WO2025166314A1

WO2025166314A1 - Ex vivo delivery of lipid nanoparticles for delivery of gene modifying systems to t cells

Info

Publication number: WO2025166314A1
Application number: PCT/US2025/014224
Authority: WO
Inventors: Kassi Taylor STEIN; Jason John RODRIGUEZ
Original assignee: Tessera Therapeutics Inc
Current assignee: Tessera Therapeutics Inc
Priority date: 2024-02-02
Filing date: 2025-01-31
Publication date: 2025-08-07
Anticipated expiration: 2026-08-02

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 CELLS

CROSS 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 edited

1 cells 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: ox

3

4

[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 20x10⁶ cells/mL to about 10Ox10⁶ cells/mL. In some aspects, wherein the blood fraction comprises a cell density of about 20x10⁶ cells/mL to about 100x10⁶ cells/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 Table

6 E6. 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 10⁶ cells to about 5 μg of the LNP per 1x10⁶ cells. In some aspects, the blood-LNP composition comprises about 20 cells/mL to about 100 x 10⁶ cells/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 10⁶ cells to about 10 μg of the RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg of the RNA per lx 10⁶ cells to about 5 μg of the RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 1 μg of the RNA per lx 10⁶ cells to about 5 μg of the RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 2 μg of the RNA per lx 10⁶ cells to about 5 μg of the RNA per 1x10⁶ cells.

[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 LNPs

8 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.

[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. In

9 some 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 modifying

10 polypeptide 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 20x10⁶ cells/mL to about 100x10⁶ cells/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 10⁶ cells - 5 μg LNP per 1x10⁶ cells; 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 20x10⁶ cells/mL to about 200x10⁶ cells/mL or wherein the concentration of lymphocytes is around 100x10⁶ cells/mL to about 200x10⁶ cells/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 10⁶ cells - 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. In

11 some 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 LNPs

13 at 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 2x10⁵ cells), 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 a

17 patient 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, an

19 endonuclease 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 (a

21 point 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-specific

22 expression-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 a

25 receptor 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, 26 one 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 28 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: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 29 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: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^

30 signaling 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 region

31 comprising 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 an

32 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: 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., at

33 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: 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 34 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: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 35 the 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 least

36 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: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 37 comprising 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 38 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: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 of

39 SEQ 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 41 embodiments, 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-H2

42 comprising 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 43 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: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., at

44 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: 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 47 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: 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, 48 207, 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 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.

49 The 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 50 antibody, 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)_U ALEA(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)_U ALEA(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)_U ALEA(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 53 modified 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 54 targeting 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 C_L.-C_HI 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 of

55 type 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:

[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 a

57 modified 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,

59 increasing 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-reactive

60 group 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, the

61 cysteine 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).

62 In 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 63 comprise 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 LN

C. 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 include

64 phytosterols, 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 20

65 mm 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 may

66 be 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 67 neutral 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.

68

Lipid 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 Lipid

Structure

ID

69 [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;

R¹ is C_1-4alkyl;

R² is C_1-4alkyl; n is 1, 2, 3, or 4;

R³ is C_4-1oalkyl;

R⁴ is C_4-10alkyl; p is 2, 3, 4, 5, or 6;

R⁵ is C_4-10alkyl; and

R⁶ is C_4-10alkyl.

[0213] In some embodiments, compounds of formula (IV) are compounds of formula (IV-A): o O

N O.

O' ‘O'

O.

‘O O o ‘0 p

R⁵ R⁶

(IV-A), or a pharmaceutically acceptable salt thereof, wherein: p is 2, 3, 4, 5, or 6;

77 R⁵ is C_4-10alkyl; and

R⁶ is C_4-10alkyl.

[0214] In some embodiments, compounds of formula (IV) are compounds of formula (IV-B):

O

.0. R⁵ o R⁶

(IV-B), or a pharmaceutically acceptable salt thereof, wherein:

R⁵ is C_4-6alkyl; and

R⁶ is C_4-6alkyl.

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

Table L3: Exemplary Ionizable Lipids

Compound Structure

78

[0216] In some embodiments, the ionizable lipid has one of the structures depicted below:

Lipid 154

80

Lipid 163

81

82

Lipid 183 o o o o o

O'

.0. o

Lipid 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;

83 I, 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,

84 17-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 LNP

85 is 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 to

86 enantiomers, 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,

87 dilinoleoylphosphatidylcholine, 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 targeted

88 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%, 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 89 1 : 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, is

90 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%.

[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 some

91 embodiments, 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 92 embodiments, 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 least

96 about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about

99% identity thereto).

Table Rl: Retrotransposase systems with improved integration activity

B. Gene modifying polypeptide

RT 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%,

120 at 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 some

124 embodiments, 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-

125 enriched 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 at

126 least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about

98%, 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 in

Table 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 BMC

Bioinformat 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 be

131 derived 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 132 sequence 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]-¹ or [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 to

133 preserve 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 optimal

134 packaging 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)_n

Interns

[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 separate

138 genes, 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 G

Promoters

[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 enhancer

140 that 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 141 as 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 occurring

142 promoters. 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 the

CMV 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 some

143 embodiments, 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.

144 C. Template Nucleic Acid

Template 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,

145 a 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 derived

146 from 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 a

147 secondary 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 148 RNA 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 in

149 combination 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 object

150 sequence, 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%, 152 or 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 153 template 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, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 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 154 target site of at least about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 or about 10 kilobases (and optionally no more than about 1, about 5, about 10, or about 20 kilobases). In some embodiments, a gene modifying system is capable of producing a deletion of at least about 81, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 nucleotides (and optionally no more than about 500, about 400, about 300, or about 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least about 81, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 nucleotides (and optionally no more than about 500, about 400, about 300, or about 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, or about 200 nucleotides (and optionally no more than about 500, about 400, about 300, or about 200 nucleotides). In some embodiments, a gene modifying system is capable of producing a deletion of at least about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 or about 10 kilobases (and optionally no more than about 1, about 5, about 10, or about 20 kilobases).

Heterologous Object sequence - Chimeric Antigen Receptors

[0337] In some embodiments, the heterologous object sequence encodes a membrane protein, e.g., a CAR or a membrane protein other than a CAR, and/or an endogenous human membrane protein. In some embodiments, the heterologous object sequence encodes an extracellular protein. In some embodiments, the heterologous object sequence encodes an enzyme, a structural protein, a signaling protein, a regulatory protein, a transport protein, a sensory protein, a motor protein, a defense protein, or a storage protein. Other exemplary proteins that may be encoded by a heterologous object sequence include, without limitation, an immune receptor protein, e.g. a synthetic immune receptor protein such as a chimeric antigen receptor protein (CAR), a T cell receptor, a B cell receptor, or an antibody.

155 [0338] In some embodiments, the heterologous object sequence encodes a chimeric antigen receptor (CAR) comprising an antigen binding domain. In some embodiments, the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a single intracellular signaling domain. In some embodiments, the CAR is or comprises a second generation CAR comprising an antigen binding domain, a transmembrane domain, and two intracellular signaling domains (e.g., a first intracellular signaling domain and a second intracellular signaling domain). In some embodiments, the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three intracellular signaling domains. In some embodiments, a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four intracellular signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, the antigen binding domain is or comprises an scFv, Fab, a diabody, a D domain binder, centyrins (e.g., antibody-like scaffolds, e.g., a CARTyrin), one or more single domain antibodies such as VHH domains (e.g., comprises two VHH binding domains).

[0339] In some embodiments, a CAR antigen binding domain is or comprises an antibody or antigen-binding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab. In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, a cell surface antigen is characteristic of one type of cell. In some embodiments, a cell surface antigen is characteristic of more than one type of cell. In some embodiments, the CAR antigen binding domain binds to two epitopes of the target antigen (e.g., is a biepitopic binding domain). In some embodiments, the CAR comprises two antigen binding domains, such that each antigen binding domain binds to a different target antigen on a cell, e.g., a neoplastic cell.

[0340] In some embodiments, the antigen binding domain targets an antigen characteristic of a T-cell. In some embodiments, the antigen characteristic of a T-cell is selected from an exemplary T cell antigen listed in Table 4, or an antigenic fragment thereof.

Table 4: Exemplary T-cell Antigens

[0341] In some embodiments, the CAR comprises at least one signaling domain selected from one or more intracellular signaling domains listed in Table 5, or a functional fragment thereof. In some embodiments, the CAR comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the first intracellular signaling domain mediates downstream signaling during T-cell activation. In some embodiments, the second intracellular signaling domain is a costimulatory domain.

Table 5: Exemplary Intracellular Signaling Domains

[0342] In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a

CD28 domain or functional variant thereof; and (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof. In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof, and/or (iii) a 4- IBB domain, or a CD 134 domain, or functional variant thereof. In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (IT AM), or functional variant thereof; (ii) a

158 CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0343] In some embodiments, the CAR comprises a CD28z co-stimulatory domain.

[0344] In some embodiments, the CAR comprises a CD3z signaling domain.

[0345] In some embodiments, intracellular signaling domain comprises an intracellular signaling domain listed in Table 6, or sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

Table 6: Sequences of Exemplary Intracellular Signaling Domains

[0346] In some embodiments, the CAR further comprises one or more spacers, e.g., wherein the spacer is a first spacer between the antigen binding domain and the transmembrane domain. In some embodiments, the first spacer includes at least a portion of an immunoglobulin constant region or variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and a signaling domain. In some embodiments, the second spacer is an oligopeptide, e.g., wherein the oligopeptide comprises glycine-serine doublets. In some embodiments, the CAR further comprises a hinge domain. In some embodiments, the hinge domain is a CDS hinge domain. In some embodiments, the CDS hinge domain has an amino acid sequence of a CDS hinge domain in Table 7, or sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

Table 7: Sequences of Exemplary Hinge Domains

[0347] In some embodiments, the CAR comprises a sequence of a CAR listed in Table 8, or sequence having at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity thereto.

Table 8: Sequences of Exemplary CAR Molecules

IV. HETEROLOGOUS GENE MODIFYING SYSTEM

[0348] In some embodiments, the gene modifying system is a heterologous gene modifying system. In some embodiments, the gene modifying polypeptide comprises a Cas domain and a

165 reverse transcriptase domain. In some embodiments, the gene modifying polypeptide comprises 1) a Cas domain (e.g., a Cas nickase domain, e.g., a Cas9 nickase domain); 2) a reverse transcriptase (RT) domain disclosed herein, or a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% identity thereto, wherein the RT domain is C-terminal of the Cas domain; and a linker disposed between the RT domain and the Cas domain.

Cas domain

[0349] In some embodiments, the Cas domain can direct the heterologous gene modifying polypeptide to a target site specified by a gRNA spacer, thereby modifying a target nucleic acid sequence in “cis”. In some embodiments, a heterologous gene modifying polypeptide is fused to a Cas domain. In some embodiments, a heterologous gene modifying polypeptide comprises a CRISPR/Cas domain (also referred to herein as a CRISPR-associated protein). In some embodiments, a CRISPR/Cas domain comprises a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., a Cas protein, and optionally binds a guide RNA, e.g., single guide RNA (sgRNA).

[0350] CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e.g., Cas9 or Cpfl) to cleave foreign DNA. For example, in a typical CRISPR- Cas system, an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “spacer” sequence, a typically about 20-nucleotide RNA sequence that corresponds to target DNA sequence (“protospacer”). In the wild-type system, and in some engineered systems, crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure that is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid molecule. A crRNA/tracrRNA hybrid then directs the Cas endonuclease to recognize and cleave a target DNA sequence. A target DNA sequence is generally adjacent to a “protospacer adjacent motif’ (“PAM”) that is specific for a given Cas endonuclease and required for cleavage activity at a target site matching the spacer of the crRNA. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements, e.g., examples of PAM sequences

166 include 5'-NGG (Streptococcus pyogenes), 5'-NNAGAA (Streptococcus thermophilus CRISPR1), 5'-NGGNG (Streptococcus thermophilus CRISPR3), and 5'-NNNGATT (Neisseria meningiditis). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5'-NGG, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5' from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpfl, which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpfl -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpfl system, in some embodiments, comprises only Cpfl nuclease and a crRNA to cleave a target DNA sequence. Cpfl endonucleases, are typically associated with T-rich PAM sites, e. g., 5'-TTN. Cpfl can also recognize a 5' -CT A PAM motif. Cpfl typically cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5' overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3' from) from a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759 - 771.

[0351] A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpfl, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In some embodiments, a DNA-binding domain or endonuclease domain includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram-positive bacteria or a gram-negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus (e.g., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., anN. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.

167 [0352] In some embodiments, a heterologous gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 557 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 557 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the N-terminal end of the heterologous gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 557 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the N-terminal end of the heterologous gene modifying polypeptide.

Exemplary N-terminal NLS-Cas9 domain

MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI

KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE

SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK

FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE

NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG

DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL

PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR

TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW

MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE

LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLF

DDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVI

EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNG

RDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVK

KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL

DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV

GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLA

NGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK

RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER

SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK

YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVL

SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI

TGLYETRIDLSQLGGDGG (SEQ ID NO: 557)

168 [0353] In some embodiments, a heterologous gene modifying polypeptide may comprise the amino acid sequence of SEQ ID NO: 127 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto. In embodiments, the amino acid sequence of SEQ ID NO: 127 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned at the C-terminal end of the heterologous gene modifying polypeptide. In embodiments, the amino acid sequence of SEQ ID NO: 127 below, or the sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto, is positioned within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids of the C-terminal end of the heterologous gene modifying polypeptide.

Exemplary C-terminal sequence comprising an NLS

AGKRTADGSEFEKRTADGSEFESPKKKAKVE (SEQ ID NO: 127)

Exemplary benchmarking sequence

MPAAKRVKLDGGDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSI

KKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEE

SFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK

FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE

NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG

DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQL

PEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQR

TFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW

MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNE

LTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISG

VEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLF

DDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVI

EMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNG

RDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVK

KMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQIL

DSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV

GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLA NGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPK RNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMER

SSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK

169 YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVL SAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDGGSGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGTLNIEDEY RLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVSIKQYPMSQ EARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPT

VPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTR LPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTL GNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFL GKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLT

KPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLT KDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVA

LNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKA GAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIH GEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNRMADQ AARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFEAGKRTADGSEFEKRTADGSEFESP

KKKAKVE (SEQ ID NO: 558)

[0354] In some embodiments, a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or function. In some embodiments, the PAM is or comprises, from 5' to 3', NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G. In some embodiments, a Cas protein is a protein listed in Table 7 or 8. In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises DI 135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions. Exemplary advances in the engineering of Cas enzymes to recognize altered PAM sequences are reviewed in Collias et al Nature Communications 12:555 (2021), incorporated herein by reference in its entirety.

170 [0355] In some embodiments, the Cas protein is catalytically active and cuts one or both strands of the target DNA site. In some embodiments, cutting the target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by the cellular repair machinery.

[0356] In some embodiments, the Cas protein is modified to deactivate or partially deactivate the nuclease, e.g., nuclease-deficient Cas9. Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 that has been partially deactivated generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, dCas9 binding to a DNA sequence may interfere with transcription at that site by steric hindrance. In some embodiments, dCas9 binding to an anchor sequence may interfere with (e.g., decrease or prevent) genomic complex (e.g., ASMC) formation and/or maintenance. In some embodiments, a DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9. Many catalytically inactive Cas9 proteins are known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A or N863A mutations.

[0357] In some embodiments, a catalytically inactive, e.g., dCas9, or partially deactivated Cas9 protein comprises a DI 1 mutation (e.g., DI 1 A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises aH969 mutation (e.g., H969A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises aN995 mutation (e.g., N995A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises mutations at one, two, or three of positions Dll, H969, and N995 (e.g., Dll A, H969A, and N995A mutations) or analogous substitutions to the amino acids corresponding to said positions.

[0358] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a DIO mutation (e.g., a D10A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g.,

171 dCas9, comprises a DIO mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or analogous substitutions to the amino acids corresponding to said positions.

[0359] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises aN863 mutation (e.g., aN863A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a DIO mutation (e.g., D10A), aD839 mutation (e.g., D839A), aH840 mutation (e.g., H840A), and aN863 mutation (e.g., N863A) or analogous substitutions to the amino acids corresponding to said positions.

[0360] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises an E993 mutation (e.g., a E993A mutation) or an analogous substitution to the amino acid corresponding to said position.

[0361] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D917 mutation (e.g., a D917A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a a E1006 mutation (e.g., a E1006A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917 mutation (e.g., D917A), a E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or analogous substitutions to the amino acids corresponding to said positions.

[0362] In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D16 mutation (e.g., a D16A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution to the amino acid

172 corresponding to said position. In some embodiments, a partially deactivated Cas domain has nickase activity. In some embodiments, a partially deactivated Cas9 domain is a Cas9 nickase domain. In some embodiments, the catalytically inactive Cas domain or dead Cas domain produces no detectable double strand break formation. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises aH588 mutation (e.g., aH588A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N611 mutation (e.g., a N611 A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), aH588 mutation (e.g., H588A), and aN611 mutation (e.g., N611A) or analogous substitutions to the amino acids corresponding to said positions.

[0363] In some embodiments, a DNA-binding domain or endonuclease domain may comprise a Cas molecule comprising or linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid, e.g., template RNA, comprising a gRNA).

[0364] In some embodiments, an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a modified SpCas9. In embodiments, the modified SpCas9 comprises a modification that alters protospacer-adjacent motif (PAM) specificity. In embodiments, the PAM has specificity for the nucleic acid sequence 5'-NGT-3'. In embodiments, the modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of positions Lilli, D1135, G1218, E1219, A1322, of R1335, e.g., selected from Lil HR, D1135V, G1218R, E1219F, A1322R, R1335V. In embodiments, the modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, e.g., selected from LI 111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto. In embodiments, the modified SpCas9 comprises: (i) one or more amino acid substitutions selected from DI 135L, SI 136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more amino acid substitutions selected from Lil HR, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto.

173 [0365] In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas domain, e.g., a Cas9 domain. In embodiments, the endonuclease domain or DNA binding domain comprises a nuclease-active Cas domain, a Cas nickase (nCas) domain, or a nucleaseinactive Cas (dCas) domain. In embodiments, the endonuclease domain or DNA binding domain comprises a nuclease-active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nucleaseinactive Cas9 (dCas9) domain. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 (e.g., dCas9 and nCas9), Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, the endonuclease domain or DNA binding domain comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference. In some embodiments, the endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and/or the RuvCl subdomain of a Cas, e.g., Cas9, e.g., as described herein, or a variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, or Casl2i. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas polypeptide (e.g., enzyme), or a functional fragment thereof. In embodiments, the Cas polypeptide (e.g., enzyme) is selected from Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, CasSa, Cas8b, Cas8c, Cas9 (e.g., Csnl or Csxl2), Cas10, Cas10d, Casl2a/Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2d/CasY, Casl2e/CasX, Casl2g, Casl2h, Casl2i, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, Cse5e, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl l, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARE, DinG, Cpfl, Casl2b/C2cl, Casl2c/C2c3, Casl2b/C2cl, Casl2c/C2c3, SpCas9(K855A), eSpCas9(l.l), SpCas9-HFl, hyper accurate Cas9 variant (HypaCas9), homologues thereof, modified or engineered versions thereof, and/or functional fragments thereof. In embodiments, the Cas9 comprises one or more substitutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, DI 125A, W1126A, and DI 127 A. In embodiments, the Cas9 comprises one or more mutations at positions selected from: DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987, e.g., one or more 174 substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas (e.g., Cas9) sequence from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or a fragment or variant thereof.

[0366] In some embodiments, the endonuclease domain or DNA binding domain comprises a Cpfl domain, e.g., comprising one or more substitutions, e.g., at position D917, E1006A, D1255 or any combination thereof, e.g., selected from D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, and D917A/E1006A/D1255A.

[0367] In some embodiments, the endonuclease domain or DNA binding domain comprises spCas9, spCas9-VRQR, spCas9- VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9- LRVSQL.

[0368] In some embodiments, a heterologous gene modifying polypeptide has an endonuclease domain comprising a Cas9 nickase, e.g., Cas9 H840A. In embodiments, the Cas9 H840A has the following amino acid sequence:

Cas9 nickase (H840A) :

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEA

TRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGN

IVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDV

DKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLI

ALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAG

YIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAI

LRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVV

DKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS

GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGW

GRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS

LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSR

ERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYD

175 VDATVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQR

KFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVK

VITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDY

KVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV

WDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKY

GGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV

KKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP

EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENII

HLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

(SEQ IDNO: 559)

[0369] In some embodiments, a heterologous gene modifying polypeptide comprises a dCas9 sequence comprising a D10A and/or H840A mutation, e.g., the following sequence:

SMDKKYSIGLAIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET

AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPI

FGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDN

SDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLF

GNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS

DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKN

GYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLG

ELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNF

EEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRK

PAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD

LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRY

TGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQ

GDSLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQK

NSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS

DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI

TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVY

GDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETG

EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPK

KYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK

EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG

SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE

NIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

(SEQ IDNO: 560)

RT domain

[0370] In some embodiments, the RT domain comprises an RT catalytic portion and RNA- binding region (e.g., a region that binds the template RNA).

176 [0371] In some embodiments, a nucleic acid encoding 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 retrovirus. In some embodiments, the RT domain has been mutated from its original amino acid sequence, e.g., has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions. In some embodiments, the RT domain is derived from the RT of a retrovirus, e.g., HTV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, or Rous Sarcoma Virus (RSV) RT.

[0372] In some embodiments, the retroviral reverse transcriptase (RT) domain exhibits enhanced stringency of target-primed reverse transcription (TPRT) initiation, e.g., relative to an endogenous RT domain. In some embodiments, the RT domain initiates TPRT when the 3 nt in the target site immediately upstream of the first strand nick, e.g., the genomic DNA priming the RNA template, have at least 66% or 100% complementarity to the 3 nt of homology in the RNA template. In some embodiments, the RT domain initiates TPRT when there are less than 5 nt mismatched (e.g., less than 1, 2, 3, 4, or 5 nt mismatched) between the template RNA homology and the target DNA priming reverse transcription. In some embodiments, the RT domain is modified such that the stringency for mismatches in priming the TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in the priming region relative to a wild-type (e.g., unmodified) RT domain. In some embodiments, the RT domain comprises aHIV-1 RT domain. In embodiments, the HIV-1 RT domain initiates lower levels of synthesis even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described by Jamburuthugoda and Eickbush J Mol Biol 407(5): 661-672 (2011); incorporated herein by reference in its entirety). 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, e.g., is derived from a virus wherein it functions as a monomer. In embodiments, the RT domain is selected from an RT domain from murine leukemia virus (MLV; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), Avian reticuloendotheliosis virus (AVIRE) (e.g., UniProtKB accession: P03360); Feline leukemia virus (FLV or FeLV) (e.g., e.g., UniProtKB accession: P10273); Mason-Pfizer monkey virus (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt P03361), human T-cell leukemia virus- 1 (HTLV-1) (e.g., UniProt P03362), human foamy virus (HFV) (e.g., UniProt

177 P14350), simian foamy virus (SFV) (e.g., SFV3L) (e.g., UniProt P23074 or P27401), or bovine foamy /syncytial virus (BFV/BSV) (e.g., UniProt 041894), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto). In some embodiments, an RT domain is dimeric in its natural functioning. In some embodiments, the RT domain is derived from a virus wherein it functions as a dimer. In embodiments, the RT domain is selected from an RT domain from avian sarcoma/leukemia virus (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis virus (AMV) (e.g., UniProt Q83133), human immunodeficiency virus type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt Pl 5833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency virus (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency virus (FIV) (e.g., UniProt P16088) (Herschhom and Hizi Cell Mol Life Sci 67(16):2717-2747 (2010)), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto). 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.

[0373] In some embodiments, a system described herein comprises an integrase domain, e.g., wherein the integrase domain may be part of the RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an integrase domain. In some embodiments, an RT domain (e.g., as described herein) lacks an integrase domain, or comprises an integrase domain that has been inactivated by mutation or deleted. In some embodiment, a system described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of the RT domain. In some embodiments, the RNase H domain is not part of the RT domain and is covalently linked via a flexible linker. 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, the polypeptide comprises an inactivated endogenous RNase H domain. In some embodiments, an endogenous RNase H domain from one of the other domains

178 of the polypeptide is genetically removed such that it is not included in the polypeptide, e.g., the endogenous RNase H domain is partially or completely truncated from the comprising 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 (1988) (incorporated herein by reference in its entirety), e.g., lower by at least

10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is abolished.

[0374] 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 motif (SEQ ID NO: 563) 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).

[0375] In some embodiments, reverse transcriptase domains are modified, for example by sitespecific mutation. In some embodiments, reverse transcriptase domains are engineered to have improved properties, e.g. SuperScript IV (SSIV) reverse transcriptase derived from the MMLV RT. In some embodiments, the reverse transcriptase domain may be engineered to have lower error rates, e.g., as described in W02001068895, incorporated herein by reference. In some embodiments, the reverse transcriptase domain may be engineered to be more thermostable. In some embodiments, the reverse transcriptase domain may be engineered to be more processive. In some embodiments, the reverse transcriptase domain may be engineered to have tolerance to inhibitors. In some embodiments, the reverse transcriptase domain may be engineered to be faster. In some embodiments, the reverse transcriptase domain may be engineered to better tolerate modified nucleotides in the RNA template. In some embodiments, the reverse transcriptase domain may be engineered to insert modified DNA nucleotides. In some embodiments, the reverse transcriptase domain is engineered to bind a template RNA. In some embodiments, one or more mutations are chosen from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, H8Y, T306K, or D653N in the RT domain of murine leukemia virus reverse transcriptase or a corresponding mutation at a corresponding position of another RT domain.

179 [0376] In some embodiments, a heterologous gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., a wild-type M-MLV RT, e.g., comprising the following sequence:

M-MLV (WT):

TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI

SGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQ

GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK

TPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTA PALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLR MVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD

RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSS LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR YAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEA

RGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO: 564)

[0377] In some embodiments, a heterologous gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., an M-MLV RT, e.g., comprising the following sequence:

TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS

IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN

KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI

SGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQ

GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK

TPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQALLTA

PALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLR

MVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD

RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSS

LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR

YAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEA

RGNRMADQAARKAAITETPDTSTLL (SEQ ID NO: 565)

180 [0378] In some embodiments, a heterologous gene modifying polypeptide comprises the RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659-1329 of NP 057933. In embodiments, the heterologous gene modifying polypeptide further comprises one additional amino acid at the N-terminus of the sequence of amino acids 659-1329 of NP 057933, e.g., as shown below:

TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS

IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN

KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDP

EMGISGQLTWTRLPQGFKNSPTLFDEALHRDLADFRIQHPDLILLQYVDDLLLAAT

SELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKE

TVMGQPTPKTPRQLREFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAY

QEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPV

AAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTH

YQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDAD

HTWYTDGSSLLOEGORKAGAAVTTETEVIWAKALPAGTSAORAELIALTQALKMAEGK

KLNVYTDSRYAFATAHIHGEIYRRRGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPG

HQKGHSAEARGNRMADQAARKAA (SEO ID NO: 566)

Core RT (bold), annotated per above RNAseH (underlined), annotated per above

[0379] In embodiments, the heterologous gene modifying polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP 057933. In embodiments, the heterologous gene modifying polypeptide comprises an RNaseHl domain (e.g., amino acids 1178-1318 of NP_057933).

[0380] In some embodiments, a retroviral reverse transcriptase domain, e.g., M-MLV RT, may comprise one or more mutations from a wild-type sequence that may improve features of the RT, e.g., thermostability, processivity, and/or template binding. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, e.g., a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and W313F. In some embodiments, an M-MLV RT used herein comprises the mutations D200N, L603W, T330P, T306K, and W313F. In embodiments, the mutant M-MLV RT comprises the following amino acid sequence:

M-MLV (PE2):

TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIPLKATSTPVS

181 IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN

KRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI

SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQ

GTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPK

TPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTA

PALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLR

MVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTD

RVQFGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSS

LLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR

YAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEA

RGNRMADQAARKAAITETPDTSTLLI (SEQ ID NO: 567)

[0381] In some embodiments, a writing domain (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 of the writing domain.

[0382] In some embodiments, the reverse transcription domain only recognizes and reverse transcribes a specific template, e.g., a template RNA of the system. In some embodiments, the template comprises a sequence or structure that enables recognition and reverse transcription by a reverse transcription domain. In some embodiments, the template comprises a sequence or structure that enables association with an RNA-binding domain of a polypeptide component of a genome engineering system described herein. In some embodiments, the genome engineering system reverse preferably transcribes a template comprising an association sequence over a template lacking an association sequence.

[0383] The writing domain may also comprise DNA-dependent DNA polymerase activity, e.g., comprise enzymatic activity capable of writing DNA into the genome from a template DNA sequence. In some embodiments, DNA-dependent DNA polymerization is employed to complete second-strand synthesis of a target site edit. In some embodiments, the DNA- dependent DNA polymerase activity is provided by a DNA polymerase domain in the polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second-strand synthesis. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a second polypeptide of the system. In some embodiments, the DNA-

182 dependent DNA polymerase activity is provided by an endogenous host cell polymerase that is optionally recruited to the target site by a component of the genome engineering system.

[0384] In some embodiments, the reverse transcriptase domain has a lower probability of premature termination rate (Poff) in vitro relative to a reference reverse transcriptase domain. In some embodiments, the reference reverse transcriptase domain is a viral reverse transcriptase domain, e.g., the RT domain from M-MLV.

[0385] In some embodiments, the reverse transcriptase domain has a lower probability of premature termination rate (Poff) in vitro of less than about 5 x 10"³/nt, 5 x 10"⁴/nt, or 5 x 10"⁶/nt, e.g., as measured on a 1094 nt RNA. In embodiments, the in vitro premature termination rate is determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated by reference herein its entirety).

[0386] In some embodiments, the reverse transcriptase domain is able to complete at least about 30% or 50% of integrations in cells. The percent of complete integrations can be measured by dividing the number of substantially full-length integration events (e.g., genomic sites that comprise at least 98% of the expected integrated sequence) by the number of total (including substantially full-length and partial) integration events in a population of cells. In embodiments, the integrations in cells is determined (e.g., across the integration site) using long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety).

[0387] In embodiments, quantifying integrations in cells comprises counting the fraction of integrations that contain at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the DNA sequence corresponding to the template RNA (e.g., a template RNA having a length of at least 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, or 5 kb, e.g., a length between 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 1.0-1.2, 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2-3, 3-4, or 4-5 kb).

[0388] In some embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro. In embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro at a rate between 0.1 - 50 nt/sec (e.g., between 0.1-1, 1-10, or 10-50 nt/sec). In embodiments, polymerization of dNTPs by the reverse transcriptase domain is measured by a single-molecule assay, e.g., as described in Schwartz and Quake (2009) PNAS 106(48): 20294- 20299 (incorporated by reference in its entirety).

183 [0389] In some embodiments, the reverse transcriptase domain has an in vitro error rate (e.g., misincorporation of nucleotides) of between 1 x 10"³ - 1 x 10"⁴ or 1 x 10"⁴ - 1 x 10"⁵ substitutions/nt , e.g., as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153 (incorporated herein by reference in its entirety). In some embodiments, the reverse transcriptase domain has an error rate (e.g., misincorporation of nucleotides) in cells (e.g., HEK293T cells) of between 1 x 10"³ - l x 10"⁴ or 1 x 10"⁴ - l x 10"⁵ substitutions/nt, e.g., by long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety).

[0390] In some embodiments, the reverse transcriptase domain is capable of performing reverse transcription of a target RNA in vitro. In some embodiments, the reverse transcriptase requires a primer of at least 3 nucleotides to initiate reverse transcription of a template. In some embodiments, reverse transcription of the target RNA is determined by detection of cDNA from the target RNA (e.g., when provided with a ssDNA primer, e.g., which anneals to the target with at least 3, 4, 5, 6, 7, 8, 9, or 10 nt at the 3' end), e.g., as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated herein by reference in its entirety).

[0391] In some embodiments, the reverse transcriptase domain performs reverse transcription at least 5 or 10 times more efficiently (e.g., by cDNA production), e.g., when converting its RNA template to cDNA, for example, as compared to an RNA template lacking the protein binding motif (e.g., a 3' UTR). In embodiments, efficiency of reverse transcription is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2): 147-153 (incorporated by reference herein in its entirety).

[0392] In some embodiments, the reverse transcriptase domain specifically binds a specific RNA template with higher frequency (e.g., about 5 or 10-fold higher frequency) than any endogenous cellular RNA, e.g., when expressed in cells (e.g., HEK293T cells). In embodiments, frequency of specific binding between the reverse transcriptase domain and the template RNA are measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(ll):5490-5501 (incorporated herein by reference in its entirety).

Template nucleic acids for use with heterologous gene modifying polypeptides

[0393] The heterologous gene modifying systems described herein can modify a host target DNA site using a template nucleic acid sequence. In some embodiments, the systems described herein transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription (TPRT). By modifying DNA sequence(s) via reverse transcription of the RNA

184 sequence template directly into the host genome, the 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. The system can also delete a sequence from the target genome or introduce a substitution using an object sequence. Therefore, the 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.

[0394] In some embodiments, the template nucleic acid comprises one or more sequence (e.g., 2 sequences) that binds the heterologous gene modifying polypeptide.

[0395] 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). For example, a system described herein comprises a first RNA comprising (e.g., from 5' to 3') a sequence that binds the heterologous gene modifying polypeptide (e.g., the DNA-binding domain and/or the endonuclease domain, e.g., a gRNA) and a sequence that binds a target site (e.g., a second strand of a site in a target genome), and a second RNA (e.g., a template RNA) comprising (e.g., from 5 ' to 3') optionally a sequence that binds the heterologous gene modifying polypeptide (e.g., that specifically binds the RT domain), a heterologous object sequence, and a PBS sequence. 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. For example, in some embodiments a first RNA comprises a first conjugating domain and a second RNA comprises a second conjugating domain, and the first and second conjugating domains are capable of hybridizing to one another, e.g., under stringent conditions. In some embodiments, the stringent conditions for hybridization include hybridization in 4x sodium chloride/sodium citrate (SSC), at about 65 C, followed by a wash in IxSSC, at about 65 C.

[0396] In some embodiments, the template nucleic acid comprises RNA. In some embodiments, the template nucleic acid comprises DNA (e.g., single stranded or double stranded DNA).

[0397] In some embodiments, the template nucleic acid comprises one or more (e.g., 2) homology domains that have homology to the target sequence. In some embodiments, the homology domains are about 10-20, 20-50, or 50-100 nucleotides in length.

185 [0398] In some embodiments, a template RNA can comprise a gRNA sequence, e.g., to direct the heterologous gene modifying polypeptide to a target site of interest. In some embodiments, a template RNA comprises (e.g., from 5' to 3') (i) optionally a gRNA spacer that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a gRNA scaffold that binds a polypeptide described herein (e.g., a heterologous gene modifying polypeptide or a Cas polypeptide), (iii) a heterologous object sequence comprising a mutation region (optionally the heterologous object sequence comprises, from 5’ to 3’, a first homology region, a mutation region, and a second homology region), and (iv) a primer binding site (PBS) sequence comprising a 3' target homology domain.

[0399] The template nucleic acid (e.g., template RNA) component of a genome editing system described herein typically is able to bind the heterologous gene modifying polypeptide of the system. In some embodiments the template nucleic acid (e.g., template RNA) has a 3 ' region that is capable of binding a heterologous gene modifying polypeptide. 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 heterologous gene modifying polypeptide 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 heterologous gene modifying polypeptide (e.g., specifically bind to the RT domain). In some embodiments, the template nucleic acid (e.g., template RNA) may associate with the DNA binding domain of the polypeptide, e.g., a gRNA associating with a Cas9-derived DNA binding domain. In some embodiments, the binding region may also provide DNA target recognition, e.g., a gRNA hybridizing to the target DNA sequence and binding the polypeptide, e.g., a Cas9 domain. In some embodiments, the template nucleic acid (e.g., template RNA) may associate with multiple components of the polypeptide, e.g., DNA binding domain and reverse transcription domain.

[0400] 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.

[0401] In some embodiments, the template nucleic acid is a template RNA. In some embodiments, the template RNA comprises one or more modified nucleotides. For example, in some embodiments, the template RNA comprises one or more deoxyribonucleotides. In some embodiments, regions of the template RNA are replaced by DNA nucleotides, e.g., to enhance

186 stability of the molecule. For example, the 3' end of the template may comprise DNA nucleotides, while the rest of the template comprises RNA nucleotides that can be reverse transcribed. For instance, in some embodiments, the heterologous object sequence is primarily or wholly made up of RNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% RNA nucleotides). In some embodiments, the PBS sequence is primarily or wholly made up of DNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% DNA nucleotides). In other embodiments, the heterologous object sequence for writing into the genome may comprise DNA nucleotides. In some embodiments, the DNA nucleotides in the template are copied into the genome by a domain capable of DNA-dependent DNA polymerase activity. In some embodiments, the DNA- dependent DNA polymerase activity is provided by a DNA polymerase domain in the polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second strand synthesis. In some embodiments, the template molecule is composed of only DNA nucleotides.

[0402] In some embodiments, a system described herein comprises two nucleic acids which together comprise the sequences of a template RNA described herein. In some embodiments, the two nucleic acids are associated with each other non-covalently, e.g., directly associated with each other (e.g., via base pairing), or indirectly associated as part of a complex comprising one or more additional molecule.

[0403] A template RNA described herein may comprise, from 5’ to 3’: (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) a primer binding site (PBS) sequence. Each of these components is now described in more detail. gRNA spacer and gRNA scaffold

[0404] A template RNA described herein may comprise a gRNA spacer that directs the heterologous gene modifying system to a target nucleic acid, and a gRNA scaffold that promotes association of the template RNA with the Cas domain of the heterologous gene modifying polypeptide. The systems described herein can also comprise a gRNA that is not part of a template nucleic acid. For example, a gRNA that comprises a gRNA spacer and gRNA scaffold, but not a heterologous object sequence or a PBS sequence, can be used, e.g., to induce second strand nicking, e.g., as described in the section herein entitled “Second Strand Nicking”.

[0405] In some embodiments, the gRNA is a short synthetic RNA composed of a scaffold sequence that participates in CRISPR-associated protein binding and a user-defined ~20

187 nucleotide targeting sequence for a genomic target. The structure of a complete gRNA was described by Nishimasu et al. Cell 156, P935-949 (2014). The gRNA (also referred to as sgRNA for single-guide RNA) consists of crRNA- and tracrRNA-derived sequences connected by an artificial tetraloop. The crRNA sequence can be divided into guide (20 nt) and repeat (12 nt) regions, whereas the tracrRNA sequence can be divided into anti-repeat (14 nt) and three tracrRNA stem loops (Nishimasu et al. Cell 156, P935-949 (2014)). In practice, guide RNA sequences are generally designed to have a length of between 17 - 24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to a targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. In some embodiments, the gRNA comprises two RNA components from the native CRISPR system, e.g. crRNA and tracrRNA. As is well known in the art, the gRNA may also comprise a chimeric, single guide RNA (sgRNA) containing sequence from both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing/binding). Chemically modified sgRNAs have also been demonstrated to be effective for use with CRISPR-associated proteins; see, for example, Hendel et al. (2015) Nature Biotechnol., 985 - 991. In some embodiments, a gRNA spacer comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene.

[0406] In some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA adopts an underwound ribbon-like structure of gRNA bound to target DNA (e.g., as described in Mulepati et al. Science 19 Sep 2014:Vol. 345, Issue 6203, pp. 1479- 1484). Without wishing to be bound by theory, this non-canonical structure is thought to be facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid. Thus, in some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA may tolerate increased mismatching with the target site at some interval, e.g., every sixth base. In some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA comprising homology to the target site may possess wobble positions at a regular interval, e.g., every sixth base, that do not need to base pair with the target site.

[0407] In some embodiments, the template nucleic acid (e.g., template RNA) has at least 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 bases of at least 80%, 85%, 90%, 95%, 99%, or 100% homology to the target site, e.g., at the 5’ end, e.g., comprising a gRNA spacer sequence of length appropriate to the Cas9 domain of the heterologous gene modifying polypeptide (Table 8A).

[0408] In some embodiments, a Cas9 derivative with enhanced activity may be used in the gene modification polypeptide. In some embodiments, a Cas9 derivative may comprise mutations that

188 improve activity of the HNH endonuclease domain, e.g., SpyCas9 R221K, N394K, or mutations that improve R-loop formation, e.g., SpyCas9 L1245V, or comprise a combination of such mutations, e.g., SpyCas9 R221K/N394K, Spy Cas9 N394K/L 1245V, SpyCas9 R221K/L1245V, or SpyCas9 R221K/N394K/L1245V (see, e.g., Spencer and Zhang Sci Rep 7: 16836 (2017), the Cas9 derivatives and comprising mutations of which are incorporated herein by reference). In some embodiments, a Cas9 derivative may comprise one or more types of mutations described herein, e.g., PAM-modifying mutations, protein stabilizing mutations, activity enhancing mutations, and/or mutations partially or fully inactivating one or two endonuclease domains relative to the parental enzyme (e.g., one or more mutations to abolish endonuclease activity towards one or both strands of a target DNA, e.g., a nickase or catalytically dead enzyme). In some embodiments, a Cas9 enzyme used in a system described herein may comprise mutations that confer nickase activity toward the enzyme (e.g., SpyCas9 N863A or H840A) in addition to mutations improving catalytic efficiency (e.g., SpyCas9 R221K, N394K, and/or L1245V). In some embodiments, a Cas9 enzyme used in a system described herein is a SpyCas9 enzyme or derivative that further comprises an N863A mutation to confer nickase activity in addition to R221K and N394K mutations to improve catalytic efficiency.

[0409] Table 12 provides parameters to define components for designing gRNA and/or Template RNAs to apply Cas variants listed in Table 8 A for gene modifying. The cut site indicates the validated or predicted protospacer adjacent motif (PAM) requirements, validated or predicted location of cut site (relative to the most upstream base of the PAM site). The gRNA for a given enzyme can be assembled by concatenating the crRNA, Tetraloop, and tracrRNA sequences, and further adding a 5' spacer of a length within Spacer (min) and Spacer (max) that matches a protospacer at a target site. Further, the predicted location of the ssDNA nick at the target is important for designing a PBS sequence of a Template RNA that can anneal to the sequence immediately 5' of the nick in order to initiate target primed reverse transcription. In some embodiments, a gRNA scaffold described herein comprises a nucleic acid sequence comprising, in the 5’ to 3’ direction, a crRNA of Table 12, a tetraloop from the same row of Table 12, and a tracrRNA from the same row of Table 12, or a sequence having at least 70%, 80%, 85%, 90%, 95%, or 99% identity thereto. In some embodiments, the gRNA or template RNA comprising the scaffold further comprises a gRNA spacer having a length within the Spacer (min) and Spacer (max) indicated in the same row of Table 12. In some embodiments, the gRNA or template RNA having a sequence according to Table 12 is comprised by a system that further comprises a heterologous gene modifying polypeptide, wherein the heterologous gene modifying polypeptide comprises a Cas domain described in the same row of Table 12.

189

[0410] Herein, when an RNA sequence (e.g., a template RNA sequence) is said to comprise a particular sequence (e.g., a sequence of Table 12 or a portion thereof) that comprises thymine

(T), it is of course understood that the RNA sequence may (and frequently does) comprise uracil

(U) in place of T. For instance, the RNA sequence may comprise U at every position shown as T in the sequence in Table 12. More specifically, the present disclosure provides an RNA sequence according to every gRNA scaffold sequence of Table 12, wherein the RNA sequence has a U in place of each T in the sequence in Table 12. Additionally, it is understood that terminal Us and Ts may optionally be added or removed from tracrRNA sequences and may be modified or unmodified when provided as RNA. Without wishing to be bound by example, versions of gRNA scaffold sequences alternative to those exemplified in Table 12 may also function with the different Cas9 enzymes or derivatives thereof exemplified in Table 8A, e.g., alternate gRNA scaffold sequences with nucleotide additions, substitutions, or deletions, e.g., sequences with stem-loop structures added or removed. It is contemplated herein that the gRNA scaffold sequences represent a component of systems that can be similarly optimized for a given system, Cas-RT fusion polypeptide, indication, target mutation, template RNA, or delivery vehicle.

Heterologous object sequence

[0411] A template RNA described herein may comprise a heterologous object sequence that the heterologous gene modifying polypeptide can use as a template for reverse transcription, to write a desired sequence into the target nucleic acid. In some embodiments, the heterologous object sequence comprises, from 5’ to 3’, a post-edit homology region, the mutation region, and a preedit homology region. Without wishing to be bound by theory, an RT performing reverse transcription on the template RNA first reverse transcribes the pre-edit homology region, then

194 the mutation region, and then the post-edit homology region, thereby creating a DNA strand comprising the desired mutation with a homology region on either side.

[0412] In some embodiments, the heterologous object sequence is at least 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,

90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, or 1,000 nucleotides

(nts) in length, or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases in length. In some embodiments, the heterologous object sequence is no more than 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, 1,000, or

2000 nucleotides (nts) in length, or no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 kilobases in length. In some embodiments, the heterologous object sequence is 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 74-1000, 75-1000, 76-1000, 77-1000, 78-1000, 79-1000, 80-1000, 85-1000, 90-1000, 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40- 500, 50-500, 60-500, 70-500, 74-500, 75-500, 76-500, 77-500, 78-500, 79-500, 80-500, 85-500, 90-500, 100-500, 120-500, 140-500, 160-500, 180-500, 200-500, 30-200, 40-200, 50-200, 60- 200, 70-200, 74-200, 75-200, 76-200, 77-200, 78-200, 79-200, 80-200, 85-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, 30-100, 40-100, 50-100, 60-100, 70-100, 74-100, 75-100, 76-100, 77-100, 78-100, 79-100, 80-100, 85-100, or 90-100 nucleotides (nts) in length, or 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, 6-7, 7-20, 7-15, 7-10, 7-9, 7-8, 8-20, 8-15, 8- 10, 8-9, 9-20, 9-15, 9-10, 10-15, 10-20, or 15-20 kilobases in length. In some embodiments, the heterologous object sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10- 20 nt in length, e.g., 10-80, 10-50, or 10-20 nt in length, e.g., about10-20 nt in length. In some embodiments, the heterologous object sequence is 8-30, 9-25, 10-20, 11-16, or 12-15 nucleotides in length, e.g., is 11-16 nt in length. Without wishing to be bound by theory, in some embodiments, a larger insertion size, larger region of editing (e.g., the distance between a first edit/substitution and a second edit/substitution in the target region), and/or greater number of desired edits (e.g., mismatches of the heterologous object sequence to the target genome), may result in a longer optimal heterologous object sequence.

195 [0413] In certain embodiments, the template nucleic acid comprises a customized RNA sequence template which 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, e.g., leading to exon skipping of one or more exons; causing disruption of an endogenous gene, e.g., creating a genetic knockout; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up-regulation of one or more operably linked genes, e.g., leading to gene activation or overexpression; causing down-regulation of one or more operably linked genes, e.g., creating a genetic knock-down; etc. In certain embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and/or transgenes, provide binding sites for transcription factor activators, repressors, enhancers, etc., and combinations thereof. In some embodiments, a customized template can be engineered to encode a nucleic acid or peptide tag to be expressed in an endogenous RNA transcript or endogenous protein operably linked to the target site. In other embodiments, the coding sequence can be further customized with splice donor sites, splice acceptor sites, or poly-A tails.

[0414] The template nucleic acid (e.g., template RNA) of the system typically comprises an object sequence (e.g., a heterologous object sequence) for writing a desired sequence into a target DNA. The object sequence may be coding or non-coding. 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 introduce 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 introduce 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.

196 [0415] In some embodiments, writing of an object sequence into a target site results in the substitution of nucleotides, e.g., where the full length of the object sequence corresponds to a matching length of the target site with one or more mismatched bases. In some embodiments, a heterologous object sequence may be designed such that a combination of sequence alterations may occur, e.g., a simultaneous addition and deletion, addition and substitution, or deletion and substitution.

[0416] In some embodiments, the heterologous object sequence may contain an open reading frame or a fragment of an open reading frame. In some embodiments the heterologous object sequence has a Kozak sequence. In some embodiments the heterologous object sequence has an internal ribosome entry site. In some embodiments the heterologous object sequence has a selfcleaving peptide such as a T2A or P2A site. In some embodiments the heterologous object sequence 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. 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).

[0417] In some embodiments, the heterologous object sequence may contain a non-coding sequence. For example, the template nucleic acid (e.g., template RNA) may comprise a regulatory element, e.g., a promoter or enhancer sequence or miRNA binding site. In some embodiments, integration of the object sequence at a target site will result in upregulation of an endogenous gene. In some embodiments, integration of the object sequence at a target site will result in downregulation of an endogenous gene. In some embodiments the template nucleic acid (e.g., 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

197 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.

[0418] In some embodiments, the template nucleic acid (e.g., template RNA) comprises a site that coordinates epigenetic modification. In some embodiments, the template nucleic acid (e.g., template RNA) comprises a chromatin insulator. For example, the template nucleic acid (e.g., template RNA) comprises a CTCF site or a site targeted for DNA methylation.

[0419] In some embodiments, the template nucleic acid (e.g., 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).

[0420] In some embodiments, the heterologous object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome in an endogenous intron. In some embodiments, the heterologous object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome and thereby acts as a new exon. In some embodiments, the insertion of the heterologous object sequence into the target genome results in replacement of a natural exon or the skipping of a natural exon.

[0421] 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 object sequence, wherein the reverse transcription will result in insertion of the heterologous object 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

198 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.

[0422] In some embodiments, the pre-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.

[0423] In some embodiments, the post-edit homology domain comprises a nucleic acid sequence having 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule.

PBS sequence

[0424] In some embodiments, a template nucleic acid (e.g., template RNA) comprises a primer binding site (PBS) sequence. In some embodiments, a PBS sequence is disposed 3 ’ of the heterologous object sequence and is complementary to a sequence adjacent to a site to be modified by a system described herein, or comprises no more than 1, 2, 3, 4, or 5 mismatches to a sequence complementary to the sequence adjacent to a site to be modified by the system/heterologous gene modifying polypeptide. In some embodiments, the PBS sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in the target nucleic acid molecule. In some embodiments, binding of the PBS sequence to the target nucleic acid molecule permits initiation of target-primed reverse transcription (TPRT), e.g., with the 3 ’ homology domain acting as a primer for TPRT. In some embodiments, the PBS sequence is 3-5, 5-10, 10-30, 10-25, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-30, 11-25, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-30, 12-25, 12-20, 12- 19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-30, 13-25, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-30, 14-25, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-30, 15-25, 15-20, 15- 19, 15-18, 15-17, 15-16, 16-30, 16-25, 16-20, 16-19, 16-18, 16-17, 17-30, 17-25, 17-20, 17-19, 17-18, 18-30, 18-25, 18-20, 18-19, 19-30, 19-25, 19-20, 20-30, 20-25, or 25-30 nucleotides in length, e.g., 10-17, 12-16, or 12-14 nucleotides in length. In some embodiments, the PBS sequence is 5-20, 8-16, 8-14, 8-13, 9-13, 9-12, or 10-12 nucleotides in length, e.g., 9-12 nucleotides in length.

[0425] The template nucleic acid (e.g., template RNA) may have some homology to the target DNA. In some embodiments, the template nucleic acid (e.g., template RNA) PBS sequence domain may serve as an annealing region to the target DNA, such that the target DNA is positioned to prime the reverse transcription of the template nucleic acid (e.g., template RNA).

199 In some embodiments the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of exact homology to the target DNA at the 3' end of the RNA. In some embodiments the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8,

9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the target DNA, e.g., at the 5' end of the template nucleic acid (e.g., template RNA).

[0426] The template RNA sequences may be customized depending on the cell being targeted. For example, in some embodiments it is desired to inactivate a PAM sequence upon editing (e.g., using a “PAM-kill” modification) to decrease the potential for further gene editing (e.g., by Cas retargeting) following the initial edit. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the PAM of the target site, such that upon editing, the PAM site will be mutated to a sequence no longer recognized by the heterologous gene modifying polypeptide. Thus, a mutation region within the heterologous object sequence of the template RNA may comprise a PAM-kill sequence. Without wishing to be bound by theory, in some embodiments, a PAM-kill sequence prevents re-engagement of the heterologous gene modifying polypeptide upon completion of a genetic modification, or decreases re-engagement relative to a template RNA lacking a PAM-kill sequence. In some embodiments, a PAM-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the PAM-kill sequence results in a silent mutation. In other embodiments, it is desired to leave the PAM sequence intact (no PAM-kill).

[0427] Similarly, in some embodiments, to decrease the potential for further gene editing (e.g., by Cas retargeting) following the initial edit, it may be desirable to alter the first three nucleotides of the RT template sequence via a “seed-kill” motif. Consequently, certain template RNAs described herein are designed to write a mutation (e.g., a substitution) into the portion of the target site corresponding to the first three nucleotides of the RT template sequence, such that upon editing, the target site will be mutated to a sequence with lower homology to the RT template sequence. Thus, a mutation region within the heterologous object sequence of the template RNA may comprise a seed-kill sequence. Without wishing to be bound by theory, in some embodiments, a seed-kill sequence prevents re-engagement of the heterologous gene modifying polypeptide upon completion of genetic modification, or decreases re-engagement relative to an otherwise similar template RNA lacking a seed-kill sequence. In some

200 embodiments, a seed-kill sequence does not alter the amino acid sequence encoded by a gene, e.g., the seed-kill sequence results in a silent mutation. In other embodiments, it is desired to leave the seed region intact, and a seed-kill sequence is not used.

[0428] In further embodiments, to optimize or improve gene editing efficiency, it may be desirable to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand. In some embodiments, multiple silent mutations (for example, silent substitutions) may be introduced within the RT template sequence to evade the target cell’s mismatch repair or nucleotide repair pathways or to bias the target cell’s repair pathways toward preservation of the edited strand.

Target Nucleic Acid Site

[0429] In some embodiments, after gene modification, the target site surrounding the edited sequence contains a limited number of insertions or deletions, for example, in less than about 50% or 10% of editing events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety). In some embodiments, the target site does not show multiple consecutive editing events, e.g., head-to-tail or head-to-head duplications, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In some embodiments, the target site does not contain insertions resulting from endogenous RNA in more than about 1% or 10% of events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains the integrated sequence corresponding to the template RNA.

[0430] In certain aspects of the present invention, the host DNA-binding site integrated into by the 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 polypeptide may bind to one or more than one host DNA sequence.

[0431] In some embodiments, a system is used to edit a target locus in multiple alleles. In some embodiments, a system is designed to edit a specific allele. For example, a heterologous gene modifying polypeptide may be directed to a specific sequence that is only present on one allele, 201 e.g., comprises a template RNA with homology to a target allele, e.g., a gRNA or annealing domain, but not to a second cognate allele. In some embodiments, a heterologous gene modifying system can alter a haplotype-specific allele. In some embodiments, a heterologous 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.

Second Strand Nicking

[0432] In some embodiments, a heterologous gene modifying system described herein comprises a nickase activity (e.g., in the heterologous gene modifying polypeptide) that nicks the first strand, and a nickase activity (e.g., in a polypeptide separate from the heterologous gene modifying polypeptide) that nicks the second strand of target DNA. As discussed herein, without wishing to be bound by theory, nicking of the first strand of the target site DNA is thought to provide a 3' OH that can be used by an RT domain to reverse transcribe a sequence of a template RNA, e.g., a heterologous object sequence. Without wishing to be bound by theory, it is thought that introducing an additional nick to the second strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence. In some embodiments, the additional nick to the second strand is made by the same endonuclease domain (e.g., nickase domain) as the nick to the first strand. In some embodiments, the same heterologous gene modifying polypeptide performs both the nick to the first strand and the nick to the second strand. In some embodiments, the heterologous gene modifying polypeptide comprises a CRISPR/Cas domain and the additional nick to the second strand is directed by an additional nucleic acid, e.g., comprising a second gRNA directing the CRISPR/Cas domain to nick the second strand. In other embodiments, the additional second strand nick is made by a different endonuclease domain (e.g., nickase domain) than the nick to the first strand. In some embodiments, that different endonuclease domain is situated in an additional polypeptide (e.g., a system of the invention further comprises the additional polypeptide), separate from the heterologous gene modifying polypeptide. In some embodiments, the additional polypeptide comprises an endonuclease domain (e.g., nickase domain) described herein. In some embodiments, the additional polypeptide comprises a DNA binding domain, e.g., described herein.

[0433] It is contemplated herein that the position at which the second strand nick occurs relative to the first strand nick may influence the extent to which one or more of: desired gene modifying DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired

202 insertions occur, or undesired deletions occur. Without wishing to be bound by theory, second strand nicking may occur in two general orientations: inward nicks and outward nicks.

[0434] In some embodiments, in the inward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) away from the second strand nick. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are positioned between the first PAM site and second PAM site (e.g., in a scenario wherein both nicks are made by a polypeptide (e.g., a heterologous gene modifying polypeptide) comprising a CRISPR/Cas domain). When there are two PAMs on the outside and two nicks on the inside, this inward nick orientation can also be referred to as “P AM-out”. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are between the sites where the polypeptide and the additional polypeptide bind to the target DNA. In some embodiments, in the inward nick orientation, the location of the nick to the second strand is positioned between the binding sites of the polypeptide and additional polypeptide, and the nick to the first strand is also located between the binding sites of the polypeptide and additional polypeptide. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are positioned between the PAM site and the binding site of the second polypeptide which is at a distance from the target site.

[0435] An example of a heterologous gene modifying system that provides an inward nick orientation comprises a heterologous gene modifying polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are between the PAM sites of the sites to which the two gRNAs direct the heterologous gene modifying polypeptide. As a further example, another heterologous gene modifying system that provides an inward nick orientation comprises a heterologous gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are between the PAM site and the site to which the zinc finger molecule binds. As a further example, another heterologous gene

203 modifying system that provides an inward nick orientation comprises a heterologous gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are between the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds.

[0436] In some embodiments, in the outward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) toward the second strand nick. In some embodiments, in the outward nick orientation when both the first and second nicks are made by a polypeptide comprising a CRISPR/Cas domain (e.g., a heterologous gene modifying polypeptide), the first PAM site and second PAM site are positioned between the location of the nick to the first strand and the location of the nick to the second strand. When there are two PAMs on the inside and two nicks on the outside, this outward nick orientation also can be referred to as “PAM-in”. In some embodiments, in the outward nick orientation, the polypeptide (e.g., the heterologous gene modifying polypeptide) and the additional polypeptide bind to sites on the target DNA between the location of the nick to the first strand and the location of the nick to the second. In some embodiments, in the outward nick orientation, the location of the nick to the second strand is positioned on the opposite side of the binding sites of the polypeptide and additional polypeptide relative to the location of the nick to the first strand. In some embodiments, in the outward orientation, the PAM site and the binding site of the second polypeptide which is at a distance from the target site are positioned between the location of the nick to the first strand and the location of the nick to the second strand.

[0437] An example of a heterologous gene modifying system that provides an outward nick orientation comprises a heterologous gene modifying polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are outside of the PAM sites of the sites to which the two gRNAs direct the heterologous gene modifying polypeptide (i.e., the PAM sites are between the location of the first nick and the location of the second nick). As a further example, another heterologous

204 gene modifying system that provides an outward nick orientation comprises a heterologous gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are outside the PAM site and the site to which the zinc finger molecule binds (i.e., the PAM site and the site to which the zinc finger molecule binds are between the location of the first nick and the location of the second nick). As a further example, another heterologous gene modifying system that provides an outward nick orientation comprises a heterologous gene modifying polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are outside the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds (i.e. , the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds are between the location of the first nick and the location of the second nick).

[0438] Without wishing to be bound by theory, it is thought that, for heterologous gene modifying systems where a second strand nick is provided, an outward nick orientation is preferred in some embodiments. As is described herein, an inward nick may produce a higher number of double-strand breaks (DSBs) than an outward nick orientation. DSBs may be recognized by the DSB repair pathways in the nucleus of a cell, which can result in undesired insertions and deletions. An outward nick orientation may provide a decreased risk of DSB formation, and a corresponding lower amount of undesired insertions and deletions. In some embodiments, undesired insertions and deletions are insertions and deletions not encoded by the heterologous object sequence, e.g., an insertion or deletion produced by the double-strand break repair pathway unrelated to the modification encoded by the heterologous object sequence. In some embodiments, a desired gene modification comprises a change to the target DNA (e.g., a substitution, insertion, or deletion) encoded by the heterologous object sequence (e.g., and achieved by the heterologous gene modifying writing the heterologous object sequence into the

205 target site). In some embodiments, the first strand nick and the second strand nick are in an outward orientation.

[0439] In addition, the distance between the first strand nick and second strand nick may influence the extent to which one or more of: desired heterologous gene modifying system DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by theory, it is thought the second strand nick benefit, the biasing of DNA repair toward incorporation of the heterologous object sequence into the target DNA, increases as the distance between the first strand nick and second strand nick decreases. However, it is thought that the risk of DSB formation also increases as the distance between the first strand nick and second strand nick decreases.

Correspondingly, it is thought that the number of undesired insertions and/or deletions may increase as the distance between the first strand nick and second strand nick decreases. In some embodiments, the distance between the first strand nick and second strand nick is chosen to balance the benefit of biasing DNA repair toward incorporation of the heterologous object sequence into the target DNA and the risk of DSB formation and of undesired deletions and/or insertions. In some embodiments, a system where the first strand nick and the second strand nick are at least a threshold distance apart has an increased level of desired heterologous gene modifying system modification outcomes, a decreased level of undesired deletions, and/or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than a threshold distance apart. In some embodiments the threshold distance(s) is given below.

[0440] In some embodiments, the first nick and the second nick are at least 20, 25, 30, 35, 40,

45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or

200 nucleotides apart. In some embodiments, the first nick and the second nick are no more than

25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 250 nucleotides apart. In some embodiments, the first nick and the second nick are 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-

200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 20-190, 30-190, 40-190,

50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-

190, 160-190, 170-190, 180-190, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-

180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 20-170, 30-

170, 40-170, 50-170, 60-170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170,

140-170, 150-170, 160-170, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160,

206 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 20-150, 30-150, 40-150, 50-150, 60-

150, 70-150, 80-150, 90-150, 100-150, 110-150, 120-150, 130-150, 140-150, 20-140, 30-140,

40-140, 50-140, 60-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 20-130,

30-130, 40-130, 50-130, 60-130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 20-120,

30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120, 110-120, 20-110, 30-110,

40-110, 50-110, 60-110, 70-110, 80-110, 90-110, 100-110, 20-100, 30-100, 40-100, 50-100, 60-

100, 70-100, 80-100, 90-100, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 20-80, 30-80, 40-

80, 50-80, 60-80, 70-80, 20-70, 30-70, 40-70, 50-70, 60-70, 20-60, 30-60, 40-60, 50-60, 20-50, 30-50, 40-50, 20-40, 30-40, or 20-30 nucleotides apart. In some embodiments, the first nick and the second nick are 40-100 nucleotides apart.

[0441] Without wishing to be bound by theory, it is thought that, for heterologous gene modifying systems where a second strand nick is provided and an inward nick orientation is selected, increasing the distance between the first strand nick and second strand nick may be preferred. As is described herein, an inward nick orientation may produce a higher number of DSBs than an outward nick orientation, and may result in a higher amount of undesired insertions and deletions than an outward nick orientation, but increasing the distance between the nicks may mitigate that increase in DSBs, undesired deletions, and/or undesired insertions. In some embodiments, an inward nick orientation wherein the first nick and the second nick are at least a threshold distance apart has an increased level of desired heterologous gene modifying system modification outcomes, a decreased level of undesired deletions, and/or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the a threshold distance apart. In some embodiments the threshold distance is given below.

[0442] In some embodiments, the first strand nick and the second strand nick are in an inward orientation. In some embodiments, the first strand nick and the second strand nick are in an inward orientation and the first strand nick and second strand nick are at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 nucleotides apart, e.g., at least 100 nucleotides apart, (and optionally no more than 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 nucleotides apart). In some embodiments, the first strand nick and the second strand nick are in an inward orientation and the first strand nick and second strand nick are 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-

200, 190-200, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-

190, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 100-170, 110-

207 170, 120-170, 130-170, 140-170, 150-170, 160-170, 100-160, 110-160, 120-160, 130-160, 140-

160, 150-160, 100-150, 110-150, 120-150, 130-150, 140-150, 100-140, 110-140, 120-140, 130- 140, 100-130, 110-130, 120-130, 100-120, 110-120, or 100-110 nucleotides apart.

V. EX VIVO DELIVERY WITH AN ECD DEVICE

[0443] The method disclosed here is capable of delivering any lipids, LNPs, conjugates, or compositions described herein to a system for ex vivo administration, such as in an extracorporeal system. The extracorporeal system for use in the provided method may include a combination of various machine hardware components (i.e., apheresis and blood processing machines), a software control module, and/or a sensor module in-line to ensure monitor the process such as to assess efficiency of transduction, cell health and other aspects related to accuracy and safety of the dosing, and the use of replacement fluids designed to fully exploit the design of the system according to the present methods. It is understood that components described for one system according to the present invention can be implemented within other systems (e.g., blood processing systems) according to the present invention as well. In some embodiments, the method is performed in-line. In some embodiments, the method is performed in a closed fluid circuit, or functionally closed fluid circuit. In some embodiments, various components of the system of administration for use in the provided embodiments are operably connected to the patient, and/or to each other.

[0444] In some embodiments, a lymphocyte is contacted with any lipids, LNPs, conjugates, or compositions described to create a blood-LNP composition. In some embodiments, ex vivo administration takes place in an extra corporeal device (ECD). In some embodiments, the cell is a lymphocyte. In some embodiments, the ex vivo administration takes place in 4 hours. In some embodiments, the lymphocyte is a T cell. In some embodiments, the ex vivo administration takes place in less than 4 hours. In some embodiments, the ex vivo administration takes place as little as 1 hour.

208 A. Collecting the blood fraction

[0445] In some embodiments, any lipids, LNPs, conjugates, or compositions described herein may be contacted with lymphocytes ex vivo. In some embodiments, ex vivo administration comprises collecting a blood fraction and delivering LNPs described herein.

[0446] In some embodiments, the method comprises collecting a blood fraction from a patient. In some embodiments, the patient is a human. In some embodiments, the patient is mammal. In some embodiments, the patient has cancer. In some embodiments, the patient has leukemia or lymphoma.

[0447] In some embodiments, the blood fraction is whole blood, PBMCs, or lymphocytes. In some embodiments, collecting the blood fraction is done by leukapheresis. In some embodiments, the blood fraction comprises lymphocytes from the patient. In some embodiments, the blood fraction comprises T-cells from the patient. In some embodiments, the blood fraction is a leukapheresis composition obtained from whole blood by leukapheresis.

[0448] In some embodiments, the method comprises obtaining a blood fraction from a patient. In some embodiments, the method includes inserting a venous-access device into a patient and withdrawing whole blood from the patient. In some embodiments, the blood is withdrawn from the patient through a draw line, which is optionally operably connected to a blood processing set described below. In some embodiments, a draw line pump controls the flow through the draw line. In some embodiments, an anticoagulant is introduced into the withdrawn blood through an anticoagulant line. In some embodiments, the anticoagulant line pump controls the flow through the anticoagulant line.

[0449] In some embodiments, the collection of the blood fraction is performed in a blood processing set. A suitable blood processing set in some embodiments has at least one blood treatment device, such as a hemofilter or dialyzer. In some embodiments, the blood processing set has a blood chamber and a dialysate chamber separated from the blood chamber by a membrane. In some embodiments, the blood processing set has at least one sensor, module, control or regulating unit.

[0450] In some embodiments, the blood processing set comprises a fluid circuit, such as a closed in-line circuit. In some embodiments, the blood processing set is connected in a fluid connection with any of the disclosed units. In some embodiments, the blood processing set is connected in a fluid connection with any of the disclosed devices. In some embodiments, the blood processing

209 set is connected in a signal connection with any of the disclosed units. In some embodiments, the blood processing set is connected in a single connection with any of the disclosed devices.

[0451] In some embodiments, collecting a blood fraction comprises collecting a whole blood and further isolating one or more components from the whole blood. In some embodiments, the method further comprises isolating peripheral blood mononuclear cells (PBMCs) or precursors thereof from whole blood. In some embodiments, the method further comprises isolating mononuclear cells or precursors thereof from whole blood. In some embodiments, the mononuclear cells are isolated via apheresis from whole blood. In some embodiments, the PBMCs are isolated via apheresis from whole blood. In some embodiments, the method further comprises isolating the collection of leukocytes or precursors thereof from whole blood. In some embodiments, cells are isolated via apheresis from whole blood. In some embodiments, leukocytes or precursors thereof are isolated via apheresis from whole blood. In some embodiments, the leukocytes or precursors thereof are isolated via leukapheresis. In some embodiments, the mononuclear cells or precursors thereof are isolated via mononuclear collection (MNC) or continuous MNC (CMNC). In some embodiments, the leukocytes (white blood cells) include lymphocytes (e.g. T cells, NK cells and B cells), monocytes, macrophages and granulocytes (e.g. neutrophils, eosinophils and basophils). In some embodiments, the isolated cells may also include red blood cells.

[0452] In some embodiments, the blood fraction is isolated from whole blood with apheresis. In some embodiments, apheresis is a process wherein whole blood is (a) withdrawn (b) separated into two or more fractions (i.e., components); and (c) at least one of the separated blood components is retransfused (reinfused) into the patient. In some aspects, types of apheresis procedures include "leukapheresis" (wherein leukocytes are separated from the whole blood) and "thrombocytapheresis" (wherein platelets are separated from the whole blood). In some embodiments, the method comprises a step of leukapheresis. In some aspects, apheresis procedures are performed through the use of automated apheresis instruments. In some aspects, apheresis procedures are performed through the use of electronically-controlled apheresis instruments. In some aspects, apheresis procedures are performed through the use of automated and electronically-controlled apheresis instruments.

[0453] In some embodiments, the isolation of the blood fraction is via separation into one or more blood fractions in a separation chamber. In some of any of the provided embodiments, the fraction of blood containing leukocyte components or precursors thereof is collected via a separation chamber. In some embodiments, the separation chamber is configured to separate the 210 PBMCs from whole blood by filtration, such as by membrane filtration, hi some embodiments, the separation chamber is configured to separate the PBMCs from whole blood by centrifugation. In some embodiments, the remaining blood components (e.g. plasma, red blood cells and/or platelets) may be returned into the blood stream of the patient.

[0454] In some embodiments, the separation chamber comprises a centrifuge in which PBMCs are separated by centrifugation. With centrifugation, blood components are separated in order of increasing density as follows: plasma, platelets, lymphocytes and monocytes, granulocytes, and red blood cells. Once blood components are separated, outlet tubes placed within the separation chamber (e.g. apheresis system) allow specific components (e.g. PBMCs) to be selectively removed from the patient based on the density variation into a container. The other components can be returned to the patient and, optionally, are mixed with replacement fluids, such as colloids and crystalloids, during return. In some embodiments, a packing factor (PF) for centrifugation is chosen to achieve the desired separation of cells. The packing factor is characterized by the g- force associated with the centrifugations, the sedimentation velocity at 1 g, the residence time in the separation chamber, and the distance over which sedimentation occurs. The packing factor provides a measure of the radial migration compared to the width of the centrifuge chamber, with adequate cell separation obtained when P > 1. In some embodiments, the rotational speed of the centrifuge is from 800 rpm to 2400 rpm. For instance, the packing factor can depend on factors such as the particular apheresis device being used, the centrifugal speed, the residence time of cells in the chamber and other factors. In some embodiments, the packing factor is between 2 and 20.

[0455] In some embodiments, the separation chamber separates the drawn blood into at least a first blood fraction, and a second blood fraction. In some embodiments, the separation chamber separates the drawn blood into at least a first blood fraction containing leukocytes or precursors thereof, and a second blood fraction (e.g. red blood cells and/or plasma). In some embodiments, the separation chamber may be configured such that the blood fractions are sent to a first and second blood bag, respectively. In some embodiments, the blood component separation device also has an outlet and may optionally alternate between discharging the first blood component (i.e., leukocytes or precursors thereof) and the second blood. In some embodiments, the separation chamber is a centrifuge, optionally a centrifuge bowl. In some embodiments, the centrifuge may separate the drawn blood into a third blood component in addition to the first blood component and the second blood component blood component. In some embodiments, the second and/or third blood fraction may be returned to the patient in addition to the first blood

211 fraction via the retur line. In some embodiments, the first blood fraction may comprise leukocytes or precursors thereof and/or the second blood fraction may be comprised of red blood cells, and/or the third blood component can be plasma and/or platelets. In some embodiments, the separation chamber separates the whole blood into a first blood fraction (e.g., containing leukocytes or precursors thereof) and a second blood fraction, optionally wherein the whole blood is separated into a first, second, and third blood fraction. In some embodiments, the separation chamber extracts the first blood fraction from the separation chamber. In some embodiments, the separation chamber extracts leukocytes or precursors thereof from the separation chamber. In some embodiments, the second blood (e.g. red blood cells) and/or third blood fraction (e.g. plasma and/or platelets) is returned to the patient through the return line. In some embodiments, the return line operably connects to the venous-access device at a point between the draw line pump and the venous-access device.

[0456] In some embodiments, the separation chamber is an apheresis device. In some embodiments, the separation chamber is an apheresis device which separates cells based on their respective density. For example, a device which uses differential centrifugation to separate the most dense red blood cells, from the less dense cell components of (i) plasma and (ii) the “huffy coat”. In some embodiments, the collecting cells by separation of the blood is collecting cells of the “huffy coat”. In some embodiments, the “huffy coat” layer comprises lymphocytes (e.g., T, B, and NK cells) as well as monocytes and granulocytes. In some embodiments, the “huffy coat” layer comprises and/or further comprises PBMCs. In some embodiments, the “huffy coat” layer comprises HSCs.

[0457] In some embodiments, the separation chamber is an apheresis device that separates cells based on their respective density with the use of a density gradient reagent. For example, a device which uses differential centrifugation to separate the most dense red blood cells and gradient reagent, from the less dense cell components of (i) plasma, and (ii) PBMCs. In some embodiments, the collecting cells by separation of the blood is collecting the PBMC. In some embodiments, the cells of the PBMC layer comprises lymphocytes (e.g., T, B, and NK cells), optionally wherein the cells of the PBMCs layer further comprise monocytes. In some embodiments, the cells of the PBMCs comprise HSCs. Any density reagent known in the art is suitable for use in the method, for example sucrose, Percoll, and/or Ficoll can be used to perform density based differential centrifugation in a separation chamber (i.e., apheresis device).

[0458] In some embodiments, the separation chamber is a leukapheresis device. In some embodiments, the separation chamber is a leukapheresis device that separates cells based on their 212 respective density with the use of a density gradient reagent. For example, a device which uses differential centrifugation to separate the most dense red blood cells and gradient reagent, from the less dense cell components of (i) plasma, and (ii) leukocytes and/or precursors thereof. In some embodiments, the collecting cells by separation of the blood is collecting the leukocytes. In some embodiments, the cells of the leukocyte layer comprises lymphocytes (e.g., T, B, and NK cells), optionally wherein the cells of the leukocyte layer further comprise monocytes.

[0459] In some embodiments, the separated cells are collected into a container (also called a “collection container”). The container may be in different forms, including a flexible bag, similar to an IV bag, or a rigid container similar to a cell culture vessel. In particular embodiments, the container is a collection bag. Generally, the composition of the container will be any suitable, biologically inert material, such as glass or plastic, including polypropylene, polyethylene, etc. In particular embodiments, the container is sterile, such as a sterile bag. In some embodiments, the container includes one or more ports such that the cells or reagents can be introduced into or transferred out of the container. For instance, the container may include one or more ports so that reagents for transfection of cells (e.g. a composition comprising LNPs or conjugates described herein) can be introduced to cells within the container. In some cases more than one port may be present for the introduction of one or more reagents, media, etc. and/or for transferring out the cells.

[0460] In some embodiments, the blood fraction is about 285 mL to about 350 mL. In some embodiments, the blood fraction is about 200 mL to about 400 mL or about 250 mL to about 350 mL.

[0461] In some embodiments, the blood fraction comprises PBMCs. In some embodiments, the blood fraction comprises lymphocytes. In some embodiments, the blood fraction comprises enriched leukocytes. In some embodiments, the blood fraction comprises cells that are not leukocytes. In some embodiments, the blood fraction comprises leukocyte precursors, such as hematopoietic stem cells. In some embodiments, the blood fraction comprises stem cells. In some embodiments, the blood fraction comprises hematopoietic stem cells (HSCs). In some embodiments, the blood fraction comprises T cells, such as CD4+ or CD8+ T cells. In some embodiments, the blood fraction comprises Natural Killer cells (NK cells). In some embodiments, the blood fraction comprises B cells. In some embodiments, the blood fraction comprises macrophages. In some embodiments, the blood fraction comprises myeloid derived suppressor cells. In some embodiments, the blood fraction comprises a leukocyte selected from

213 the group consisting of monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages.

[0462] In some embodiments, the methods disclosed herein comprise performing a wash to remove platelets from the blood fraction. In some embodiments, the methods disclosed herein comprise a separation method comprising a spinning membrane separation to remove platelets from the blood fraction. In some embodiments, the methods disclosed herein comprise a separation method comprising continuous counterflow centrifugation to remove platelets from the blood fraction. In some embodiments, the methods disclosed herein comprise a separation method comprising use of a device comprising a centrifugation chamber to remove platelets from the blood fraction.

[0463] In some embodiments, the blood fraction comprises a lymphocyte concentration of about 20 cells/mL to about 200x10⁶ cells/mL. In some embodiments, the blood fraction comprises a lymphocyte concentration of about 20 cells/mL to about 200x10⁶ cells/mL. In some embodiments, the blood fraction comprises a lymphocyte concentration of about 20 cells/mL to about 100x10⁶ cells/mL. In some embodiments, the blood fraction comprises a lymphocyte concentration of about 100 cells/mL to about 200x10⁶ cells/mL. In some embodiments, the blood fraction comprises a lymphocyte concentration of about 100 cells/mL to about 200x10⁶ cells/mL. In some embodiments, the blood fraction comprises a lymphocyte concentration of about 20x10⁶ cells/mL to about 100x10⁶ cells/mL. In some embodiments, the blood fraction comprises a lymphocyte concentration of about 10x10⁶ cells/mL to about 10Ox10⁶ cells/mL, about 20x10⁶ cells/mL to about 10Ox10⁶ cells/mL, about 30x10⁶ cells/mL, about 10Ox10⁶ cells/mL, about 40x10⁶ cells/mL to about 10Ox10⁶ cells/mL, about 50x10⁶ cells/mL- about 10Ox10⁶ cells/mL, about 60x10⁶ cells/mL to about 10Ox10⁶ cells/mL, about 70x10⁶ cells/mL- about 10Ox10⁶ cells/mL, about 80x10⁶ cells/mL- about 10Ox10⁶ cells/mL, or about 90x10⁶ cells/mL to about 10Ox10⁶ cells/mL. In some embodiments, the blood fraction comprises a lymphocyte concentration of about 10x10⁶ cells/mL, about 20x10⁶ cells/mL, about 30x10⁶ cells/mL, about 40x10⁶ cells/mL, about 50x10⁶ cells/mL, about 60x10⁶ cells/mL, about 70x10' cells/mL, about 80x10⁶ cells/mL, about 90x10⁶ cells/mL, or about 100x10⁶ cells/mL.

[0464] In some embodiments, the blood fraction comprises cell density of about 10 cells/mL to about 100x10⁶ cells/mL. In some embodiments, the blood fraction comprises cell density of about 10 cells/mL to about 200x10⁶ cells/mL. In some embodiments, the blood fraction comprises cell density of about 100 cells/mL to about 200x10⁶ cells/mL. In some embodiments, the blood fraction comprises cell density of about 20 cells/mL to about 100x10⁶ cells/mL. In 214 some embodiments, the blood fraction comprises cell density of about 20x10⁶ cells/mL to about 100x10⁶ cells/mL. In some embodiments, the blood fraction comprises cell density of about 20x10⁶ cells/mL to about 100x10⁶ cells/mL, about 30x10⁶ cells/mL to about 100x10⁶ cells/mL, about 40x10⁶ cells/mL to about 100x10⁶ cells/mL, about 50x10⁶ cells/mL to about 100x10⁶ cells/mL, about 60x10⁶ cells/mL to about10Ox10⁶ cells/mL, about 70x10⁶ cells/mL to about 10Ox10⁶ cells/mL, about 80x10⁶ cells/mL to about10Ox10⁶ cells/mL, or about 90x10⁶ cells/mL to about 100x10⁶ cells/mL. In some embodiments, the blood fraction comprises cell density of about 20x10⁶ cells/mL, about 30x10⁶ cells/mL, about 40x10⁶ cells/mL, about 50x10⁶ cells/mL, about 60x10⁶ cells/mL, about 70x10⁶ cells/mL, about 80x10⁶ cells/mL, about 90x10⁶ cells/mL, or about 100x10⁶ cells/mL.

[0465] In some embodiments, the percentage of viable cells in the blood fraction is at least 80%. In some embodiments, the percentage of viable cells in the blood fraction is at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, or at least 10%. In some embodiments, the percentage of viable cells in the blood fraction is between 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, or 90% and 100%.

B. Contacting the LNPs "with the blood fraction

[0466] In some embodiments, the method comprises contacting the blood fraction with any lipid, LNP conjugate, or composition. In some embodiments, lymphocytes are contacted with any lipids, LNPs, conjugates, or compositions. In some embodiments, lymphocytes are contacted with any lipids, LNPs, conjugates, or compositions ex vivo to create a blood-LNP composition. In some embodiments, ex vivo contacting takes place in a device. In some embodiments, the device is an ECD device.

[0467] In some embodiments, the collected blood fraction is pumped into an ECD device. In some embodiments, the ECD device comprises at least one delivery chamber. In some embodiments, the blood fraction is contacted with any lipids, LNPs, conjugates, or compositions in a contacting chamber. In some aspects, any suitable contacting chamber known in art may be used in the provided methods. In some embodiments, the contacting chamber is made from hard plastic and comprises a lumen with a set volume. In some embodiments, the contacting chamber is not made from hard plastic and comprises a lumen with a variable volume. In some embodiments, the contacting chamber is made from a flexible plastic such as polyvinyl chloride.

215 In some contacting, the delivery chamber is a blood bag. In some embodiments, the ECD device is a Fresenins Cue device. In some embodiments, the ECD device may be the Sefra Select System (Cytiva), the CliniMACS Prodigy Instrument (Miltenyi Biotec), the Lovo Cell Processing Systems (Fresenius Kabi AG), or the CTS Rotea Counterflow Centrifugation System (ThermoFisher Scientific).

[0468] In some embodiments, the contacting chamber is open along at least one wall. In some embodiments, the contacting chamber comprises at least one opening (e.g. inlet) capable of permitting the aspiration of liquid in and out of the internal cavity. In some embodiments, the contacting chamber is closed. In some embodiments, the contacting chamber is sterile.

[0469] In some embodiments, the method comprises contacting the blood fraction with LNPs encapsulating a gene modifying system or a heterologous gene modifying system, as described herein. In some embodiments, the LNPs comprise one or more targeting moieties, as described herein, e.g., one or more targeting moieties selected from an anti-CD2 targeting moiety, an anti- CD3 targeting moiety, an anti-CD4 targeting moiety, an anti-CD5 targeting moiety, an anti-CD6 targeting moiety, an anti-CD7 targeting moiety, an anti-CD8 targeting moiety and an anti-CD28 targeting moiety. In some embodiments, the LNPs comprise two or more targeting moieties, e.g., two or more targeting moieties selected from an anti-CD2 targeting moiety, an anti-CD3 targeting moiety, an anti-CD4 targeting moiety, an anti-CD5 targeting moiety, an anti-CD6 targeting moiety, and anti-CD7 targeting moiety, an anti-CD8 targeting moiety, and an anti- CD28 targeting moiety. In some embodiments, the targeting moiety targets T cells. In some embodiments, the targeting moiety targets CD2, CD3, CD4, CD5, CD6, CD7, CD8, or CD28. In some embodiments, the targeting moiety comprises an anti-CD3 moiety. In some embodiments, the anti-CD3 moiety comprises any of the sequences set forth in Table E7.

[0470] In some embodiments, the method comprises contacting the blood fraction with LNPs encapsulating a gene modifying system comprising a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide and a template nucleic acid. In some embodiments, the method comprises contacting the blood fraction with one or more LNPs. In some embodiments, the method comprises contacting the blood fraction with LNPs comprising a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide. In some embodiments, the method comprises contacting the blood fraction with a LNPs encapsulating a template nucleic acid. In some embodiments, the method comprises contacting the blood fraction with LNPs comprising a gene modifying polypeptide or a nucleic acid encoding the gene modifying polypeptide and a template nucleic acid. In some embodiments, the method comprises 216 contacting the blood fraction with LNPs encapsulating a gene modifying polypeptide or a nucleic acid encoding a gene modifying polypeptide and LNPs encapsulating a template nucleic acid, separately.

[0471] In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of from 1 :2 to about 1:50. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of from 1 :3 to about 1:25. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of from 1 :2 to about 1:10. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of from 1 :3 to about 1:5. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of about 1:25. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of about 1:15. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of about 1:10. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of about 1:5. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of about 1:4. In some embodiments, the method comprises contacting the blood fraction with LNPs (e.g., tLNPs) encapsulating a gene modifying polypeptide, or a nucleic acid encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of about 1:3. In some embodiments, the method comprises contacting the blood fraction with LNPs encapsulating a gene modifying polypeptide, or a nucleic acid

217 encoding a gene modifying polypeptide, and a template nucleic acid at a ratio of about 1:5, about 1:10, about 1:15, about 1:20, about 1:30, or about 1:35.

[0472] In some embodiments, the gene modifying system comprises a gene modifying polypeptide or a nucleic acid encoding a gene modifying polypeptide. In some embodiments, the gene modifying polypeptide comprises a retrotransposon 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. In some embodiments, the gene modifying polypeptide comprises an amino acid sequence provided in Table R1 or Table R2. In some embodiments, the template RNA of a gene modifying system comprises a sequence provided in Table Rl.

[0473] In some embodiments, gene modifying system comprises a heterologous object sequence. In some embodiments, the template RNA of a gene modifying system comprises a heterologous object sequence. In some embodiments, the heterologous object sequence comprises a chimeric antigen sequence (CAR). In some embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, a first intracellular signaling domain, and a second intracellular signaling domain. In so some embodiments, the CAR comprises an antigen domain that binds to one of more antigens of a blood cancer (e.g. leukemia or lymphoma), wherein optionally the antigen is a B cell antigen (ii) the CAR comprises an antigen domain that binds to one of more antigens of a solid tumor; (iii) the CAR comprises an antigen binding domain of any one described herein, (iv) the CAR comprises a linker domain, (v) the CAR comprises a transmembrane domain (vi) the CAR comprises a hinge domain (vii), the CAR comprises an intracellular signaling domain, (viii) the CAR comprises a costimulatory domain , (IX), the CAR comprises an antigen binding domain which comprises an scFv; or (X) the CAR comprises an amino acid sequence according to those described herein; (xi) wherein the CAR comprises a first intracellular signaling domain and a second intracellular signaling domain. In some embodiments, the template RNA sequence encodes a CAR, wherein the template RNA sequence comprises any of the sequences provided in Tables 6, 7 and 8.

[0474] In some embodiments, the method comprises contacting the blood fraction with LNPs. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 5 μg LNP per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 4.5 μg LNP per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 4 μg LNP per 1x10⁶ cells. In some embodiments, the method 218 comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 3.5 μg LNP per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 3 μg LNP per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 2.5 μg LNP per 1x10⁶ cells. In some embodiments, the LNP does is about 0.1 μg LNP per 1x10⁶ cells, about 0.2 μg LNP per 1x10⁶ cells, about 0.3 μg LNP per 1x10⁶ cells, about 0.4 μg LNP per 1x10⁶ cells, about 0.5 μg LNP per 1x10⁶ cells, about 0.6 μg LNP per 1x10⁶ cells, about 0.7 μg LNP per 1x10⁶ cells, about 0.8 μg LNP per 1x10⁶ cells, about 1 μg LNP per 1x10⁶ cells, about 1.2 μg LNP per 1x10⁶ cells, about 1.4 μg LNP per 1x10⁶ cells, about 1.6 μg LNP per 1x10⁶ cells, about 1.7 μg LNP per 1x10⁶ cells, about 1.8 μg LNP per 1x10⁶ cells, about 1.9 μg LNP per 1x10⁶ cells, about 2 μg LNP per 1x10⁶ cells, about 2.1 μg LNP per 1x10⁶ cells, about 2.2 μg LNP per 1x10⁶ cells, about 2.3 μg LNP per 1x10⁶ cells, about 2.4 μg LNP per /1x10⁶ cells, or about 2.5 μg LNP per 1x10⁶ cells. In some embodiments, the LNPs dose is between about 0.5 to about 2.5 μg LNP per 1x10⁶ cells, about 1 to about 2.5 μg LNP per 1x10⁶ cells, about 1.5 to about 2.5 μg LNP per 1x10⁶ cells, or about 2 to about 2.5 μg LNP per 1x10⁶ cells.

[0475] In some embodiments, the method comprises contacting the blood fraction with LNPs. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose calculated based on the quantity of the gene modifying system encapsulated by the LNPs. In some embodiments, the gene modifying system comprises nucleic acids. In some embodiments, the nucleic acids are RNAs (e.g. mRNAs). In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose calculated based on the quantity of the RNAs (e.g. mRNAs). encapsulated by the LNPs. In some embodiments, the nucleic acids encapsulated by the LNPs comprise nucleic acids encoding the gene modifying polypeptide and the template nucleic acids. In some embodiments, the RNAs encapsulated by the LNPs comprise mRNA encoding the gene modifying polypeptide and the template RNA.

[0476] In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 5 μg RNA per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with RNA at a dose of about 0.1 to about 4.5 μg RNA per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 4 μg RNA per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 3.5 μg RNA per 1x10⁶ cells. In some embodiments, the method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 3 μg RNA per 1x10⁶ cells. In some embodiments, the

219 method comprises contacting the blood fraction with LNPs at a dose of about 0.1 to about 2.5 μg RNA per 1x10⁶ cells. In some embodiments, the LNP does is about 0.1 μg RNA per 1x10⁶ cells, about 0.2 μg RNA per 1x10⁶ cells, about 0.3 μg RNA per 1x10⁶ cells, about 0.4 μg RNA per 1x10⁶ cells, about 0.5 μg RNA per 1x10⁶ cells, about 0.6 μg RNA per 1x10⁶ cells, about 0.7 μg RNA per 1x10⁶ cells, about 0.8 μg RNA per 1x10⁶ cells, about 1 μg RNA per 1x10⁶ cells, about 1.2 μg RNA per 1x10⁶ cells, about 1.4 μg RNA per 1x10⁶ cells, about 1.6 μg RNA per 1x10⁶ cells, about 1.7 μg RNA per 1x10⁶ cells, about 1.8 μg RNA per 1x10⁶ cells, about 1.9 μg RNA per 1x10⁶ cells, about 2 μg LNP per 1x10⁶ cells, about 2.1 μg RNA per 1x10⁶ cells, about 2.2 μg RNA per 1x10⁶ cells, about 2.3 μg RNA per 1x10⁶ cells, about 2.4 μg RNA per /1x10⁶ cells, or about 2.5 μg RNA per 1x10⁶ cells. In some embodiments, the RNA dose is between about 0.5 to about 2.5 μg RNA per 1x10⁶ cells, about 1 to about 2.5 μg RNA per 1x10⁶ cells, about 1.5 to about 2.5 μg RNA per 1x10⁶ cells, or about 2 to about 2.5 μg RNA per 1x10⁶ cells.

[0477] In some embodiments, the method comprises contacting the blood fraction with the LNPs for between about 0.5 hours to about 4 hours. In some embodiments, the method comprises contacting the blood fraction with the LNPs for between about 0.75 hours to about 4 hours, about 1 hours to about 4 hours, about 1.25 hours to about 4 hours, about 1.5 hours to about 4 hours, about 1.75 hours to about 4 hours, about 2 hours to about 4 hours, about 2.25 hours to about 4 hours, about 2.5 hours to about 4 hours, about 2.75 hours to about 4 hours, about 3 hours to about 4 hours, about 3.25 hours to about 4 hours, about 3.5 hours to about 4 hours, about 3.75 hours to about 4 hours. In some embodiments, the method comprises contacting the blood fraction with the LNPs for about 0.5 hours, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75, about 3 hours, about 3.25 hours, about 3.5 hours, about 3.75 hours, and about 4 hours.

[0478] In some embodiment, the method comprises stimulating the lymphocytes in the blood fraction with a T-cell stimulating agent. In some embodiments, the stimulating takes place before contacting the LNPs with the blood fraction. In some embodiments, the stimulating takes place after contacting the LNPs with the blood fraction. In some embodiments, the stimulation and the LNPs contacting are done simultaneously. In some embodiments the T-cell stimulating reagent comprises a CD3 agonist and/or a CD28 agonist. In some embodiments, the T-cell stimulating reagent comprises a colloidal polymeric nanomatrix conjugated to a CD3 agonist and a CD28 agonist.

[0479] In some embodiment, the method comprises stimulating the lymphocytes in the blood fraction with a T-cell stimulating agent by incubating the T-cell stimulating agent with the blood 220 fraction. In some embodiment, the method comprises incubating the T-cell stimulating agent with the blood fraction for between about 0.5 and about 4 hours. In some embodiments, the T- cell stimulating agent is incubated with the blood fraction for about 0.75 to about 4 hours, about 1 hour to about 4 hours, about 1.25 hours to about 4 hours, about 1.5 hours to about 4 hours, about 1.75 hours to about 4 hours, about 2 hours to about 4 hours, about 2.25 hours to about 4 hours, about 2.5 hours to about 4 hours, about 2.75 hours to about 4 hours, about 3 hours to about 4 hours, about 3.25 hours to about 4 hours, about 3.5 hours to about 4 hours, about 3.75 hours to about 4 hours. In some embodiments, the T-cell stimulating agent is incubated with the blood fraction for about 0.5 hours, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, about 2 hours, about 2.25 hours, about 2.5 hours, about 2.75, about 3 hours, about 3.25 hours, about 3.75 hours, and about 4 hours.

[0480] In some embodiments, the lymphocytes in the blood fraction are stimulated with a T-cell stimulating agent by incubating the T-cell stimulating agent with the blood fraction. In some embodiments, the method comprises incubating the T-cell stimulating agent with the blood fraction at a concentration of between about 54 μL/mL to about 18 μL/mL. In some embodiments, the method comprises incubating the T-cell stimulating agent with the blood fraction at a concentration of between about 54 μL/mL to about 6.7 μL/mL. In some embodiments, the method comprises incubating the T-cell stimulating agent with the blood fraction at a concentration of between about 55 μL/mL to about 5 μL/mL, about 50 μL/mL to about 10 μL/mL, about 45 μL/mL to about 15 μL/mL, about 40 μL/mL to about 20 μL/mL, or about 35 μL/mL to about 25 μL/mL. In some embodiments, the method comprises incubating the T-cell stimulating agent with the blood fraction at a concentration of between about 55 μL/mL to about 20 μL/mL, about 50 μL/mL to about 25 μL/mL, about 45 μL/mL to about 30 μL/mL, or about 40 μL/mL to about 35 μL/mL.

[0481] In some embodiments, the LNPs comprise a gene modifying system. In some embodiments, the components of the gene modifying system are encapsulated in one LNP. In some embodiments, the components of the gene modifying system are encapsulated in multiple LNPs. In some embodiments, the gene modifying system comprises a gene modifying polypeptide. In some embodiments, the gene modifying system comprises a nucleic acid encoding a gene modifying polypeptide. In some embodiments, the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule. In some embodiments, the nucleic acid encoding the gene modifying polypeptide is a DNA molecule.

221 [0482] In some embodiments, the gene modifying system comprises a nucleic acid encoding a retrotransposon element. In some embodiments, the retrotransposon element is selected from a group consisting of Vingi-l Acar, CR1-1 PH, RTE-1 MD, and RTE-3 BF. In some embodiments, the retrotransposon element is selected from the retrotransposons disclosed in Table Rl. In some embodiments, the gene modifying system comprises a template nucleic acid for use with the retrotransposon element. In some embodiments, the gene modifying system comprises the retrotransposon element and template nucleic acid at a ratio between about 1:4 and about 1:25. In some embodiments, the gene modifying system comprises the retrotransposon element and template nucleic acid at a ratio of about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:11, about 1:12, about 1:13, about 1:14, about 1:15, about 1:16, about 1:17, about 1:18, about 1:19, about 1:20, about 1:21, about 1:22, about 1:23, about 1:24, or about 1:25. In some embodiments, the gene modifying system comprises the retrotransposon element and template nucleic acid a ratio between about 1:5 and about 1:25, or about 1:10 and about 1:25.

[0483] In some embodiments, the method further comprises mixing the blood-LNP composition. In some embodiments, mixing may agitate the blood-LNP composition. In some embodiments, mixing the blood-LNP composition may comprise rocking.

[0484] In some embodiments, the blood-LNP composition comprises about 0.1 μg LNP per lx 10⁶ cells to about 2.5 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.5 μg LNP per 1x10⁶ cells to about 2.5 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 1 μg LNP per 1x10⁶ cells to about 2.5 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 1.5 μg LNP per 1x10⁶ cells to about 2.5 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 2 μg LNP per lx 10⁶ cells to about 2.5 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg LNP per lx 10⁶ cells to about 2.0 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg LNP per lx 10⁶ cells to about 1.5 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg LNP per lx 10⁶ cells to about 1 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg LNP per lx 10⁶ cells to about .5 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.5 μg LNP per lx 10⁶ cells to about 2 μg LNP per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 1 μg LNP per 1x10⁶ cells to about 1.5 μg LNP per 1x10⁶ cells.

222 [0485] In some embodiments, the blood-LNP composition comprises LNPs encompassing a gene modifying system. In some embodiments, the gene modifying system comprises RNA (e.g. mRNA and template RNA). In some embodiments, the blood-LNP composition comprises about 0.1 μg RNA per lx 10⁶ cells to about 2.5 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.5 μg RNA per 1x10⁶ cells to about 2.5 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 1 μg RNA per 1x10⁶ cells to about 2.5 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 1.5 μg RNA per 1x10⁶ cells to about 2.5 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 2 μg RNA per lx 10⁶ cells to about 2.5 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg RNA per lx 10⁶ cells to about 2.0 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg RNA per lx 10⁶ cells to about 1.5 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg RNA per lx 10⁶ cells to about 1 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.1 μg RNA per lx 10⁶ cells to about .5 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 0.5 μg RNA per lx 10⁶ cells to about 2 μg RNA per 1x10⁶ cells. In some embodiments, the blood-LNP composition comprises about 1 μg RNA per 1x10⁶ cells to about 1.5 μg RNA per 1x10⁶ cells.

[0486] In some embodiments, the blood-LNP composition comprises about 20 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 30 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 40 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 50 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 60 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 70 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 80 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 90 x 10⁶ cells/mL to about 100 x 10⁶ cells/mL and between about 223 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood- LNP composition comprises about 30x 10⁶ cells/mL to about 90 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 40 x 10⁶ cells/mL to about 80 x 10⁶ x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. In some embodiments, the blood-LNP composition comprises about 50 x 10⁶ cells/mL to about 70 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent. . In some embodiments, the blood-LNP composition comprises about 20 x 10⁶ cells/mL to about 50 x 10⁶ cells/mL and between about 54 μL/mL to about 6.7 μL/mL of T cell stimulating reagent.

C. Edited Cells

[0487] In some embodiments, the methods comprise contacting the blood fraction with the LNPs 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. In some embodiments, the gene modifying polypeptide integrates the heterologous object sequence into the genome of at least one lymphocyte to produce at least one edited lymphocyte. In some embodiments, the edited lymphocytes are edited T Cells. In some embodiments, the edited lymphocytes are CAR-T Cells.

[0488] In some embodiments, the methods comprise contacting blood fraction with the LNPs ex vivo. In some embodiments, the LNPs comprise a gene modifying system. In some embodiments, after contacting the blood fraction with the LNPs, the blood-LNP composition comprises edited cells. In some embodiments, the edited cells are edited lymphocytes. In some embodiments, the edited cells are edited T Cells.

[0489] In some embodiments, the edited lymphocytes comprise the heterologous object sequence integrated in genomic DNA, e.g., at a genomic locus. In some embodiments, the heterologous object sequence is a CAR. In some embodiments, the edited lymphocytes express a CAR. In some embodiments, the edited lymphocytes are CAR-T cells.

[0490] In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 0% to about 50% of the lymphocytes are edited lymphocytes. In some embodiments, the 224 blood-LNP composition comprises lymphocytes wherein about 0% to about 30% of the lymphocytes are edited lymphocytes. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 5% to about 25% of the lymphocytes are edited lymphocytes. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 10% to about 20% of the lymphocytes are edited lymphocytes. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about

19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about

27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% of the lymphocytes are edited lymphocytes.

[0491] In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 0% to about 50% of the lymphocytes are CAR-T cells. In some embodiments, the blood- LNP composition comprises lymphocytes wherein about 0% to about 30% of the lymphocytes are CAR-T cells. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 5% to about 25% of the lymphocytes are CAR-T cells. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 10% to about 20% of the lymphocytes are CAR-T cells. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, %, about 31%, about 32%, about 33%, about 34%, or about 35% of the lymphocytes are CAR-T cells.

D. Creating a Therapeutic Composition

[0492] In some embodiments the methods provided herein comprise contacting a blood fraction LNPs encapsulating a gene modifying system for a period of time to allow editing of the lymphocytes and removing residual to create a therapeutic composition comprising the edited lymphocytes. In some embodiments, the edited lymphocytes comprise a CAR. In some embodiments, the edited lymphocytes express a CAR.

[0493] In some embodiments, the LNPs are optionally removed from the blood-LNP composition by processing the blood-LNP through a spinning membrane. In some embodiments,

225 the cellular components of the blood-LNP are retained and the unbound LNPs pass through the membrane.

[0494] In some embodiments, the method further comprises assaying the therapeutic composition to determine the number of lymphocytes. In some embodiments, the method further comprises assaying the therapeutic composition to determine the number of edited lymphocytes. In some embodiments, the method further comprises assaying the therapeutic composition to determine the percentage of edited lymphocytes. In some embodiments, the concentration or number of edited cells can be measured using any method known in the art suitable for assessing the concentrations of cells or particular cells expressing a CAR from a sample of blood. For example, nucleic acid based methods, such as quantitative PCR (qPCR) or flow cytometry-based methods, or other assays, such as an immunoassay, ELISA, or chromatography /mass spectrometry -based assays can be used.

[0495] In some embodiments, the therapeutic composition comprises lymphocytes wherein about 0% to about 50% of the lymphocytes are edited lymphocytes. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 0% to about 30% of the lymphocytes are edited lymphocytes. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 5% to about 25% of the lymphocytes are edited lymphocytes. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 10% to about 20% of the lymphocytes are edited lymphocytes. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% of the lymphocytes are edited lymphocytes.

[0496] In some embodiments, the therapeutic composition comprises lymphocytes wherein about 0% to about 50% of the lymphocytes are CAR-T cells. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 0% to about 30% of the lymphocytes are CAR-T cells. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 5% to about 25% of the lymphocytes are CAR-T cells. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 10% to about 20% of the lymphocytes are CAR-T cells. In some embodiments, the therapeutic composition comprises lymphocytes wherein about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, 226 about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, or about 35% of the lymphocytes are CAR-T cells.

[0497] In some embodiments, the therapeutic composition further comprises a pharmaceutically acceptable buffer. In some embodiments, pharmaceutically acceptable buffer can include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of pharmaceutically acceptable buffers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey). In some embodiments, the buffers may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. In some embodiments, the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some embodiments, prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some embodiments, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. In one embodiment, the pharmaceutically acceptable buffer is not DMSO alone.

[0498] In some embodiments, the method further comprises sterility testing. In some embodiments, the method comprises performing a preliminary rapid-read sterility assay. In some embodiments, the rapid-read sterility assay is used to asses microbial contaminants, e.g., endotoxin levels, in the therapeutic composition. In some embodiments, the therapeutic composition does not comprise LNPs. In some embodiments, the therapeutic composition does not comprise microbial contaminants. In some embodiments, the therapeutic composition does not comprise microbial contaminants, e.g., endotoxins.

E. Reinfusing the therapeutic composition into the patient

[0499] In some embodiments, the methods further comprise reinfusing the therapeutic composition described herein into the patient within about 10 hours of collecting the blood

227 fraction, e.g., within about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours of collecting the blood fraction.

[0500] In some embodiments, reinfusing the therapeutic composition comprises transferring the therapeutic composition from a contacting chamber to a transfer container. In some embodiments, the therapeutic composition is moved from the contacting chamber to the transfer container via one or more operably connected tubing lines. In some embodiments, the transfer container is a bag. In some embodiments, the transfer container is a rigid container. In some embodiments, the transfer container is opaque or partially opaque.

[0501] In some embodiments, the therapeutic composition is connected to a return processing unit via an operable connection, optionally with a tube, line, valve, luer port, or spike. In some embodiments, the therapeutic composition is pumped (i.e., via an in-line pump as described above) directly into the lumen of the return processing unit.

[0502] In some of any of the provided embodiments, the reinfusion of the therapeutic composition is via a return processing unit. In some embodiments, the return processing unit returns therapeutic composition to the patient. In some embodiments, the return processing unit device has an inlet. In some embodiments, the return processing unit is comprised in a fluid circuit, optionally a closed in-line circuit. In some embodiments, the return processing unit can be operably connected in a fluid and/or signal connection with any of the disclosed units and/or devices, or in a fluid and/or signal connection with such units and/or devices. In some embodiments, the operable connection via at least one connector selected from the group consisting of valves, luer ports and spikes. In some embodiments, one or more of these connectors are disposable. In some embodiments, one or more components of the return processing unit set is disposable. In some embodiments, the return processing unit is disposable. In some embodiments, the return processing unit is sterile.

[0503] In some embodiments, the edited cells expand in vivo after the therapeutic composition is reinfused into the patient. In some embodiments, the edited cells expand for between about 1 and about 10 days. In some embodiments, the edited cells expand for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0504] In some embodiments, the edited lymphocytes expand in vivo after the therapeutic composition is reinfused into the patient. In some embodiments, the edited lymphocytes expand

228 for between about 1 and about 10 days. In some embodiments, the edited lymphocytes expand for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0505] In some embodiments, the CAR-T cells expand in vivo after the therapeutic composition is reinfused into the patient. In some embodiments, the CAR-T cells expand for between about 1 and about 10 days. In some embodiments, the CAR-T cells expand for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days.

[0506] In some embodiments, about 7 days after reinfusion, about 0-20% of patient lymphocytes are edited lymphocytes. In some embodiments, about 7 days after reinfusion, about 0-20% of patient lymphocytes are edited lymphocytes. In some embodiments, after 7 days, about 2-20%, about 2-19%, about 2-18%, about 2-17%, about 2-16%, about 2-15%. about 2-14%, about 2- 13%, about 2-12%, about 2-11%, about 2-10%, or about 2-5% of patient lymphocytes are edited lymphocytes. In some embodiments, about 7 days after reinfusion, about 1-19%, about 1-18%, about 1-17%, about 1-16%, about 1-15%, about 1-14%, about 2-13%, about 3-12%, about 4- 11%, about 5-10%, about 6-9%, or about 7-8% of patient lymphocytes are edited lymphocytes. In some embodiments, about 7 days after reinfusion, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% of patient lymphocytes are edited lymphocytes.

[0507] In some embodiments, about 7 days after reinfusion, about 0-20% of patient T cells are CAR-T cells. In some embodiments, about 7 days after reinfusion, about 0-20% of patient T cells are CAR-T cells. In some embodiments, after 7 days, about 2-20%, about 2-19%, about 2- 18%, about 2-17%, about 2-16%, about 2-15%. about 2-14%, about 2-13%, about 2-12%, about 2-11%, about 2-10%, or about 2-5% of patient T cells are CAR-T cells. In some embodiments, about 7 days after reinfusion, about 1-19%, about 1-18%, about 1-17%, about 1-16%, about 1- 15%, about 1-14%, about 2-13%, about 3-12%, about 4-11%, about 5-10%, about 6-9%, or about 7-8% of patient T cells are CAR-T cells. In some embodiments, about 7 days after reinfusion, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% of patient T cells are CAR-T cells.

229 [0508] In some embodiments, about 7 days after reinfusion, the patient T cells comprise more than about 30 million CAR-T cells. In some embodiments, about 7 days after reinfusion, the patient T cells comprise more than about 100 million CAR-T cells, or more than about 300 million CAR-T cells. In some embodiments, about 7 days after reinfusion, the patient T cells comprise between about 30 million and about 1 billion CAR-T cells, between about 30 million and 950 million CAR-T cells, between about 30 million and 930 million CAR-T cells, between about 30 million and 900 million CAR-T cells, between about 30 million and 850 million CAR- T cells, between about 30 million and 800 million CAR-T cells, between about 30 million and 750 million CAR-T cells, between about 30 million and 700 million CAR-T cells, between about 30 million and 650 million CAR-T cells, between about 30 million and 600 million CAR-T cells, between about 30 million and 550 million CAR-T cells, between about 30 million and 500 million CAR-T cells, between about 30 million and 450 million CAR-T cells, between about 30 million and 400 million CAR-T cells, between about 30 million and 350 million CAR-T cells, between about 30 million and 300 million CAR-T cells, between about 30 million and 250 million CAR-T cells, between about 30 million and 200 million CAR-T cells, between about 30 million and 150 million CAR-T cells between about 30 million and 100 million CAR-T cells, or between about 30 million and 50 million CAR-T cells.

[0509] In some embodiments, the therapeutic composition is reinfused into the patient within about 10 hours of removing the blood fraction. In some embodiments, the therapeutic composition is reinfused into the patient within about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour of removing the blood fraction. In some embodiments, the therapeutic composition is reinfused into the patient in between about 1 and about 10 hours, about 1 and about 5 hours about 2 and about 9 hours, about 3 and about 8 hours, about 4 and about 7 hours, about 5 and about 6 hours of removing the blood fraction. In some embodiments, the therapeutic composition is reinfused into the in about 1-9 hours, 1-5 hours, about 2-8 hours, about 3-7 hours, about 4-6 hours, or about 4 hours of removing the blood fraction.

VI. SYSTEM

[0510] 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 230 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.

[0511] In some embodiments, the system comprises an incoming processing unit. In some embodiments, the incoming processing unit is a leukapheresis device.

[0512] In some embodiments, the system comprises a chamber for contacting LNPs encapsulating components of a gene modifying system with a blood fraction. In some embodiments, the gene modifying system comprises a gene modifying polypeptide or a nucleic acid encoding a gene modifying polypeptide. In some embodiments, the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl. In some embodiments, the gene modifying polypeptide is a synthetic fusion molecule comprising a Cas domain and a reverse transcriptase domain. In some embodiments, the gene modifying polypeptide comprises an amino acid sequence set forth in Table R2 and Table E3.

[0513] In some embodiments, the gene modifying system comprises a nucleic acid encoding the gene modifying polypeptide. In some embodiments, the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule. In some embodiments, the nucleic acid encoding the gene modifying polypeptide is a DNA molecule. In some embodiments, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth in Table Rl or Table E3. In some embodiments, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

[0514] In some embodiments, the system comprises a chamber for contacting LNPs with the blood fraction to create a blood-LNP composition. In some embodiments, the LNPs comprise a targeting moiety. In some embodiments, the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7, CDS or CD28. In some embodiments, the LNPs comprise a targeting moiety. In some embodiments, the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7.

[0515] In some embodiments, the system comprises a transfer container for reinfusing the therapeutic composition into the same subject within about 10 hours of removing the blood

231 fraction. In some embodiments, the system comprises a transfer container for reinfusing the therapeutic composition into the same subject within about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, or about 1 hour of removing the blood fraction. In some embodiments, the system comprises a transfer container for reinfusing the therapeutic composition into the same subject within between about 1 and about 10 hours, about 2 and about 9 hours, about 3 and about 8 hours, about 4 and about 7 hours, about 5 and about 6 hours of removing the blood fraction. In some embodiments, the system comprises a transfer container for reinfusing the therapeutic composition into the same subject within between about 1-9 hours, about 2-8 hours, about 3-7 hours, about 4-6 hours, or about 4 hours of removing the blood fraction.

VII. BLOOD-LNP COMPOSITION

[0516] Disclosed herein is a blood-LNP composition comprising: (a) lymphocytes; wherein the concentration of lymphocytes is around 20x10⁶ cells/mL to about 10Ox10⁶ cells/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 10⁶ cells - 5 μg LNP per 1x10⁶; and (c) optionally, a T-cell stimulating reagent.

[0517] In some embodiments, the blood-LNP composition comprises LNPs encompassing a gene modifying system. In some embodiments, the gene modifying system comprises RNA (e.g. mRNAs and template RNA). In some embodiments, disclosed herein is a blood-LNP composition comprising: (a) lymphocytes; wherein the concentration of lymphocytes is around 20x10⁶ cells/mL to about 10Ox10⁶ cells/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 RNA per lx 10⁶ cells - 5 μg RNA per 1x10⁶; and (c) optionally, a T-cell stimulating agent.

232 [0518] In some embodiments, the LNPs encompass a gene modifying system. In some embodiments, the gene modifying system comprises a gene modifying polypeptide. In some embodiments, the gene modifying polypeptide comprises a retrotransposon element set forth in Table Rl. In some embodiments, the gene modifying polypeptide is a synthetic fusion molecule comprising a Cas domain and a reverse transcriptase domain. In some embodiments, the gene modifying polypeptide comprises an amino acid sequence set forth in Table R2 or Table E3.

[0519] In some embodiments, the gene modifying system comprises a nucleic acid encoding the gene modifying polypeptide. In some embodiments, the nucleic acid encoding the gene modifying polypeptide is a mRNA molecule. In some embodiments, the nucleic acid encoding the gene modifying polypeptide is a DNA molecule. In some embodiments, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid sequence set forth in Table Rl or Table E3. In some embodiments, the nucleic acid encoding the gene modifying polypeptide comprises a nucleic acid encoding a retrotransposon element as set forth in Table Rl.

10520] In some embodiments, the blood-LNP comprises LNPs. In some embodiments, the LNPs comprise a targeting moiety. In some embodiments, the targeting moiety binds to CD2, CD3, CD5, CD6, CDS, or CD28. In some embodiments, the targeting moiety binds to CD2, CD3, CD5, CD6, or CD7.

[0521] In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 0% to about 50% of the lymphocytes are edited lymphocytes. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 0% to about 30% of the lymphocytes are edited lymphocytes. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 5% to about 25% of the lymphocytes are edited lymphocytes. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 10% to about 20% of the lymphocytes are edited lymphocytes. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% of the lymphocytes are edited lymphocytes.

[0522] In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 0% to about 50% of the lymphocytes are CAR-T cells. In some embodiments, the blood-

233 LNP composition comprises lymphocytes wherein about 0% to about 30% of the lymphocytes are CAR-T cells. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 5% to about 25% of the lymphocytes are CAR-T cells. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 10% to about 20% of the lymphocytes are CAR-T cells. In some embodiments, the blood-LNP composition comprises lymphocytes wherein about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30% of the lymphocytes are CAR-T cells.

EXAMPLES

[0523] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and are not to be construed as limiting the scope or content of the invention in any way.

Example 1: In vitro screen for anti-CD3 targeting moieties

[0524] A screen was conducted to identify anti-CD3 targeting moieties that can be conjugated to the surface of an LNP to create targeted LNPs (tLNPs) capable of enhanced delivery of a payload (e.g., a therapeutic agent) to T cells. A base LNP was conjugated to select anti-CD3 targeting moieties to generate tLNPs. The capability of these tLNPs to deliver an mRNA encoding a GFP reporter to activated or resting T cells, either in the presence or absence of serum, was assayed to determine the potential suitability of each targeting moiety for ex vivo and/or in vivo T cell delivery applications.

T Cell Activation and Culture

[0525] Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume. Cells were counted on a Cellometer K2 Cell Counter then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10⁶ cells/mL. The complete T cell media was comprised of CTS AIM V SFM basal media with 5% human serum AB and the following cytokines: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL and human IL-15 at 5ng/mL.

234 Trans Act™, a polymeric nanomatrix comprising human CD3 and CD28, was added to cell suspension at a volume of 10uL per 1x10⁶ cells. Activated cells were transferred to flasks and incubated at 37°C, 5% CO₂ for 72 hours (“Activated” condition).

[0526] At 48 hours post-activation, additional T cells from the same donor were thawed and resuspended in complete T cell media as above, without TransAct™ stimulation. These cells were transferred to flasks and incubated at 37°C, 5% CO₂ for 24 hours (“Rested” condition).

Preparation ofT Cells for Transfection

[0527] Following the incubation periods above, the activated and rested T cells were collected separately and counted. Two aliquots of 1.5x10⁷ cells each were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated. The cell pellets were resuspended in CTS AIM V SFM at a concentration of 1.33x10⁶ cells/mL, supplemented with human IL-2, human IL7, and human IL-15, at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.

[0528] One aliquot per condition (activated and rested) was resuspended in basal media without serum, while the second aliquot was resuspended in basal media with 5% fetal bovine serum. Previous data (not shown) demonstrated that transfection of activated T cells with non-targeted base LNPs was enhanced in the presence of FBS, while select targeted LNPs (notably LNPs with the anti-CD3-8 clone (see Table El) demonstrated higher levels of transfection of activated T cells in the absence of serum (see FIG. 1). All candidate anti-CD3 targeting moieties were screened both with and without FBS present in the media during transfection.

[0529] The resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates at a density of 2x10⁵ cells (150uL) per well, and returned to incubation at 37°C, 5% CO₂ while the LNPs were prepared.

Preparation of Targeted LNPs for Transfection

[0530] Targeted LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (V003), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA (encoding eGFP) dissolved in 25 mM acetate buffer. The two phases were mixed using a microfluidic device to formulate LNP-TCO at a total flow rate of 18 ml/min (aqueous phase: ethanol phase=3: 1). 3x volume of CBS was added to LNP solution and the resulting solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes. Following this, the anti-CD3 targeting moiety was added

235 to the LNP solution at 1 : 1 ratio of mRNA: Antibody. The mixture was incubated at 25 C for 2 hours, followed by overnight incubation at 4 C. The targeted LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation

(100k MWCO).

[0531] Table 19 provides the anti-CD3 targeting moieties that were tested..Each anti-CD3 targeting moiety was produced with an additional sortase tag (LPETG) on the C terminus of the heavy chain. IOUM anti-CD3 Fab-LPETG and ImM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaC12, pH 7.4). 2uM

Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for

3 hours with shaking at 800 rpm. Excess reagents were removed, and buffer was exchanged to

PBS containing 10mM EDTA by a 1 OK Amicon centrifugal filter. To confirm a DOL (degree of labeling) of close to ImeTz per antibody, sortase modified anti-CD3 targeting moiety was allowed to react with 5eq AF594 containing TCO group in PBS for 2 hours at room temperature.

After the reaction, excess dye was removed by two runs with Zeba desalting column. DOL was measured by absorbances at A280 and A590 using DOL calculator. The resulting meTz- modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding eGFP) of 1:1, except where otherwise noted (Table El). The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane. The resulting antibody-LNP product was further concentrated using an amicon column.

[0532] Targeted LNPs were screened with a dose titration ranging from 0.125ug/lx10⁶ cells up to 4ug/lx10⁶ cells. tLNPs were diluted to a top concentration of 16ug/mL in basal CTS AIM V

SFM media (with no serum or cytokines), then serially diluted 1: 1 in additional basal CTS AIM

V SFM media to the lowest concentration of 0.5ug/mL. tLNPs were further diluted 1 in 4 upon addition to the plated cells, for the final concentration range of 0.125ug/mL to 4ug/mL.

LNP Transfection

240 [0533] 50uL of diluted tLNPs were added to plated activated and rested T cells, in singlicate, and mixed gently by pipetting. After addition of the tLNPs, all plated cells were at a final density of 1x10⁶ cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. Transfected cells were returned to incubation at 37°C, 5% CO₂ overnight.

Flow Cytometry

[0534] Following overnight incubation, cells in plates were pelleted by centrifugation at 800xg for 2 minutes, washed once with 10OuL of IX PBS, then resuspended in 50uL PBS containing LIVE/DEAD™ Fixable Near-IR Dead Cell Stain at 1: 1,000 dilution, plus the following antibodies: mouse anti-human CD3 APC, mouse anti-human CD25 Alexa Fluor 700, and mouse anti-human LDLR PE. All antibodies were used at a 1:200 final dilution. Cells were stained on ice for 20 minutes, then centrifuged, washed as above and resuspended in 10OuL of IX PBS containing 2% FBS.

[0535] Flow cytometry data was acquired on a NovoCyte 3000RYB, then analyzed for the proportion of live cells expressing GFP as well as the median fluorescence intensity (MFI) of GFP signal in live cells.

Cytokine Quantification

[0536] Supernatants from transfected T cells were collected and assayed using the MSD MultiSpot Assay for human pro-inflammatory panel 1. Supernatants were diluted 1:50 in Diluent 2 and assayed in duplicate according to the manufacturer’s directions.

Results

[0537] 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). The tLNPs with the anti-CD3-8 targeting moiety at a 1 : 1 ratio of targeting moiety to mRNA boosted GFP expression by 75-fold relative to non-targeted LNPs (FIG.2C) and increased the proportion of transfected cells from 53.6% to 99.4% of cells at the highest doses tested (FIG. 2B). The tLNPs conjugated to the other tested anti-CD3 targeting moieties also increased both GFP expression and the proportion of GFP+ cells relative to the base LNP, with the tLNPs having the anti-CD3-5 targeting moiety

241 exhibiting the second highest GFP expression levels after the anti-CD3-8 tLNPs (FIGs. 2B and 2C).

[0538] As shown by FIGs. 2A and 2D, when FBS was present, the transfection efficiency in activated T cells appeared more normalized across the anti-CD3 targeting moieties. The increase in MFI between anti-CD3-8 tLNPs and non-targeted base LNPs was less than 4-fold higher. Additionally, FIG. 2D shows that the overall levels of expression were enhanced in the presence of serum for most anti-CD3 targeting moieties screened, except for the anti-CD3-8 and anti- CD3-5 targeting moieties.

[0539] 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. As in activated cells, rested cells transfected with tLNPs conjugated to the anti-CD3-8 or the anti-CD3-5 targeting moieties had higher GFP expression in serum-free conditions compared to cells transfected in the presence of FBS. 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 LNPs at 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). This trend was similar for the tLNPs conjugated to the anti-CD3-5 targeting moiety.

[0540] The anti-CD3 targeting moieties induced variable expression of the T cell activation marker CD25 (FIG. 4A). Anti-CD3-2 was minimally activating, even at the highest dose tested, while anti-CD3-4 and anti-CD3-6 induced high levels of CD25 expression. Targeted LNPs comprising the anti-CD3-8 targeting moiety induced moderately high levels of CD25 expression, even at reduced targeting moiety densities.

Example 2: Screening for LNPs comprising novel ionizable lipids that enhance delivery to activated and resting T cells

[0541] A screen was conducted to identify novel ionizable lipids that can be formulated in LNPs to enhance delivery of a payload to human T cells. Base LNPs (lacking targeting moieties) were formulated with Lipid092, Lipidl10, Lipidl33, Lipidl34, or Lipidl54, and an mRNA encoding GFP, and then the transduction efficiency of each LNP was tested in activated and resting T cells. LNPs containing the V003, SM102, or Lipidl77 ionizable lipids were also generated to establish baseline transduction controls for the purpose of comparing transduction efficiency.

242 T Cell Activation and Culture

[0542] Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume. Cells were counted on a Cellometer K2 Cell Counter, then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10⁶ cells/mL. The complete T cell media was comprised of CTS AIM V SFM basal media with 5% human serum AB and the following cytokines: human IL-2 at 20ng/mL human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. TransAct™, a polymeric nanomatrix comprising human CD3 and CD28, was added to cell suspension at a volume of 10uL per 1x10⁶ cells. Activated cells were transferred to flasks and incubated at 37°C, 5% CO₂ for 72 hours (“Activated” condition).

[0543] At 48 hours post-activation, additional T cells from the same donor were thawed and resuspended in complete T cell media as above, without TransAct™ stimulation. These cells were transferred to flasks and incubated at 37°C, 5% CO₂ for 24 hours (“Rested” condition).

Preparation ofT Cells for Transfection

[0544] Following the incubation periods above, the activated and rested T cells were collected separately and counted. 2.0x10⁷ cells were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated. The cell pellets were resuspended in CTS AIM V SFM at a concentration of 1.33x10⁶ cells/mL, supplemented with 6.67% fetal bovine serum, and human IL-2, human IL7, and human IL-15, at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.

[0545] The resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates at a density of 2x10⁵ cells (150uL) per well, and returned to incubation at 37°C, 5% CO₂ while the LNPs were prepared.

Preparation of Base LNPs for Transfection

[0546] LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid092, Lipidl10, Lipidl33, Lipidl34, Lipidl54, Lipidl77, V003, or SM102), 8% DSPC, 42.5% cholesterol, 2% DMG-PEG2K and 0.5% DSPE-PEG2K- TCO. An aqueous phase was composed of mRNA (encoding eGFP) dissolved in 25 mM acetate buffer. The two phases were mixed using a microfluidic device to formulate LNP-TCO at a total flow rate of 18 ml/min (aqueous phase: ethanol phase=3:l). 3x volume of CBS was added to LNP solution and the resulting solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes. The LNPs were then subjected to overnight dialysis in Tris/sucrose buffer at 4 C

243 followed by centrifugation (100k MWCO) and/or via Tangential Flow Filtration (TFF) to the desired concentration.

LNP Transfection

[0547] 50uL of diluted LNPs were added to plated activated and rested T cells at 10Ong, 200ng, 400ng, 600ng and 800ng of LNP per 2 x10⁵ cells, and mixed gently by pipetting. After addition of the LNPs, all plated cells were at a final density of 1x10⁶ cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. Transfected cells were returned to incubation at 37°C, 5% CO₂ overnight.

Flow Cytometry

[0548] Following overnight incubation, flow cytometry was conducted on the cells to detect GFP as described in Example 1.

Results

[0549] The base LNPs formulated with Lipid092 and Lipidl54 exhibited the highest transfection levels in activated T cells out of all the LNPs tested. 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. The MFI levels increased in a dose-dependent fashion for these LNPs.

Activated T cells transfected with the other LNPs exhibited much lower GFP expression levels at all doses tested.

[0550] The base LNPs formulated with Lipid092 and Lipidl54 also exhibited the highest transfection levels in rested T cells out of all the LNPs tested. FIG. 5C shows that Lipid092 and Lipidl54 LNPs transfected the largest numbers of cells at the 10Ong to 400 ng doses (per 2x10⁵ cells), 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. By comparison, LNPs formulated with V003 induced the lowest GFP expression levels out of all LNPs tested.

244 Example 3: Testing of ionizable lipids for LNP delivery of a gene modifying system to activated T cells in vitro

[0551] The Lipid092 and Lipidl54 ionizable lipids that exhibited enhanced delivery to T cells in the lipid screen described in Example 3 were next formulated in base LNPs with a gene modifying system comprised of mRNA encoding a gene modifying polypeptide and a template RNA encoding GFP for use with the gene modifying polypeptide. Activated T cells were then transfected to determine whether these base LNPs could deliver the gene modifying system to activated T cells to enable insertion of the heterologous GFP gene into the genomic DNA of the cells. The gene writing results were compared to baseline writing results using a base LNP formulated with the V003 ionizable lipid.

T Cell Activation and Culture

[0552] Cryopreserved primary T cells from a single human donor (HemaCare, donor D328798) were thawed and transferred into PBS with 2% BSA by volume. Cells were counted on a Cellometer K2 Cell Counter, then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10⁶ cells/mL. The complete T cell media was comprised of CTS AIM V SFM basal media with 5% human serum AB (Gemini Bio #100- 512) and the following cytokines: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. TransAct™, a polymeric nanomatrix comprising human CD3 and CD28, was added to cell suspension at a volume of 10uL per 1x10⁶ cells. Activated cells were transferred to flasks and incubated at 37°C, 5% CO₂ for 72 hours (“Activated” condition).

Preparation ofT Cells for Transfection

[0553] Following the incubation periods above, the activated were collected separately and counted. 2.0x10⁷ cells were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated. The cell pellets were resuspended in CTS AIM V SFM at a concentration of 1.33x10⁶ cells/mL, supplemented with 6.67% fetal bovine serum, and human IL-2, human IL7, and human IL-15, at concentrations of 26.6ng/mL, 13.3ng/mL and 6.6ng/mL, respectively.

[0554] The resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates at a density of 2x10⁵ cells (150uL) per well, and returned to incubation at 37°C, 5% CO₂ while the LNPs were prepared.

245 Preparation of Base LNPs for Transfection

[0555] LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipid092, Lipidl54, or V003), 8% DSPC, 42.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA encoding the RTE1 MD retrotransposon (driver) and a template RNA encoding GFP dissolved in 25 mM acetate buffer. The two phases were mixed using a microfluidic device to formulate LNP-TCO at a total flow rate of 18 ml/min (aqueous phase: ethanol phase=3:l). 3x volume of CBS was added to LNP solution and the resulting solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes. The LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).

[0556] The gene modifying system formulated in the LNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprised the following components.

[0557] The gene modifying polypeptide comprised an RTE1 MD polypeptide having the amino acid sequence of:

[0558] The gene modifying polypeptide comprised a GGS Linker having an amino acid sequence of:

246 GGS

[0559] The gene modifying polypeptide comprised an SV40 NTS having an amino acid sequence of:

PKKKRKV (SEQ ID NO: 345)

[0560] The gene modifying polypeptide comprised an XTEN linker having an amino acid sequence of:

SGSETPGTSESATPES (SEQ ID NO: 417)

[0561] The gene modifying polypeptide comprised an HiBit tag having an amino acid sequence of

VSGWRLFKKIS (SEQ ID NO: 568)

[0562] The full-length gene modifying polypeptide with all components was structured as follows: NLS-GGS linker-RTE MD-XTEN linker-HiBit tag. The full-length sequences are provided in Table E2.

Table E2: Nucleic Acid and Amino Acid Sequences of the RTE1 Gene Modifying Polypeptide

[0563] The gene modifying system further comprised a template RNA comprising the following sequences from 5’ to 3’: 5’UTR_retro, GFP reporter, MND promoter, 3’UTR_retro.

The template RNA comprised an RTE1 MD 5’UTR having the sequence of:

[0564] The template RNA comprised an RTE1 MD 3’UTR having the sequence of: [0565] The template RNA comprised a nucleic acid sequence encoding a GFP protein having a protein sequence of:

LNP Transfection and Flow Cytometry

[0566] 50uL of diluted LNPs were added to plated activated T cells at 0.25, 0.5, 1, 2.5, 5, or 10 μg of LNP per 1 x10⁶ cells, and mixed gently by pipetting. After addition of the LNPs, all plated cells were at a final density of 1x10⁶ cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL- 15 at 5ng/mL. Transfected cells were returned to incubation at 37°C, 5% CO₂ for 4 hours. After the 4 hour tLNP incubation, cells were centrifuged at 350xg for 7min, supernatant aspirated, and cells resuspended in complete T cell media as described above at le6 cells/mL in flat bottom plates. Flow cytometry was conducted on the cells, as described in Example 1, at 4 days, 7 days, and 10 days following tLNP transfection.

Results

[0567] Base LNPs formulated with Lipid 1092 and Lipid 154 both delivered the gene modifying system more effectively to activated T cells than the baseline LNP formulated with the V003 lipid. 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. The peak dose for the LNPs was at 2.5 μg per 1 x10⁶ cells. The GFP expression was reliant on the gene modifying polypeptide- mediated insertion of the GFP sequence into the genomic DNA of cells since base LNPs carrying the template RNA only (without the gene modifying polypeptide mRNA) did not create GFP positive cells when administered to the activated T cells. 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.

250 [0568] The results show that LNPs formulated with the Lipid092, Lipidl54 or V003 ionizable lipids were all capable of delivering an all-RNA gene modifying system to activated T cells to insert a heterologous gene encoded by template RNA. However, LNPs formulated with Lipid092 or Lipidl54 can deliver a gene modifying system more effectively to activated T cells to enable higher insertion rates of a heterologous gene into genomic DNA.

Example 4: Improved LNP formulation for delivery of a gene modifying system to activated T cells

[0569] Alternative formulations of targeted LNPs were tested to determine whether they could improve delivery of a gene modifying system payload to activated T cells, leading to increased insertion of an exogenous gene in the cells. Targeted LNPs were formulated with the Lipidl54 ionizable lipid and a higher amount of helper lipid (22%) relative to the LNPs in prior examples (at 8% helper lipid) and then conjugated to the anti-CD3-8 targeting moiety, as described in Example 1. The LNPs were formulated with an all-RNA gene modifying system composed of an mRNA encoding a gene modifying polypeptide and a template RNA encoding GFP. A baseline control tLNP comprising Lipidl54 and 8% helper lipid (8% DSPC) was also prepared as described in prior examples. Negative control tLNPs were formulated to cany either the gene modifying polypeptide mRNA payload only or a GFP template RNA pay load only.

T Cell Activation and Culture

[0570] Cryopreserved primary T cells from two human donors (HemaCare, donor D328798 and donor DXXX405) were thawed and transferred into PBS with 2% BSA by volume. Cells were counted on a Cellometer K2 Cell Counter, then pelleted by centrifugation at 400xg for 5 minutes and resuspended in complete T cell media at a density of 1x10⁶ cells/mL. The complete T cell media was comprised of CTS AIM V SFM basal media with 5% human serum AB and the following cytokines: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL- 15 at 5ng/mL. TransAct™, a polymeric nanomatrix comprising human CD3 and CD28,was added to cell suspension at a volume of 10uL per 1 x10⁶ cells. Activated cells were transferred to flasks and incubated at 37°C, 5% CO₂ for 72 hours.

Preparation of T Cells for Transfection

[0571] Following the incubation periods above, the activated were collected separately and counted. 2.0x10⁷ cells were taken from each activation condition, then pelleted by centrifugation, and the supernatant aspirated. The cell pellets were resuspended in CTS AIM V SFM at a

251 concentration of 2x10⁶ cells/mL, supplemented with 10% fetal bovine serum, and human IL-2, human IL7, and human IL- 15, at concentrations of 40ng/mL, 20ng/mL and 10ng/mL, respectively.

[0572] The resuspended T cells were plated in non-treated 96-well U-bottom polystyrene plates at a density of 2x10⁵ cells (10OuL) per well, and returned to incubation at 37°C, 5% CO₂ while the LNPs were prepared.

Preparation of Targeted LNPs for Transfection

[0573] LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipidl54), 22% helper lipid (DSPC or egg sphingomyelin), 28.5% cholesterol, 2% DMG-PEG-2K and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA encoding the gene modifying polypeptide and a template RNA encoding GFP dissolved in 25 mM acetate buffer. The two phases were mixed using a microfluidic device to formulate LNP-TCO at a total flow rate of 18 ml/min (aqueous phase: ethanol phase=3: 1). 3x volume of CBS was added to LNP solution and the resulting solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes. The LNPs were then buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO). The control tLNP was prepared using the same approach, except the ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipidl54), 8% DSPC, 42.5% cholesterol, 2% DMG-PEG2K and 0.5% DSPE-PEG2K-TCO. For the negative control tLNPs, an aqueous phase containing either the gene modifying polypeptide mRNA only or the GFP template RNA only was mixed with the ethanol phase followed by the process described earlier.

[0574] The anti-CD3-8 targeting moiety was produced with an additional sortase tag (LPETG) on the C terminus of the heavy chain. IOUM anti-CD3 Fab-LPETG and ImM Triglycine methyltetrazine (GGG-meTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaC12, pH 7.4). 2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 1 OK Amicon centrifugal filter. To confirm a DOL (degree of labeling) of close to ImeTz per antibody, sortase modified anti-CD3 targeting moiety was allowed to react with 5eq AF594 containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed by two runs with Zeba desalting column. DOL was measured by absorbances at A280 and A590 using DOL calculator. The resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP 252 solution (as prepared above) for antibody-LNP conjugation, with a mass ratio between antibody and mRNA (encoding eGFP) of 1 : 1, except where otherwise noted (Table 19). The solution was incubated at room temperature for 2 hours and then at 4°C overnight. Any unreacted anti-CD3 targeting moiety was removed by a 300K MPES TFF membrane. The resulting antibody-LNP product was further concentrated using an arnicon column.

[0575] The gene modifying system formulated in the LNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprised the following components.

[0576] The gene modifying polypeptide comprised an RTE1 MD polypeptide having the amino acid sequence of:

[0577] The gene modifying polypeptide comprised a GGS Linker having an amino acid sequence of:

GGS

[0578] The gene modifying polypeptide comprised an SV40 NLS having an amino acid sequence of:

PKKKRKV (SEQ ID NO: 345)

253 [0579] The gene modifying polypeptide comprised an XTEN linker having an amino acid sequence of:

SGSETPGTSESATPES (SEQ ID NO: 417)

[0580] The gene modifying polypeptide comprised an HiBit tag having an amino acid sequence of:

VSGWRLFKKIS (SEQ ID NO: 568)

[0581] The full-length gene modifying polypeptide with all components was structured as follows: NLS-3GS linker-RTE-MD-XTEN linker-HiBit tag. The full-length sequences are provided in Table 21 (Example 4).

[0582] The gene modifying system further comprised a template RNA comprising the following sequences from 5’ to 3’: 5’UTR_retro, GFP reporter, MND promoter, 3’UTR_retro.

[0583] The template RNA comprised an RTE1 MD 5’UTR having the sequence of:

GGGUGUAUGGGGUGCUCAGGGAGGGGUAGUAUCUCUGGUAUGGAGGGCUUGUCG

UGCCCUCCUAGGGCAGCUCUCCAGCCUCUGACCCCCACCUGACACCCAGCUCUCAC

UUGUGGCUCCCAGUAGCUGCUAGCAUGUGGCAGCGGCCACACCCCGGGCAACGGC

UUCGACAGGCCGGCUAAACCUUGUGAGGGUAGCCAUCGGGUCAUCGACCCCUGGU

GAACCAGGGCUUUGCUCACCCAGCAUGUGAAGACUGCUUCGGCUGAACAGACGGA

AGAAACCAAUAAGAAGGUUCAACGGCUGAGAGGGCGACGCAGCAAAGCACUGUG

GAGUGCUUAGGGCGUGUUGGAGCACAAAGGACAACACGGCCAUCCAAUGCAGCU

GAGGAAGUCUCCAGAUGUAACAAUUUUUCGUGCCACUGGACCCAGGCUUCCAACG

CCGAGAGAGUGGGACUGUCUCUGUGCAUCGGCUUUUCCACUUAAAUCUCUUUCAC

GCACAAGUAUCUUUGUGCACACUCAUCUAUCCUAACCCCGUCCACCCUCUUCAAG

ACCUGCGGCGAUGGGGGAGUGGCGACGCAACAGGUGGAGGUGACCACUGGCAGU

UGUAGUCACGAUCCUGCACGUAGGCGGCCCACGGACCAGUGGUCGCUCGGCCCUG

UGGGCAGCAGGGACGUUCGGCAGCAUCCUGGGCGACUGAGCAGCCCUCUCUAG

(SEQ ID NO: 569)

[0584] The template RNA comprised an RTE1 MD 3’UTR having the sequence of:

UGAAACUGCACAAAGACAAUAGUCAUUCUCGAUCACCGAGAGACUACCAC

U (SEQ ID NO: 570)

254 [0585] The template RNA comprised a nucleic acid sequence encoding a GFP protein having a protein sequence of:

MVSKGEELFTGWPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP

TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEG

DTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGS

VQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGM

DELYK (SEQ ID NO: 571)

LNP Transfection and Flow Cytometry

[0586] 50uL of diluted tLNPs were added to plated activated T cells at 0.25, 0.5, 1, 2.5, 5, or 10 μg of LNP per 1 x10⁶ cells, and mixed gently by pipetting. Negative control tLNPs were dosed at 2.5 μg of LNP per 1 x10⁶ cells. After addition of the tLNPs, all plated cells were at a final density of 1x10⁶ cells/mL in 200uL, with a final concentration of 5% FBS (for serum-containing conditions) and cytokines at the following concentrations: human IL-2 at 20ng/mL, human IL-7 at 10ng/mL, and human IL-15 at 5ng/mL. Transfected cells were returned to incubation at 37°C, 5% CO₂ for 4 hours. After the 4 hour tLNP incubation, cells were centrifuged at 350xg for 7min, supernatant aspirated, and cells resuspended in complete T cell media as described above at le6 cells/mL in flat bottom plates. Flow cytometry was conducted on the cells, as described in Example 1, at 4 days, 7 days, and 10 days following tLNP transfection.

Results

[0587] FIG. 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. The differences in percent GFP+ cells and MFI were especially large at lower doses. The tLNPs formulated with 22% egg sphingomyelin also generated more cells expressing GFP relative to the tLNPs formulated with 8% DSPC at 4 days and 7 days post-transfection, but the expression differences were not as when cells were transfected with tLNPs comprising 22% DSPC. The negative control tLNPs comprising the gene modifying polypeptide mRNA alone or the GFP template RNA alone did not produce any GFP -expressing cells. Thus, delivery of a gene modifying system to activated T cells using tLNPs comprising higher helper lipid levels can increase the number of cells that are engineered

255 by the gene modifying system to comprise genomic integration of an exogenous nucleic acid sequence.

Example 5: Evaluating Tumor Cell Killing in Cancer-bearing Mice by BCMA CAR- T Cells Generated Using an Exemplary Retrotransposon-based Gene Modifying System

[0588] This Example demonstrates that a targeted LNP comprising an all-RNA gene modifying system can transduce enriched T cells to generate functional CAR-T cells in a timeframe suitable for same-day extracorporeal manufacturing. In this instance, BCMA-targeted CAR-T cells were generated and shown to be capable of killing human cancer cells (multiple myeloma tumor cells) in mouse models.

Preparation of Targeted LNPs for Transfection

[0589] LNPs were formulated as follows. An ethanol phase was prepared with five lipids, containing 47% ionizable lipid (Lipidl54), 22% DSPC, 28.5% cholesterol, 2% PEG-DMG and 0.5% DSPE-PEG2K-TCO. An aqueous phase was composed of mRNA encoding the gene modifying polypeptide or a template RNA encoding the CAR dissolved in 25 mM acetate buffer. The two phases were mixed using a microfluidic device to formulate LNP-TCO at a total flow rate of 18 ml/min (aqueous phase: ethanol phase=3:l). 3x volume of CBS was added to LNP solution and the resulting solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes.

[0590] An anti-CD3 targeting moiety (a Fab fragment) was produced with an additional sortase tag (LPETG) on the C terminus of the heavy chain. IOUM anti-CD3 Fab-LPETG and ImM Triglycine methyltetrazine (GGG-MeTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaC12, pH 7.4). 2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 1 OK centrifugal filter. To confirm a DOL (degree of labeling) of close to IMeTz per antibody, sortase modified anti- CD3 targeting moiety was allowed to react with 5 equivalents of AF594, a fluorescent dye, containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed by two runs with a desalting column. DOL was measured by absorbances at A280 and A590 using DOL calculator. The resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for Fab-LNP conjugation, with a mass ratio between the Fab and RNA of 1 : 1. The solution was incubated at room temperature for 2 hours and then at 4°C overnight. The unreacted antibody was removed and the LNPs were 256 buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).

[0591] The gene modifying system formulated in the tLNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprising the following components.

[0592] The gene modifying polypeptide comprised an SV40 NLS having an amino acid sequence of:

PKKKRKV (SEQ ID NO: 345)

[0593] The gene modifying polypeptide comprised a GGS Linker having an amino acid sequence of:

GGS

[0594] The gene modifying polypeptide comprised an RTE1 MD polypeptide having the amino acid sequence of:

MDSTAHPNQGRGLEKVSQTLPALQTPGQHTAAGGSSPLSGRNQRKNTKKLLLGAWNIR

TLLDRENTPRPERRTALIGKELARYNIDIAALSETRLPEEGSLSEPTTGYTFFWKGRASNE

DRIHGVGLAIKTSLLKQLPDLPVGISERLMKIRLPLSKDRYATIISAYAPTLTSTEETIEQFY

SDLSAVLHSVPTNDKLILLGDFNARVGQDHERWKGVLGKHGVGKMNNNGLLLLSKCS

EFELTITNTVFRMANKYKTTWMHPRSKQWHLIDYIIVRRRDIQDVKITRAMRGAECWT

DHRLVRATLQMRIAPRHPKRAQTVRAFYNVSRLRDPSYLQTFQSCLDDKLSAKGPLTGS

STEKWNQFRDAVKETSKAVLGPKQRNHQDWFDENNTAIEDLLSKKNKAFMEWQNNPN

SAPKKDRFKSLQATAQREIRKMQDRWWEKKAEEIQRFADMKNYKQFFSALKTVYGPL

KPTTTPLLSSDGDTLIKDKKGISNRWKEHFSQLLNRPSSVDQSALDQIPQNRTIEQLDVPP

SIEEVQKAIKQMSAGKAPGKDGIPTEVYKALNGKALQAFHIVLTSIWEEEDMPPELRDAS

IVALYKNKGSRAACDNYRGISLLSTAGKILARVILNRLLSSVSEQNLPESQCGFRPDRSTI

DMVFTVRQMQEKCLEQNLSLYIVFIDLTKAFDTVNRDALWVILSKLGCPAKFVKLIQLF

HVDMTGEVLSGGETSDRFNISNGVKQGCVLAPVLFNLFFTQVLRHAVMDLDLGVYIKY

RLDGSLFDLRRLTAKTKTTERLILEALFADDCALMAHQENHLQTTVDRFSTATKLFGLTI

SLSKTEVLFQPAPGRPTNQPCITIDGTQLSNVNTFKYLGSTIANDGSLDHEINARIQKASQ

ALGRLRCKVLQHRGVSTATKLKVYNAVVLSSLLYGCETWTLYRKHMKQLEQFHQRSL

RSIMRIRWQDRITNQEVLDRANSTSIEVMVLKTQLRWSGHVIRMDPQRIPRQVFYGELS

AGLRKQGRPKKRFKDQLKSNLKWAGITPKQLELAASDRSSWRTHINHAATTFEDERRR

RLAAARERRHQATTAPPVTTGVPCPMCHKLCASAFGLQSHMRVHRR (SEQ ID NO: 32)

[0595] The gene modifying polypeptide comprised an XTEN linker having an amino acid sequence of:

SGSETPGTSESATPES (SEQ ID NO: 417)

257 [0596] The gene modifying polypeptide comprised an HiBit tag having an amino acid sequence of:

VSGWRLFKKIS (SEQ ID NO: 568)

[0597] The full-length gene modifying polypeptide with all components was structured as follows: NLS-3GS linker-RTE-MD-XTEN linker-HiBit tag. The full-length sequences are provided in Table E3.

Table E3: Nucleic Acid and Amino Acid Sequences of the RTE1 Gene Modifying Polypeptide

[0598] The gene modifying system further comprised a template RNA with the following sequences from 5’ to 3’: 5’UTR_retro, BCMA CAR, MND promoter, 3’UTR_retro.

[0599] The template RNA comprised an RTE1 MD 5’UTR having the sequence of:

[0600] The template RNA comprised an RTE1 MD 3’UTR having the sequence of:

UGAAACUGCACAAAGACAAUAGUCAUUCUCGAUCACCGAGAGACUACCACU (SEQ

ID NO: 570)

[0601] The template RNA comprised a nucleic acid sequence encoding a BCMA-CAR fusion polypeptide, wherein the nucleic acid sequence was: [0602] The BCMA-CAR fusion polypeptide comprised an amino acid sequence of:

MALPVTALLLPLALLLHAARPDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK

PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQKYDLLTFGGG

TKVEIKGSTSGSGKPGSGEGSTKGQLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWG

WIRQPPGKGLEWIGSISYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC

ARDRGDTILDVWGQGTMVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRP

AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPF

MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE

YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD

GLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 429)

[0603] The BCMA-CAR fusion polypeptide comprised a CDS signal peptide domain having an amino acid sequence of:

MALPVTALLLPLALLLHAARP (SEQ ID NO: 456)

[0604] The BCMA-CAR fusion polypeptide comprised a CDS hinge domain having an amino acid sequence of:

FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 425)

[0605] The BCMA-CAR fusion polypeptide comprised a CDS transmembrane domain having an amino acid sequence of:

IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 573)

[0606] The BCMA-CAR fusion polypeptide comprised a 4- IBB signaling domain having an amino acid sequence of:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 423)

[0607] The BCMA-CAR fusion polypeptide comprised a CD3z signaling domain having an amino acid sequence of:

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG

LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 422)

262 T Cell Thaw and Overnight Rest

[0608] Cryopreserved T cells from a single human donor (Charles River, donor D342744) were thawed and transferred into cytokine-free T cell medium comprised of TexMACS™ cell culture medium (Miltenyi #170-076-307) with 5% human AB serum by volume. Cells were counted then pelleted by centrifugation at 500xg for 5 minutes and resuspended in cytokine-free medium at a target concentration of 3.57x10⁶ cells/mL. Cells were transferred to cell culture flasks to target 1x6 cells/cm², and placed inside of a cell culture incubator at 37°C and 5% CO₂ for overnight rest.

LNP Transfection

[0609] After overnight rest, cells were removed from the incubator and combined into a 500 mL centrifuge tube. A sample was obtained for cell count. Cells were then pelleted by centrifugation at 500xg for 5 minutes and resuspended in transfection medium comprised of Plasma-Lyte A solution (Baxter #2B2544) with 1% Human Serum Albumin by volume at a target cell stock concentration of 5.0x10⁶ cells/mL. Cell stock, TransAct™, LNP, and additional transfection medium were added together in T25 culture flasks, and mixed gently by pipetting. MACS® GMP T Cell TransAct™, a polymeric nanomatrix comprising human CD3 and CD28, was added to the transfection medium at a final volume dilution of 1 : 17.5. For the transfected condition, LNPs comprising an mRNA encoding the gene modifying polypeptide and the template RNA were dosed at a total concentration of 2.5 ug LNPs per million cells, and. a ratio of one part gene modifying polypeptide LNPs to 25 parts template LNPs. For the untransfected condition, no LNPs were added. The final target cell concentration for transfection was 20x10⁶ cells/mL and 12.5x10⁶ cells/cm². Incubation occurred for four hours at room temperature. After the 4-hour incubation, cells were counted and separated into two portions: one portion for ex vivo culture and another portion for in vivo mouse injection. The portion for ex vivo culture was washed and resuspended in complete T cell medium at a target cell concentration of 1x10⁶ celis/mL , then placed inside of a cell culture incubator at 37°C and 5% CO₂. Complete T cell medium was comprised of TexMACS cell culture medium (Miltenyi #170-076-307), 5% human AB serum by volume, 100 lU/mL IL-2 , 10 ng/mL IL-7, and 5 ng/mL IL-15. The portion for in vivo mouse injection was washed and resuspended in RPMI 1640 medium at target injection densities described in Section 1.5.

263 Culture ex vivo

[0610] For the cell portion that was cultured ex vivo, cell counts were performed on days 2, 4, and 7 following transfection day. Complete T cell medium was added as needed to maintain cultures at 1x10⁶ cells/mL. Samples were obtained for flow cytometry to measure CAR frequency on days 4 and 7.

[0611] For the staining procedure, 0.5x10⁶ cells were first quenched with stain buffer (BD #554657), pelleted by centrifugation at 500xg for 5 minutes, resuspended in 100 μL of stain solution containing 0.125 μL of rh-BCMA Fc Chimera AF647, and incubated for 30 minutes in the dark at 2-8°C. Following incubation, cells were quenched with stain buffer, pelleted by centrifugation at 500xg for 5 minutes, then resuspended in 245 μL of stain buffer containing 0.01% Pluronic F-68 and 5 μL of propidium iodide live/dead stain. Flow cytometry data was acquired on a CytoFLEX flow cytometer (Beckman Coulter), then analyzed for the proportion of live cells expressing anti-BCMA CAR.

[0612] FIG. 8A and 8B show cell culture viabilities and population doubling levels for ex vivo cultures. FIG 9 shows CAR frequencies for ex vivo cultures.

Murine in vivo Tumor Killing

[0613] NSG-MHC VII DKO mice (NOD.Cg-Prkdc^scid H2-K1^b-^tm1BpeH2-Abl^g7-em1MvwH2-D1^b- tm1Bpe II2rg^tm1wjl/SzJ) , 8-10 weeks of age (The Jackson Laboratory) were implanted via subcutaneous injection in the right flank with 5 x 10⁶ RPMI-8226 human multiple myeloma tumor cells (ATCC). Tumors were measured twice weekly with digital calipers and the tumor volume was calculated using the formula Volume (mm3) = (L x W²)/2 where L = length and W = Width. Ten days following tumor engraftment, mice were treated with either 3x10⁶ or 10x10⁶ total viable cells from either transfected T cell portions (Anti-BCMA CAR+ T cell groups) or untransfected T cell portions (UTX control groups) via intravenous injection. A separate vehicle control group was injected only with injection medium. The groups are outlined in Table E4.

Table E4: Murine Model Testing groups

[0614] FIGs. 10A-15C compares individual animal RPMI-8226 tumor growth kinetics in treatment groups 1, 3, and 5 (vehicle treated in FIG. 10A, untransfected T cells treated in FIG. 10B, and BCMA CAR-T cells treated in FIG. 10C). By Day 31 (21 days post-treatment), 2 out of 4 mice treated with anti-BCMA CAR-T cells had cleared their tumors and the tumors that were not yet cleared at that timepoint were substantially reduced in size. By Day 38 (28 days post-treatment) all mice that received the anti-BCMA CAR-T cells had cleared their tumors. In contrast, animals treated with vehicle control or UTX T cells experienced continued tumor growth.

[0615] These data demonstrate that anti-BCMA CAR-T cells generated from enriched T cells using a gene modifying system delivered via targeted LNPs in a timeframe for same-day extracorporeal manufacturing and delivery are able to expand in vivo and are effective at clearing tumors. These results also demonstrated tumor clearance at a low dose of CAR-T cells

(estimated as 380,000 CAR-T cells.

Example 6: CAR expression visible in T cells in as little as 1 hour

[0616] This Example demonstrates that a targeted LNP comprising an all-RNA gene modifying system can transduce enriched T cells to generate functional CAR-T cells in within 1 hour.

[0617] LNPs and T cells were prepared according to Example 5. The cell stock was divided into three aliquots and incubated according to protocol described in Example 5. Table E4 describes the incubation conditions of the three cell stocks.

Table E4: Incubation conditions

[0618] Following incubation, the percent of T cells expressing the CAR-T was measured using flow cytometry. FIG. 11 shows CAR expression was visible in T cells in as little as 1 hour of incubation time.

Example 7: CAR expression visible in T cells in as little as 1 hour

[0619] This Example demonstrates that an anti-CD3 targeted LNP comprising an all-RNA gene modifying system can transduce T cells in peripheral blood mononuclear cells (PBMCs), a complex mixture of cells comprising lymphocytes, to generate functional CAR-T cells in a timeframe suitable for same-day extracorporeal manufacturing. In this instance, BCMA-targeted CAR-T cells were generated and shown to be capable of killing human cancer cells (multiple myeloma tumor cells) in vitro.

Preparation of Targeted LNP s for Transfection

[0620] LNPs were formulated as follows. The inter-chain disulfide bond of the anti-CD3-9 Fab fragment (sequences provided in Table E5) was specifically reduced before conjugation to an LNP.

Table E5

[0621] A solution containing the Fab fragment was buffer exchanged with PBS containing 10mM EDTA. Ten equivalents of TCEP were added to each solution and the reduction was carried out for 1 hour at room temperature. Complete reduction of the Fab fragment was confirmed by SDS PAGE gels.

[0622] Base LNPs were prepared at a molar ratio of 47% ionizable lipid (Lipidl 54): 28.5% cholesterol: 22% DSPC: 2.0% PEG-DMG: 0.5% DSPE-PEG2000-maleimide at anN/P of 6 in the ethanol phase. The aqueous phase was composed of mRNA encoding the exemplary gene modifying polypeptide and the exemplary template RNA encoding the CAR at a mass ratio of 1:25 in 25 mM acetate buffer. The two phases were mixed using a T-mixer or microfluidic device to formulate base LNP (aqueous phase: ethanol phase=3:l). 3x volume of citrate- buffered saline was added to LNP solution and resulted solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes.

[0623] After the base LNP was formulated, the reduced anti-CD3-9 Fab fragment was added to the LNP-maleimide solution at specified mass ratio to the total RNA mass (example 0.4: 1). The solution was incubated at room temperature for 2 hours for conjugation and then stored at 4°C overnight. The unreacted maleimide was quenched by addition of excess cysteine (3 equivalent) for 1 hour at room temperature. The LNPs were purified and concentrated by a centrifugal filter (100K MWCO) or via TFF. The resulting tLNP were characterized by Zetasizer for size and PDI and by Ribogreen assays for mRNA concentration and encapsulation efficiency.

[0624] The gene modifying system formulated in the tLNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprising the following components.

[0625] The gene modifying polypeptide comprised an SV40 NLS having an amino acid sequence of:

267 PKKKRKV (SEQ ID NO: 345)

[0626] The gene modifying polypeptide comprised a GGS Linker having an amino acid sequence of:

GGS

[0627] The gene modifying polypeptide comprised an RTE1 MD polypeptide having the amino acid sequence of:

Table E6: Nucleic Acid and Amino Acid Sequences of the RTE1 Gene Modifying Polypeptide

[0628] The gene modifying system further comprised a template RNA with the following sequences from 5’ to 3’: 5’UTR_retro, BCMA CAR, MND promoter, 3’UTR_retro.

[0629] The template RNA comprised an RTE1 MD 5’UTR having the sequence of:

[0630] The template RNA comprised an RTE1 MD 3’UTR having the sequence of:

270

[0631] The template RNA comprised a nucleic acid sequence encoding a BCMA-CAR fusion polypeptide, wherein the nucleic acid sequence was:

[0632] The BCMA-CAR fusion polypeptide comprised an amino acid sequence of:

[0633] The BCMA-CAR fusion polypeptide comprised a CDS signal peptide domain having an amino acid sequence of:

[0634] The BCMA-CAR fusion polypeptide comprised a CDS hinge domain having an amino acid sequence of:

[0635] The BCMA-CAR fusion polypeptide comprised a CDS transmembrane domain having an amino acid sequence of:

272 [0636] The BCMA-CAR fusion polypeptide comprised a 4- IBB signaling domain having an amino acid sequence of:

[0637] The BCMA-CAR fusion polypeptide comprised a CD3z signaling domain having an amino acid sequence of:

Peripheral Blood Monocyte Cells (PBMCs) Thaw and Overnight Rest

[0638] Cryopreserved PBMCs from two donors (Hemacare, donor 448 and 354) were thawed at 37°C and washed in T cell medium comprised of TexMACS™ cell culture medium (Miltenyi #170-076-307) with 5% human AB serum (Gemini Bio 100-152) . Cells were counted after pelleting by centrifugation at 300xg for 5 minutes and resuspended in cytokine-free medium at a target concentration of 1 x10⁶ cells/mL. Cells were transferred to cell culture flasks and maintained at a target cell concentration of 1 x10⁶ cells/mL in TexMACs + 5% human AB serum + IL-7(Miltenyi-l 30-095-362, 10ng/mL) and IL-15(Miltenyi-130-095-764, 5ng/mL) and placed inside of a cell culture incubator at 37°C and 5% CO₂ for overnight rest.

LNP Transfection

[0639] After overnight rest, cells were harvested and combined into a 50 mL centrifuge tube.

The flask was washed with PBS and transferred to a conical tube. 15 mL of versene

(FisherSci: 15-040-066) was added to the flask to detach adherent cells. The cells were then incubated at 37°C until they detached, and then transferred to a conical tube with PBMCs and spun cells for 7 minutes at 350xg. The cells were then resuspended in Plasma-Lyte A(Baxter #2B2544) plus 2% Human Serum Albumin (AkronBio: AK8228-0100) for cell counting. The cells were counted and plated for final resuspension at 40e6 cells/mL in a 96- well cell repellant U- bottom plate (FisherSci:07-000-612).

[0640] TransAct™ and the tLNPs were mixed together in Plasma-Lyte A and added to the cell suspension , such that the final cell concentration was 20e6 cells/mL in Plasma-Lyte A 1% Human Serum Albumin. MACS® GMP T Cell TransAct™, a polymeric nanomatrix comprising human CD3 and CD28, was added to the transfection medium at a final volume dilution of 1:17.5. For the transfected condition, LNPs comprising an mRNA encoding the gene modifying 273 polypeptide and the template RNA were dosed at a total concentration of 5 ug tLNPs per million cells, and a ratio of one part gene modifying polypeptide tLNPs to 25 parts template tLNPs. For the untransfected condition, no tLNPs were added. Incubation occurred for four hours at room temperature. After the 4-hour incubation, cells were washed by spinning at 350xg for 7 minutes at room temperature. The supernatant was pipetted out, and the cells were then resuspended in complete T cell medium comprised of TexMACS cell culture medium, 5% human AB serum by volume, 100 lU/mL IL-2 , 10 ng/mL IL-7, and 5 ng/mL IL-15 at a cell concentration of 1x10⁶ cells/mL in a 96 well flat bottom plate (Greiner Bio-One: 655970).

Culture ex vivo

[0641] Complete T cell medium was added as needed to maintain cultures at 1x10⁶ cells/mL.

Samples were obtained for flow cytometry to measure CAR frequency on days 5 and 7.

[0642] For the staining procedure, 0.5x10⁶ cells were first quenched with stain buffer (BD #554657), pelleted by centrifugation at 500xg for 5 minutes, resuspended in 100 μL of stain solution containing 0.125 μL of rh-BCMA Fc Chimera AF647, and incubated for 30 minutes in the dark at 2-8°C. Following incubation, cells were quenched with stain buffer, pelleted by centrifugation at 500xg for 5 minutes, then resuspended in 200 μL of stain buffer 7-AAD (Invitrogen #A1310) live/dead stain at 1:1000 ratio. Flow cytometry data was acquired on a NovoCyte flow cytometer (Agilent), then analyzed for the proportion of live cells expressing anti-BCMA CAR.

In vitro tumor killing assay

[0643] On day 10, CAR-T cells were harvested, and function was assessed via a BCMA target cell killing assay. Briefly, BCMA-CART cells were co-cultured for 16 hours with BCMA- positive tumor cell lines (MM1.S), stably transduced with ffLuc, at serially decreasing CAR+T cell (effector) to tumor cell ratios. The CellTiter-Glo® ((Promega #G7572) Luminescent Cell Viability Assay, homogeneous method of determining the number of viable cells based on quantitation of the ATP present, an indicator of metabolically active cells, where % cytotoxicity = (BLI Mock- BLI sampie)/BLI Mock, BLI Mock = mean target cell alone value of that experiment. Additionally, after 16 hours of co-culture, supernatants were assessed their IFN-y cytokine levels relative to control T cells via ELISA(MSD). The experiment was also conducted using a noncodon optimized version of the template RNA (see sequence in example 9 below), the results were not as strong (data not shown).

274 [0644] FIG. 12 shows that the anti-CD3 tLNPs described in this Example were capable of delivering an exemplary gene modifying system to T cells present in donor PBMCs to generate CAR-T cells. An average of about 42% of the T cells expressed the CAR. FIG. 13 shows that the CAR-T cells generated using the methods described herein killed BCMA tumor cells in a cytotoxicity assay. FIG. 14 shows that these CAR-T cells also expressed much higher levels of the IFN-y cytokine relative to control T cells that did not receive the anti-CD3 tLNPs formulated with the gene modifying system, demonstrating functional potency of the CAR-T cells.

Example 8: Testing Different Anti-CD3 Targeting Moieties in ECD Conditions [0645] This Example provides the results of a screen to identify anti-CD3 targeting moieties suitable for targeting an LNP to T cells. In particular, the LNP can be used to deliver a gene modifying system to generate functional CAR-T cells in extra corporeal device (ECD) conditions and in a timeframe suitable for same-day extracorporeal manufacturing. In this instance, BCMA-targeted CAR-T cells were generated and shown to be capable of killing human cancer cells (multiple myeloma tumor cells) in vitro.

Preparation of Targeted LNPs for Transfection

[0646] A solution containing the anti-CD3 Fab fragment to be tested was buffer exchanged with PBS containing 10mM EDTA. Ten equivalents of TCEP were added to each solution and the reduction was carried out for 1 hour at room temperature. Complete reduction of the Fab fragment was confirmed by SDS PAGE gels.

[0647] Base LNPs were prepared at a molar ratio of 47% ionizable lipid (Lipidl54): 28.5% cholesterol: 22% DSPC: 2.0% PEG-DMG: 0.5% DSPE-PEG2000-maleimide at anN/P of 6 in the ethanol phase. The aqueous phase was composed of mRNA encoding the exemplary gene modifying polypeptide and the exemplary template RNA encoding the CAR at a mass ratio of 1 : 1 in 25 mM acetate buffer. The two phases were mixed using a T-mixer or microfluidic device to formulate base LNP (aqueous phase: ethanol phase=3:l). 3x volume of citrate-buffered saline was added to LNP solution and resulted solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes.

[0648] After the base LNP was formulated, the reduced anti-CD3 Fab fragment was added to the LNP-maleimide solution at specified mass ratio to the total RNA mass (example 0.4:1). The solution was incubated at room temperature for 2 hours for conjugation and then stored at 4°C overnight. The unreacted maleimide was quenched by addition of excess cysteine (3 equivalent) for 1 hour at room temperature. The LNPs were purified and concentrated by a centrifugal filter 275 (100K MWCO) or via TFF. The resulting-tLNP were characterized by Zetasizer for size and PDI and by Ribogreen assays for mRNA concentration and encapsulation efficiency.

[0649] The sequences for each tested anti-CD3 targeting moiety (each Fab fragment; in presented in Table E7.

Table E7: Exemplary anti-CD3 targeting moiety sequences [0650] 10uM anti-CD3 Fab-LPETG and ImM Triglycine methyltetrazine (GGG-MeTz) were prepared in sortase buffer (50 mM Tris, 150 mM NaCl, 10 mM CaC12, pH 7.4). 2uM Sortase A5 was then added to initiate the reaction. The above solution was incubated at 30°C for 3 hours with shaking at 800 rpm. Excess reagents were removed, and buffer was exchanged to PBS containing 10mM EDTA by a 10K centrifugal filter. To confirm a DOL (degree of labeling) of close to IMeTz per antibody, sortase modified anti-CD3 targeting moiety was allowed to react with 5 equivalents of AF594, a fluorescent dye, containing TCO group in PBS for 2 hours at room temperature. After the reaction, excess dye was removed by two runs with a desalting column. DOL was measured by absorbances at A280 and A590 using DOL calculator. The resulting meTz-modified anti-CD3 targeting moiety was added to the TCO modified-LNP solution (as prepared above) for Fab-LNP conjugation, with a mass ratio between the Fab and RNA of 1 : 1. The solution was incubated at room temperature for 2 hours and then at 4°C overnight. The unreacted antibody was removed and the LNPs were buffer exchanged to the desired concentration either via Tangential Flow Filtration (TFF) and/or centrifugation (100k MWCO).

[0651] The gene modifying system formulated in the tLNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprising the following components.

[0652] The gene modifying polypeptide comprised an SV40 NLS having an amino acid sequence of:

PKKKRKV (SEQ ID NO: 345)

[0653] The gene modifying polypeptide comprised a GGS Linker having an amino acid sequence of:

GGS

[0654] The gene modifying polypeptide comprised an RTE1 MD polypeptide having the amino acid sequence of:

[0655] The full-length gene modifying polypeptide with all components was structured as follows: NLS-3GS linker-RTE-MD-XTEN linker-HiBit tag. The full-length sequences are provided in Table E6.

[0656] The gene modifying system further comprised a template RNA with the following sequences from 5’ to 3’: 5’UTR_retro, BCMA CAR, MND promoter, 3’UTR_retro.

[0657] The template RNA comprised an RTE1 MD 5’UTR having the sequence of:

[0658] The template RNA comprised an RTE1 MD 3’UTR having the sequence of:

279 [0659] The template RNA comprised a codon optimized nucleic acid sequence encoding a

BCMA-CAR fusion polypeptide, wherein the nucleic acid sequence was SEQ ID NO: 575.

SEQ ID 575:

[0660] The BCMA-CAR fusion polypeptide comprised an amino acid sequence of:

[0661] The BCMA-CAR fusion polypeptide comprised a CDS signal peptide domain having an amino acid sequence of:

MALPVTALLLPLALLLHAARP (SEQ ID NO: 456)

[0662] The BCMA-CAR fusion polypeptide comprised a CDS hinge domain having an amino acid sequence of:

[0663] The BCMA-CAR fusion polypeptide comprised a CDS transmembrane domain having an amino acid sequence of:

[0664] The BCMA-CAR fusion polypeptide comprised a 4- IBB signaling domain having an amino acid sequence of: [0665] The BCMA-CAR fusion polypeptide comprised a CD3z signaling domain having an amino acid sequence of:

LNP Transfection

[0666] Fresh leukopaks from two different donors were processed using a CUE instrument (Fresnius). The platelet-washed and concentrated leukapheresis material was then pelleted by centrifugation at 350xg for 7 minutes and resuspended in transfection medium comprised of Plasma-Lyte A solution (Baxter #2B2544) with 2% Human Serum Albumin by volume at a target cell stock concentration of 40 x10⁶ cells/mL. Cells were plated in a 96- well U-bottom plate at 2e6 cells/well. For the transfected condition, LNPs comprising an mRNA encoding the gene modifying polypeptide and the template RM A were dosed at a total concentration of 5ug and 2.5 ug LNPs per million cells, diluted in Plasma-Lyte A solution, and a ratio of one part gene modifying polypeptide LNPs to four parts template LNPs. For the untransfected condition, no LNPs were added. MACS® GMP T Cell TransAct™, a polymeric nanomatrix comprising human CD3 and CD28, was added to the LNP transfection medium at a final volume dilution of 1:8.75. The final target cell concentration for transfection was 20x10⁶ cells/mL with 1:17.5 dilution of MACS® GMP T Cell TransAct™ in 1% Human Serum Albumin by volume. Incubation occurred for four hours incubated at 25C with 5% CO₂. After the 4-hour incubation, cells were washed with equal volume PBS and resuspended in complete T cell medium at a target cell concentration of 1x10⁶ cells/mL , in a 24 well G-rex plate, then placed inside of a cell culture incubator at 37°C and 5% CO₂. Complete T cell medium was comprised of TexMACS cell culture medium (Miltenyi #170-076-307), 5% human AB serum by volume, 100 lU/mL IL-2, 10 ng/mL IL-7, and 5 ng/mL IL-15.

Culture ex vivo

[0667] Complete T cell medium was added as needed to maintain cultures at 1x10⁶ cells/mL.

Samples were obtained for flow cytometry to measure CAR frequency on day 7.

[0668] For the staining procedure, 0.5x10⁶ cells were first quenched with stain buffer (BD #554657), pelleted by centrifugation at 500xg for 5 minutes, resuspended in 100 μL of stain solution containing 0.125 μL of rh-BCMA Fc Chimera AF647, and incubated for 30 minutes in

282 the dark at 2-8°C. Following incubation, cells were quenched with stain buffer, pelleted by centrifugation at 500xg for 5 minutes, then resuspended in 100 μL of stain buffer containing and 5 μL of 7-AAD at a dilution of 1 : 1000. stain. Flow cytometry data was acquired on a Novocyte Quanteon flow cytometer (Agilent), then analyzed for the proportion of live cells expressing anti-BCMA CAR.

In vitro tumor killing assay

[0669] On day 8, CART cells were harvested, and function was assessed via a BCMA target cell killing assay using live cell imaging. Briefly, BCMA-CART cells were co-cultured for 24 hours with BCMA-positive tumor cell lines (MM.lS-GFP-fluc) at serially decreasing T cell (effector) to tumor cell ratios and tracked over 48 hours, these data represent the 24-hour read where optimal killing window was determined. Tumor targets were tracked via GFP expression and cell death was tracked via expression of Annexin V. Tumor cell death was quantified by coexpression of both markers and normalized to a positive tumor cell only control treated with Camptothecin to induce apoptosis.

[0670] The experiment was also conducted using a non-codon optimized version of the template RNA (see sequence in example 9 below), the results were not as strong (data not shown).

[0671] FIG. 15 shows that tLNPs conjugated to anti-CD3-9, anti-CD3-10, anti-CD3-ll or anti- CD3-12 targeting moieties (fab fragments) were all capable of delivering an exemplary gene modifying system to T cells in CUE materials to generate CAR-T cells under conditions suitable for extracorporeal manufacturing of CAR-T cells using patient lymphocytes. For all four anti- CD3 tLNPs tested, delivery of an exemplary gene modifying system resulted in an average of over 10% of CD4+ CD8+ T cells expressing CAR at both dose levels. FIG. 16 shows the CAR expression levels (MFI) in the CAR-T cells generated by each of the anti-CD3 tLNPs. FIG. 17 shows that CAR-T cells generated using each of the anti-CD3 tLNPs comprising an exemplary gene modifying system were capable of killing BCMA-expressing tumor cells in a cytotoxicity assay.

Example 9: Comparing Generation of CAR-T Cells using LNP Delivery of a Gene Modifying System to Lentivirus Delivery

[0672] This Example demonstrates that an anti-CD3 targeted LNP comprising an all-RNA gene modifying system can transduce T cells in peripheral blood mononuclear cells (PBMCs), a complex mixture of cells comprising lymphocytes, to generate functional CAR-T cells at

283 comparable frequencies as a lentivirus approach. BCMA-targeted CAR-T cells were generated using the exemplary gene modifying system and showed improved killing of tumor cells (multiple myeloma tumor cells) relative to the lentivirus-induced T cells in vitro.

Preparation of Targeted LNPs for Transfection

[0673] LNPs were formulated as follows. The inter-chain disulfide bond of the anti-CD3-9 Fab fragment (sequences provided in Table E5) was specifically reduced before conjugation to an LNP.

[0674] A solution containing the Fab fragment was buffer exchanged with PBS containing 10mM EDTA. Ten equivalents of TCEP were added to each solution and the reduction was carried out for 1 hour at room temperature. Complete reduction of the Fab fragment was confirmed by SDS PAGE gels.

[0675] Base LNPs were prepared at a molar ratio of 47% ionizable lipid (Lipidl 54): 28.5% cholesterol: 22% DSPC: 2.0% PEG-DMG2000: 0.5% DSPE-PEG2000-maleimide at anN/P of 6 in the ethanol phase. The aqueous phase was composed of mRNA encoding the exemplary gene modifying polypeptide and the exemplary template RNA encoding the CAR at a mass ratio of 1 : 1 in 25 mM acetate buffer. The two phases were mixed using a microfluidic device to formulate base LNP at a total flow rate of 18 mL/min (aqueous phase: ethanol phase=3: 1). 3x volume of citrate-buffered saline was added to LNP solution and resulted solution was neutralized by 1 vol% of IM Tris-HCl, pH 8 after 15 minutes.

[0676] After the base LNP was formulated, the reduced anti-CD3-9 Fab fragment was added to the LNP-maleimide solution. The solution was incubated at room temperature for 2 hours for conjugation and then stored at 4°C overnight. The unreacted maleimide was quenched by cysteine for 1 hour at room temperature. The LNP was concentrated and unbound Fab fragments were removed by a centrifugal filter (100K MWCO). The solution was exchanged with Tris buffer containing 9% sucrose during the process. The resulting dual tLNP was characterized by Zetasizer for size and PDI and by Ribogreen assays for mRNA concentration.

[0677] The gene modifying system formulated in the tLNPs comprised an mRNA encoding an exemplary gene modifying polypeptide comprising the following components.

[0678] The gene modifying polypeptide comprised an SV40 NLS having an amino acid sequence of:

284 PKKKRKV (SEQ ID NO: 345)

[0679] The gene modifying polypeptide comprised a GGS Linker having an amino acid sequence of:

GGS

[0680] The gene modifying polypeptide comprised an RTE1 MD polypeptide having the amino acid sequence of:

[0681] The gene modifying polypeptide comprised an XTEN linker having an amino acid sequence of:

[0682] The gene modifying polypeptide comprised an HiBit tag having an amino acid sequence of:

[0683] The full-length gene modifying polypeptide with all components was structured as follows: NLS-3GS linker-RTE-MD-XTEN linker-HiBit tag. The full-length sequences are provided in Table E3.

[0684] The gene modifying system further comprised a template RNA with the following sequences from 5’ to 3’: 5’UTR_retro, BCMA CAR, MND promoter, 3’UTR_retro.

285 [0685] The gene modifying system further comprised a template RNA with the following sequences from 5’ to 3’: 5’UTR_retro, BCMA CAR, MND promoter, 3’UTR_retro.

[0686] The template RNA comprised an RTE1 MD 5’UTR having the sequence of:

[0687] The template RNA comprised an RTE1 MD 3’UTR having the sequence of:

UGAAACUGCACAAAGACAAUAGUCAUUCUCGAUCACCGAGAGACUACCACU (SEQ

ID NO: 570)

[0688] The template RNA comprised a nucleic acid sequence encoding a BCMA-CAR fusion polypeptide, wherein the nucleic acid sequence was:

[0689] The BCMA-CAR fusion polypeptide comprised an amino acid sequence of:

[0690] The BCMA-CAR fusion polypeptide comprised a CDS signal peptide domain having an amino acid sequence of:

[0691] The BCMA-CAR fusion polypeptide comprised a CDS hinge domain having an amino acid sequence of:

[0692] The BCMA-CAR fusion polypeptide comprised a CDS transmembrane domain having an amino acid sequence of:

[0693] The BCMA-CAR fusion polypeptide comprised a 4- IBB signaling domain having an amino acid sequence of:

[0694] The BCMA-CAR fusion polypeptide comprised a CD3z signaling domain having an amino acid sequence of:

Peripheral Blood Monocyte Cells (PBMCs) Thaw and Overnight Rest

[0695] Ciyopreserved PBMCs from two donors (Hemacare, donor 448 and donor 354) were thawed at 37°C and washed in a T cell medium comprised of TexMACS™ cell culture medium (Miltenyi #170-076-307) with 5% human AB serum (Gemini Bio 100-152). Cells were counted after pelleting by centrifugation at 300xg for 5 minutes and resuspended in cytokine-free medium at a target concentration of le6 x10⁶ cells/mL. Cells were transferred to cell culture flasks and maintained at a target cell concentration of a le⁶ cells/mL , in TexMACs + 5%

288 human AB serum + IL-7(Miltenyi-130-095-362, 10ng/mL) and IL-15(Miltenyi-130-095-764, 5ng/mL) and placed inside of a cell culture incubator at 37°C and 5% CO₂ for overnight rest.

[0696] After overnight rest, cells were harvested into a 50 mL centrifuge tube, washed and then transferred to a conical tube. 15 mL of versene (FisherSci: 15-040-066) was added to the flask to detach adherent cells. The cells were then incubated at 37°C until they detached and were then transfered to a conical tube. The cells were spun for 7 minutes at 350xg and resuspended in Plasma-Lyte A (Baxter #2B2544) +2% Human Serum Albumin (AkronBio: AK8228-0100) for cell counting. The cells were then divided for lentivirus transduction or LNP transfection.

LNP Transfection

[0697] The cells were counted and plated for final resuspension at 40x10⁶ cells/mL in a 96- well cell repellant U- bottom plate (FisherSci:07-000-612). TransAct™, and the tLNPs, were mixed in Plasma-Lyte A and added to the cell suspension, such that the final cell concentration was 20x10⁶ cells/mL in Plasma-Lyte A 1% Human Serum Albumin. The MACS® GMP T Cell TransAct™, a polymeric nanomatrix comprising human CD3 and CD28, was added to the transfection medium at a final volume dilution of 1 : 17.5. For the transfected condition, tLNPs comprising an rnRNA encoding the gene modifying polypeptide and the template RNA were dosed at a total concen tration of 5 ug LNPs per million cells, and a ratio of one part gene modifying polypeptide LNPs to 25 pails template LNPs. For the untransfected condition, no tLNPs were added. Incubation occurred for four hours at room temperature. After tiie 4-hour incubation, cells were washed by spinning at 350xg for 7 minutes at room temperature. The supernatant was pipetted out and the cells were then resuspended in Complete T cell medium comprised of TexMACS cell culture medium, 5% human AB serum by volume, 100 lU/mL IL- 2, 10 ng/mL IL-7, and 5 ng/mL IL-15 at a cell concentration of 1x10⁶ cells/mL in a 96 well flat bottom plate (Greiner Bio-One: 655970).

Lentivirus Administration

[0698] Cells were counted and plated at le6 cells/well in TexMACs, 2% Human Serum Albumin (HSA) in a 96-well cell repellent plate. The BCMA CAR lentivirus (Lentigen, MND- CAR085-WPRE, Lot: PC-0328-0923-2913) was thawed briefly in bead bath and then diluted in TexMACs media such that the final multiplicity of infection (MOI) was 6 per well. Transduction enhancer, LentiBoost, was added to the diluted virus at a concentration of 1 : 1000. The Lentivirus Master Mix was added to each corresponding well.

289 [0699] After a 4-hour incubation, cells were washed by spinning at 350xg for 7 minutes at room temperature. The supernatant was pipetted out and the cells were then resuspended in Complete T cell medium comprised of TexMACS cell culture medium, 5% human AB serum by volume, 100 UJ/mL IL-2, 10 ng/mL IL-7, and 5 ng/mL IL-15 at a cell concentration of 1x10⁶ cells/mL in a 96 well flat bottom plate (Greiner Bio-One: 655970).

Culture ex vivo

[0700] Complete T cell medium was added as needed to maintain cultures at 1x10⁶ cells/mL.

[0701] For the staining procedure, 0.5x10⁶ cells were first quenched with stain buffer (BD #554657), pelleted by centrifugation at 500xg for 5 minutes, resuspended in 100 μL of stain solution containing 0.125 μL of rh-BCMA Fc Chimera AF647, and incubated for 30 minutes in the dark at 2-8°C. Following incubation, cells were quenched with stain buffer, pelleted by centrifugation at 500xg for 5 minutes, then resuspended in 200 μL of stain buffer 7-AAD live/dead stain at 1 : 1000 ratio (Invitrogen #A1310). Flow cytometry data was acquired on a NovoCyte (Agilent) flow cytometer, then analyzed for the proportion of live cells expressing anti-BCMA CAR.

In vitro tumor killing assay

[0702] On day 10, CART cells were harvested, and function was assessed via a BCMA target cell killing assay. Briefly, BCMA-CAR-T cells were co-cultured for 16 hours with BCMA- positive tumor cell lines (MM1.S), stably transduced with ffLuc, at serially decreasing CAR+ T cell (effector) to tumor cell ratios. Controls included no LNP/Virus treated T cells. Cell Titre Gio, The CellTiter-Glo® ((Promega #G7572) Luminescent Cell Viability Assay, homogeneous method of determining die number of viable cells based on quantitation of the ATP present, an indicator of metabolically active cells, where % cytotoxicity = (BLI Mock- BLI sam_Pie)/BLI Mock, BLI Mock = mean target cell alone value of that experiment. Additionally, after 16 hours of coculture supernatants were assessed their IFN-y cytokine levels relative to control T cells via ELISA(MSD).

[0703] FIG. 18 shows that the anti-CD3 tLNPs comprising an exemplary gene modifying system can generate CAR-T cells when administered to donor PBMCs at levels almost as high as when introducing a CAR transgene via lentivirus transduction. FIG. 19 shows that CAR-T cells generated using the gene modifying system exhibited improved killing of BCMA-expressing tumor cells in a cytotoxicity assay compared to lentivirus-induced CAR-T cells.

290

Claims

1. 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;

291 wherein 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;

(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:

292

293

294

6. 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:

295

9. 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 20x10⁶ cells/mL to about 200x10⁶ cells/mL or from about 20x10⁶ cells/mL to about 100x10⁶ cells/mL.

17. The method of any of claims 1-16, wherein the blood fraction comprises a cell density of from about 20x10⁶ cells/mL to about 200x10⁶ cells/mL or from about 20x10⁶ cells/mL to about 100x10⁶ cells/mL.

296

18. 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.

297

34. 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 10⁶ cells to about 5 μg of the LNPs per 1x10⁶ cells.

39. The method of any of claims 31-38, wherein the blood-LNP composition comprises about 20 cells/mL to about 100 x 10⁶ cells/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.

298

51. 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.

299

62. 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.

300

78. 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 .

301

88. 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 20x10⁶ cells/mL to about 200x10⁶ cells/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:

(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 10⁶ cells - 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.

302

98. 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