fab targeting moiety. For instance, specific Fab fragments for targeting immune cells or hematopoietic stem cells (HSCs) as disclosed herein. A schematic of an LNP site-specifically conjugated to a Fab fragment is shown in FIG. 25.56

[0172] In one embodiment, Fab fragments used for conjugation may be used by recombinant methods. In particular embodiments, the Fab fragments generated recombinantly are designed not to include a hinge region at the C -terminus. Therefore, the recombinantly generated Fab fragments include only one disulfide bond between the CL-CH1 and domains. As set forth herein, the CL-CH1 can then be reduced and the resultant free thiol groups can be used as anchors to conjugate the Fab fragment to the surface of an LNP.

[0173] In some embodiments, the Fab fragment is of the IgG class, the IgM class, or the IgA class. In some embodiments, the Fab fragment is of the IgG class and has an IgGl, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fab fragment is a native protein. In some embodiments, the Fab fragment is an engineered protein.

[0174] In one aspect, the disclosure provides methods of making a targeted LNP, said method comprising:(i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chain domain 1 (CHI), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and(ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.

[0175] In some aspects, the disclosure provides a conjugate produced by a method comprising:(i) contacting a composition comprising a Fab fragment with a reducing reagent, wherein the Fab fragment comprises a heavy chain and a light chain and an interchain disulfide bond linking the constant light chain domain (CL) and the constant heavy chain domain 1 (CHI), whereby the reducing reagent reduces the interchain disulfide bond of the Fab fragment to generate two free cysteine residues; and(ii) contacting the product of step (i) with a precursor LNP comprising a plurality of thiol-reactive groups covalently bonded to one or more lipids of the precursor LNP, thereby forming a targeted LNP.

[0176] In some embodiments, the thiol-reactive group (e.g., maleimide, pyridyl disulfide, 2,3- dibromomaleimide, or haloacetyl) is chemically reacted with a lipid molecule to create a57modified lipid wherein the thiol-reactive group is covalently attached to the lipid where it is capable of reacting with at least one free cysteine residue of the reduced Fab fragment (either on the heavy or light chain of the Fab fragment). The reaction between the thiol-reactive group and the at least one free cysteine residue can be completed prior to or after formation of the LNP with the modified lipid. For instance, the various components (e.g., lipids) comprising the LNP and a therapeutic payload can be mixed with lipid molecules, including one or more lipids that comprise a thiol-reactive group, thus generating an LNP that comprises a plurality of thiolreactive groups. The thiol-reactive group can then be reacted with at least one free cysteine residue of the Fab fragment, hence generating a conjugate. Alternatively, a lipid that has been modified with the thiol-reactive group can be directly reacted with at least one free cysteine residue of a Fab fragment. The resultant modified lipid attached to the Fab fragment can then be inserted into a pre-formed LNP that has not yet been surface modified. This procedure allows for the reaction to be performed on an individual lipid molecule rather than on the surface of the LNP.

[0177] Any suitable reducing reagent can be used to reduce the interchain disulfide bond of the Fab fragment. Examples of reducing reagents include, but are not limited to, 2-mercaptoethanol, 2-mercaptoethylamine, dithiothreitol (DTT), dithioerythritol (DTE), and tris(carboxyethyl)phosphine (TCEP), and combinations thereof. In some embodiments, the reducing reagent is a mild reducing reagent. Examples of mild reducing reagents include, e.g., DTT, TCEP, and DTE. In some embodiments, the reducing reagent is TCEP. Any suitable reaction conditions can be used for the reduction of the interchain disulfide bond in step (i). In some embodiments, the reduction reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reduction reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reduction reaction is performed at physiological temperature (e.g. , about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reduction reaction is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the reduction reaction is performed at about 0° C to about 4° C.

[0178] In some embodiments, excess reducing agent is removed following step (i), prior to conjugation to the precursor LNP. In some embodiments, excess reducing agent is not removed following step (i), prior to conjugation to the precursor LNP.58

[0179] In some embodiments of the method or process, the thiol-reactive groups on the LNP (or lipid to be post-inserted into an LNP) comprises any suitable reactive group, including but not limited to, maleimide, pyridyl disulfide, 2,3-dibromomaleimide, or haloacetyl.

[0180] In some embodiments, the thiol-reactive group is maleimide. In some embodiments, maleimide reacts with one of the two free cysteine residues of the Fab fragment (either on the heavy or light chain) to form a thiosuccinimide moiety. In some embodiments, maleimide reacts with a free cysteine residue on the heavy chain of the Fab fragment. In some embodiments, maleimide reacts with a free cysteine residue on the light chain of the Fab fragment. In some embodiments, two maleimide groups each react with the Fab fragment, wherein one maleimide reacts with a free cysteine residue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.

[0181] Any suitable conditions can be used for the reaction between the thiol-reactive group and at least one of the two free cysteine residues of the Fab fragment in step (ii). In some embodiments, the reaction can occur in water, aqueous buffer, or cell culture media. In some embodiments, the reaction is performed at physiological pH (e.g., about 7.4). In some embodiments, the reaction is performed at physiological temperature (e.g., about 37° C). In some embodiments, the reduction reaction is performed between 0°C and 40°C, e.g., between 10°C and 35°C, between 15°C and 30°C, between 20°C and 30°C, or between 20°C and 25°C. In some embodiments, the reaction is performed at ambient temperature (e.g., about 23 to about 25° C). In some embodiments, the reaction is performed at about 0° C to about 4° C.

[0182] In some embodiments, a Fab fragment comprising an interchain disulfide bond between the heavy and light chain is contacted with a reducing reagent, whereby the reducing reagent reduces the interchain disulfide to generate two free cysteine residues (step (i)). In step (ii), the reduced Fab fragment is reacted with an LNP comprising a plurality of thiol-reactive groups (e.g., maleimide or DBM) conjugated to the surface of the LNP, whereby the thiol-reactive groups react with the free cysteine residues of the reduced Fab fragment. The Fab fragment is site-specifically conjugated to the surface of the LNP via a linkage through at least one of the free cysteine residues of the Fab fragment.

[0183] Following reaction of the reduced Fab fragment with the thiol-reactive group of the LNP, either the heavy chain, light chain or both the heavy chain and light chain of the Fab fragment are conjugated to the surface of the LNP The concentration of the thiol-reactive group (e.g., maleimide) will likely determine which orientation is dominant. In some embodiments,59increasing the number of thiol-reactive groups on the LNP increases the number of Fab fragments conjugated to two thiol-reactive groups. In some embodiments, decreasing the number of thiol-reactive groups on the LNP decreases the number of Fab fragments conjugated to two thiol-reactive groups. Regardless of the orientation, the heavy chain and the light chain remain intact on the surface of the LNP, thus forming a functional Fab fragment that is capable of engaging with a receptor on a targeted cell.

[0184] In some embodiments, maleimide reacts with one of the two free cysteine residues of the antibody or antigen-binding fragment thereof. In some embodiments, maleimide reacts with one of the two free cysteine residues of the Fab fragment to form a thiosuccinimide moiety. In some embodiments, maleimide reacts with a free cysteine residue on the heavy chain of the Fab fragment. In some embodiments, maleimide reacts with a free cysteine residue on the light chain of the Fab fragment. In some embodiments, two maleimide groups on the LNP each react with the Fab fragment, wherein one maleimide reacts with a free cysteine residue on the light chain and the other maleimide reacts with a free cysteine residue on the heavy chain.

[0185] In some embodiments, the thiol-reactive group is 2,3-dibromomaleimide (DBM). Following reduction of the disulfide bond, the reduced Fab fragment is added to DBM covalently bonded to a lipid. As set forth above, the lipid may be part of an LNP or may be post-inserted into an LNP following reaction with the Fab fragment. Both of the free cysteine residues displace the two bromine groups of DBM, hence generating a dithiomalemide. The dithiolmalemide can be converted to the corresponding maleamic acid via hydrolysis. DBM reacts with a free cysteine residue on the heavy chain and a free cysteine residue on the light chain of the Fab fragment to form a bridge between the cysteine residues. Accordingly, the heavy and light chain of the Fab fragment are effectively bridged together following reaction with DBM.

[0186] In all embodiments discussed above, the thiol-reactive group can be introduced onto any of the lipids comprising the LNP. In some embodiments, the conjugate can comprise one or more pegylated lipid molecules. In some embodiments, the thiol-reactive group is covalently bonded to at least one of the pegylated lipid molecules, hence generating the structure Lipid- PEGx-thiol-reactive group, wherein x is 2-120 ethylene glycol units. In such embodiments, at least one free cysteine residue of an Fab fragment reacts with the thiol-reactive group bonded to the one or more of the pegylated lipids comprising the LNP. In some embodiments, the LNP comprises from about 0.05 mol % to about 2 mol % of the pegylated lipid bonded to the thiolreactive group. In some embodiments, the PEG spacer between the lipid and the thiol-reactive60group comprises at least about 5, 10, 20, 30, 50, 50, 60, 70, 80, 90, 200, or 110 ethylene glycol units. In some embodiments, the PEG spacer comprises about 10-120 ethylene glycol units. In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 500 (i.e., PEG500) to about 5,000 (i.e., PEG5000). In some embodiments, the molecular weight of the pegylated lipid bonded to the thiol-reactive group is from about 1,000 (i.e., PEG1000) to about 3,000 (i.e., PEG5300). In some embodiments, the thiol-reactive group is bonded to at least one of the non-pegylated phospholipids comprising the LNP. In some embodiments, the thiol-reactive group is bonded to at least one of the ionizable lipids comprising the LNP. In some embodiments, the thiol-reactive group is bonded to at least one of the sterol molecules comprising the LNP. In some embodiments, the lipid portion of the pegylated lipid bonded to the thiol-reactive group is selected from DMG, DPG, DSG, DTA, DOPE, DPPE, DMPE, DSPE, sphingosine, sphingomyelin, and stearic acid.

[0187] The disclosed methods provide stable conjugates that display excellent ability to transduce specific targeted cells. In some aspects, the disclosure provides a conjugate comprising an LNP and a Fab fragment, wherein the LNP is covalently bonded to either or both a first cysteine residue in the constant region of the heavy chain of the Fab fragment and a second cysteine residue in the constant region of light chain of the Fab fragment. In some embodiments, the Fab fragment does not comprise a disulfide bond linking the constant region of the heavy chain of the Fab fragment and the constant region of the light chain of the Fab fragment. In some embodiments, both the constant region of the heavy chain constant region of the heavy chain and the constant region of the light chain of the Fab fragment are covalently bonded to the LNP. In some embodiments, only the constant region of the heavy chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the light chain remains associated with the covalently bond heavy chain on the surface of the LNP. In some embodiments, only the constant region of the light chain of the Fab fragment is covalently bonded to the LNP. In some such embodiments, the heavy chain remains associated with the covalently bond light chain on the surface of the LNP. In some of the foregoing embodiments, the Fab fragment is linked to the LNP through a thiosuccinimide moiety. In other of the foregoing embodiments, the Fab fragment is linked to the LNP through a dithiomalemide moiety. In still other of the foregoing embodiments, the Fab fragment is linked to the LNP through a maleamic acid moiety.

[0188] In some embodiments, the Fab fragment conjugated to the LNP is an IgGl Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment is covalently bonded to the LNP. In other such embodiments, the61cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 233 (Kabat numbering) of the heavy chain of the IgGl Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgGl Fab fragment are covalently bonded to the LNP.

[0189] In some embodiments, the Fab fragment conjugated to the LNP is an IgG2 Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment is covalently bonded to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG2 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG2 Fab fragment are covalently bonded to the LNP.

[0190] In some embodiments, the Fab fragment conjugated to the LNP is an IgG4 Fab fragment. In some such embodiments, the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment is covalently to the LNP. In other such embodiments, the cysteine at position 127 (Kabat numbering) of the heavy chain of the IgG4 Fab fragment and the cysteine at position 214 (Kabat numbering) of the light chain of the IgG4 Fab fragment is covalently to the LNP.

[0191] The processes described herein also enable the ability to conjugate two or more different Fab fragments to the surface of an LNP. In some embodiments, two Fab fragments (Fabl and Fab2) are reduced (step (i)) and reacted (step (ii)) with a precursor LNP comprising a thiolreactive group (e.g., maleimide or DBM). Following the reaction in step (ii), both Fabl and Fab2 are conjugated to the surface of the LNP. Despite reduction of the disulfide bonds in Fabl and Fab2, the heavy chain and light chain in Fabl and the heavy and light chain in Fab2 remain together on the surface of the LNP. In other words, neither the heavy chain or light chain of Fabl associate with the heavy or light chain of Fab2 on the surface of the LNP.

[0192] In some embodiments involving conjugating two Fab fragments (i.e., a first Fab fragment and a second Fab fragment) to the surface of an LNP, the first Fab fragment and the second Fab fragment can be reduced in the same reaction (e.g., the first and second Fab fragments are mixed in a reaction vessel and contacted with the same reducing reagent). In some embodiments, the first Fab fragment and the second Fab fragment are reduced separately (e.g., the first and second Fab fragments thereof are each contacted with a reducing reagent in separate reaction vessels).62In some embodiments, the first Fab fragment is contacted with the reducing reagent prior to step (ii) (wherein the reduced first Fab fragment is conjugated to the LNP surface). In some embodiments, the second Fab fragment is contacted with the reducing reagent after step (ii) (wherein the reduced second Fab fragment is conjugated to the LNP surface). In some embodiments, the reduced first Fab fragment and the reduced second Fab fragment are contacted with the LNP simultaneously. In some embodiments, the reduced first Fab fragment thereof and the reduced second Fab fragment are contacted with the LNP sequentially (in either order). It can be contemplated that any number of Fab fragments thereof can be implemented in the method or process (e.g., a third, fourth, fifth, etc. Fab fragment). In some embodiments, a total of three different Fab fragments can be conjugated to the surface of the LNP. In some embodiments, a total of four different Fab fragments can be conjugated to the surface of the LNP.

[0193] In some embodiments, the reaction between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP forms at least one covalent bond. In some embodiments, the formation of at least one covalent bond between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is reversible. In some embodiments, the formation of at least one covalent bond between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is irreversible. In some embodiments, the reaction efficiency between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is greater than 5%, greater than 10%, greater than 25%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. In some embodiments, the reaction efficiency between at least one of the two free cysteine residues of the Fab fragment and the thiol-reactive group of the LNP is from about 5% to about 30%, about 10% to about 20%, about 25% to about 50%, about 30% to about 40%, about 50% to about 80%, about 60% to about 70%, about 70% to about 95%, or about 80% to about 90%. In some embodiments, the conjugate product of the disclosed method can be purified from remaining intermediate product using any suitable technique such as, but not limited to, ultrafiltration and diafiltration.

[0194] In some embodiments, conjugates prepared by the method or process disclosed herein have a high density of the Fab fragment on the surface of the LNP. For instance, the conjugate can comprise a plurality of Fab fragments conjugated to the LNP surface. In some embodiments, the conjugate can comprise more than 10 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 20 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 30 Fab fragments. In some embodiments, the conjugate can 63comprise more than 50 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 75 Fab fragments per LNP. In some embodiments, the conjugate can comprise more than 100 Fab fragments. In some embodiments, the conjugate can comprise from about 50 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 200 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 100 to about 230 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 150 Fab fragments per LNP. In some embodiments, the conjugate can comprise from about 10 to about 30 Fab fragments per LNC. Lipid Nanoparticles (LNP)

[0195] Lipid nanoparticles, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids), also referred to herein as helper lipids; one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.

[0196] Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example, those described in Table 4 of WO2019217941, which is incorporated by reference — e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in table 5 of WO2019217941, incorporated by reference.

[0197] In some embodiments, conjugated lipids, when present, can include one or more of PEG- diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypoly ethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.

[0198] In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in W02009 / 127060 or US2010 / 0130588, which are incorporated by reference. Additional exemplary sterols include64phytosterols, including those described in Eygeris et al (2020), dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0199] In some embodiments, the lipid particle comprises an ionizable lipid, anon-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 35 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid (e.g., comprising the therapeutic agent and / or encoding the gene modifying polypeptide, template nucleic acid, or gene modifying system) can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10: 1 to about 30: 1.

[0200] In some embodiments, the average LNP diameter of the targeted LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the targeted LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the targeted LNP formulation ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 2065mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.

[0201] An LNP described herein, e.g., a targeted LNP, may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a poly dispersity index from about 0 to about 0.25, such as about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.10, about 0.11, about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, or about 0.25. hi some embodiments, the poly dispersity index of a LNP may be from about 0.10 to about 0.20.

[0202] The zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of a LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV, from about -5 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0203] The efficiency of encapsulation of a protein and / or nucleic acid (e.g., an mRNA encoding a polypeptide), describes the amount of protein and / or nucleic acid that is encapsulated or otherwise associated with an LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and / or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and / or nucleic acid may66be at least about 50%, for example about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In some embodiments, the encapsulation efficiency may be at least about 80%. In some embodiments, the encapsulation efficiency may be at least about 90%. In some embodiments, the encapsulation efficiency may be at least about 95%.

[0204] An LNP of the disclosure may optionally comprise one or more coatings. In some embodiments, an LNP may be formulated in a capsule, film, or tablet having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density.

[0205] Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by W02020061457, which is incorporated herein by reference in its entirety.

[0206] In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Minis Bio). In certain embodiments, LNPs are formulated using the GenVoy ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, targeted LNPs of the disclosure are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.

[0207] Additional specific LNP formulations useful for delivery of nucleic acids are described in US8158601 and US8168775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.Ionizable Lipids

[0208] The LNPs of the disclosure (e.g., targeted LNPs) comprise one or more ionizable lipids. In some embodiments, an ionizable lipid may be a cationic lipid, an ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s), which bear the positive charge. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of 67neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid, and a therapeutic agent as described herein (e.g., one or more nucleic acids (e.g., RNA) comprising a gene modifying system) encapsulated within or associated with the lipid nanoparticle. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) is co-formulated with the cationic lipid. The therapeutic agent (e.g., one or more nucleic acids) may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the therapeutic agent (e.g., one or more nucleic acids) may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described hereinencapsulates at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 92%, at least about 95%, at least about 97%, at least about 98% or about 100% of an RNA molecule (e.g., an mRNA molecule).

[0209] In some embodiments, the lipid to nucleic acid ratio (mass / mass ratio; w / w ratio) can be in the range of from about 1 : 1 to about 25: 1, from about 10: 1 to about 14: 1, from about 3 : 1 to about 15: 1, from about 4: 1 to about 10: 1, from about 5: 1 to about 9: 1, or about 6: 1 to about 9: 1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N / P ratio, for example, N / P ratio of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or higher. Generally, the lipid nanoparticle formulation’s overall lipid content can range from about 5 mg / ml to about 30 mg / mL.

[0210] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid V003, depicted below. V003 is described in U.S. Patent No. 10,059,655.68Lipid V003

[0211] In some embodiments, the LNP (e.g., targeted LNP) comprises the ionizable lipid shown in Table LI. In some cases, an LNP containing an ionizable lipid of Table LI exhibits higher levels of transduction in immune cells (e.g., T cells)and / or higher expression of a payload protein in immune cells (e.g., T cells) relative to an LNP that contains V003 as the ionizable lipid. In other embodiments, Lipid 092 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 093 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 153 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipidl54 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 155 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 162 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipidl63 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 169 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 176 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 178 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells). In other embodiments, Lipid 183 is used as an ionizable lipid to generate LNPs for delivery to immune cells (e.g., T cells).Table LI: Exemplary Ionizable Lipids LipidStructureID69

[0212] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid having a structure of formula (IV):or a pharmaceutically acceptable salt thereof, wherein:X is -O- or -CH2-; m is 0, 1, 2, or 3;R1is C1-4alkyl;R2is C1-4alkyl; n is 1, 2, 3, or 4;R3is C4-1oalkyl;R4is C4-10alkyl; p is 2, 3, 4, 5, or 6;R5is C4-10alkyl; andR6is C4-10alkyl.

[0213] In some embodiments, compounds of formula (IV) are compounds of formula (IV-A): o ON O.O' ‘O'O.‘O O o ‘0 pR5R6(IV-A), or a pharmaceutically acceptable salt thereof, wherein: p is 2, 3, 4, 5, or 6;77R5is C4-10alkyl; andR6is C4-10alkyl.

[0214] In some embodiments, compounds of formula (IV) are compounds of formula (IV-B):O.0. R5o R6(IV-B), or a pharmaceutically acceptable salt thereof, wherein:R5is C4-6alkyl; andR6is C4-6alkyl.

[0215] In some embodiments, the compound of formula (I) is a compound selected from the exemplary compounds of Table L3.Table L3: Exemplary Ionizable LipidsCompound Structure78

[0216] In some embodiments, the ionizable lipid has one of the structures depicted below:Lipid 15480Lipid 1638182Lipid 183 o o o o oO'.0. oLipid 232

[0217] Other exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016 / 0311759; I of US20150376115 or in US2016 / 0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013 / 0178541; I of US2013 / 0303587 or US2013 / 0123338; I of US2015 / 0141678; II, III, IV, or V of US2015 / 0239926; I of US2017 / 0119904; I or II of WO2017 / 117528; A of US2012 / 0149894; A of US2015 / 0057373; A of WO2013 / 116126; A of US2013 / 0090372; A of US2013 / 0274523; A of US2013 / 0274504; A of US2013 / 0053572; A of W02013 / 016058; A of W02012 / 162210; I of US2008 / 042973; I, II, III, or IV of US2012 / 01287670; I or II of US2014 / 0200257; I, II, or III of US2015 / 0203446; I or III of US2015 / 0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014 / 0308304; of US2013 / 0338210; I, II, III, or IV of W02009 / 132131; A of US2012 / 01011478; I or XXXV of US2012 / 0027796; XIV or XVII of US2012 / 0058144; of US2013 / 0323269; I of US2011 / 0117125; I, II, or III of US2011 / 0256175;83I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012 / 0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011 / 0076335; I or II of US2006 / 008378; I of US2013 / 0123338; I or X-A-Y-Z of US2015 / 0064242; XVI, XVII, or XVIII of US2013 / 0022649; I, II, or III of US2013 / 0116307; I, II, or III of US2013 / 0116307; I or II of US2010 / 0062967; I-X of US2013 / 0189351; I of US2014 / 0039032; V of US2018 / 0028664; I of US2016 / 0317458; I of US2013 / 0195920; 5, 6, or 10 of US10,221,127; III-3 of WO2018 / 081480; 1-5 or 1-8 of W02020 / 081938; 18 or 25 of US9,867,888; A of US2019 / 0136231; II of W02020 / 219876; 1 of US2012 / 0027803; OF-02 of US2019 / 0240349; 23 of US10,086,013;CKK-E12 / A6 of Miao et al (2020); C12-200 of WO2010 / 053572; 7C1 of Dahlman et al (2017); 304-013 or 503-013 of Whitehead et al; TS-P4C2 of US9,708,628; I of W02020 / 106946; I of W02020 / 106946.

[0218] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3 lZ)-heptatriaconta- 6,9,28,3 l-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3- nonyldocosa-13, 16-dien-l-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of US9,867,888(incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of W02015 / 095340(incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-l-yl) 9-((4- dimethylamino)butanoyl)oxy)heptadecanedioate (L319), , e.g. as synthesized in Example 7, 8, or 9 of US2012 / 0027803(incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is l,l'-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxy dodecyl) amino)ethyl)piperazin-l-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of W02010 / 053572(incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is; Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17- ((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,8417-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl 3-(lH-imidazol-4-yl)propanoate, e.g., Structure (I) from W02020 / 106946 (incorporated by reference herein in its entirety).

[0219] In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 25% to about 65%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 35% to about 60%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 40% to about 50%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 45% to about 50%. %. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 25% to about 40%. In some embodiments, the mol% of the ionizable lipid in the LNP, e.g., targeted LNP, is from about 15% to about 30%.

[0220] In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 40%. In some of the foregoing embodiments, the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).

[0221] In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 25% to about 35%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 25% to about 35%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP85is from about 25% to about 35%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 25% to about 35%. In some of the foregoing embodiments, the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).

[0222] In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%). In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 20% to about 30% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%m about 34% or about 35%). In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 25% to about 40% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%). In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 28% to about 32% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 30% to about 35% (e.g., about 31%, about 32%, about 33%, about 34% or about 35%). In some of the foregoing embodiments, the mol% of cholesterol in the targeted LNP is from about 30%-40% or from about 32% to about 37% (e.g., about 33%, about 34%, about 35% or about 36%).

[0223] In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 60% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 60% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 30% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 50% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the ionizable lipid in the targeted LNP is from about 35% to about 50% and the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 30% to about 40%. In some of the foregoing embodiments, the mol% of cholesterol in the targeted LNP is from about 25%-40%,

[0224] The compounds disclosed herein (e.g., lipids in Table LI, Table L3), or their pharmaceutically acceptable salts, may include an asymmetric center and may thus give rise to86enantiomers, diastereomers, and other stereoisomeric forms. The present disclosure contemplates various stereoisomers and mixtures thereof and includes “enantiomers,” which refers to two stereoisomers whose molecules are nonsuperimposeable mirror images of one another and “diastereomers,” which refers to stereoisomers that have at least two asymmetric atoms, but which are not mirror-images of each other. The present disclosure is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), or (7?)- and (S)- isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation / isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers.Helper Lipids

[0225] The LNPs, e.g., targeted LNPs, of the disclosure comprise one or more ionizable lipids. Exemplary helper lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), 1,2- dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane- 1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, l-stearoyl-2- oleoyl- phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine, egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine,87dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10- C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org / 10.1021 / acs.nanolett.0c01386, incorporated herein by reference.

[0226] Other examples of non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017 / 099823 or US patent publication US2018 / 0028664, the contents of which is incorporated herein by reference in their entirety. In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018 / 0028664, incorporated herein by reference in its entirety.

[0227] In some embodiments, the helper lipid is a sphingolipid. In some such embodiments, the non-pegylated lipid is a sphingomyelin. In some embodiments, the sphingomyelin has a head group selected from, phosphocholine, phosphoethanolamine or ceramide. In some embodiments, the sphingomyelin is egg sphingomyelin.

[0228] In some embodiments, the helper lipid comprises about 5-40% (mol), about 8%-30%, about 10%-28%, about 20%-36%, about 22%-32%, or about 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).

[0229] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 21% to about 23%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted88LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, or about 32%. As set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs (e.g., targeted LNPs) with these percentages of helper lipids provides enhanced transduction and expression of the pay loads relative to targeted LNPs with smaller or larger quantities of helper lipid.

[0230] In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 40%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 22% to about 36%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 18% to about 32%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is from about 25% to about 30%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNPis from about 30% to about 35%. In some embodiments, the mol% of the helper lipid (e.g., DSPC or sphingomyelin) in the targeted LNP is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34% or about 35%. As set forth in the examples below, in vivo delivery of certain payloads following administration of the disclosed LNPs with these percentages of helper lipids provides enhanced transduction and / or expression of the pay loads relative to LNPs with smaller or larger quantities of helper lipid. It will be understood that mol% of the helper lipid as used herein refers to the mol% of the total lipid component of the LNP, which does not include the therapeutic agent (i.e., payload) or the targeting moiety.

[0231] In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) in the LNP, e.g., targeted LNP, is from about 1:1 to about 7:1. In some embodiments, the molar ratio between the ionizable lipid and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1 : 1 to about 4:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1 : 1 to about 3:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 891 : 1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1 : 1 to about 2:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC) is from about 1.5:1 to about 2.5:1. In some embodiments, the molar ratio between the ionizable lipid and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2: 1 to about 2.5:1.

[0001] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table LI or Table L3 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid in Table LI or Table L3 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the LNP is DPPE-PEG2000 or DPG-PEG2000.

[0232] In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid of Formula I and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises an ionizable lipid of Formula I and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP is from about 22% to about 28%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%. In some embodiments, the targeted LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the LNP is DPPE-PEG2000 or DPG-PEG2000.

[0233] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 154 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 154 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is90from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.

[0234] In some embodiments, the LNP, e.g., targeted LNP comprises Lipid 232 and DSPC. In some embodiments, the LNP, e.g., targeted LNP, comprises Lipid 232 and a sphingomyelin. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 25%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of the DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is from about 20% to about 30%. In some embodiments, the mol% of DSPC or sphingomyelin in the LNP, e.g., targeted LNP, is about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 25%, about 27%, about 28%, about 29%, or about 30%.Sterols

[0235] In some embodiments, the LNPs, e.g., targeted LNPs, of the disclosure can comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53-coprostanol, choiesteiyl-(2’-hydroxy)-ethyl ether, choiesteiyl-(4'- hydroxy )-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p- cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., choiesteryl-(4 '-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT publication W02009 / 127060 and US patent publication US2010 / 0130588, each of which is incorporated herein by reference in its entirety.

[0236] In some embodiments, the component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30- 40% (mol) of the total lipid content of the lipid nanoparticle.

[0237] In some embodiments, the molar ratio between the cholesterol molecule and the non- pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 6: 1 to about 0.5:1. In some91embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 3:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 2: 1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1.5:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:1 to about 0.5:1. In some embodiments, the ratio between the cholesterol molecule and the non-pegylated helper lipid (e.g., DSPC or sphingomyelin) is from about 1:2 to about 0.8:1.Pegylated lipids

[0238] In some embodiments, the LNPs, e.g., targeted LNPs, of the disclosure can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.

[0239] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidy lethanoloamine (PEG-PE), 1,2-dimyristoyl-sn-glycerol, methoxypoly ethylene glycol (DMG-PEG-2K), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'- di(tetradecanoyloxy)propyl-l-0-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-l,2-distearoyl-sn- glycero-3-phosphoethanolamine sodium salt, or a mixture thereof. Additional exemplary PEG- lipid conjugates are described, for example, in US5,885,613, US6,287,591,

[0240] US2003 / 0077829, US2003 / 0077829, US2005 / 0175682, US2008 / 0020058,US2011 / 0117125, US2010 / 0130588, US2016 / 0376224, US2017 / 0119904, and US / 099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018 / 0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016 / 0376224, the content of both of which is incorporated herein by reference in its entirety. In some 92embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilatuylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (l-[8'-(Cholest-5-en-3[beta]- oxy)carboxamido-3',6'-dioxaoctanyl] carbamoyl- [omega] -methyl-poly (ethylene glycol), PEG- DMB (3,4-Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] . In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2- dimyristoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000], In some embodiments, the PEG-lipid comprises a structure selected from:

[0241] In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.93

[0242] Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9 and in W02020106946A1, the contents of all of which are incorporated herein by reference in their entirety.

[0243] In some embodiments, the pegylated lipid has at least one Cl 6 (palmitoyl) PEG lipid anchor. In some embodiments, the pegylated lipid has two Cl 6 PEG lipid anchors (i.e., dialkyl chains of 16 carbons long). In some embodiments, the pegylated lipid is 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000.). In some embodiments, the pegylated lipid is l,2-Dipalmitoyl-rac-glycero-3-methylpolyoxyethylene (DPG-PEG2000). In some embodiments, the pegylated lipid is Cl 6 PEG ceramide. In some embodiments, the targeted LNPs comprising the C 16 pegylated lipids show reduced liver uptake than otherwise identical LNPs comprising C14 pegylated lipids.

[0244] In some embodiments, the LNP further comprises a pegylated lipid comprising at least one C14 alkyl chain (e.g., two C14 alkyl chains). In some such embodiments, the pegylated lipid is DMG-PEG2000.

[0245] In some embodiments, the pegylated lipid has at least one Cl 8 PEG lipid anchor. In some embodiments, the pegylated lipid has two Cl 8 PEG lipid anchors (i.e., dialkyl chains of 18 carbons long). In some embodiments, the C18 pegylated lipid is l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the C18 pegylated lipid is distearoyl-rac-glycerol-PEG2000 (DSG-PEG2000).

[0246] In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5- 10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. m. GENE MODIFYING SYSTEM

[0247] The disclosure provides delivery of gene modifying systems by LNPs. The disclosure provides delivery of gene modifying systems by targeted LNPs (conjugates). This section describes aspects of gene modifying systems to be site specifically delivered to cells. Section II describes particular LNPs and conjugates of the disclosure that are capable of delivering the gene modifying systems to cells. Section II also describes particular lipids that can be used to construct the LNP component of the conjugates of the disclosure.94

[0248] In some embodiments, provided herein are systems used to insert a heterologous object sequence (e.g., a CAR) into the genome of a cell. In some embodiments, the system comprises: (A) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, wherein the gene modifying polypeptide comprises: (i) an endonuclease and / or DNA binding domain; and (ii) a reverse transcriptase (RT) domain, where (i) and (ii) are both derived from a retrotransposon (e.g., from the same retrotransposon or different retrotransposons); and (B) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence. A gene modifying polypeptide, in some embodiments, acts as a substantially autonomous protein machine capable of integrating a template nucleic acid sequence into a target DNA molecule (e.g., in a mammalian host cell, such as a genomic DNA molecule in the host cell), substantially without relying on host machinery. The heterologous object sequence may include, e.g., a coding sequence, a regulatory sequence, or a gene expression unit.A. Retrotransposon

[0249] In some embodiments, the gene modifying systems comprises a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide. In some embodiments, the gene modifying polypeptide may comprise a retrotransposon. In some embodiments, the nucleic acid encoding a gene modifying polypeptide comprise a sequence encoding a retrotransposon. In some embodiments, the retrotransposon may be selected from a group consisting of RTE (e.g., RTE-1 MD, RTE-3 BF, and RTE-25_LMi), CR1 (e.g., CR1-1_PH), Crack (e.g., Crack-28_RF), L2 (e.g., L2-2_Dre and L2-5 GA), and Vingi (e.g., Vingi-l Acar) retrotransposons.

[0250] As described herein, the elements of such retrotransposons can be functionally modularized and / or modified to target, edit, modify or manipulate a target DNA sequence, e.g., to insert an object (e.g., heterologous) nucleic acid sequence into a target genome, e.g., a mammalian genome, by reverse transcription. In some embodiments, a gene modifying system comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a retrotransposase reverse transcriptase domain, and (ii) a retrotransposase endonuclease domain that contains DNA binding functionality; and (B) a template RNA (or DNA encoding the template RNA) comprising (i) a sequence that binds the polypeptide and (ii) a heterologous object sequence. The RNA template element of a gene modifying system is typically heterologous to the polypeptide element and provides an object sequence to be inserted (reverse transcribed) into the host genome.95

[0251] In some embodiments, the gene modifying system comprises a retrotransposase sequence of an element listed in any one of Table 10, Table 11, Table X, Table Z1 Table 3A, or 3B of PCT Pub. No.: WO / 2021 / 178717, which are incorporated herein by reference as they relate to domains from retrotransposons.

[0252] In some embodiments, an amino acid sequence encoded by an element of Table Rl is an amino acid sequence encoded by the full length sequence of an element listed in Table Rl, or a sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, the full- length sequence of an element listed in Table Rl may comprise one or more (e.g., all of) of a 5’ UTR, polypeptide-encoding sequence, or 3’ UTR of a retrotransposon as described herein. In some embodiments, an amino acid sequence of Table Rl is an amino acid sequence encoded by the full length sequence of an element listed in Table Rl, or a sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, a 5’ UTR of an element of Table Rl comprises a 5’ UTR of the full length sequence of an element listed in Table Rl, or a sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto. In some embodiments, a 3’ UTR of an element of Table Rl comprises a 3’ UTR of the full length sequence of an element listed in Table Rl, or a sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto.

[0253] Table Rl and Table R2 provides gene modifying polypeptides comprising retrotransposon elements, altered for improved efficiency of integration into the human genome. Retrotransposase polypeptides were improved through consensus mapping to re-derive the optimal amino acid sequence. Template molecules for use with cognate retrotransposase enzymes were mapped back to their host genomes and flanking genomic DNA used to elucidate target site motifs. When detectable, conserved sequence motifs from the flanking genomic DNA of endogenous occurrences of an element were aligned to the human genome, and new sequences were derived from the human genome as 5’ or 3’ “Human Homology Arms.” In some embodiments, a template RNA described herein comprises one or both of a first homology domain comprising a sequence of a 5' Human Homology Arm of Table Rl or Table R2 (or a sequence having at least about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto) and a second homology domain comprising a sequence of a 3' Human Homology Arm of Table Rl or Table R2 (or a sequence having at least96about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about99% identity thereto).Table Rl: Retrotransposase systems with improved integration activityB. Gene modifying polypeptideRT Domain

[0254] In certain aspects of the present invention, the reverse transcriptase domain of the gene modifying polypeptide is based on a reverse transcriptase domain of an APE-type or RLE-type non-LTR retrotransposon, or of a PLE-type retrotransposon. A wild-type reverse transcriptase domain of an APE-type, RLE-type, or PLE-type retrotransposon can be used in a gene modifying system or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) to alter the reverse transcriptase activity for target DNA sequences. In some embodiments, the reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the reverse transcriptase domain is a heterologous reverse transcriptase from a different LTR-retrotransposon, non-LTR retrotransposon, or other source. In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a RTE (e.g., RTE-1 MD, RTE- 3_BF, and RTE-25_LMi), CR1 (e.g., CR1-1_PH), Crack (e.g., Crack-28_RF), L2 (e.g., L2- 2_Dre and L2-5 GA), and Vingi (e.g., Vingi-l Acar) retrotransposon.

[0255] In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a retrotransposon listed in Table 10, Table 11, Table X, Table Zl, Table Z2, or Table 3A or 3B of PCT Pub. No.: WO / 2021 / 178717, which are incorporated herein by reference as they relate to domains from retrotransposons.

[0256] In certain embodiments, a gene modifying system includes a polypeptide that comprises a reverse transcriptase domain of a retrotransposon listed in Table Rl. In some embodiments, the amino acid sequence of the reverse transcriptase domain of a gene modifying system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%,120at least about 99% identical to the amino acid sequence of a reverse transcriptase domain of a retrotransposon whose DNA sequence is referenced in Table Rl. Reverse transcriptase domains can be identified, for example, based upon homology to other known reverse transcription domains using routine tools as Basic Local Alignment Search Tool (BLAST). In some embodiments, reverse transcriptase domains are modified, for example by site-specific mutation. In some embodiments, the reverse transcriptase domain is engineered to bind a heterologous template RNA.

[0257] In some embodiments, a polypeptide (e.g., RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence. In some embodiments, a template RNA comprises an RNA sequence that is specifically bound by the RNA-binding domain.

[0258] In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, the RT domain is monomeric. In some embodiments, an RT domain naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer. Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, the RT function of the system is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, the multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.

[0259] In some embodiment, a gene modifying polypeptide described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of the RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, mutation of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16(l):265-277 (1988) (incorporated herein by reference in its entirety), e.g., lower by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is abolished.121

[0260] In some embodiments, an RT domain is mutated to increase fidelity compared to an otherwise similar domain without the mutation. For instance, in some embodiments, a YADD (SEQ ID NO: 561) or YMDD (SEQ ID NO: 563) motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD (SEQ ID NO: 562). In embodiments, replacement of the YADD (SEQ ID NO: 561) or YMDD (SEQ ID NO: 563) or YVDD (SEQ ID NO: 562) results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; incorporated herein by reference in its entirety).Endonuclease domain:

[0261] In some embodiments, the gene modifying polypeptide comprises an endonuclease domain (e.g., a heterologous endonuclease domain). In certain embodiments, the endonuclease / DNA binding domain of an APE-type retrotransposon, the endonuclease domain of an RLE-type retrotransposon, or the endonuclease domain of a PLE-type retrotransposon can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein. In some embodiments, the endonuclease domain or endonuclease / DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments, the endonuclease element is a heterologous endonuclease element. The amino acid sequence of an endonuclease domain of a gene modifying system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table X, Zl, Z2, 3 A, or 3B of PCT Pub. No: WO / 2021 / 178717, which are incorporated herein by reference as they relate to domains from retrotransposons.

[0262] In certain embodiments, a gene modifying system includes a polypeptide that comprises an endonuclease domain of a retrotransposon listed in Table Rl. In some embodiments, the amino acid sequence of the endonuclease domain of a gene modifying system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of a endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table Rl. Endonuclease domains can be identified, for example, based upon homology to other known endonuclease domains using tools as Basic Local Alignment Search Tool (BLAST).122

[0263] In some embodiments, a gene modifying polypeptide possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA) binding domain. In certain embodiments, the endonuclease / DNA binding domain of an APE-type retrotransposon or the endonuclease domain of an RLE-type retrotransposon can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a gene modifying system described herein.Template nucleic acid binding domain

[0264] A gene modifying polypeptide typically contains regions capable of associating with the template nucleic acid (e.g., template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs, e.g., secondary structures present in the 3’ UTR in non-LTR retrotransposons. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) RNA binding domain is contained within the reverse transcription domain, e.g., the reverse transcriptase-derived component has a known signature for RNA preference, e.g., secondary structures present in the 3’ UTR in non-LTR retrotransposons.DNA Binding Domain

[0265] In certain aspects, the DNA-binding domain of a gene modifying polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence. In certain embodiments, the DNA-binding domain of the engineered retrotransposon is a heterologous DNA-binding protein or domain relative to a native retrotransposon sequence. In certain embodiments, the heterologous DNA-binding domain is a DNA binding domain of a retrotransposon described in Table R1 herein or in Table X, Table Zl, Table Z2, or Table 3 A or 3B of PCT Pub. No.: WO / 2021 / 178717. In some embodiments, DNA binding domains can be identified based upon homology to other known DNA binding domains using tools as Basic Local Alignment Search Tool (BLAST). In still other embodiments, DNA-binding domains are modified, for example by site-specific mutation. In some embodiments, the DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells.123

[0266] In embodiments, the DNA binding domain comprises one or more modifications relative to a wild-type DNA binding domain, e.g., a modification via directed evolution, e.g., phage- assisted continuous evolution (PACE).

[0267] In certain aspects of the present invention, the host DNA-binding site integrated into by the gene modifying system can be in a gene, in an intron, in an exon, an ORF, outside of a coding region of any gene, in a regulatory region of a gene, or outside of a regulatory region of a gene. In other aspects, the engineered retrotransposon may bind to one or more than one host DNA sequence. In other aspects, the engineered retrotransposon may have low sequence specificity, e.g., bind to multiple sequences or lack sequence preference.

[0268] In some embodiments, a gene modifying system is used to edit a target locus in multiple alleles. In some embodiments, a gene modifying system is designed to edit a specific allele. For example, a gene modifying polypeptide may be directed to a specific sequence that is only present on one allele, e.g., comprises a template RNA with homology to a target allele, e.g., an annealing domain, but not to a second cognate allele. In some embodiments, a gene modifying system can alter a haplotype-specific allele. In some embodiments, a gene modifying system that targets a specific allele preferentially targets that allele, e.g., has at least a 2, 4, 6, 8, or 10-fold preference for a target allele.RNA Binding Domain

[0269] In some embodiments, the RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, the reference RNA binding domain is an RNA binding domain from R2 BM of B. mori. In some embodiments, the RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM - 10 nM (e.g., between 100 pM-1 nM or 1 nM - 10 nM). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in cells (e.g., by FRET or CLIP-Seq).

[0270] In some embodiments, the RNA binding domain is associated with the template RNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(ll):5490-5501 (incorporated by reference herein in its entirety). In some124embodiments, the RNA binding domain is associated with the template RNA in cells (e.g., in HEK293T cells) at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra.Localization sequences for gene modifying systems

[0271] In certain embodiments, a gene modifying system comprises an RNA. In some embodiments, the gene modifying system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence.

[0272] The nuclear localization sequence may be an RNA sequence that promotes the import of the RNA into the nucleus. In certain embodiments, the nuclear localization signal is located on the template RNA. In certain embodiments, the retrotransposase polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nuclear localization signal is located on the template RNA and not on an RNA encoding the retrotransposase polypeptide. While not wishing to be bound by theory, in some embodiments, the RNA encoding the retrotransposase is targeted primarily to the cytoplasm to promote its translation, while the template RNA is targeted primarily to the nucleus to promote its retrotransposition into the genome. In some embodiments, the nuclear localization signal is at the 3’ end, 5’ end, or in an internal region of the template RNA. In some embodiments the nuclear localization signal is 3’ of the heterologous sequence (e.g., is directly 3’ of the heterologous sequence) or is 5’ of the heterologous sequence (e.g., is directly 5’ of the heterologous sequence). In some embodiments, the nuclear localization signal is placed outside of the 5’ UTR or outside of the 3’ UTR of the template RNA. In some embodiments the nuclear localization signal is placed between the 5’ UTR and the 3’ UTR, wherein optionally the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is an anti -sense orientation or is downstream of a transcriptional termination signal or polyadenylation signal). In some embodiments, the nuclear localization sequence is situated inside of an intron. In some embodiments a plurality of the same or different nuclear localization signals are in the RNA, e.g., in the template RNA. In some embodiments, the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 bp in legnth. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences, which drive RNA localization into the nucleus. In some embodiments, the nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments, the nuclear localization signal binds a nuclear-125enriched protein. In some embodiments, the nuclear localization signal binds the HNRNPK protein. In some embodiments the nuclear localization signal is rich in pyrimidines, e.g., is a C / T rich, C / U rich, C rich, T rich, or U rich region. In some embodiments, the nuclear localization signal is derived from a long non-coding RNA. In some embodiments, the nuclear localization signal is derived from MALAT1 long non-coding RNA or is the 600 nucleotide M region of MALAT1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments, the nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments, the nuclear localization sequence is described in Shukla et al., The EMBO Journal e98452 (2018). In some embodiments, the nuclear localization signal is derived from a non-LTR retrotransposon, an LTR retrotransposon, retrovirus, or an endogenous retrovirus.

[0273] In some embodiments, a polypeptide described herein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example, a nuclear localization sequence (NLS), e.g., as described above. In some embodiments, the NLS is a bipartite NLS. In some embodiments, an NLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, the NLS is fused to the N-terminus of a gene modifying polypeptide described herein. In some embodiments, the NLS is fused to the C-terminus of the gene modifying polypeptide. In some embodiments, a linker sequence is disposed between the NLS and the neighboring domain of the gene modifying polypeptide.

[0274] In some embodiments, an NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 9), PKKRKVEGADKRTADGSEFESPKKKRKV(SEQ ID NO: 10), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 11) KRTADGSEFESPKKKRKV(SEQ ID NO: 12), KKTELQTTNAENKTKKL (SEQ ID NO: 13), or KRGINDRNFWRGENGRKTR (SEQ ID NO: 14), KRPAATKKAGQAKKKK (SEQ ID NO: 15), PAAKRVKLD (SEQ ID NO: 344), KRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 414), KRTADGSEFE (SEQ ID NO: 415), KRTADGSEFESPKKKAKVE (SEQ ID NO: 416),AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 127), or a functional fragment or variant thereof.

[0275] In some embodiments, a gene modifying polypeptide comprises an NLS as comprised in SEQ ID NO: 557 and / or SEQ ID NO: 127, or an NLS having an amino acid sequence having at126least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about98%, or about 99% identity thereto.

[0276] Exemplary NLS sequences are also described in PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises an amino acid sequence as disclosed inTable 8. An NLS of this table may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C -terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMCBioinformat 13:157 (2012), incorporated herein by reference in its entirety).

[0277] In some embodiments, the NLS is a bipartite NLS. A bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length). A monopartite NLS typically lacks a spacer. An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 15), wherein the spacer is bracketed. Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 16). Exemplary NLSs are described in International Application W02020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences.

[0278] In certain embodiments, a gene modifying system polypeptide further comprises an intracellular localization sequence, e.g., a nuclear localization sequence and / or a nucleolar localization sequence. The nuclear localization sequence and / or nucleolar localization sequence may be amino acid sequences that promote the import of the protein into the nucleus and / or nucleolus, where it can promote integration of heterologous sequence into the genome. In certain embodiments, a gene modifying system polypeptide (e.g., a retrotransposase, e.g., a polypeptide according to Table R1 herein) further comprises a nucleolar localization sequence. In certain embodiments, the retrotransposase polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nucleolar localization signal is encoded on the RNA encoding the retrotransposase polypeptide and not on the template RNA. In some embodiments, the nucleolar localization signal is located at the N-terminus, C -terminus, or in an internal region of the polypeptide. In some embodiments, a plurality of the same or different nucleolar localization signals are used. In some embodiments, the nuclear localization signal is less than 5, 10, 25, 50, 75, or 100 amino acids in length. Various polypeptide nucleolar localization signals can be used. For example, Yang et al., Journal of Biomedical Science 22, 33 (2015), describe a nuclear localization signal that also functions as a nucleolar localization signal. In some embodiments, the nucleolar localization signal may also be a nuclear localization signal. In some embodiments, the nucleolar localization signal may overlap with a nuclear localization signal. In some embodiments, the nucleolar localization signal may comprise a stretch of basic residues. In some embodiments, the nucleolar localization signal may be rich in arginine and lysine residues. In some embodiments, the nucleolar localization signal may be131derived from a protein that is enriched in the nucleolus. In some embodiments, the nucleolar localization signal may be derived from a protein enriched at ribosomal RNA loci. In some embodiments, the nucleolar localization signal may be derived from a protein that binds rRNA. In some embodiments, the nucleolar localization signal may be derived from MSP58. In some embodiments, the nucleolar localization signal may be a monopartite motif. In some embodiments, the nucleolar localization signal may be a bipartite motif. In some embodiments, the nucleolar localization signal may consist of a multiple monopartite or bipartite motifs. In some embodiments, the nucleolar localization signal may consist of a mix of monopartite and bipartite motifs. In some embodiments, the nucleolar localization signal may be a dual bipartite motif. In some embodiments, the nucleolar localization motif may be a KRASSQALGTIPKRRSSSRFIKRKK (SEQ ID NO: 17). In some embodiments, the nucleolar localization signal may be derived from nuclear factor-KB-inducing kinase. In some embodiments, the nucleolar localization signal may be an RKKRKKK motif (SEQ ID NO: 18) (described in Birbach et al., Journal of Cell Science, 117 (3615-3624), 2004).

[0279] In some embodiments, a nucleic acid described herein (e.g., an RNA encoding a gene modifying polypeptide, or a DNA encoding the RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a gene modifying system. For instance, the microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the RNA encoding the gene modifying polypeptide is present in a non-target cell, it would be bound by the miRNA, and when the RNA encoding the gene modifying polypeptide is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the RNA encoding the gene modifying polypeptide may reduce production of the gene modifying polypeptide, e.g., by degrading the mRNA encoding the polypeptide or by interfering with translation. Accordingly, the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells. A system having a microRNA binding site in the RNA encoding the gene modifying polypeptide (or encoded in the DNA encoding the RNA) may also be used in combination with a template RNA that is regulated by a second microRNA binding site, e.g., as described herein in the section entitled ‘Template RNA component of gene modifying system.”

[0280] In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide 132sequence of multiple retrotransposons. In some embodiments, a 5’ or 3’ untranslated region for use in any of the systems described herein can be a molecular reconstruction based upon the aligned 5’ or 3’ untranslated region of multiple retrotransposons. Based on the Accession numbers, polypeptides or nucleic acid sequences can be aligned, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD-Search for conserved domain analysis. Molecular reconstructions can be created based upon sequence consensus, e.g. using approaches described in Ivies et at, Cell 1997, 501 - 510 ; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99. In some embodiments, the retrotransposon from which the 5’ or 3’ untranslated region or polypeptide is derived is a young or a recently active mobile element, as assessed via phylogenetic methods such as those described in Boissinot et al., Molecular Biology and Evolution 2000, 915-928.Linker

[0281] In some embodiments, domains of the compositions and systems described herein (e.g., the endonuclease and reverse transcriptase domains of a polypeptide or the DNA binding domain and reverse transcriptase domains of a polypeptide) may be joined by a linker. A composition described herein comprising a linker element has the general form S1-L-S2, wherein SI and S2 may be the same or different and represent two domain moieties (e.g., each a polypeptide or nucleic acid domain) associated with one another by the linker. In some embodiments, a linker may connect two polypeptides. In some embodiments, a linker may connect two nucleic acid molecules. In some embodiments, a linker may connect a polypeptide and a nucleic acid molecule. A linker may be a chemical bond, e.g., one or more covalent bonds or non-covalent bonds. A linker may be flexible, rigid, and / or cleavable. In some embodiments, the linker is a peptide linker. Generally, a peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length, e.g., 2-50 amino acids in length, 2-30 amino acids in length.

[0282] Some commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). Flexible linkers may be useful for joining domains that require a certain degree of movement or interaction and may include small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce unfavorable interactions between the linker and the other moieties. Examples of such linkers include those having the structure [GGS]-1or [GGGS]-1(SEQ ID NO: 545). Rigid linkers are useful to keep a fixed distance between domains and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of the domains is critical to133preserve the stability or bioactivity of one or more components in the agent. Rigid linkers may have an alpha helix-structure or Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu. Cleavable linkers may release free functional domains in vivo. In some embodiments, linkers may be cleaved under specific conditions, such as the presence of reducing reagents or proteases. In vivo cleavable linkers may utilize the reversible nature of a disulfide bond. One example includes a thrombin-sensitive sequence (e.g., PRS) between the two Cys residues. In vitro thrombin treatment of CPRSC (SEQ ID NO: 546) results in the cleavage of the thrombin-sensitive sequence, while the reversible disulfide linkage remains intact. Such linkers are known and described, e.g., in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivo cleavage of linkers in compositions described herein may also be carried out by proteases that are expressed in vivo under pathological conditions (e.g. cancer or inflammation), in specific cells or tissues, or constrained within certain cellular compartments. The specificity of many proteases offers slower cleavage of the linker in constrained compartments.

[0283] In some embodiments the amino acid linkers are (or are homologous to) the endogenous amino acids that exist between such domains in a native polypeptide. In some embodiments, the endogenous amino acids that exist between such domains are substituted but the length is unchanged from the natural length. In some embodiments, additional amino acid residues are added to the naturally existing amino acid residues between domains.

[0284] In some embodiments, the amino acid linkers are designed computationally or screened to maximize protein function (Anad et al., FEES Letters, 587:19, 2013).

[0285] In addition to being fully encoded on a single transcript, a polypeptide can be generated by separately expressing two or more polypeptide fragments that reconstitute the holoenzyme. In some embodiments, the gene modifying polypeptide is generated by expressing as separate subunits that reassemble the holoenzyme through engineered protein-protein interactions. In some embodiments, reconstitution of the holoenzyme does not involve covalent binding between subunits. Peptides may also fuse together through trans-splicing of inteins (Tomabene et al. Sci Transl Med 11, eaav4523 (2019)). In some embodiments, the gene modifying holoenzyme is expressed as separate subunits that are designed to create a fusion protein through the presence of split inteins (e.g., as described herein) in the subunits. In some embodiments, the gene modifying holoenzyme is reconstituted through the formation of covalent linkages between subunits. In some embodiments, the breaking up of a gene modifying polypeptide into subunits may aid in delivery of the protein by keeping the nucleic acid encoding each part within optimal134packaging limits of a viral delivery vector, e.g., AAV (Tomabene et al. Sci Transl Med 11, eaav4523 (2019)). In some embodiments, the gene modifying polypeptide is designed to be dimerized through the use of covalent or non-covalent interactions as described above.Exemplary Linkers are shown in Table Linker 1 below.

[0286] In some embodiments, a linker of a gene modifying polypeptide comprises a motif chosen from: (SGGS)n(SEQ ID NO: 25), (GGGS)n(SEQ ID NO: 26), (GGGGS)n(SEQ ID NO: 1), (G)n, (EAAAK)n(SEQ ID NO: 3), (GGS)n, or (XP)nInterns

[0287] In some embodiments, the gene modifying system comprises an intein. Generally, an intein comprises a polypeptide that has the capacity to join two polypeptides or polypepide fragments together via a peptide bond. In some embodiments, the intein is a trans-splicing intein that can join two polypeptide fragments, e.g., to form the polypeptide component of a system as described herein. In some embodiments, an intein may be encoded on the same nucleic acid molecule encoding the two polypeptide fragments. In certain embodiments, the intein may be translated as part of a larger polypeptide comprising, e.g., in order, the first polypeptide fragment, the intein, and the second polypeptide fragment. In embodiments, the translated intein may be capable of excising itself from the larger polypeptide, e.g., resulting in separation of the attached polypeptide fragments. In embodiments, the excised intein may be capable of joining the two polypeptide fragments to each other directly via a peptide bond. In some embodiments, as described in more detail below, Intein-N may be fused to the N-terminal portion of a first domain described herein, and and intein-C may be fused to the C-terminal portion of a second domain described herein for the joining of the N-terminal portion to the C-terminal portion, thereby joining the first and second domains. In some embodiments, the first and second domains are each independent chosen from a DNA binding domain, an RNA binding domain, an RT domain, and an endonuclease domain.

[0288] As used herein, "intein" refers to a self-splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). An intein may, in some instances, comprise a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as "protein introns." The process of an intein excising itself and joining the remaining portions of the protein is herein termed "protein splicing" or "intein- mediated protein splicing." In some embodiments, an intein of a precursor protein (an intein containing protein prior to intein-mediated protein splicing) comes from two genes. Such intein is referred to herein as a split intein (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate138genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be herein referred as "intein-N." The intein encoded by the dnaE-c gene may be herein referred as "intein-C."

[0289] Use of inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem.289(21); 14512-9 (2014) (incorporated herein by reference in its entirety). For example, when fused to separate protein fragments, the inteins IntN and IntC may recognize each other, splice themselves out, and / or simultaneously ligate the flanking N- and C- terminal exteins of the protein fragments to which they were fused, thereby reconstituting a full- length protein from the two protein fragments.

[0290] In some embodiments, a synthetic intein based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pair, is used. Examples of such inteins have been described, e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5 (incorporated herein by reference in its entirety). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference.

[0291] In some embodiments, a protein fragment ranges from about 2-1000 amino acids (e.g., between 2-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids) in length. In some embodiments, a protein fragment ranges from about 5-500 amino acids (e.g., between 5-10, 10-50, 50-100, 100-200, 200-300, 300-400, or 400-500 amino acids) in length. In some embodiments, a protein fragment ranges from about 20-200 amino acids (e.g., between 20-30, 30-40, 40-50, 50-100, or 100-200 amino acids) in length.

[0292] In some embodiments, a portion or fragment of a gene modifying polypeptide is fused to an intein. The nuclease can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein and the C-terminus of the intein is fused to the N-terminus of an AAV capsid protein.

[0293] In some embodiments, an endonuclease domain is fused to intein-N and a polypeptide comprising an RT domain is fused to an intein-C.139

[0294] Exemplary nucleotide and amino acid sequences of interns are provided below: G A G G T L G A C A A C L GT GPromoters

[0295] In some embodiments, one or more promoter or enhancer elements are operably linked to a nucleic acid encoding a gene modifying protein or a template nucleic acid, e.g., that controls expression of the heterologous object sequence. In certain embodiments, the one or more promoter or enhancer elements comprise cell-type or tissue specific elements. In some embodiments, the promoter or enhancer is the same or derived from the promoter or enhancer140that naturally controls expression of the heterologous object sequence. For example, the ornithine transcarbomylase promoter and enhancer may be used to control expression of the ornithine transcarbomylase gene in a system or method provided by the invention for correcting ornithine transcarbomylase deficiencies. In some embodiments, a promoter for use in the invention is for a gene described in Table 33 or 34, e.g., which may be used with an allele of the reference gene, or, in other embodiments, with a heterologous gene. In some embodiments, the promoter is a promoter of Table 33 or a functional fragment or variant thereof.

[0296] Exemplary tissue specific promoters that are commercially available can be found, for example, at a uniform resource locator (e.g., invivogen.com / tissue-specific-promoters). In some embodiments, a promoter is a native promoter or a minimal promoter, e.g., which consists of a single fragment from the 5’ region of a given gene. In some embodiments, a native promoter comprises a core promoter and its natural 5’ UTR. In some embodiments, the 5’ UTR comprises an intron. In other embodiments, these include composite promoters, which combine promoter elements of different origins or were generated by assembling a. distal enhancer with a minimal promoter of the same origin.

[0297] Exemplary cell or tissue specific promoters are provided in the tables, below, and exemplary nucleic acid sequences encoding them are known in the art and can be readily accessed using a variety of resources, such as the NCBI database, including RefSeq, as well as the Eukaryotic Promoter Database (epd.epfl.ch / / index.php).Table 33. Exemplary cell or tissue-specific promoters

[0298] Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544; incorporated herein by reference in its entirety).

[0299] In some embodiments, a nucleic acid encoding a gene modifying polypeptide or template nucleic acid is operably linked to a control element, e.g., a transcriptional control element, such 141as a promoter. The transcriptional control element may, in some embodiments, be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell). In some embodiments, a nucleotide sequence encoding a polypeptide is operably linked to multiple control elements, e.g., that allow expression of the nucleotide sequence encoding the polypeptide in both prokaryotic and eukaryotic cells.Nonlimiting Exemplary Cell-Specific Promoters

[0300] Cell-specific promoters known in the art may be used to direct expression of a gene modifying protein, e.g., as described herein. Nonlimiting exemplary mammalian cell-specific promoters have been characterized and used in mice expressing Cre recombinase in a cellspecific manner. Certain nonlimiting exemplary mammalian cell-specific promoters are listed in Table 1 of US9845481, incorporated herein by reference.

[0301] In some embodiments, a vector as described herein comprises an expression cassette. The term “expression cassette”, as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention. Typically, an expression cassette comprises the nucleic acid molecule of the instant invention operatively linked to a promoter sequence. The term “operatively linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (e.g., the coding sequence is under the transcriptional control of the promoter). Encoding sequences can be operatively linked to regulatory sequences in sense or antisense orientation. In certain embodiments, the promoter is a heterologous promoter. The term “heterologous promoter”, as used herein, refers to a promoter that is not found to be operatively linked to a given encoding sequence in nature. In certain embodiments, an expression cassette may comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck response element (WRE), and / or other elements known to affect expression levels of the encoding sequence. A“promoter” typically controls the expression of a coding sequence or functional RNA. In certain embodiments, a promoter sequence comprises proximal and more distal upstream elements and can further comprise an enhancer element. An “enhancer” can typically stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. In certain embodiments, the promoter is derived in its entirety from a native gene. In certain embodiments, the promoter is composed of different elements derived from different naturally occurring142promoters. In certain embodiments, the promoter comprises a synthetic nucleotide sequence. It will be understood by those skilled in the art that different promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or to the presence or the absence of a drug or transcriptional co-factor. Ubiquitous, cell-type-specific, tissue-specific, developmental stage-specific, and conditional promoters, for example, drug-responsive promoters (e.g ., tetracycline-responsive promoters) are well known to those of skill in the art. Examples of promoter include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (composite of theCMV enhancer the chicken beta actin promoter (CBA) and the rabbit beta globin intron.), NSE (neuronal specific enolase), synapsin orNeuN promoters, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), SFFV promoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. Other promoters can be of human origin or from other species, including from mice. Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [beta]- actin, rat insulin promoter, the phosphoglycerate kinase promoter, the human alpha- 1 antitrypsin (hAAT) promoter, the transthyretin promoter, the TBG promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EFl -alpha promoter, the CAG promoter and other constitutive promoters, hybrid promoters with multi-tissue specificity, promoters specific for neurons like synapsin and glyceraldehyde-3 - phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, CA). Additional exemplary promoter sequences are described, for example, in WO2018213786A1 (incorporated by reference herein in its entirety).

[0302] In some embodiments, a vector described herein is a multicistronic expression construct. Multicistronic expression constructs include, for example, constructs harboring a first expression cassette, e.g. comprising a first promoter and a first encoding nucleic acid sequence, and a second expression cassette, e.g. comprising a second promoter and a second encoding nucleic acid sequence. Such multicistronic expression constructs may, in some instances, be particularly useful in the delivery of non-translated gene products, such as hairpin RNAs, together with a polypeptide, for example, a gene modifying polypeptide and gene modifying template. In some143embodiments, multicistronic expression constructs may exhibit reduced expression levels of one or more of the included transgenes, for example, because of promoter interference or the presence of incompatible nucleic acid elements in close proximity. If a multicistronic expression construct is part of a viral vector, the presence of a self-complementary nucleic acid sequence may, in some instances, interfere with the formation of structures necessary for viral reproduction or packaging.

[0303] In some embodiments, the sequence encodes an RNA with a hairpin. In some embodiments, the hairpin RNA is a guide RNA, a template RNA, shRNA, or a microRNA. In some embodiments, the first promoter is an RNA polymerase I promoter. In some embodiments, the first promoter is an RNA polymerase II promoter. In some embodiments, the second promoter is an RNA polymerase III promoter. In some embodiments, the second promoter is a U6 or Hl promoter. In some embodiments, the nucleic acid construct comprises the structure of AAV construct Bl or B2.

[0304] Without wishing to be bound by theory, multicistronic expression constructs may not achieve optimal expression levels as compared to expression systems containing only one cistron. One of the suggested causes of lower expression levels achieved with multicistronic expression constructs comprising two ore more promoter elements is the phenomenon of promoter interference (see, e.g., Curtin J A, Dane A P, Swanson A, Alexander I E, Ginn S L. Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Then 2008 March; 15(5):384-90; and Martin- Duque P, Jezzard S, Kaftansis L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2004 October; 15(10):995-1002; both references incorporated herein by reference for disclosure of promoter interference phenomenon). In some embodiments, the problem of promoter interference may be overcome, e.g., by producing multicistronic expression constructs comprising only one promoter driving transcription of multiple encoding nucleic acid sequences separated by internal ribosomal entry sites, or by separating cistrons comprising their own promoter with transcriptional insulator elements. In some embodiments, single-promoter driven expression of multiple cistrons may result in uneven expression levels of the cistrons. In some embodiments, a promoter cannot efficiently be isolated and isolation elements may not be compatible with some gene transfer vectors, for example, some retroviral vectors.144C. Template Nucleic AcidTemplate RNA component of gene modifying system

[0305] The gene modifying system comprises a template nucleic acid, comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous sequence. In some embodiments, the template nucleic acid is a template RNA. In some embodiments, template RNA works with the gene modifying polypeptide to transcribe an RNA sequence template into the lymphocytes DNA sites by targeted-primed reverse transcription. By writing DNA sequence(s) via reverse transcription of the RNA sequence template directly into the host genome, the gene modifying system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step. Therefore, the gene modifying system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and / or function information.

[0306] In some embodiments, the template RNA encodes a gene modifying protein in cis with a heterologous object sequence. Various cis constructs were described, for example, in Kuroki- Kami et al (2019) Mobile DNA 10:23 (incorporated by reference herein in its entirety), and can be used in combination with any of the embodiments described herein. For instance, in some embodiments, the template RNA comprises a heterologous object sequence, a sequence encoding a gene modifying protein (e.g., a protein comprising (i) a reverse transcriptase domain and (ii) an endonuclease domain, e.g., as described herein), a 5’ untranslated region, and a 3’ untranslated region. The components may be included in various orders. In some embodiments, the gene modifying protein and heterologous object sequence are encoded in different directions (sense vs. anti-sense), e.g., using an arrangement shown in Figure 3 A of Kuroki-Kami et al, Id. In some embodiments, the gene modifying protein and heterologous object sequence are encoded in the same direction. In some embodiments, the nucleic acid encoding the polypeptide and the template RNA or the nucleic acid encoding the template RNA are covalently linked, e.g., are part of a fusion nucleic acid, and / or are part of the same transcript. In some embodiments, the fusion nucleic acid comprises RNA or DNA.

[0307] The nucleic acid encoding the gene modifying polypeptide may, in some instances, be 5’ of the heterologous object sequence. For example, in some embodiments, the template RNA comprises, from 5’ to 3’, a 5’ untranslated region, a sense-encoded gene modifying polypeptide,145a sense-encoded heterologous object sequence, and 3’ untranslated region. In some embodiments, the template RNA comprises, from 5’ to 3’, a 5’ untranslated region, a sense- encoded gene modifying polypeptide, anti-sense-encoded heterologous object sequence, and 3’ untranslated region.

[0308] It is understood that, when a template RNA is described as comprising an open reading frame or the reverse complement thereof, in some embodiments the template RNA must be converted into double stranded DNA (e.g., through reverse transcription) before the open reading frame can be transcribed and translated.

[0309] In certain embodiments, customized RNA sequence template can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure / altemative splicing; causing disruption of an endogenous gene; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up- or down-regulation of operably liked genes, etc. In certain embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and / or transgenes, provide for binding sites to transcription factor activators, repressors, enhancers, etc., and combinations of thereof. In other embodiments, the coding sequence can be further customized with splice acceptor sites, poly-A tails. In certain embodiments the RNA sequence can contain sequences coding for an RNA sequence template homologous to the retrotransposase, be engineered to contain heterologous coding sequences, or combinations thereof.

[0310] The template RNA may have some homology to the target DNA. In some embodiments the template RNA has at least 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 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 175, about 200 or more bases of exact homology to the target DNA at the 3’ end of the RNA. In some embodiments the template RNA has at least 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 20, about 25, about 30, about 35, about 40, about 45, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 175, about 180, or about 200 or more bases of at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100% homology to the target DNA, e.g., at the 5’ end of the template RNA. In some embodiments, the template RNA has a 3’ untranslated region derived146from a retrotransposon, e.g. a retrotransposons described herein. In some embodiments the template RNA has a 3’ region of at least about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 160, about 180, about 200 or more bases of at least about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99% or about 100% homology to the 3’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g. a retrotransposon in Table Rl. In some embodiments, the template RNA has a 5’ untranslated region derived from a retrotransposon, e.g. a retrotransposons described herein. In some embodiments the template RNA has a 5’ region of at least about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 160, about 180, or about 200 or more bases of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or greater homology to the 5’ sequence of a retrotransposon, e.g., a retrotransposon described herein, e.g. a retrotransposon described in Table Rl.

[0311] The template RNA component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template RNA has a 3’ region that is capable of binding a gene modifying genome editing protein. The binding region, e.g., 3’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system.

[0312] The template RNA component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template RNA has a 5’ region that is capable of binding a gene modifying protein. The binding region, e.g., 5’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system. In some embodiments, the 5’ untranslated region comprises a pseudoknot, e.g., a pseudoknot that is capable of binding to the gene modifying protein.

[0313] In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a stem-loop sequence. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a hairpin. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a helix. In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 5’ untranslated region) comprises a psuedoknot. In some embodiments, the template RNA comprises a ribozyme. In some embodiments the ribozyme is similar to a hepatitis delta virus (HDV) ribozyme, e.g., has a147secondary structure like that of the HDV ribozyme and / or has one or more activities of the HDV ribozyme, e.g., a self-cleavage activity. See, e.g., Eickbush et al., Molecular and Cellular Biology, 2010, 3142-3150.

[0314] In some embodiments, the template RNA (e.g., an untranslated region of the hairpin RNA, e.g., a 3’ untranslated region) comprises one or more stem-loops or helices. Exemplary structures of R2 3’ UTRs are shown, for example, in Ruschak et al. “Secondary structure models of the 3' untranslated regions of diverse R2 RNAs” RNA. 2004 Jun; 10(6): 978-987, e.g., at Figure 3, therein, and in Eikbush and Eikbush, “R2 and R2 / R1 hybrid non-autonomous retrotransposons derived by internal deletions of full-length elements” Mobile DNA (2012) 3:10; e.g., at Figure 3 therein, which articles are hereby incorporated by reference in their entirety.

[0315] In some embodiments, a template RNA described herein comprises a sequence that is capable of binding to a gene modifying protein described herein. For instance, in some embodiments, the template RNA comprises an MS2 RNA sequence capable of binding to an MS2 coat protein sequence in the gene modifying protein. In some embodiments, the template RNA comprises an RNA sequence capable of binding to a B-box sequence. In some embodiments, in addition to or in place of a UTR, the template RNA is linked (e.g., covalently) to a non-RNA UTR, e.g., a protein or small molecule.

[0316] In some embodiments, the template RNA has a poly-A tail at the 3’ end. In some embodiments, the template RNA does not have a poly-A tail at the 3’ end.

[0317] In some embodiments the template RNA has a 5’ region of at least about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 160, about 180, about 200 or more bases of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or greater homology to the 5’ sequence of a retrotransposon, e.g., a retrotransposon described herein.

[0318] The template RNA of the system typically comprises an object sequence for insertion into a target DNA. The object sequence may be coding or non-coding.

[0319] In some embodiments, a system or method described herein comprises a single template RNA. In some embodiments, a system or method described herein comprises a plurality of template RNAs.

[0320] In some embodiments, the object sequence may contain an open reading frame. In some embodiments, the template RNA has a Kozak sequence. In some embodiments, the template 148RNA has an interal ribosome entry site. In some embodiments, the template RNA has a selfcleaving peptide such as a T2A or P2A site. In some embodiments, the template RNA has a start codon. In some embodiments, the template RNA has a splice acceptor site. In some embodiments, the template RNA has a splice donor site. Exemplary splice acceptor and splice donor sites are described in WO2016044416, incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those of skill in the art and include, by way of example only, CTGACCCTTCTCTCTCTCCCCCAGAG (from human HBB gene) and TTT CTCTCCCACAAG (from human immunoglobulin-gamma gene). In some embodiments the template RNA, has a microRNA binding site downstream of the stop codon. In some embodiments, the template RNA has a poly A tail downstream of the stop codon of an open reading frame. In some embodiments, the template RNA comprises one or more exons. In some embodiments, the template RNA comprises one or more introns. In some embodiments, the template RNA comprises a eukaryotic transcriptional terminator. In some embodiments, the template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments, the RNA comprises the human T-cell leukemia virus (HTLV-1) R region. In some embodiments, the RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WERE). In some embodiments, in the template RNA, the heterologous object sequence encodes a polypeptide and is coded in an antisense direction with respect to the 5’ and 3’ UTR. In some embodiments, in the template RNA, the heterologous object sequence encodes a polypeptide and is coded in a sense direction with respect to the 5’ and 3’ UTR.

[0321] In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a gene modifying system. For instance, the microRNA binding site can be chosen on the basis that is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the template RNA is present in a non-target cell, it would be bound by the miRNA, and when the template RNA is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the template RNA may interfere with insertion of the heterologous object sequence into the genome. Accordingly, the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells. A system having a microRNA binding site in the template RNA (or DNA encoding it) may also be used in149combination with a nucleic acid encoding a gene modifying polypeptide, wherein expression of the gene modifying polypeptide is regulated by a second microRNA binding site, e.g., as described herein, e.g., in the section entitled “Polypeptide component of gene modifying system.”

[0322] In some embodiments, the object sequence may contain a non-coding sequence. For example, the template RNA may comprise a promoter or enhancer sequence. In some embodiments, the template RNA comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional. In some embodiments, the promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments, the promoter comprises a TATA element. In some embodiments, the promoter comprises a B recognition element. In some embodiments, the promoter has one or more binding sites for transcription factors. In some embodiments, the non-coding sequence is transcribed in an antisense-direction with respect to the 5’ and 3’ UTR. In some embodiments, the non-coding sequence is transcribed in a sense direction with respect to the 5’ and 3’ UTR.

[0323] In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a promoter sequence, e.g., a tissue specific promoter sequence. In some embodiments, the tissue-specific promoter is used to increase the target-cell specificity of a gene modifying system. For instance, the promoter can be chosen on the basis that it is active in a target cell type but not active in (or active at a lower level in) a non-target cell type. Thus, even if the promoter integrated into the genome of a non-target cell, it would not drive expression (or only drive low-level expression) of an integrated gene. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a microRNA binding site, e.g., in the template RNA or a nucleic acid encoding a gene modifying protein, e.g., as described herein. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a DNA encoding a gene modifying polypeptide, driven by a tissue-specific promoter, e.g., to achieve higher levels of gene modifying protein in target cells than in non-target cells.

[0324] In some embodiments, a heterologous object sequence comprised by a template RNA (or DNA encoding the template RNA) is operably linked to at least one regulatory sequence. In some embodiments, the heterologous object sequence is operably linked to a tissue-specific promoter, such that expression of the heterologous object sequence, e.g., a therapeutic protein, is upregulated in target cells, as above. In some embodiments, the heterologous object sequence is operably linked to a miRNA binding site, such that expression of the heterologous object150sequence, e.g., a therapeutic protein, is downregulated in cells with higher levels of the corresponding miRNA, e.g., non-target cells, as above.

[0325] In some embodiments, the template RNA comprises a microRNA sequence, a siRNA sequence, a guide RNA sequence, a piwi RNA sequence.

[0326] In some embodiments, the template RNA comprises anon-coding heterologous object sequence, e.g., a regulatory sequence. In some embodiments, integration of the heterologous object sequence thus alters the expression of an endogenous gene. In some embodiments, integration of the heterologous object sequence upregulates expression of an endogenous gene. In some embodiments, integration of the heterologous object sequence downregulated expression of an endogenous gene.

[0327] In some embodiments, the template RNA comprises a site that coordinates epigenetic modification. In some embodiments, the template RNA comprises an element that inhibits, e.g., prevents, epigenetic silencing. In some embodiments, the template RNA comprises a chromatin insulator. For example, the template RNA comprises a CTCF site or a site targeted for DNA methylation.

[0328] In order to promote higher level or more stable gene expression, the template RNA may include features that prevent or inhibit gene silencing. In some embodiments, these features prevent or inhibit DNA methylation. In some embodiments, these features promote DNA demethylation. In some embodiments, these features prevent or inhibit histone deacetylation. In some embodiments, these features prevent or inhibit histone methylation. In some embodiments, these features promote histone acetylation. In some embodiments, these features promote histone demethylation. In some embodiments, multiple features may be incorporated into the template RNA to promote one or more of these modifications. CpG dinculeotides are subject to methylation by host methyl transferases. In some embodiments, the template RNA is depleted of CpG dinucleotides, e.g., does not comprise CpG nucleotides or comprises a reduced number of CpG dinucleotides compared to a corresponding unaltered sequence. In some embodiments, the promoter driving transgene expression from integrated DNA is depleted of CpG dinucleotides.

[0329] In some embodiments, the template RNA comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. The effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA).151

[0330] In some embodiments, the object sequence of the template RNA is inserted into a target genome in an endogenous intron. In some embodiments, the object sequence of the template RNA is inserted into a target genome and thereby acts as a new exon. In some embodiments, the insertion of the object sequence into the target genome results in replacement of a natural exon or the skipping of a natural exon.

[0331] In some embodiments, the object sequence of the template RNA is inserted into the target genome in a genomic safe harbor site, such as AAVS1, CCR5, or ROSA26. In some embodiments, the object sequence of the template RNA is inserted into the albumin locus. In some embodiments, the object sequence of the template RNA is inserted into the TRAC locus. In some embodiments, the object sequence of the template RNA is added to the genome in an intergenic or intragenic region. In some embodiments, the object sequence of the template RNA is added to the genome 5’ or 3’ within about 0.1 kb, about 0.25 kb, about 0.5 kb, about 0.75 kb, about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 7.5 kb, about 10 kb, about 15 kb, about 20 kb, about 25 kb, about 50, about 75 kb, or about 100 kb of an endogenous active gene. In some embodiments, the object sequence of the template RNA is added to the genome 5’ or 3’ within about 0.1 kb, about 0.25 kb, about 0.5 kb, about 0.75 kb, about 1 kb, about 2 kb, about 3 kb, about 4 kb, about 5 kb, about 7.5 kb, about 10 kb, about 15 kb, about 20 kb, about 25 kb, about 50 kb, about 75 kb, or about 100 kb of an endogenous promoter or enhancer. In some embodiments, the object sequence of the template RNA can be, e.g., about 50-50,000 base pairs (e.g., between about 50-40,000 bp, between about 500-30,000 bp between about 500-20,000 bp, between about 100-15,000 bp, between about 500-10,000 bp, between about 50-10,000 bp, between about 50-5,000 bp. In some embodiments, the heterologous object sequence is less than about 1,000, about 1,300, about 1,500, about 2,000, about 3,000, about 4,000, about 5,000, or about 7,500 nucleotides in length.

[0332] In some embodiments, a system or method described herein results in insertion of a heterologous sequence into a target site in the human genome. In some embodiments, the target site in the human genome has sequence similarity to the corresponding target site of the corresponding wild-type retrotransposase (e.g., the retrotransposase from which the gene modifying polypeptide was derived) in the genome of the organism to which it is native. For instance, in some embodiments, the identity between the 40 nucleotides of human genome sequence centered at the insertion site and the 40 nucleotides of native organism genome sequence centered at the insertion site is less than about 99.5%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 60%, or about 50%, or is between about 50-60%, about 60-70%, about 70-80%, about 80-90%, 152or about 90-100%. In some embodiments, the identity between the 100 nucleotides of human genome sequence centered at the insertion site and the 100 nucleotides of native organism genome sequence centered at the insertion site is less than about 99.5%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 60%, or about 50%, or is between about 50-60%, about 60-70%, about 70-80%, about 80- 90%, or about 90-100%. In some embodiments, the identity between the 500 nucleotides of human genome sequence centered at the insertion site and the 500 nucleotides of native organism genome sequence centered at the insertion site is less than about 99.5%, about 99%, about 98%, about 97%, about 96%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 60%, or about 50%, or is between about 50-60%, about 60-70%, about 70- 80%, about 80-90%, or about 90-100%.

[0333] The template nucleic acid (e.g., template RNA) component of a gene modifying system described herein typically is able to bind the gene modifying protein of the system. In some embodiments, the template nucleic acid (e.g., template RNA) has a 3’ region that is capable of binding a gene modifying protein. The binding region, e.g., 3’ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the gene modifying protein of the system. The binding region may associate the template nucleic acid (e.g., template RNA) with any of the polypeptide modules. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with an RNA-binding domain in the polypeptide. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with the reverse transcription domain of the polypeptide (e.g., specifically bind to the RT domain). For example, where the reverse transcription domain is derived from a non-LTR retrotransposon, the template nucleic acid (e.g., template RNA) may contain a binding region derived from a non-LTR retrotransposon, e.g., a 3’ UTR from a non- LTR retrotransposon. In some embodiments a system or method described herein comprises a single template nucleic acid (e.g., template RNA). In some embodiments a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs). In some embodiments, when the system comprises a plurality of nucleic acids, each nucleic acid comprises a conjugating domain. In some embodiments, a conjugating domain enables association of nucleic acid molecules, e.g., by hybridization of complementary sequences.

[0334] In some embodiments, the template nucleic acid may comprise one or more UTRs (e.g., a 5’ UTR or a 3’ UTR, e.g., from an R2-type retrotransposon). In some embodiments, the UTR facilitates interaction of the template with the reverse transcriptase domain of the polypeptide. In some embodiments, the template possesses one or more sequences aiding in association of the 153template with the gene modifying polypeptide. In some embodiments, these sequences may be derived from retrotransposon UTRs. In some embodiments, the UTRs may be located flanking the desired insertion sequence. In some embodiments, a sequence with target site homology may be located outside of one or both UTRs. In some embodiments, the sequence with target site homology can anneal to the target sequence to prime reverse transcription. In some embodiments, the 5’ and / or 3’ UTR may be located terminal to the target site homology sequence. In some embodiments, the gene modifying system may result in the insertion of a desired payload without any additional sequence (e.g., a gene expression unit without UTRs used to bind the gene modifying protein).

[0335] The template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein the reverse transcription will result in insertion of the heterologous sequence into the target DNA. In other embodiments, the RNA template may be designed to write a deletion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In other embodiments, the template nucleic acid (e.g., template RNA) may be designed to write an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.

[0336] In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 nucleotides (and optionally no more than about 500, about 400, about 300, about 200, or about 100 nucleotides). In some embodiments, a gene modifying system is capable of producing an insertion into the target site of at least about 1, about 2, about 3, about 4, about 5, about 6, abo...

Claims

CLAIMS1. A method for administering a therapeutic composition to a patient, comprising:(a) collecting a blood fraction comprising lymphocytes from the patient;(b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a gene modifying system to create a blood-LNP composition, wherein the gene modifying system comprises:(i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and(ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequence into the genome of at least one lymphocyte to produce at least one edited lymphocyte;(c) optionally, removing residual LNPs from the blood-LNP composition to create a therapeutic composition comprising the at least one edited lymphocyte; and(d) reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction.

2. A method for ex vivo gene editing of patient lymphocytes, comprising:(a) collecting a blood fraction comprising lymphocytes from a patient;(b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a gene modifying system to create a blood-LNP composition, wherein the gene modifying system comprises:(i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and(ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequence into the genome of at least one lymphocyte to produce at least one edited lymphocyte;291wherein following the contacting for at least about one hour, at least 1% of the lymphocytes in the blood-LNP composition are edited.

3. A method for treating cancer in a patient comprising:(a) collecting a blood fraction comprising lymphocytes from the patient;(b) contacting the blood fraction with lipid nanoparticles (LNPs) encapsulating a gene modifying system to create a blood-LNP composition, wherein the gene modifying system comprises:(i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and(ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence, wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the gene modifying polypeptide integrates the heterologous object sequence into the genome of at least one lymphocyte to produce at least one edited lymphocyte;(c) optionally, removing residual LNPs from the blood-LNP composition to create a therapeutic composition comprising the at least one edited lymphocytes;(d) reinfusing the therapeutic composition into the patient within about 10 hours of collecting the blood fraction. wherein the edited lymphocytes target cancer cells.

4. The method of any of claims 1-3, wherein the LNPs comprise an ionizable lipid and a helper lipid, wherein the ionizable lipid is selected from lipids in Table LI and Table L3.

5. The method of claim 4. wherein the ionizable lipid has one of the following structures:2922932946. The method of claim 4, wherein the ionizable lipid has the structure:

7. The method of claim 4, wherein the ionizable lipid has the structure:

8. The method of claim 4, wherein the ionizable lipid has the structure:2959. The method of claim 4, wherein the ionizable lipid has the structure:

10. The method of any one of claims 1-3, wherein the ionizable lipid has the structure:

11. The method of any of claims 1-10, wherein the blood fraction is collected using leukapheresis.

12. The method of any of claims 1-11, wherein the blood fraction comprises peripheral blood mononuclear cells (PBMCs).

13. The method of any of claims 1-12, further comprising performing a wash to remove platelets from the blood fraction.

14. The method of any of claims 1-13, further comprising a spinning membrane separation to remove the platelets.

15. The method of claim 1-13, further comprising using a device comprising a centrifugation chamber to remove the platelets.

16. The method of any of claims 1-15, wherein the blood fraction comprises a lymphocyte concentration of from about 20x106cells / mL to about 200x106cells / mL or from about 20x106cells / mL to about 100x106cells / mL.

17. The method of any of claims 1-16, wherein the blood fraction comprises a cell density of from about 20x106cells / mL to about 200x106cells / mL or from about 20x106cells / mL to about 100x106cells / mL.29618. The method of any of claims 1-17, wherein the LNP is contacted with the blood fraction ex vivo.

19. The method of any of claims 1-18, wherein the LNP is contacted with the blood fraction ex vivo using an extra-corporeal delivery device.

20. The method of any one of claims 1-19, wherein the gene modifying system comprises the gene modifying polypeptide.

21. The method of claim 20, wherein the gene modifying polypeptide comprises a nickase domain, a DNA binding domain, a RNA binding domain, and a reverse transcriptase domain.

22. The method of claim 20, wherein the gene modifying polypeptide comprises an amino acid sequence set forth Table R2 or Table E3.

23. The method of claim 20, wherein the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl.

24. The method of any one of claims 1-19, wherein the gene modifying system comprises a nucleic acid encoding the gene modifying polypeptide.

25. The method of claim 24, wherein the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule.

26. The method of claim 24, wherein the nucleic acid encoding the gene modifying polypeptide is a DNA molecule.

27. The method of any of claims 25 or 26, wherein the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth Table E3 or Table E6.

28. The method of claim of any of claims 25 or 26, wherein the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

29. The method of any of claims 1-28, wherein the blood fraction is further contacted with a LNP comprising a heterologous gene modifying system.

30. The method of any of claims 1-29, further comprising stimulating the lymphocytes in the blood fraction with a T-cell stimulating reagent.

31. The method of claim 30, wherein the stimulating takes place before the contacting the LNP with the blood fraction.

32. The method of claim 31, wherein the stimulating takes place concurrently with the contacting the LNP with the blood fraction.

33. The method of any of claims 30-32, wherein the T-cell stimulating reagent comprises a CD3 agonist and / or a CD28 agonist.29734. The method of claims 30-33, wherein the T-cell stimulating reagent comprises a colloidal polymeric nanomatrix conjugated to a CD3 agonist and a CD28 agonist.

35. The method of any of claims 30-34, wherein the lymphocytes are stimulated for about 30 minutes to about 4 hours.

36. The method of any of claims 1-35, wherein the LNPs are contacted with the blood fraction less than 10 hours or less than 4 hours.

37. The method of any of claims 1-35, wherein the LNPs are contacted with the blood fraction for about 30 minutes to about 4 about hours.

38. The method of any of claims 1-37, wherein the blood-LNP composition comprises about 0.1 μg of the LNPs per lx 106cells to about 5 μg of the LNPs per 1x106cells.

39. The method of any of claims 31-38, wherein the blood-LNP composition comprises about 20 cells / mL to about 100 x 106cells / mL and about 54μL / mL to about 6.7μL / mL of T cell stimulating reagent.

40. The method of any of claims 1-39, wherein the heterologous object sequence, encodes a chimeric antigen receptor (CAR).

41. The method of claim 40, wherein the edited lymphocytes comprise the CAR integrated within genomic DNA.

42. The method of any of claims 1-41, wherein the edited lymphocytes express a CAR.

43. The method of any of claims 1-42, wherein about 1% to about 30% of lymphocytes in the therapeutic composition are edited lymphocytes.

44. The method of any of claims 1 or 3-43, wherein the therapeutic composition further comprises a pharmaceutically acceptable buffer.

45. The method of any of claims 1 or 3-44, further comprising performing sterility testing before reinfusion.

46. The method of any of claims 1 or 3-45, further comprising assaying the therapeutic composition to determine the number or percentage of edited lymphocytes.

47. The method of any of claims 1 or 3-46, the therapeutic composition does not comprise microbial contaminants.

48. The method of any of claims 1 or 3-47, wherein the therapeutic composition is reinfused into the patient within about 1 hour to about 9 hours.

49. The method of any of claims 1-48, wherein the edited lymphocytes expand in-vivo after the therapeutic composition is reinfused into the patient.

50. The method of any of claims 1-49, wherein about 7 days after reinfusion, about 0% - about 20% of the patient’s lymphocytes are edited lymphocytes.29851. The method of any of claims 1-50, where about 7 days after reinfusion, the patient T cells comprise between about 30 million and about 1 billion CAR-T cells.

52. The method of any of claims 1 or 3-51, wherein the method is carried out in a single inline procedure to maintain a closed or functionally closed fluid circuit.

53. The method of any of claims 1-52, wherein (i) the gene modifying polypeptide, or the nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence, are encapsulated in separate LNPs.

54. The method of claim 53, wherein the blood fraction is contacted with the LNPs encapsulating (i) the gene modifying polypeptide, or the nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence at a ratio of between about 1:2 to about 1:25.

55. The method of any of claims 1-52, wherein the (i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence, are encapsulate in the same LNP.

56. The method of claim 55, wherein the blood fraction is contacted with the LNP encapsulating (i) the gene modifying polypeptide, or the nucleic acid encoding the gene modifying polypeptide, and (ii) the template nucleic acid comprising (1) the sequence that binds to the gene modifying polypeptide and (2) the heterologous object sequence at a ratio of between about 1:2 to about 1:25.

57. The method of any of claims 1-56, wherein the template nucleic acid is a RNA molecule.

58. The method of any of claims 1-57, wherein the template nucleic acid comprises the sequence set forth in SEQ ID NO: 575.

59. The method of any of claims 1-58, wherein the LNPs comprise a targeting moiety.

60. The method of claim 59, wherein the targeting moiety is conjugated to the LNPs through a linker, and wherein the linker comprises an enzyme recognition sequence and a Click product formed from a Click reaction between a first Click handle on the targeting moiety and a second Click handle on the LNPs.

61. The method claim 60, wherein the Click reaction is an inverse electron demand Diels- Adler reaction between a trans-cyclooctene (TCO) moiety on the first or second Click handle and a tetrazine ring on the first or second Click handle.29962. The method of any of claims 59-61, wherein the targeting moiety binds to a surface protein on T cells.

63. The method of any of claims 59-62, wherein the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7.

64. The method of any of claims 59-63, wherein the targeting moiety comprises an anti-CD3 moiety.

65. The method of claim 64, wherein the anti-CD3 moiety comprises any of the sequences set forth in Table E7.

66. The method of any of claims 40-65 wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a first intracellular signaling domain, and a second intracellular signaling domain.

67. The method of any of claims 40-66, wherein the CAR comprises an antigen-binding domain that binds to one or more antigens of a blood cancer.

68. The method of claim 67, wherein the blood cancer is leukemia, lymphoma, or multiple myeloma.

69. The method of claim 66 or 67, wherein the one or more antigens is a B cell antigen.

70. The method of claim 66, wherein the antigen binding domain binds to one or more antigens of a solid tumor.

71. The method of any of claims 66-70, wherein the antigen binding domain comprises an amino acid sequence or an antigen binding domain set forth in Table 4.

72. The method of claim any of claims 66-71, wherein the antigen binding domain comprises an scFv.

73. The method of any of claims 40-72, wherein the CAR comprises a linker domain comprising an amino acid sequence of a linker domain set forth in Table Linkerl.

74. The method of any of claims 40-73, wherein the CAR comprises a hinge domain.

75. The method of any of claims 65-74, wherein the first intracellular signaling domain comprises an amino acid sequence of an intracellular signaling domain set forth in Table 5 or Table 6.

76. The method of any of claims 65-75, wherein the second intracellular signaling domain comprises an amino acid sequence of an intracellular signaling domain set forth in Table 5 or Table 6.

77. The method of any of claims 40-76, wherein the CAR comprises a costimulatory domain comprising an amino acid sequence of a costimulatory domain set forth in Table 5 or Table 6.30078. A system for administering a therapeutic composition to a patient the system comprising:(a) an incoming processing unit for collecting a blood fraction from the subject;(b) a chamber for contacting lipid nanoparticles (LNPs) encapsulating components of a gene modifying system with the blood fraction to create a blood- LNP composition, wherein the gene modifying system comprises:(i) a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide, and(ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence; wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs,(c) optionally, a processing unit for removing residual LNPs from the blood-LNP composition to create a therapeutic composition; and(d) a transfer container for reinfusing the therapeutic composition into the same subject within 10 hours of removing the blood fraction.

79. The system of claim 78, wherein the incoming processing unit is a leukapheresis device.

80. The system of claim 78 or 79, wherein the gene modifying system comprises the gene modifying polypeptide.

81. The system of claim 80, wherein the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl.

82. The system of any of claims 78-81, wherein the blood fraction is further contacted with a LNP comprising a heterologous gene modifying system.

83. The system of claim 79 or 80, wherein the gene modifying polypeptide comprises an amino acid sequence set for in Table R2, Table E3, or Table E6.

84. The system of claim 78 or 79, wherein the gene modifying system comprises the nucleic acid encoding the gene modifying polypeptide.

85. The system of claim 84, wherein the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule.

86. The system of claim 84, wherein the nucleic acid encoding the gene modifying polypeptide is a DNA molecule.

87. The system of claims 85 or 86, wherein the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth in Table Rl, Table E3, or Table E6 .30188. The system of claim of any of claims 85 or 86, wherein the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

89. The system of any of claims 78-88, wherein the LNPs comprise a targeting moiety.

90. The system of claim 89, wherein the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7.

91. The system of any of claims 89-90, wherein the targeting moiety comprises an anti-CD3 moiety.

92. The system of claim 91, wherein the anti-CD3 moiety comprises any of the sequences set forth in Table E7.

93. A blood-LNP composition comprising:(a) lymphocytes; wherein the concentration of lymphocytes is around 20x106cells / mL to about 200x106cells / mL;(b) lipid nanoparticles (LNPs) encapsulating a gene modifying system with the blood fraction to create a blood-LNP composition, wherein the gene modifying system comprises:(i) a gene modifying polypeptide, or a nucleic acid encoding the gene modifying polypeptide, and(ii) a template nucleic acid comprising (1) a sequence that binds to the gene modifying polypeptide and (2) a heterologous object sequence; and wherein (i) and (ii) are encapsulated together in the same LNP or are encapsulated in separate LNPs, wherein the concentration of the LNPs is around 0.1 μg LNP per lx 106cells - 5 μg LNP per 1x106; and(c) optionally, a T-cell stimulating reagent.

94. The blood-LNP composition of claim 93, wherein the gene modifying system comprises the gene modifying polypeptide.

95. The blood-LNP composition of claim 94, wherein the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl.

96. The blood-LNP composition of any of claims 93-95, further comprising LNPs encapsulating a heterologous gene modifying system.

97. The blood-LNP composition of claim 93 or 94, wherein the gene modifying polypeptide comprises an amino acid sequence set forth in Table R2, Table E3, or Table E6.30298. The blood-LNP composition of claim 93, wherein the gene modifying system comprises the nucleic acid encoding the gene modifying polypeptide.

99. The blood-LNP composition of claim 98, wherein the nucleic acid encoding the gene modifying polypeptide is a DNA molecule.

100. The blood -LNP composition of claim 98, wherein the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule.

101. The blood -LNP composition of claim 99 or 100, wherein the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth in Table Rl, Table E3, or Table E6.

102. The blood-LNP composition of any of claims 98-100, wherein the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

103. The blood-LNP composition of any of claims 93-102, wherein the LNP comprises a targeting moiety.

104. The blood-LNP composition of claim 103, wherein the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7.

105. The blood-LNP composition of any of claims 103-104, wherein the targeting moiety comprises an anti-CD3 moiety.

106. The blood-LNP composition of claim 105, wherein the anti-CD3 moiety comprises any of the sequences set forth in Table E7.303

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