Lipid-based formulations for delivery of nucleic acids for engineering immune cells

Abstract

This disclosure relates to lipid-based formulations such as lipid nanoparticle (LNP) formulations for delivery of an mRNA encoding a large serine recombinase (LSR), a DNA comprising a donor attachment site (AttD) and a sequence of interest, a targeting moiety directed to the immune cell, and wherein the formulations comprise an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor, wherein the formulation is capable of integrating the sequence of interest into the genome of the immune cell, optionally wherein the immune cell is a human immune cell, and optionally wherein (i) the targeting moiety is an activation factor, (ii) the activation factor is encoded by the mRNA encoding the LSR, or (iii) the formulation comprises an additional mRNA encoding the activation factor.

Description

Attorney Docket No: 01373-0004-00PCTLIPID-BASED FORMULATIONS FOR DELIVERY OF NUCLEIC ACIDS FOR ENGINEERING IMMUNE CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to US Provisional Application No.63 / 742,265, filed January 6, 2025, the entire contents of which are incorporated by reference herein.REFERENCE TO THE SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on January 2, 2026, is named “01373-0004-00PCT.xml” and is 10,051 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.FIELD OF THE DISCLOSURE

[0003] This disclosure relates to lipid-based formulations such as lipid nanoparticle (LNP) formulations for delivery to an immune cell of an mRNA encoding a large serine recombinase (LSR), a DNA comprising a donor attachment site (AttD) and a sequence of interest, a targeting moiety directed to the immune cell, and an activation factor for the immune cell, optionally wherein the targeting moiety is the activation factor for the immune cell. In some cases, the mRNA encoding the LSR and the DNA comprising the AttD site and sequence of interest are co-encapsulated in the lipid-based formulation. The disclosure further relates to delivery systems comprising such lipid-based formulations, methods of integrating the DNA into immune cells such as T cells, methods of depleting B cells in subjects, and methods of treating subjects with conditions such as B cell cancers.BACKGROUND

[0004] Large serine recombinases (LSRs) are a class of enzymes within the DNA integrase family that precisely catalyze the recombination of DNA in a controllable way. Generally found in phage and bacteria, these enzymes recognize pairs of distinct attachment sites and facilitate the integration of genetic elements into bacterial genomes via recombination at these cognate attachment sites. Historically, the attachment site within the bacterial genome is called AttB, while the cognate phage attachment site is called AttP. Recombinant attachment sites for LSRs are also referred to as a donor attachment site AttD and an acceptor attachment site AttA. Upon insertion of the genetic elements, the attachment sites are incorporated at the boundaries of theAttorney Docket No: 01373-0004-00PCTintegrated nucleic acids, forming sequences referred to as AttL and AttR. Unlike other classes of integrases that permit bidirectional integration, LSRs typically drive unidirectional integration of nucleic acids between the cognate attachment sites.

[0005] In recent years, a number of proteins that can site-specifically modify DNA, such as TALENs, Cas proteins, transposases, and integrases, have been explored as genome editing tools with many scientific applications. Genome editing is particularly desirable as a therapeutic, as it has the potential to treat and even cure disease at the genetic level, as opposed to merely managing symptoms. Initial research on several LSR systems has been reported for genome modification purposes, but a number of limitations have emerged, including inefficient integration, lack of specificity, and the need to separately engineer a “landing pad” into human cells that can act as an appropriate attachment site for the LSR. Moreover, because LSRs function to integrate DNA into genomes, genome editing systems must be optimized for not only mRNA encoding the LSR, but also DNA to be integrated into the cellular genome. Current genome editing systems using LNPs and AAVs have traditionally been optimized for delivery of DNA or mRNA only, and not combinations of mRNA and DNA, as is needed here.

[0006] While advances in genome editing have opened new and exciting avenues for treatment of genetic-based diseases and disorders, integration of DNA has proven difficult e.g., due to cellular repair enzymes that efficiently repair DNA breaks before integration can occur, and low efficiency integration of DNA from plasmid double-stranded DNA in many cell types. See e.g., Iyer et al. (2022) The CRISPR Journal 5(5) 685-701 at Abstract.

[0007] Accordingly, there remains an unmet need for genome editing systems that can provide efficient means to genetically alter genomes by insertion of DNA. In addition, there remains an unmet need for improved methods of editing immune cells such as T cells for use in treatment of diseases in subjects, for example, by engineering T cells to express pharmaceutically useful proteins of interest.SUMMARY

[0008] In some embodiments, the disclosure provides an embodiment as follows below:

[0009] 1. A lipid-based formulation for nucleic acid delivery to an immune cell, the formulation comprising:a. an mRNA encoding a large serine recombinase (LSR);b. a DNA comprising a donor attachment site (AttD) and a sequence of interest;andc. a targeting moiety for the immune cell;Attorney Docket No: 01373-0004-00PCTwherein, the formulation comprises an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor for the immune cell, optionally wherein (i) the targeting moiety is an activation factor, (ii) the activation factor is encoded by the mRNA of (a) encoding the LSR, or (iii) the formulation comprises an additional mRNA encoding the activation factor; andwherein the formulation is capable of integrating the sequence of interest into the genome of the immune cell, optionally wherein the immune cell is a human immune cell; and optionally wherein the lipid-based formulation comprises a lipid nanoparticle (LNP). Optionally, in some embodiments of the lipid-based formulation of embodiment 1, the ratio of the DNA comprising the donor attachment site (AttD) and sequence of interest to the mRNA encoding the LSR is from 3:1 to 1:3, or from 2: 1 to 1 :2, or is 1 : 1.

[0010] 2. The lipid-based formulation of embodiment 1, wherein the immune cell is a T cell, B cell, NK cell, lymphocyte, or myeloid cell, optionally wherein the cell is a human cell.

[0011] 3 The lipid-based formulation of any one of the preceding embodiments, wherein the sequence of interest encodes a chimeric antigen receptor (CAR), T cell receptor (TCR), B cell receptor (BCR), an immune cell activation or inhibitory receptor, a growth factor ligand, a transcription factor, or a checkpoint inhibitor or agonist.

[0012] 4. The lipid-based formulation of any of the preceding embodiments, wherein the immune cell is a T cell and the sequence of interest encodes a CAR, such as a CD 19 CAR or CD20 CAR.

[0013] 5. The lipid-based formulation of any one of the preceding embodiments, wherein the targeting moiety is an activation factor for the immune cell.

[0014] 6. The lipid-based formulation of any one of embodiments 1-4, wherein the targeting moiety is not an activation factor for the immune cell.

[0015] 7. The lipid-based formulation of any one of the preceding embodiments, wherein the formulation comprises an LNP and wherein the activation factor is on the surface of LNPs in the formulation.

[0016] 8. The lipid-based formulation of any one of the preceding embodiments, wherein the activation factor is encoded by an additional mRNA in the formulation.

[0017] 9. The lipid-based formulation of any one of the preceding embodiments, wherein the activation factor is encoded by the mRNA encoding the LSR.

[0018] 10. The lipid-based formulation of any one of the preceding embodiments, wherein the activation factor is antigen-dependent.Attorney Docket No: 01373-0004-00PCT

[0019] 11. The lipid-based formulation of embodiment 10, wherein the activation factor is a CAR.

[0020] 12. The lipid-based formulation of embodiment 11, wherein the activation factor is a CD 19 CAR or CD20 CAR.

[0021] 13. The lipid-based formulation of any one of embodiments 1-9, wherein the activation factor is antigen independent.

[0022] 14. The lipid-based formulation of embodiment 13, wherein the activation factor is selected from any one of: IL-12B, BATF, LTBR, JUNB, constitutively active beta-catenin, TCF7, FOXO1, IRF4, CARD11-PIK3R3, CD79A / CD40, ITK-SYK, FYN-TRAF3IP2, KHDRBS1-LCK, SIN3A-FOXO1, RNMT, RAS, a BRAF mutant such as BRAF-G469A, a PLCG1 mutant such as PLCG1-D1165H or PLCG1-R48W or PLCG1-E1164K or PLCG1-S520F, a RASGRP1 mutant such as RASGRP1-M2611, a CARD11 mutant such as CARD11-Y361C or CARD11-S615F or CARD11-D357N, TNFRSF1B-T3771, CD28 antibody, CD80, soluble CD58, SLC7A5, SLC1A5, SLC7A1, SLC38A1, SLC38A2, GLUT1 (SLCA1), GLUT3 (SLCA3), MCT1 (SLC16A1), MCT4 (SLC16A3), phospholipase Cyl), IP3, IP3R, P2X7, CD40, CD86, 0X40, 4-1BBL, AHCY, CDK1, CDK2, AKR1C4, ATF6B, ITM2A, AHNAK, or FOXQ1, or wherein the activation factor is a chimeric molecule comprising a T cell activating domain and a dimer or multimerization motif, optionally wherein the T cell activating domain comprises CD28, CD3zeta, or an ITAM, and optionally wherein the dimer or multimerization motif is located extracellularly, in the transmembrane domain, or intracellularly.

[0023] 15. The lipid-based formulation of any one of the preceding embodiments, wherein the DNA is circular single-stranded DNA (cssDNA).

[0024] 16. The lipid-based formulation of any one of embodiments 1-14, wherein the DNA is double stranded DNA.

[0025] 17. The lipid-based formulation of any one of the preceding embodiments, wherein the targeting moiety targets CD3 (e.g., on the surface of T cells), optionally wherein the lipid-based formulation comprises an LNP wherein the targeting moiety is on the surface of the LNP, and further optionally wherein the targeting moiety is at a density of from 0.125 mol% to 0.75 mol% per LNP, or from 0.125 mol% to 0.5 mol% per LNP, or from 0.25 mol% to 0.75 mol% per LNP, or from 0.25 mol% to 0.5 mol% per LNP.

[0026] 18. The lipid-based formulation of embodiment 17, wherein the targeting moiety is an anti-CD3 antibody or antigen binding domain.Attorney Docket No: 01373-0004-00PCT

[0027] 19. The lipid-based formulation of any one of the preceding embodiments, wherein the sequence of interest encodes a CAR that binds to B cells or malignant B cells, optionally wherein the B cells or malignant B cells are human B cells or human malignant B cells.

[0028] 20. The lipid-based formulation of any one of the preceding embodiments, wherein the sequence of interest binds a CAR that binds to plasma cells or malignant plasma cells, optionally wherein the plasma cells or malignant plasma cells are human plasma cells or human malignant plasma cells.

[0029] 21. The lipid-based formulation of embodiment 19 or 20, wherein the sequence of interest encodes a CAR, optionally a CD 19 CAR or a CD20 CAR.

[0030] 22. The lipid-based formulation of any one of the preceding embodiments, wherein the formulation comprises an LNP and wherein the LNP comprises a cationic lipid, helper lipid, cholesterol, and a PEG-lipid.

[0031] 23. The lipid-based formulation of any one of the preceding embodiments, wherein the formulation comprises an LNP and wherein the targeting moiety is covalently attached to at least one lipid of the LNP, optionally to a PEG-lipid.

[0032] 24. The lipid-based formulation of any one of the preceding embodiments, wherein the mRNA encodes a nuclear localization signal (NLS).

[0033] 25. The lipid-based formulation of any one of the preceding embodiments, wherein the DNA of (b) is modified.

[0034] 26. The lipid-based formulation of any one of the preceding embodiments, wherein the mRNA of (a) is modified.

[0035] 27. A method for integration of a chimeric antigen receptor (CAR) into a cellular genome of an immune cell, comprising contacting the immune cell with the lipid-based formulation of any one of embodiments 1-26, wherein the sequence of interest encodes the CAR, such that the CAR is integrated into the cellular genome of the immune cell, optionally wherein the immune cell is a T cell, a B cell, an NK cell, a lymphocyte, or a myeloid cell, optionally wherein the cell is a human cell.

[0036] 28. A method for introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into an immune cell, comprising contacting the immune cell with the lipid-based formulation of any one of embodiments 1-26, optionally wherein the immune cell is a T cell, a B cell, an NK cell, a lymphocyte, or a myeloid cell, optionally wherein the cell is a human cell.

[0037] 29. An engineered immune cell expressing a chimeric antigen receptor (CAR) encoded by the DNA of the lipid-based formulation of any one of embodiments 1-26,Attorney Docket No: 01373-0004-00PCToptionally wherein the engineered immune cell is a T cell, a B cell, an NK cell, a lymphocyte, or a myeloid cell, optionally wherein the cell is a human cell.

[0038] 30. An engineered immune cell modified by contact with the lipid-based formulation of any one of claims 1-26, optionally wherein the immune cell is a T cell, a B cell, an NK cell, a lymphocyte, or a myeloid cell, optionally wherein the cell is a human cell.

[0039] 31. A method for treating a disease in a subject, comprising administering to the subject the lipid-based formulation of any one of claims 1-26 or the engineered immune cell of embodiment 29 or 30.

[0040] 32. The lipid-based formulation of any one of embodiments 1-26 for use in treating a disease in a subject.

[0041] 33. Use of the lipid-based formulation of any one of embodiments 1-26 in the preparation of a medicament for treating a disease in a subject.

[0042] 34. The method, lipid-based formulation, or use of any one of embodiments 31-33, wherein the disease is a B cell cancer.

[0043] 35. The method, lipid-based formulation, or use of embodiment 34, wherein the B cell cancer is a plasma cell neoplasm, a plasmacytoma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphocytic leukemia (ALL), Hairy cell leukemia, multiple myeloma, B cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia / small lymphoplasmacytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma (DLBL), follicular lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, or marginal zone lymphoma.

[0044] 36. The method, lipid-based formulation, or use of any one of embodiments 31-35, wherein the administration of the lipid-based formulation or engineered immune cell results in depletion of B cells in the subject.

[0045] 37. The method, lipid-based formulation, or use of any one of embodiments 31-36, wherein the subject is a human.

[0046] 38. A lipid-based formulation for nucleic acid delivery to an immune cell, the formulation comprising:a. an mRNA encoding a genome editing component, optionally wherein the genome editing component comprises a large serine recombinase (LSR); andb. a targeting moiety for the immune cell;wherein the formulation comprises an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor for the immune cell, optionally wherein (i) the targeting moiety is an activation factor, (ii) the activation factor is encoded by the mRNA of (a) encodingAttorney Docket No: 01373-0004-00PCTthe genome editing component, or (iii) the formulation comprises an additional mRNA encoding the activation factor; andwherein the formulation is capable of increasing the protein expression of the genome editing component as compared to a formulation otherwise identical but without the activation factor; and optionally wherein the formulation comprises an LNP.

[0047] 39. The lipid-based formulation of embodiment 38, wherein the lipid-based formulation further comprises a DNA encoding a sequence of interest.

[0048] 40. The lipid-based formulation of embodiment 38 or 39, wherein the immune cell is a T cell, B cell, NK cell, lymphocyte, or myeloid cell, optionally wherein the cell is a human cell.

[0049] 41. The lipid-based formulation of any one of embodiments 38-40, wherein the activation factor is a CAR.

[0050] 42. The lipid-based formulation of embodiment 41, wherein the activation factor is a CD 19 CAR or CD20 CAR.

[0051] 43. The lipid-based formulation of any one of embodiments 38-42, wherein the activation factor is antigen independent.

[0052] 44. The lipid-based formulation of any one of embodiments 38-43, wherein the activation factor is selected from any one of: IL-12B, BATF, LTBR, JUNB, constitutively active beta-catenin, TCF7, FOXO1, IRF4, CARD11-PIK3R3, CD79A / CD40, ITK-SYK, FYN-TRAF3IP2, KHDRBS1-LCK, SIN3A-FOXO1, RNMT, RAS, a BRAF mutant such as BRAF-G469A, a PLCG1 mutant such as PLCG1-D1165H or PLCG1-R48W or PLCG1-E1164K or PLCG1-S520F, aRASGRPl mutant such asRASGRPl-M2611, aCARDll mutant such as CARD11-Y361C or CARD11-S615F or CARD11-D357N, TNFRSF1B-T3771, CD28 antibody, CD80, soluble CD58, SLC7A5, SLC1A5, SLC7A1, SLC38A1, SLC38A2, GLUT1 (SLCA1), GLUT3 (SLCA3), MCT1 (SLC16A1), MCT4 (SLC16A3), phospholipase Cyl), IP3, IP3R, P2X7, CD40, CD86, 0X40, 4-1BBL, AHCY, CDK1, CDK2, AKR1C4, ATF6B, ITM2A, AHNAK, or FOXQ1, or wherein the activation factor is a chimeric molecule comprising a T cell activating domain and a dimer or multimerization motif, optionally wherein the T cell activating domain comprises CD28, CD3zeta, or an ITAM, and optionally wherein the dimer or multimerization motif is located extracellularly, in the transmembrane domain, or intracellularly.

[0053] 45. The lipid-based formulation of any one of embodiments 38-44, wherein the targeting moiety targets CD3 (e.g., on the surface of T cells) , optionally wherein the lipid-based formulation comprises an LNP wherein the targeting moiety is on the surface of the LNP,Attorney Docket No: 01373-0004-00PCTand further optionally wherein the targeting moiety is at a density of from 0.125 mol% to 0.75 mol% per LNP, or from 0.125 mol% to 0.5 mol% per LNP, or from 0.25 mol% to 0.75 mol% per LNP, or from 0.25 mol% to 0.5 mol% per LNP.

[0054] 46. The lipid-based formulation of embodiment 45, wherein the targeting moiety is an anti-CD3 antibody or antigen binding domain.

[0055] 47. The lipid-based formulation of any one of embodiments 39-46, wherein the sequence of interest encodes a CAR that binds to B cells or malignant B cells, optionally wherein the B cells or malignant B cells are human B cells or human malignant B cells.

[0056] 48. The lipid-based formulation of any one of embodiments 39-47, wherein the sequence of interest binds a CAR that binds to plasma cells or malignant plasma cells, optionally wherein the plasma cells or malignant plasma cells are human plasma cells or human malignant plasma cells.

[0057] 49. The lipid-based formulation of embodiment 48, wherein the sequence of interest on the DNA encodes a CAR, optionally a CD 19 CAR or a CD20 CAR.

[0058] 50. The lipid-based formulation of any one of embodiments 38-49, wherein the formulation comprises an LNP, optionally wherein the LNP comprises a cationic lipid, helper lipid, cholesterol, and a PEG-lipid.

[0059] 51. The lipid-based formulation of any one of embodiments 38-50, wherein the formulation comprises an LNP, and wherein the targeting moiety is covalently attached to at least one lipid of the LNP, optionally to a PEG-lipid.

[0060] 52. The lipid-based formulation of any one of embodiments 38-51, wherein the mRNA encodes a nuclear localization signal (NLS).

[0061] 53. The lipid-based formulation of any one of embodiments 38-52, wherein the mRNA of (a) is modified.

[0062] 54. A delivery system comprising the lipid-based formulation of any one of embodiments 1-26 or 38-53, and optionally further comprising at least one carrier, excipient, diluent, or vehicle.

[0063] 55. The delivery system of claim 54, wherein the at least one carrier, excipient, diluent, or vehicle is pharmaceutically acceptable.BRIEF DESCRIPTION OF THE FIGURES

[0064] FIGS 1A-B show the ability of CD3 -targeted LNPs to deliver mRNA encoding mCherry to different cell types within PBMCs. FIG 1A shows the percent of primary human T cells (CD4+ T cells and CD8+ T cells) expressing mCherry protein after treatment with CD3-targeted LNPs, as detected by flow cytometry, either in the presence (“+Stim”) or absence (“NoAttorney Docket No: 01373-0004-00PCTStim”) of stimulation with anti-CD3 / andCD28 beads prior to addition of LNPs. FIG IB shows the efficiency of expression of mCherry protein in different cell types within PBMCs, including NK cells, B cells, monocytes, and T cells after CD3 -targeted LNP treatment. “Mock” indicates no LNP treatment, “None” indicates treatment with a non-targeting LNP, “IgG” and “Fab” indicate CD3 -targeted LNPs made with anti-CD3 antibody in either IgG or Fab format, respectively.

[0065] FIG 2 shows the efficiency of expression of mScarlet protein (mRNA reporter) or GFP protein (DNA reporter) in human T cells after dosing of humanized NSG mice with CD3-targeted LNPs versus untargeted LNPs co-encapsulating mRNA encoding mScarlet protein and DNA nanoplasmid encoding a GFP expression cassette. The percentage of CD3+ T cells in the blood with reporter expression after LNP dosing was evaluated by flow cytometry.

[0066] FIGS 3A-B show the efficiency of expression of a CD 19 CAR in primary human T cells after incubation for 7 days with CD3-targeted LNPs co-encapsulating an mRNA encoding an LSR variant and cssDNA donor template encoding the CD 19 CAR transgene by flow cytometry (FIG 3A) or integration by ddPCR (FIG 3B). CD3 -targeted LNPs encapsulated either LSR mRNA only or cssDNA donor template only, or co-encapsulated both LSR mRNA and donor template cssDNA. FIG 3A shows the percent of CAR+ T cells. FIG 3B shows the % integration in T cells at four genomic sites (GS10, GS11, GS12, and GS13).

[0067] FIGS 4A-F show in vivo CD19-CAR T cell generation and functional activity (B cell depletion) in humanized NSG mice dosed with CD3 -targeted LNPs co-encapsulating mRNA encoding an LSR variant and cssDNA donor template encoding a CD 19 CAR transgene. FIG 4A and FIG 4B show the % of human B cells or T cells, respectively, out of total live cells analyzed by flow cytometry in bone marrow, lymph node, or spleen recovered from terminal samples 8 days after dosing mice with CD3 -targeted LNPs encapsulating the indicated mRNA and / or cssDNA components. Analysis of terminal tissues by flow cytometry showed significantly reduced B cells (FIG 4A) and no significant change in T cells (FIG 4B).Scatter plots of CAR expression (Y-axis) and forward scatter (X-axis) evaluated by flow cytometry are shown in splenic T cells from a mouse dosed with CD3-targeted LNPs coencapsulating LSR mRNA + cssDNA (FIG 4C) or encapsulating cssDNA only (FIG 4D).LSR-mediated CAR integration at four genomic sites (GS10, GS11, GS12, GS13) in terminal tissues, including blood (B), bone marrow (BM), lymph node (LN) or spleen (S), was analyzed by ddPCR in terminal tissues and is indicated as an average % integration of all mice within a group (FIG 4E) or as % integration for each individual mouse dosed with CD3 -targeted LNPs with LSR mRNA and / or cssDNA donor template (as indicated) (FIG 4F).Attorney Docket No: 01373-0004-00PCT

[0068] FIGS 5A-D show anti -tumor activity is achieved with in vivo integrated CD19-CAR T cells in NSG-MHCI / II-DKO mice in testing with two different LSR variants (“LSR-A” and “LSR-B”). Tumor burden reduction was observed by IVIS imaging with CD3-targeted LNPs co-encapsulating a variant LSR mRNA and nanoplasmid DNA encoding the CD 19 CAR (solid line circles) compared to LNPs encapsulating mRNA or DNA only (hashed lines) (FIG 5A).Integration of the CD 19 CAR was observed by ddPCR in blood over time and shown as percent integration over a sum of top 8 integration sites at days 4 and 26 (FIG 5B). CD19-CAR T cells were detected in blood 10 days after LNP dosing by flow cytometry (FIG 5C). B cells from PBMCs were depleted within 10 days of LNP dosing as detected by measuring CD20 in CD45+ cells by flow cytometry (FIG 5D).

[0069] FIGS 6A-C show anti -tumor activity is achieved with in vivo integrated CD 19-CAR T cells in NSG-MHCI / II-DKO mice in testing with two different LSR variants (“LSR-A” and “LSR-B”). Tumor burden reduction was observed by IVIS imaging with CD3-targeted LNPs co-encapsulating a variant LSR mRNA and cssDNA encoding the CD 19 CAR (solid line circles) compared to LNPs encapsulating mRNA or DNA only (hashed lines) (FIG 6A).Integration of the CD 19 CAR was observed by ddPCR in blood over time and shown as percent integration over a sum of top 8 integration sites at days 4 and 26 (FIG 6B). CD 19-CAR T cells were detected in blood 10 days after LNP dosing by flow cytometry (FIG 6C).

[0070] FIG 7 shows tumor burden in NSG-MHC I / II DKO mice following inoculation with Raji cells, followed by administration of healthy human PBMCs and either a T-cell targeting LNP co-encapsulating an LSR mRNA and a cssDNA encoding a CD20 CAR (black circles), or either no LNP (grey triangles) or an LNP comprising mRNA encoding a non-functional LSR (“dead LSR”; grey squares). The figure shows significant tumor regression and low tumor burden was maintained upon administration with LNP comprising LSR mRNA and cssDNA encoding a CD20 CAR.

[0071] FIG 8 shows tumor burden in NSG-MHC FII DKO mice following inoculation with Raji cells, followed by administration of healthy human PBMCs and either a T-cell targeting LNP co-encapsulating an LSR mRNA and a double-stranded DNA nanoplasmid (dsDNA) encoding a CD20 CAR (black circles), or either no LNP (grey triangles) or an LNP comprising mRNA encoding a non-functional LSR (“dead LSR”; grey squares). The figure shows significant tumor regression and low tumor burden was maintained upon administration with LNP comprising LSR mRNA and dsDNA encoding a CD20 CAR.

[0072] FIG 9 shows fold changes in the number of B cells in naive cynomolgus monkeys after administration of CD3 -targeted LNPs co-encapsulating an mRNA expressing LSR andAttorney Docket No: 01373-0004-00PCTcssDNA encoding a CD20 CAR at LNP doses of 0.1, 0.3, or 1 mg / kg, and either “low” (0.25 mol%) or “high” (0.5 mol%) anti-CD3 Fab concentration per LNP. A PBS control group was also included.DETAILED DESCRIPTION

[0073] Further description of certain embodiments is provided below. For example, the present disclosure encompasses lipid-based formulations for nucleic acid delivery to immune cells such as T cells, in which the formulations comprise (a) an mRNA encoding a large serine recombinase (LSR); (b) a DNA comprising a donor attachment site (AttD) and a sequence of interest; and (c) a targeting moiety for the immune cells; wherein the formulation comprises an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor for the immune cell, optionally wherein (i) the targeting moiety is an activation factor, (ii) the activation factor is encoded by the mRNA of (a) encoding the LSR, or (iii) the formulation comprises an additional mRNA encoding the activation factor. In some cases, the sequence of interest is a chimeric antigen receptor (CAR) such as a CD 19 CAR or a CD20 CAR. In some cases, the immune cell is a T cell. In some cases, the targeting moiety binds to a protein on the surface of immune cells (e.g., in embodiments where the immune cells are T cells, the targeting moiety may bind to CD3). In some embodiments, the lipid-based formulation is a lipid nanoparticle (LNP), or a liposome. The disclosure herein further relates, for example, to delivery systems comprising the lipid-based formulations, and to methods of making and using the lipid-based formulations.

[0074] Description of the components of the lipid-based formulations, delivery systems comprising lipid-based formulations, and methods of making and using them, for example, is provided in the following sections.I. DEFINITIONS

[0075] Unless stated otherwise, the following terms and phrases have the meanings described below. The definitions are not meant to be limiting in nature and serve to provide a clearer understanding of certain aspects of the present disclosure.

[0076] Activation factor: As used herein, an “activation factor” or “activation factor for immune cells” refers to a factor, such as a protein that is capable of activating or stimulating proliferation of an immune cell. The presence of the activation factor in a lipid-based formulation increases the level of integration of a sequence of interest and / or the level of expression of a genome editing component in a contacted immune cell compared to the level of integration of the sequence of interest and / or level of expression of the genome editingAttorney Docket No: 01373-0004-00PCTcomponent in an immune cell that has been contacted with a lipid-based-formulation that is otherwise identical but does not include the activation factor or nucleic acid encoding the activation factor.

[0077] AttA site: As used herein, the term “AttA site” refers to the attachment site within a cellular genome to which an LSR binds. An AttA site may exist in the genome of a non-phage and non-bacterial organism, e.g., a human.

[0078] Attachment site: As used herein, the term “attachment site” refers to a nucleic acid sequence to which an LSR binds and uses for facilitating recombination of DNA sequences of interest.

[0079] AttD site: As used herein, the term “AttD site” refers to an attachment site in a DNA to which an LSR binds. AttD sites may be engineered into a plasmid DNA to facilitate recombination of a sequence of interest at a cognate AttA site.

[0080] Delivery system: A “delivery system” as used herein refers to a composition or formulation that comprises a lipid-based formulation (e.g., an LNP or liposome or the like), and optionally further ingredients such as carriers, excipients, vehicles, or diluents. Such further ingredients may, for example, be separate from the lipid-based formulation, such as added to the formulation to form the delivery system. In some embodiments, a delivery system may be provided in a container such as a vial or syringe for delivery to a subject.

[0081] Genome editing system'. As used herein, a “genome editing system” refers to a composition or formulation comprising elements sufficient for editing a genome of an immune cell, for example, comprising elements sufficient such that a sequence of interest is capable of being inserted into the genome of an immune cell.

[0082] Identity. As used herein, the term “identity” in the context of sequence comparisons refers to the number of exact matches between two different sequences in a sequence alignment. Sequence alignment techniques and software include Basic Local Alignment Search Tool (BLAST, which includes e.g., BLASTP for protein sequences and BLASTN for nucleic acid sequences), ClustalOmega, MUSCLE, and MAFFT.

[0083] Immune cell'. As used herein, “immune cell” refers to a white blood cell, including progenitors and progeny. Examples include neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer (NK) cells, and lymphocytes (B cells and T cells).

[0084] Isolated: As used herein, an “isolated” LSR refers to an LSR, or nucleic acid encoding an LSR, that is not in its native phage or bacterial environment.Attorney Docket No: 01373-0004-00PCT

[0085] Lipid-based formulation: As used herein, a “lipid-based formulation” refers to a formulation comprising one or more components such as a nucleic acid molecule or protein associated with one or more lipid carriers. In some embodiments, the formulation is a liposome or a lipid nanoparticle (LNP) or other type of lipid-based particle. In some embodiments, a lipid-based formulation may also comprise one or more non-lipid carrier, excipient, diluent, or vehicle.

[0086] Sequence of interest: A “sequence of interest” as used herein refers to a nucleic acid sequence for insertion into the genome of an immune cell. In some cases, the sequence of interest encodes a protein or RNA for expression in the immune cell.

[0087] Targeting moiety: A “targeting moiety” or “targeting moiety for an immune cell” refers to a moiety that is capable of binding to an immune cell, such as by recognizing a molecule (e.g., a cell surface receptor) on the immune cell.

[0088] Additional definitions of terms are in the sections that follow, as well as in the “General Considerations” section below.II. LIPID-BASED FORMULATIONS, DELIVERY SYSTEMS, AND GENOME EDITING SYSTEMS

[0089] Disclosed herein are lipid-based formulations and delivery systems for nucleic acid delivery to immune cells. In some embodiments, lipid-based formulations are provided comprising (a) a nucleic acid (e.g. an RNA or mRNA) encoding a large serine recombinase (LSR), (b) a DNA comprising a donor attachment site (AttD) and a sequence of interest, and (c) a targeting moiety for immune cells, wherein the formulation comprises an activation factor for the immune cells or a nucleic acid sequence encoding the activation factor, optionally wherein the targeting moiety is an activation factor, and optionally wherein the activation factor is encoded by either the nucleic acid encoding the LSR or by a separate nucleic acid in the formulation, and wherein the formulation is capable of integrating the sequence of interest into the cellular genome of an immune cell. Hence, where the formulation is capable of integrating the sequence of interest into the cellular genome of an immune cell, the formulation acts as an example of a genome editing system. In some embodiments, the targeting moiety is an activation factor. In some such cases, the targeting moiety and the activation factor are the same substance, while in other cases, they are different substances, with each acting as an activation factor. In some embodiments, the targeting moiety is not an activation factor. In some cases, the formulation comprises an activation factor that is the same or different from the targeting moiety. In some such cases, the activation factor is a protein. In other cases, the formulation comprises a nucleic acid, such as an RNA or mRNA that encodes the activation factor, such asAttorney Docket No: 01373-0004-00PCTthe nucleic acid that also encodes the LSR or a separate nucleic acid. In some embodiments, the sequence of interest encodes a chimeric antigen receptor (CAR), optionally wherein the CAR binds to B cells or malignant B cells or to plasma cells or to malignant plasma cells. In some embodiments, the CAR is a CD 19 CAR. In other embodiments, the CAR is a CD20 CAR. In some embodiments, the immune cell is a T cell. In some embodiments, the targeting moiety binds to a cell surface receptor on the immune cell, such as to CD3 on a T cell. In some embodiments, the nucleic acid encoding the LSR is an mRNA. In some cases, the DNA comprising the AttD and the sequence of interest is double stranded. In some embodiments, the DNA comprising the AttD and the sequence of interest is single stranded. In some embodiments, the DNA comprising the AttD and the sequence of interest is circular single stranded (cssDNA). The nucleic acid encoding the LSR, and the DNA comprising the AttD and sequence of interest, and optionally any separate nucleic acid encoding the activation factor, collectively form a genome editing system.

[0090] In some embodiments, a lipid-based formulation for delivering a nucleic acid to an immune cell comprises a nucleic acid such as an RNA or mRNA encoding a genome editing component, and a targeting moiety for the immune cell, wherein the formulation comprises an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor, optionally wherein the activation factor is encoded by the nucleic acid (e.g. an RNA or mRNA) encoding the genome-editing component or by a separate nucleic acid (e.g. an RNA or mRNA), or optionally wherein the targeting moiety is an activation factor, and wherein the formulation is capable of increasing the protein expression of the genome editing component as compared to a formulation otherwise identical but without the activation factor or nucleic acid sequence encoding the activation factor. In some embodiments, the genome editing component is an LSR. In some embodiments, the lipid-based formulation further comprises a DNA encoding a sequence of interest. A genome editing component, as used herein, can be used in a genome editing system, including but not limited to zinc fingers, TALENs, Cas proteins, transposases, and integrases such as LSRs.

[0091] In some cases, the lipid-based formulation is a lipid nanoparticle (LNP). As used herein, LNP, LNPs, LNP treatment, or LNP formulation are used interchangeably. In some embodiments, the formulation is hydrophobic. In some embodiments, the formulation is nonaqueous.

[0092] A lipid-based formulation herein may be comprised within a delivery system. Thus, also disclosed herein are delivery systems comprising one or more lipid-based formulations. In some embodiments, the delivery system comprises one lipid-based formulation comprising aAttorney Docket No: 01373-0004-00PCTgenome editing system comprising a nucleic acid (e.g. an RNA or mRNA) encoding an LSR and a DNA comprising an AttD and sequence of interest, and a targeting moiety for the immune cell, wherein the formulation comprises an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor. Thus, in such a case, for example, an mRNA encoding an LSR, the DNA, and targeting moiety, and optionally an activation factor or a further nucleic acid encoding the activation factor, are all comprised within one lipid-based formulation, or “co-encapsulated” in the formulation. In such embodiments, the delivery system is capable of integrating the sequence of interest into the genome of the immune cell, optionally wherein the immune cell is a human immune. As used herein, when certain elements of a formulation are “co-encapsulated,” those elements are all included within one lipid-based formulation. A delivery system prepared from one formulation that is capable of integrating the sequence of interest into the genome of an immune cell without the addition of other components need only comprise that single lipid-based formulation, optionally with an excipient, carrier, diluent, or vehicle. In contrast, in other situations, the elements of a genome editing system may be included in two different formulations, i.e., “separately encapsulated,” in which case the different formulations may then be mixed together in preparing the delivery system or may be provided as separate formulations to the immune cells (e.g., sequentially) in administering the delivery system.

[0093] For example, in preparing a single lipid-based formulation for co-encapsulating elements such as an mRNA, a DNA, and one or more of a targeting moiety and activation factor, one may prepare the lipid-based formulation from an aqueous medium comprising all of those elements and a hydrophobic medium comprising the lipid ingredients. Thus, a lipid-based formulation, made from the mixing of the aqueous medium comprising all of the components with the hydrophobic medium, should comprise all of the above components. Such a formulation, in some cases, can be analyzed after its preparation to determine that all components are included. However, while such a formulation may comprise all genome editing components, individual LNPs or liposomes or other particles within the lipid-based formulation might individually contain different mixtures of the components.

[0094] As noted above, in some embodiments, a delivery system comprises one single lipid-based formulation that is capable of integration of the sequence of interest into the genome of the immune cell. In other embodiments, a delivery system herein comprises two lipid-based formulations for nucleic acid delivery to an immune cell, which may, when mixed together in the delivery system or provided separately to the immune cell as the delivery system, comprise all of the components allowing for integration of the sequence of interest into the genome ofAttorney Docket No: 01373-0004-00PCTthe immune cell. In some embodiments, for example, a first lipid-based formulation of the delivery system is made from and thereby comprises an mRNA encoding an LSR, while a second lipid-based formulation of the delivery system is made from and thereby comprises a DNA comprising an AttD site and a sequence of interest ( / .< ., separate encapsulation); one or both lipid-based formulations may further comprise a targeting moiety for the immune cell and an activation factor for the immune cell or a nucleic acid sequence encoding the activation factor. In some embodiments, the first lipid-based formulation comprises the targeting moiety. In some embodiments, the second lipid-based formulation comprises the targeting moiety. In some cases, both lipid-based formulations comprise the targeting moiety. Similarly, in some embodiments, the first lipid-based formulation comprises the activation factor or nucleic acid sequence encoding the activation factor. In some embodiments, the second lipid-based formulation comprises the activation factor or nucleic acid sequence encoding the activation factor. These two different lipid-based formulations may then be mixed to form a delivery system comprising all of the components needed for delivery to the immune cell. In some embodiments, the two different lipid-based formulations are provided to the immune cell as separate formulations, e.g., sequentially, to form a delivery system for delivery of all of the components to the immune cell. In such embodiments, the delivery system is capable of integrating the sequence of interest into the genome of the immune cell, optionally wherein the immune cell is a human immune. In some embodiments, the ratio of a DNA encoding a sequence of interest to an mRNA encoding an LSR in the delivery system is 3:1 to 1:3. In some embodiments, the ratio is 2: 1 to 1 :2. In some embodiments, the ratio is 1:1.

[0095] The lipid-based formulations and delivery systems disclosed herein are useful for delivery the genome editing systems disclosed herein to immune cells. In some embodiments, the genome editing system comprises an additional component further to the nucleic acid encoding a large serine recombinase (LSR), the DNA comprising a donor attachment site (AttD) and a sequence of interest, the targeting moiety for immune cells, and the activation factor for immune cells (optionally wherein the activation factor is the targeting moiety or wherein the activation factor is encoded by the nucleic acid encoding the LSR or by a separate nucleic acid). In some embodiments, the additional component enhances nuclear import of the DNA comprising the AttD and sequence of interest into the nucleus of the cell. For example, in some embodiments, the additional component is a single-stranded DNA-binding protein. In some embodiments, the additional component is a cis sequence element. In some embodiments, the additional component enhances the efficiency of integration of the DNA by increasing the formation of double-stranded DNA from the DNA (e.g., from single stranded DNA) in the cell.Attorney Docket No: 01373-0004-00PCTIn some embodiments, the additional component is a nucleic acid (e.g., an oligo) that increases the formation of double-strand DNA from the single stranded DNA in the cell.A. LARGE SERINE RECOMBINASE

[0096] The present disclosure provides lipid-based formulations for nucleic acid delivery to immune cells that comprise a nucleic acid encoding an LSR, among other components. As used herein, an “LSR” refers to a protein having an amino acid sequence of a naturally-occurring LSR, as well as a protein that is a variant of such a naturally-occurring LSR, including sequence modifications, mutants, deletions, conjugates, chemical modifications, LSR-fusion proteins, truncations and the like.

[0097] Suitable LSRs for use in the disclosed genome editing systems may include LSRs known in the art, including e.g., PaOl, Bxbl, PhiC31, Pf80, Cp36, Dn29, BcelNTa, SscINTd, SacINTd, INT10, Dre, Vika, Bxbl, OpC31, RDF, ®BT1, Rl, R2, R3, R4, R5, TP901-1, Al 18, CbFCl, >C1, MRU, TGI, 0370.1, WJ3, BL3, SPBc, K38, or variants thereof. See e.g., Durrant etal., Nature Biotechnology 41, 488-499 (2023); Yarnall etal., Nature Biotechnology 41, SOO-512 (2023); Xu et al., BMC Biotechnol, 13, 87 (2013). In some embodiments, the genome editing systems comprise an LSR that exhibits recombination activity. In some embodiments, the genome editing systems comprise an LSR that exhibits genome integration activity.

[0098] In some embodiments, the LSR exhibits recombination activity in a mammalian cell. In certain circumstances, the recombination activity results in integration of a sequence of interest into the genome of the mammalian cell. In some embodiments, the mammalian cell is a non-human mammalian cell (e.g., a mouse, rat, or non-human primate cell). In some embodiments, the mammalian cell is a human cell. Assays for determining the recombination activity of an LSR can be performed using various methods. For example, an assay involving the recombination of plasmids by an LSR to introduce a reporter molecule into a cell (e.g., to induce fluorescence in a cell) can demonstrate if an LSR has activity with a pair of attachment sites. These recombination events can be evaluated by e.g., flow cytometry or fluorescent microscopy, both techniques known in the art. Flow cytometry instruments include Attune (Thermo Fisher Scientific), ID7000 spectral cell analyzer (Sony), BD FACSymphony (BD Biosciences), and CytoFLEX (Beckman Coulter). Fluorescent microscopes include EVOS fluorescent microscope (Thermo Fisher Scientific), Eclipse fluorescent microscope (Nikon), and Axiovert fluorescent microscope (Zeiss). Recombination activity can be evaluated by comparing to negative control conditions, e.g., without addition of the LSR.Attorney Docket No: 01373-0004-00PCT

[0099] Another approach for assessing LSR activity includes providing an LSR and a DNA (e.g., cssDNA) containing an associated AttD site to a cell and determining if a sequence of interest from the DNA is integrated into the genome. The resulting integration event can be measured by e.g., nucleic acid quantification or nucleic acid sequencing. Nucleic acid quantification can be performed using droplet digital PCR (ddPCR) and suitable nucleic acid quantification kits or instruments. Non-limiting examples include Qubit BR dsDNA assay (Thermo Fisher Scientific), Qubit HS dsDNA assay (Thermo Fisher Scientific), Nanodrop spectrophotometer (Thermo Fischer Scientific), Stunner spectrophotometer (Unchained Labs), and Lunatic spectrophotometer (Unchained Labs). Nucleic acid sequencing can be performed using NextSeq (Ilumina), GridlON (Oxford Nanopore Technologies), Revio System (Pacific Biosciences), and the like.

[0100] In some embodiments, the LSR is capable of integrating a sequence from a nucleic acid (e.g., double or single stranded DNA or cssDNA) into a single chromosomal location in a genome. In some embodiments, the LSR is capable of integrating a sequence from a nucleic acid into multiple chromosomal locations in a genome. In some embodiments, the LSR can integrate a sequence from a nucleic acid in a chromosomal location that is non-coding DNA. In some embodiments, the LSR can integrate a sequence from a nucleic acid in a chromosomal location with an endogenous promoter. In some embodiments, the LSR can integrate a sequence from a nucleic acid in a chromosomal location with high transcriptional activity. In some embodiments, the LSR can integrate a sequence from a nucleic acid in a chromosome location with tissue or cell-type specific regulation of gene expression. In some embodiments, the LSR can integrate a sequence from a nucleic acid into a gene to produce a non-functional gene product. In some embodiments, the LSR can integrate a sequence from a nucleic acid in a chromosomal location to disrupt the interaction between cis-regulatory transcriptional elements.

[0101] In some embodiments, the LSR of the genome editing system comprises a nuclear localization signal (“NLS”). In some embodiments, the nuclear localization signal is not native to the LSR. In some embodiments, the LSR comprises more than one NLS. In some embodiments, the LSR comprises two NLSs. In some embodiments, the LSR comprises 3 NLSs. In some embodiments, the LSR comprises more than 3 NLSs. In some embodiments, the NLS is from SV40. In some embodiments, the NLS is from myc. In some embodiments, the NLS is from p53. Exemplary sequences for NLSs are known in the art. See e.g., Lu et al., Cell Communication and Signaling 19, 60 (2021).Attorney Docket No: 01373-0004-00PCT

[0102] In some embodiments, the LSR is a fusion protein (referred to herein as an “LSR-fusion protein”) comprising an LSR and a fusion domain. In some embodiments, the fusion domain comprises a polypeptide, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein domain, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a protein, e.g., with which the LSR is not naturally linked. In some embodiments, the fusion domain comprises a cellpenetrating peptide. In some embodiments, the fusion domain comprises an arginine-rich peptide. In some embodiments, the fusion domain comprises an arginine-rich dipeptide repeat protein. In some embodiments, the fusion domain comprises a combination of a DNA-binding domain, a cell-penetrating peptide, an arginine-rich peptide, and an arginine-rich dipeptide repeat protein. In some embodiments, the LSR of the genome editing system is fused to a DNA-binding domain. In some embodiments, the DNA-binding domain comprises a catalytically inactive Cas polypeptide (dCas). In some embodiments, the dCas comprises dCas9 or dCasl2. In some embodiments, the DNA-binding domain comprises a catalytically inactive zinc finger polypeptide (ZNF). In some embodiments, the DNA-binding domain comprises a catalytically inactive transcription activator-like effector nuclease (TALEN). In some embodiments, the LSR-fusion protein comprises a linker between the LSR and fusion domain. In some embodiments, the LSR-fusion protein does not comprise a linker between the LSR and fusion domain. In each instance, the genome editing system comprises an mRNA encoding such LSR-fusion protein.

[0103] In some embodiments, the LSR is encoded by an mRNA. In some embodiments, the LSR is encoded by a self-amplifying RNA (saRNA). In some embodiments, the LSR is encoded by a circular RNA. In some embodiments, the LSR is encoded by a DNA. In some embodiments, the LSR is encoded by cssDNA.

[0104] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 2 or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 2.

[0105] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 3 or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 3.

[0106] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 4 or a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at leastAttorney Docket No: 01373-0004-00PCT95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0107] In some embodiments, the mRNA comprises a 5’ cap structure, or a modified 5’ cap structure. In some embodiments, the mRNA comprises a 5’ untranslated region (UTR). In some embodiments, the mRNA comprises a 3’ UTR. In some embodiments, the UTR comprises one or more viral internal ribosome entry sites (IRES) or eukaryotic IRES. In some embodiments, the mRNA comprises a miRNA binding site or a fragment thereof, a restriction site or a fragment thereof, an RNA editing motif or a fragment thereof, a zip code element or a fragment thereof, an RNA trafficking element or a fragment thereof, or a combination thereof. In some embodiments, the accessory element comprises a binding domain to an IRES transacting factor (ITAF). In some embodiments, the mRNA comprises a cis-regulatory element. In some embodiments, the cis-regulatory element acts as a safety switch for regulating expression of the integrated sequence. In some embodiments, the cis-regulatory element is a miRNA binding site. In some embodiments, the cis-regulatory element is a conditional activator. In some embodiments, the cis- regulatory element is a repressor. In some embodiments the cis-regulatory element is an adenosine deaminase acting on RNA (ADAR) enzyme. In some embodiments, the mRNA comprises a polyA region, a polyC region, a poly AC region, a polypyrimidine tract, or a combination or variant thereof.

[0108] The LSRs of the formulations disclosed herein can be prepared using suitable methods known in the art. In some embodiments, the mRNA is prepared by in vitro transcription.B. DNA COMPRISING THE DONOR ATTACHMENT SITE AND SEQUENCE OF INTEREST

[0109] In addition to the nucleic acid encoding the LSR, the lipid-based formulations herein also comprise a nucleic acid, generally DNA, such as double or single stranded DNA, or circular single stranded DNA (cssDNA), comprising a donor attachment site AttD and a sequence of interest.1. Double stranded, single stranded, and cssDNA

[0110] In some embodiments, the DNA comprising the AttD and sequence of interest disclosed herein (i.e., the DNA component) is a plasmid, nanoplasmid, or minicircle, wherein in each case, the plasmid, nanoplasmid, or minicircle comprises the sequence of interest and an AttD. In some embodiments, the DNA is double-stranded DNA. In some embodiments, the DNA is single-stranded DNA. In some embodiments, the DNA is circular double-strandedAttorney Docket No: 01373-0004-00PCTDNA. In some embodiments, the DNA is circular single-stranded DNA (cssDNA). In some embodiments, the DNA is Z form DNA (Z-DNA). In some embodiments, the DNA is D form DNA (D-DNA).[OHl] The DNA can be prepared using suitable methods known in the art, including in vitro synthesis. For example, a cssDNA as disclosed herein can be prepared using suitable methods known in the art, including in vitro synthesis, or production using a phagemid in bacterial systems. See e.g., Nafisi et al., Synthetic Biology 3(1): ysy015 (2018); Cha et al., Advanced Functional Materials: 2010867 (2021); Iyer et al., CRISPR J 5(5):685-701 (2022); Shepherd et al., Scientific Reports 9: 6121 (2019). Methods for in vitro synthesis include enzymatic methods including using e.g., polymerase chain reaction, rolling circle amplification, ambient ligation mediated amplification, and isothermal amplification and strand displacement.

[0112] In some embodiments, the DNA encodes a DNA nuclear targeting sequence (DTS). In some embodiments, the DTS is an enhancer region from SV40. Sequences of DTSs are known in the art and include e.g., SV40, NFKB, and GRE.

[0113] In some embodiments, the DNA comprises about 0.3 to 20 kb. In some embodiments, the DNA comprises about 0.3 to 20 kb, 0.4 to 20 kb, 0.5 to 20 kb, 0.6 to 20 kb, 0.7 to 20 kb, 0.8 to 20 kb, 0.9 to 20 kb, 1.0 to 20 kb, 1.1 to 20 kb, 1.2 to 20 kb, 1.3 to 20 kb, 1.4 to 20 kb, or 1.5 to 20 kb. In some embodiments, the DNA comprises about 1.6 to 2 kb, 1.6 to 3 kb, 1.6 to 4 kb, 1.6 to 5 kb, 1.6 to 6 kb, 1.6 to 7 kb, 1.6 to 8 kb, 1.6 to 9 kb, 1.6 to 10 kb, 1.6 to 11 kb, 1.6 to 12 kb, 1.6 to 13 kb, 1.6 to 14 kb, 1.6 to 15 kb, 1.6 to 16 kb, 1.6 to 17 kb, 1.6 to 18 kb, 1.6 to 19 kb, 1.6 to 20 kb, 2 to 3 kb, 2 to 4 kb, 2 to 5 kb, 2 to 6 kb, 2 to 7 kb, 2 to 8 kb, 2 to 9 kb, 2 to 10 kb, 2 to 11 kb, 2 to 12 kb, 2 to 13 kb, 2 to 14 kb, 2 to 15 kb, 2 to 16 kb, 2 to 17 kb, 2 to 18 kb, 2 to 19 kb, 2 to 20 kb, 3 to 4 kb, 3 to 5 kb, 3 to 6 kb, 3 to 7 kb, 3 to 8 kb, 3 to 9 kb, 3 to 10 kb, 3 to 11 kb, 3 to 12 kb, 3 to 13 kb, 3 to 14 kb, 3 to 15 kb, 3 to 16 kb, 3 to 17 kb, 3 to 18 kb, 3 to 19 kb, or 3 to 20 kb.2. AttD site

[0114] In some embodiments, the DNA component comprises an AttD site. LSRs of the disclosed genome editing systems may be associated with one or more associated pairs of cognate attachment sites (an AttD site and an AttA site). In particular, a given LSR may facilitate integration of a DNA via its associated AttD site “pairing” with a cognate AttA site in the cellular genome. In the formulations encompassed herein, the DNA comprises an AttD site that will function with the LSR to integrate a sequence of interest in the cellular genome.Attorney Docket No: 01373-0004-00PCT

[0115] In some embodiments, the DNA comprises more than one AttD site (i.e., an “array” of AttDs). In some embodiments, the DNA comprises two AttDs. In some embodiments, the DNA comprises three AttDs. In some embodiments, the DNA comprises four AttDs. In some embodiments, the DNA comprises five AttDs. In some embodiments, the DNA comprises six AttDs. In some embodiments, the DNA comprises seven AttDs. In some embodiments, the DNA comprises eight AttDs. In some embodiments, the DNA comprises nine AttDs. In some embodiments, the DNA comprises ten AttDs.

[0116] In some embodiments, an LSR disclosed herein binds or is capable of binding to an attachment site comprising 80 nucleotides (nt). In some embodiments, the LSR is capable of binding cognate attachment sites of different lengths. In some embodiments, at least one attachment site comprises a sequence less than 80 nt. In some embodiments, the cognate attachment sites each comprise 80 nt. In some embodiments, the cognate attachment sites each comprise less than 80 nt. In some embodiments, at least one of the attachment sites comprises a 52 nt sequence. In some embodiments, at least one of the attachment sites comprises a 48 nt sequence (Ghosh, et. al., (2003) Mol. Cell 12(5): 1101-11). In some embodiments, at least one of the attachment sites comprise a 39 nt sequence (Groth, et. al., (2000) Proc. Natl. Acad. Sci. U.S.A. 97(ll):5995-6000). In some embodiments, at least one of the attachment sites comprises a 36 nt sequence (Ghosh, et. al., (2003) Mol. Cell 12(5): 1101-11). In some embodiments, at least one of the attachment sites comprises a 34 nt sequence (Groth, et. al., (2000) Proc. Natl. Acad. Sci. U.S.A. 97(11):5995-6000). In some embodiments, at least one of the attachment sites comprises a 26 nt sequence (Durrant, et. al., (2022) Nat. Biotechnol.41(4):488-99). In some embodiments, the AttD site comprises fewer nucleotides than the AttA site. In some embodiments, the AttD site comprises greater nucleotides than the AttA site. In some embodiments, the AttD site comprises at least 26, 34, 36, 39, 48, or 52 nucleotides, wherein the nucleotides comprise a dinucleotide core and an even number of nucleotides directly adjacent on either side of the dinucleotide core. In some embodiments, the AttD site comprises the nucleotide sequence of SEQ ID NO: 1 or comprises a sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0117] In some embodiments, the AttD site comprises an AttP site. In some embodiments, the AttD site comprises a modified AttP site. In some embodiments, the modified AttP site has been optimized for a mammalian genome. In some embodiments, the mammalian genome is a non-human genome (e.g., a mouse, rat, or non-human primate genome). In some embodiments, the mammalian genome is a human genome. In some embodiments, the modified AttP siteAttorney Docket No: 01373-0004-00PCTcomprises a modified dinucleotide core. In some embodiments, the modified AttP sites comprise a modification outside of the dinucleotide core. In some embodiments, the modified AttP sites comprise modifications to both the dinucleotide core and the sequence outside of the dinucleotide core.

[0118] In some embodiments, the AttD site comprises an AttB site. In some embodiments, the AttD site comprises a modified AttB site. In some embodiments, the modified AttB site has been optimized for a mammalian genome. In some embodiments, the mammalian genome is a non-human genome (e.g., a mouse, rate, or non-human primate genome). In some embodiments, the mammalian genome is a human genome. In some embodiments, the modified AttB site comprises a modified dinucleotide nucleotide core. In some embodiments, the modified AttB site comprises a modification outside of the dinucleotide core. In some embodiments, the modified AttB site comprises modifications to both the dinucleotide core and the sequence outside of the dinucleotide core.3. Sequence of interest

[0119] In some embodiments, the DNA component comprises a sequence that encodes one or more sequences of interest for integrating into the cellular genome of an immune cell. In some embodiments, the DNA component comprises a sequence that encodes one or more sequences of interest for integrating into a cellular genome or for non-integrative episomal expression in a cell. In some embodiments, the sequence of interest is a non-coding sequence. In some embodiments, the sequence of interest is a coding sequence. In some embodiments, the sequence of interest is a gene or a portion of a gene. In some embodiments, the sequence of interest is a variant of a gene. In some embodiments, the sequence of interest encodes a protein, peptide, or polypeptide. In some embodiments, the sequence of interest comprises or encodes for a transcriptional or translational control element (e.g., promoter elements, activator sequences, repressor sequences). In some embodiments, the sequence of interest encodes a secreted protein or a membrane-bound protein. In some embodiments the sequence of interest comprises a sequence that encodes a therapeutic protein. In some embodiments the sequence of interest encodes a therapeutic RNA. In some embodiments, the sequence of interest comprises one or more of a sequence that comprises or encodes for a transcriptional or translational control element, and a sequence that encodes a therapeutic protein such as a secreted or membrane-bound protein. In some embodiments, the sequence of interest comprises one or more of a sequence that encodes for a transcriptional or translational control element, and a sequence that encodes a therapeutic RNA. In some embodiments, the sequence encodesAttorney Docket No: 01373-0004-00PCTan miRNA. In some embodiments, the sequence encodes an shRNA. In some embodiments, the sequence encodes a circRNA.

[0120] In some embodiments, the sequence of interest comprises a sequence that encodes a guide RNA. In some embodiments, the sequence of interest comprises a sequence that encodes a guide RNA for DNA-based Cas systems (e.g., Cas9). In some embodiments, the sequence of interest comprises a sequence that encodes a guide RNA for RNA-based Cas systems (e.g., Casl3). See e.g., Ding et al., Nature Communications 15: 1572 (2024). In some embodiments, the sequence of interest comprises a sequence that encodes a micro RNA. In some embodiments, the sequence of interest comprises a sequence that encodes a tRNA. In some embodiments, the sequence of interest comprises a sequence that encodes a long non-coding RNA (Inc RNA). In some embodiments, the sequence of interest comprises a sequence that encodes a circular RNA.

[0121] In some embodiments, the sequence of interest encodes a secreted protein. In some embodiments, the sequence of interest encodes a membrane-bound protein. In some embodiments, the sequence of interest encodes a chimeric antigen receptor (CAR). In some embodiments, the sequence of interest encodes a T cell receptor (TCR). In some embodiments, the sequence of interest encodes a B cell receptor (BCR). In some embodiments, the sequence of interest encodes an immune cell activation or inhibitory receptor. In some embodiments, the sequence of interest encodes a growth factor ligand. In some embodiments, the sequence of interest encodes a transcription factor. In some embodiments, the sequence of interest encodes a checkpoint inhibitor or agonist.

[0122] In some embodiments, the DNA comprises a sequence of interest and further comprises a promoter for expression of the sequence of interest. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the promoter is derived from a virus. In some embodiments, the promoter is derived from a mammal. In some embodiments, the promoter is a synthetic promoter. Suitable promoters are known in the art and may be used in the genome editing systems described herein. Non-limiting examples of promoters include chicken P actin (CBA), CMV early enhancer / chicken P actin (sCAG), human cytomegalovirus (hCMV), human synapsin (hSYN), spleen focus-forming virus (SFFV), human polypeptide chain elongation factor (EFla), phosphoglycerate kinase (PGK), ubiquitin C (UbiC), insulin promoter, CASI, and the human alpha- 1 -antitrypsin promoter.Attorney Docket No: 01373-0004-00PCT

[0123] In some embodiments, the sequence of interest of the DNA comprises about 0.3 to 20 kb. In some embodiments, the sequence of interest of the DNA comprises about 0.3 to 20 kb, 0.4 to 20 kb, 0.5 to 20 kb, 0.6 to 20 kb, 0.7 to 20 kb, 0.8 to 20 kb, 0.9 to 20 kb, 1.0 to 20 kb, 1.1 to 20 kb, 1.2 to 20 kb, 1.3 to 20 kb, 1.4 to 20 kb, or 1.5 to 20 kb. In some embodiments, the sequence of interest of the DNA comprises about 1.6 to 2 kb, 1.6 to 3 kb, 1.6 to 4 kb, 1.6 to 5 kb, 1.6 to 6 kb, 1.6 to 7 kb, 1.6 to 8 kb, 1.6 to 9 kb, 1.6 to 10 kb, 1.6 to 11 kb, 1.6 to 12 kb, 1.6 to 13 kb, 1.6 to 14 kb, 1.6 to 15 kb, 1.6 to 16 kb, 1.6 to 17 kb, 1.6 to 18 kb, 1.6 to 19 kb, 1.6 to 20 kb, 2 to 3 kb, 2 to 4 kb, 2 to 5 kb, 2 to 6 kb, 2 to 7 kb, 2 to 8 kb, 2 to 9 kb, 2 to 10 kb, 2 to 11 kb, 2 to 12 kb, 2 to 13 kb, 2 to 14 kb, 2 to 15 kb, 2 to 16 kb, 2 to 17 kb, 2 to 18 kb, 2 to 19 kb, 2 to 20 kb, 3 to 4 kb, 3 to 5 kb, 3 to 6 kb, 3 to 7 kb, 3 to 8 kb, 3 to 9 kb, 3 to 10 kb, 3 to 11 kb, 3 to 12 kb, 3 to 13 kb, 3 to 14 kb, 3 to 15 kb, 3 to 16 kb, 3 to 17 kb, 3 to 18 kb, 3 to 19 kb, or 3 to 20 kb.

[0124] In some embodiments, the sequence of interest encodes a CAR. As used herein a “chimeric antigen receptor” or “CAR” refers to a fusion protein comprising an extracellular domain comprising an antigen binding domain. An “antigen binding domain” may comprise the portion of an intact antibody that binds to an antigen. Examples of antigen binding domains include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies (sdAbs), such as VHH antibodies (also called nanobodies); tandem single domain antibodies (sdAbs); and multi-specific antibodies formed from antigen binding domains. In some cases, the antigen binding domain of the CAR is a single chain antigen binding domain such as an scFv. In some cases, a CAR is membrane-bound, in which case, it may act in some embodiments as a cell surface receptor. In some embodiments a CAR comprises an extracellular domain comprising an antigen binding domain as well as a transmembrane domain. In some embodiments a membrane-bound CAR further comprises one or more intracellular signaling domains. In some cases, a CAR comprises two or more intracellular signaling domains, such as CD3(^ (CD3zeta) as well as one or more co-stimulatory domains such as 41BB, OX-40, and / or CD28. In some embodiments, the CAR comprises a signal peptide, which may be cleaved to form a mature protein, and thus when encoded by a sequence of interest, the sequence of interest also encodes the signal sequence. CARs may have a variety of architectures, such as a single-chain CAR, a multi-chain CAR, a single-targeted CAR, a multi-targeted CAR, a bivalent tandem CAR, a bivalent loop CAR, a multi ci stronic CAR, a bicistronic CAR, or the like.

[0125] In some embodiments, the CAR binds to a membrane-bound protein. In some embodiments, the CAR binds to a secreted protein. In some embodiments, the CAR binds to aAttorney Docket No: 01373-0004-00PCTmembrane-bound tumor antigen. In some embodiments, the CAR binds to a secreted tumor antigen. In some embodiments, the CAR binds to a tumor stromal antigen.

[0126] In some embodiments, the CAR binds to lymphocytes, lymphocyte progenitors, or lymphocyte progeny. In some embodiments, the CAR binds to B cells (also called B lymphocytes) or malignant B cells. In some embodiments, the CAR binds to plasma cells or malignant plasma cells. In some embodiments, the CAR is a CD 19 CAR. In some embodiments, the CAR is a CD20 CAR.

[0127] As used herein, “B cells” or “B lymphocytes” refer to a class of white blood cells that, for example, can produce antibodies. Exemplary progeny or derivatives of B cells include “plasma cells” and “memory B cells”. As used herein, a “malignant B cell” refers to a cancer cell of B cell origin. A “malignant plasma cell” refers to a cancer cell of plasma cell origin. B cell and plasma cell cancers comprise cancers characterized by abnormal B cell and plasma cell development, respectively. Certain non-limiting examples of such cancers include plasma cell neoplasms, plasmacytomas, Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphocytic leukemia (ALL), Hairy cell leukemia, multiple myeloma, B cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia / small lymphoplasmacytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma (DLBL), follicular lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, and marginal zone lymphoma.C. TARGETING MOIETY

[0128] In some embodiments, the lipid-based formulations disclosed herein comprise a targeting moiety for immune cells. In some cases, the immune cell is a T cell, B cell, macrophage, myeloid cell, NK cell, dendritic cell, or hematopoietic stem cell (HSC), for example, and in such a case the targeting moiety targets (or binds to or is directed to) such a cell. For instance, in some cases, the targeting moiety targets a T cell. In some embodiments, the targeting moiety targets a B cell. In some embodiments, the targeting moiety targets a macrophage. In some embodiments, the targeting moiety targets a myeloid cell. In some embodiments, the targeting moiety targets an NK cell. In some embodiments, the targeting moiety targets a dendritic cell. In some embodiments, the targeting moiety targets an HSC. In some embodiments, the targeting moiety is an antibody or antigen binding domain. In some embodiments, the targeting moiety is a bispecific antibody or bispecific antigen binding domain. In some embodiments, the targeting moiety is a Fab. In some embodiments, the targeting moiety is an scFv. In some embodiments, the targeting moiety is a nanobody. In some embodiments, the targeting moiety is an antibody or antigen binding domain that binds to aAttorney Docket No: 01373-0004-00PCTcell surface receptor on an immune cell. In some cases, the cell surface receptor is CD3, CD4, CD5, CD7, or CD8. In some embodiments, the targeting moiety is an / ' / -acetylgalactosamine (GalNAc) targeting ligand.

[0129] In some embodiments, the targeting moiety binds to CD3 on the surface of T cells. In some embodiments, the targeting moiety is an anti-CD3 antibody, such as a bispecific antibody or bispecific antigen binding domain, such as a Fab, or scFv or nanobody targeting CD3. Anti-CD3 antibodies known in the art may be used in the disclosed embodiments. In some embodiments, that anti-CD3 antibody comprises a heavy chain sequence comprising SEQ ID NO: 5. In some embodiments, that anti-CD3 antibody comprises a heavy chain variable region sequence comprising SEQ ID NO: 6. In some embodiments, that anti-CD3 antibody comprises a light chain sequence comprising SEQ ID NO: 7. In some embodiments, that anti-CD3 antibody comprises a heavy chain variable region sequence comprising SEQ ID NO: 8.

[0130] In some embodiments, the formulation comprises more than one targeting moiety. In other embodiments, the formulation comprises only one targeting moiety. In some cases, the targeting moiety is also an activation factor for the targeted immune cell (e.g., an anti-CD3 antibody for targeting and activating T cells). In other cases, the targeting moiety is not an activation factor for the cell (e.g., an anti-CD8 antibody for targeting but not activating T cells).D. ACTIVATION FACTOR

[0131] In some embodiments, the lipid-based formulations disclosed herein comprise an activation factor for immune cells or a nucleic acid sequence encoding an activation factor for immune cells. As noted above, an “activation factor” increases the level of integration of a sequence of interest and / or the level of expression of the genome editing component in the contacted cell compared to the level of integration of the sequence of interest and / or level of expression of the genome editing component in a cell that has been contacted with a formulation that is otherwise identical but does not include the activation factor or a nucleic acid encoding the activation factor, and is a factor that acts to activate or stimulate proliferation of the immune cells to which the formulation is targeted. The level of integration can be measured by nucleic acid quantitation, such as by ddPCR and reported as percent integration at an integration site in the cellular genome. The level of expression of genome editing component can be measured by standard techniques for determining protein expression in a cell, e.g., Western blot or flow cytometry.Attorney Docket No: 01373-0004-00PCT

[0132] In some cases, the targeting moiety is an activation factor for the targeted immune cells. In some such cases, the targeting moiety is the activation factor (i.e. one moiety functions to both target the formulation to the immune cells and to act as an activation factor). In other such cases, the targeting moiety and the activation factor are separate substances, although both act as activation factors. In some cases, the targeting moiety is not an activation factor. Thus, in such cases, the targeting moiety and activation factor are different substances.

[0133] Lipid-based formulations herein may comprise an activation factor in several different ways. As noted above, in some cases the targeting factor is also the activation factor. In some cases, the activation factor is an additional component of the lipid-based formulation, in addition to the targeting moiety and the nucleic acids. In some cases, the formulation comprises a nucleic acid encoding an activation factor. Such a nucleic acid may be, for example, the nucleic acid that also encodes the LSR, or it may be a separate nucleic acid molecule.

[0134] In some cases, the activation factor is encoded by an mRNA. In some cases, the activation factor is encoded by the mRNA that also encodes the LSR. In some cases, the activation factor is encoded by a separate mRNA from the mRNA that encodes the LSR. In some cases, the activation factor is encoded by a DNA.

[0135] In some cases, the activation factor is antigen-dependent, meaning that the activity of the activation factor, for example, in increasing the level of integration of the sequence or interest, or in increasing the level of expression of a genome editing component, or in activating or stimulating proliferation of immune cells, is increased in the presence of an antigen. In some embodiments, for example, expression of an antigen-dependent activation factor, in the presence of an antigen, leads to cellular activation changes in the cell that expresses the activation factor (e.g., increase in signaling cascades, increase in cell division, increase in expression of activation markers). In some cases the activation factor may have higher activity in the presence of one or more specific antigens. In some cases, the activation factor is a CAR, such as a CD 19 CAR or a CD20 CAR.

[0136] In other cases, the activation factor is antigen-independent, meaning that its activity does not significantly change in the presence or absence of an antigen. In some cases, the activation factor is selected from any one of: IL-12B, BATF, LTBR, JUNB, constitutively active beta-catenin, TCF7, FOXO1, IRF4, CARD11-PIK3R3, CD79A / CD40, ITK-SYK, FYN-TRAF3IP2, KHDRBS1-LCK, SIN3A-FOXO1, RNMT, RAS, a BRAF mutant such as BRAF-G469A, a PLCG1 mutant such as PLCG1-D1165H or PLCG1-R48W or PLCG1-E1164K or PLCG1-S520F, aRASGRPl mutant such asRASGRPl-M2611, aCARDll mutantAttorney Docket No: 01373-0004-00PCTsuch as CARD11-Y361C or CARD11-S615F or CARDl 1-D357N, TNFRSF1B-T3771, CD28 antibody, CD80, soluble CD58, SLC7A5, SLC1A5, SLC7A1, SLC38A1, SLC38A2, GLUT1 (SLCA1), GLUT3 (SLCA3), MCT1 (SLC16A1), MCT4 (SLC16A3), phospholipase Cyl), IP3, IP3R, P2X7, CD40, CD86, 0X40, 4-1BBL, AHCY, CDK1, CDK2, AKR1C4, ATF6B, ITM2A, AHNAK, or FOXQ1. In some embodiments, the activation factor comprises a chimeric molecule comprising a T cell activating domain and a dimer or multimerization motif. For example, the T cell activating domains could comprise CD28, CD3 zeta, or ITAMs. The dimer or multimerization motifs could be located extracellularly, in the transmembrane domain of the chimeric molecule, or intracellularly.E. LIPID-BASED FORMULATION COMPONENTS

[0137] In some embodiments, the lipid-based formulations disclosed herein comprise a cationic lipid, helper lipid, cholesterol / sterol, and a PEG lipid. In some embodiments, the cationic lipid (also referred to as ionizable lipid herein) may include any cationic lipid known in the art to be useful in lipid-based formulations for delivering nucleic acids (RNA or DNA) to cells. In some embodiments, the ionizable lipid comprises DLin-MC3-DMA, SM-102, ALC-0315, DOTAP, Lipid5, LP01, CKK-E12, OF-02, ATX001, ATX100, L319, CL1, TCL053, C24, SSPalmO-Phe, C14-4, A9, MC2, DODMA, DLin-DMA, or 9A1P9.

[0138] In some embodiments, the helper lipid may include any helper lipid known in the art to be useful in lipid-based formulations in delivering nucleic acids (RNA or DNA) to cells. In some embodiments, the helper lipid is DSPC, DOPE, POPC, DOPC, or sphingomyelin.

[0139] In some embodiments, the sterol / cholesterol may include any sterol / cholesterol known in the art to be useful in lipid-based formulations in delivering nucleic acids (RNA or DNA) to cells. In some embodiments, the sterol / cholesterol is cholesterol, P-sitosterol, Sitosterol, or DC cholesterol.

[0140] In some embodiments, the PEG-lipid may include any PEG-lipid known in the art to be useful in lipid-based formulations in delivering nucleic acids (RNA or DNA) to cells. In some embodiments, the PEG-lipid comprises DMG-PEG, DPG-PEG, DSG-PEG, ALC-0159, PEG, DSPE-PEG, DSPE-PEG-X, where X is a small molecule, C14-PEG, C16-PEG, or C18-PEG. In some embodiments, the PEG molecular weight can vary between 500 Da- 10000 Da.

[0141] In some embodiments, the lipid-based formulation comprises a GalNAc lipid.

[0142] In some embodiments, the lipid-based formulation comprises between about 20-70% ionizable lipid, about 5-40% helper lipid, about 10-70% cholesterol, and about 0.5-10% PEG lipid, wherein the total percentage equals 100%. In some embodiments, the lipid-basedAttorney Docket No: 01373-0004-00PCTformulation comprises between about 35-50% ionizable lipid, about 10-20% helper lipid, about 28-48% cholesterol, and about 1-5% PEG lipid, wherein the total percentage equals 100%. In some embodiments, the lipid-based formulation comprises between about 35-50% ionizable lipid, about 10-20% helper lipid, about 28-48% cholesterol, and about 2% PEG lipid, wherein the total percentage equals 100%. In some embodiments, the lipid-based formulation comprises about 50% ionizable lipid, about 10% helper lipid, about 38% cholesterol, and about 2% PEG-lipid. In some embodiments, the lipid-based formulation comprises about 35% ionizable lipid, about 16% helper lipid, about 47% cholesterol, and about 2% PEG-lipid. In some embodiments, the lipid-based formulation comprises about 50% ionizable lipid, about 20% helper lipid, about 28% cholesterol, and about 2% PEG-lipid.

[0143] In some embodiments, the lipid-based formulation is a lipid nanoparticle (LNP). In some embodiments, the lipid-based formulation is a lipoplex. In some embodiments, the lipid-based formulation is a liposome.

[0144] In some embodiments, the lipid-based formulation is about 50-250 nm in size. In some embodiments, the lipid-based formulation is about 50-100 nm, 50-150 nm, 50-200 nm, or 50-250 nm in size. In some embodiments where two formulations are in one genome editing system, the formulations may be the same or different sizes. In some embodiments, at least one of the lipid-based formulation is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 220, 225, 230, 235, 240, 245, or 250 nm in size.

[0145] In some embodiments, the lipid-based formulation further comprises a small molecule, peptide, oligo (e.g., siRNA or ASO) encapsulated in the formulation.

[0146] In some embodiments, the lipid-based formulation comprises a targeting moiety. In some embodiments, the targeting moiety is attached covalently to one or more lipids of the lipid-based formulation. In some cases, the targeting moiety is attached noncovalently to one or more lipids of the lipid-based formulation. In some cases, the targeting moiety is attached covalently to a PEG-lipid such as a DMG-PEG or DSPE-PEG. In some embodiments, the covalent attachment to the PEG-lipid is through a conjugating group such as an azide or maleimide group. In some such cases, the targeting moiety is conjugated to the LNPs via an azide or maleimide conjugation reaction, for example, in which the at least one lipid comprises an azide or maleimide group that reacts with a functional group of the targeting moiety to form a covalent bond. Thus, when present in a lipid-based formulation, the lipid species used for conjugation may be conjugated or unconjugated to a targeting moiety, and if conjugated may comprise a functional group that links the lipid to the targeting moiety, such as the reactionAttorney Docket No: 01373-0004-00PCTproduct of an azide or maleimide conjugation reaction. For example, where a PEG-lipid such as DMG-PEG2000 and / or DSPE-PEG2000 is used for conjugation, the lipid-based formulation comprises a functional group linking the PEG-lipid such as DMG-PEG2000 and / or DSPE-PEG2000 to the targeting moiety. In some embodiments, the lipid-based formulation comprises a portion of PEG-lipid that is conjugated to the targeting moiety and a portion of PEG-lipid that is unconjugated to the targeting moiety.

[0147] In some embodiments, the lipid-based formulation is an LNP. In some embodiments, the nucleic acids may be “cargo” or “payload” of the LNP, noncovalently associated with the lipid components of the LNP, while the targeting moiety and optionally the activation factor may in some embodiments be on the surface of the LNP, such as conjugated to one or more lipid components, such as covalently conjugated to one or more lipid components such as PEG-lipids.F. ADDITIONAL COMPONENT OR COMPONENTS

[0148] In some embodiments, the lipid-based formulations or delivery systems disclosed herein further comprise an additional component separate from the activation factor that enhances integration of the DNA. In some such embodiments, an additional component enhances integration of the DNA by increasing the import of the DNA into the nucleus of the cell. In some embodiments, an additional component enhances integration of the DNA by increasing the import of the DNA into the nucleus of the cell from the lipid-based formulation. In some embodiments, an additional component enhances integration of single-stranded DNA by increasing the formation of double-stranded DNA in the cell.

[0149] Such enhancements of integration may be measured, for example, by ddPCR and reported as percent integration at an integration site in the cellular genome. An increase in nuclear import can be measured by in situ hybridization (ISH) or transgene expression ( / .< ., expression of the sequence of interest) as compared to the genome editing system without the additional component. An increase in double-strand formation can be measured using in situ hybridization (ISH) and probes designed to bind to the double-stranded form of DNA as compared to the genome editing system without the additional component.

[0150] In some embodiments, the formulation further comprises a nucleic acid encoding a single-stranded DNA-binding protein. In some embodiments, the mRNA encodes the singlestranded DNA-binding protein. In some embodiments, the mRNA encodes the single-stranded DNA-binding protein as a fusion to the LSR. In some embodiments, the mRNA encodes the single-stranded DNA-binding protein not as a fusion to the LSR. In some embodiments, theAttorney Docket No: 01373-0004-00PCTsingle-stranded DNA binding protein is a separate nucleic acid from the mRNA encoding the LSR. In some embodiments, the single-stranded DNA binding protein is encoded by a separate mRNA. In some embodiments, the single-stranded DNA binding protein is encoded by a separate DNA. In some embodiments, the single-stranded DNA binding protein is included in the formulation as a protein. In some embodiments, the single-stranded DNA binding protein comprises one or more NLSs. Non-limiting examples of single-stranded DNA binding proteins include Replication Protein A (e.g., RPA1, RP2, or RPA3), Rad51, DMC1, single-stranded DNA binding protein (SSB), RecA, UvxX, UvsY, NS1, SV40 Large T antigen, El, E2, DNA dependent Protein Kinase (DNA-PK). In some embodiments, the single-stranded DNA binding protein is a natural protein. In some embodiments, the single-stranded DNA binding protein is synthetically designed. In some embodiments, the single-stranded DNA binding protein is a synthetic protein comprising an OB-fold nucleic acid binding domain (including e.g., SSB, Exol, RecO, Red, RecG, PriA, Pol II, RuvA, RPA, hSSB, CTC1, STN1, POTI, TPP1, TAP82, BRCA2, DNA ligase 4, MCM). See e.g., Bianco, Front. Mol. Biosci. 9: 784451 (2022).

[0151] In some embodiments, the formulation or delivery system further comprises a cis sequence element that e.g., enhances nuclear import of the DNA component, such as cssDNA. In some embodiments, the DNA encodes the cis sequence element. In some embodiments, the cis sequence element is a separate nucleic acid from the DNA. In some embodiments, the cis sequence element is a separate DNA. Non-limiting examples of cis sequence elements include a G quartet, aptamer, or AART. In some embodiments, the additional component is a singlestranded DNA aptamer. In some embodiments, the DNA aptamer binds to HMGB1.

[0152] In some embodiments, where the DNA component is single stranded, such as cssDNA, the formulation or delivery system further comprises an additional nucleic acid that increases double-strand formation of the DNA in the cell. In some embodiments, the additional nucleic acid is a DNA. In some embodiments, the additional nucleic acid is a modified DNA. In some embodiments, the additional nucleic acid is an oligo. In some embodiments, the additional nucleic acid is a locked form of DNA. In some embodiments, the additional nucleic acid has a 3’ hydroxyl group. In some embodiments, the additional nucleic acid is 40 nucleotides. In some embodiments, the additional nucleic acid is less than 40 nucleotides. In some embodiments, the additional nucleic acid is less than 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 25, or 20 nucleotides.III. NUCLEIC ACID MODIFICATIONS

[0153] In some embodiments, the DNA and / or RNA in the lipid-based formulations disclosed herein is modified. Any DNA or RNA modification known to those of skill in the artAttorney Docket No: 01373-0004-00PCTis encompassed. In some embodiments, a modified RNA or DNA is synthesized with a non-canonical nucleoside or nucleotide. Modifications such as those listed herein can be combined to provide modified RNA or DNA comprising nucleosides and nucleotides that can have two, three, four, or more modifications. In some embodiments, every base of a RNA or DNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In some embodiments, modified RNA or DNA comprise at least one modified residue at or near the 5’ end and / or at the 3’ end of the RNA or DNA.

[0154] In some embodiments, an RNA (e.g., an mRNA component) is modified but DNA (e.g., a DNA component such as cssDNA, ssDNA or dsDNA) is not modified. In some embodiments, DNA (e.g., a DNA component such as cssDNA, ssDNA, or dsDNA) is modified but RNA (e.g., an mRNA component) is not modified. In some embodiments, both RNA and DNA are modified.

[0155] In some embodiments, the RNA or DNA components in the lipid-based formulation comprise one, two, three or more modified nucleosides or nucleotides. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in a modified RNA or DNA are modified nucleosides or nucleotides.

[0156] In some embodiments, the RNA modification comprises a uridine modification. In some embodiments, the RNA modification comprises 1-methyl-pseudouridine. In some embodiments, the RNA modification comprises pseudouridine. In some embodiments, the RNA modification comprises a replacement of each uridine for 1-methyl-pseudouridine. In some embodiments, the RNA modification comprises a replacement of each uridine for pseudouridine.

[0157] Some embodiments encompass a phosphate backbone modification. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The backbone can also be modified by replacement of a bridging oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. In some embodiments, the phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate,Attorney Docket No: 01373-0004-00PCThydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In some embodiments, the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[0158] Some embodiments encompass a sugar modification. For example, the 2' hydroxyl group (OH) of a ribose sugar of RNA can be modified. In some embodiments, the 2' hydroxyl group modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification. In some embodiments, the 2' hydroxyl group modification can include “locked” nucleic acids (LNA). In some embodiments, the 2' hydroxyl group modification can include "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include a methoxyethyl group. “Deoxy” 2' modifications are encompassed and can include hydrogen, a halo (e.g., bromo, chloro, fluoro, or iodo), an amino (e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, diheteroarylamino, or amino acid), NH(CH2CH2NH)nCH2CH2- amino, -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano, mercapto, alkyl-thio-alkyl, thioalkoxy, and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino. In some embodiments, the modified nucleic acid comprises arabinose. In some embodiments, the modified nucleic acid comprises an abasic sugar. In some embodiments, the modified nucleic acid comprises one or more sugars that are in the L form.IV. METHODS

[0159] In some aspects, the present disclosure provides a method for integration of a sequence of interest into a cellular genome of an immune cell comprising contacting an immune cell with a lipid-based formulation or delivery system disclosed herein such that the sequence of interest is integrated into the cellular genome of the immune cell. In some embodiments, the method occurs in vivo. In some embodiments, the method occurs in vitro. In some embodiments, the method occurs ex vivo. In some embodiments, the immune cell is, for example, a B cell, a plasma cell, or a memory B cell, a macrophage, a myeloid cell, an NK cell, a dendritic cell, a hematopoietic stem cell (HSC), or a T cell. In some cases, the cell has been engineered (e.g., an engineered B cell, or an engineered T cell). In some embodiments, the immune cell is a T cell. In some embodiments, the method comprises contacting an immuneAttorney Docket No: 01373-0004-00PCTcell with a lipid-based formulation herein or a delivery system comprising the formulation such that the sequence of interest is integrated into the cellular genome of the immune cell. In some embodiments, the method comprises contacting an immune cell with a lipid-based formulation herein or a delivery system comprising the formulation such that the protein encoded by the sequence of interest is expressed by the immune cell.

[0160] In some embodiments, a method of editing a chromosomal site in a cell such as an immune cell comprises contacting the cell with a lipid-based formulation or delivery system disclosed herein such that the recombination activity of the LSR results in the sequence of interest being integrated into the cellular genome. The location of the integration is determined by the presence of one or more AttA sites in the genome, where an AttA site together with an AttD site forms a cognate attachment site. In some embodiments, the integration occurs at a site that does depend on an endogenous promoter for expression of the sequence of interest. In some embodiments, the integration occurs at a site in which the endogenous promoter may be used for expression of the sequence of interest.

[0161] In some cases, presence of the targeting moiety targets the lipid-based formulation to the appropriate immune cell type. For example, the target immune cell may express a cell surface receptor such as CD3, CD4, CD5, CD7, or CD8 or the like to which the targeting moiety binds, thus allowing the lipid-based formulation to contact the immune cell to be engineered. In some cases, presence of an activation factor for the immune cell assists in enhancing expression of the LSR, in comparison to a formulation that is identical but without the activation factor. In some cases, the targeting moiety is the activation factor. In other cases, the lipid-based formulation comprises separate targeting moiety and activation factor components. In some cases, the formulation comprises a nucleic acid that expresses the activation factor, such as an mRNA, such that once in the immune cell, the activation factor is produced.

[0162] In some embodiments, the disclosure provides methods for integration of a CAR into a cellular genome of a T cell, comprising contacting the T cell with the lipid-based formulation or delivery system disclosed herein, wherein the sequence of interest encodes the CAR, such that the CAR is integrated into the cellular genome of the T cell. In some embodiments, the disclosure provides methods for introducing a nucleic acid encoding a CAR into a T cell, comprising contacting the T cell with a lipid-based formulation or delivery system disclosed herein. In some embodiments, the T cell is a human T cell. In some cases, the sequence of interest encodes a CD 19 CAR. In some embodiments, the targeting moiety bindsAttorney Docket No: 01373-0004-00PCTto an anti-CD3 antibody or antigen binding domain. In some embodiments, the activation factor is an anti-CD3 antibody or antigen binding domain.

[0163] The present disclosure also provides a method of treating a disease in a subject using the lipid-based formulations or delivery systems disclosed herein. In some embodiments, the disease is caused by a genetic mutation. Non-limiting examples of mutations include polymorphisms such as single nucleotide polymorphisms (SNP), point mutations, frameshift mutations, genomic rearrangements, translocations, and inversions. In some embodiments, the disease is caused by a polymorphism. In some embodiments, the disease is caused by an SNP.

[0164] In some embodiments, the method comprises administering an effective amount of a lipid-based formulation or delivery system to a subject, wherein the sequence of interest integrates into the subject’s genome, thereby treating the subject’s disease. In some embodiments, the disease is cancer. In some embodiments, the disease is a B cell cancer. For instance, B cell cancers comprise cancers characterized by abnormal B cell development. Certain non-limiting examples of such cancers include plasma cell neoplasms, plasmacytomas, Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphocytic leukemia (ALL), Hairy cell leukemia, multiple myeloma, B cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia / small lymphoplasmacytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma (DLBL), follicular lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma. In some embodiments, administration of the lipid-based formulation or engineered cells derived therefrom to a subject results in depletion of B cells in the subject. In some embodiments, administration of the lipid-based formulation or engineered cells derived therefrom to a subject results in depletion of plasma cells in the subject. In some cases, the subject is a human.

[0165] In some embodiments for treatment of a B cell cancer, the immune cells to be engineered are T cells. In some embodiments for treatment of a B cell cancer, the sequence of interest encodes a molecule that binds to B cells, such as malignant B cells. In some embodiments, the sequence of interest encodes a molecule that binds to plasma cells, such as malignant plasma cells. In some embodiments, administration of the lipid-based formulation or delivery system herein or administration of engineered cells derived therefrom to a subject results in depletion of B cells in the subject. In some embodiments, administration of the lipid-based formulation delivery system herein or of engineered cells derived therefrom to a subject results in depletion of plasma cells in the subject. In some cases, the subject is a human. In some cases, the sequence of interest encodes a CAR, such as a CD 19 CAR or CD20 CAR.Attorney Docket No: 01373-0004-00PCT

[0166] In some embodiments of methods of treating a disease, such as a B cell cancer, the sequence of interest encodes a CAR. In some embodiments, the sequence of interest encodes a T cell receptor (TCR). In some embodiments, the sequence of interest encodes a B cell receptor (BCR). In some embodiments, the sequence of interest encodes an immune cell activation or inhibitory receptor. In some embodiments, the sequence of interest encodes a growth factor ligand. In some embodiments, the sequence of interest encodes a transcription factor. In some embodiments, the sequence of interest encodes a checkpoint inhibitor or agonist.

[0167] In some embodiments, the method comprises administering the lipid-based formulation or delivery system to the subject. In other embodiments, the method comprises administering an engineered immune cell to the subject, wherein the immune cell is engineered to express the sequence of interest after contact with the lipid-based formulation or delivery system in vitro. In other embodiments, the method comprises administering an engineered immune cell to the subject, wherein the immune cell is engineered to express the sequence of interest after contact with the lipid-based formulation or delivery system ex vivo. In some cases, the subject is a human.

[0168] In some aspects, the present disclosure provides methods for treating a disease in a subject by administering any one or more of the disclosed lipid-based formulations or delivery systems to a subject in need thereof, thereby generating an engineered immune cell within the subject, such as an engineered T cell. In some aspects, the present disclosure provides methods for treating a disease in a subject by generating an engineered immune cell such as an engineered T cell using any one or more of the disclosed lipid-based formulations or delivery systems and administering the engineered cell to the subject, thereby treating the disease. In some embodiments, a method of treating a disease in a subject, comprises (i) contacting isolated immune cells with an effective amount of any one or more of the disclosed lipid-based formulations to yield engineered immune cells and (ii) administering the engineered immune cells to the subject, thereby treating the disease. In some embodiments, the immune cells are T cells. In some embodiments, the subject is a human. In some embodiments, the disease is caused by a genetic mutation. In some embodiments, the disease is a B cell cancer.

[0169] The number of administrations of treatment to a subject may vary. Introducing the lipid-based formulation or delivery system or the engineered immune cells obtained therefrom into the subject may be a one-time event; but in certain situations, such treatment may elicit improvement for a limited period of time and require an ongoing series of repeated treatments. In other situations, multiple administrations of the engineered cells or the lipid-basedAttorney Docket No: 01373-0004-00PCTformulation may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.

[0170] The present disclosure provides a lipid-based formulation or delivery system for use in treating a disease in a subject, wherein the use comprises administering an effective amount of the lipid-based formulation to the subject or contacting isolated immune cells from the subject with an effective amount of the lipid-based formulation to yield engineered cells and administering the engineered cells to the subject. In some embodiments, the disease is caused by a genetic mutation. In some embodiments, the disease is a B cell cancer. In some embodiments, the subject is a human. In some embodiments, the use comprises administering an effective amount of the lipid-based formulation or delivery system to the subject. In some embodiments, the use comprises contacting isolated cells with an effective amount of the lipid-based formulation or delivery system to yield engineered cells and administering the engineered cells to the subject.

[0171] The present disclosure provides uses of a lipid-based formulation or delivery system as disclosed herein in the manufacture of a medicament. In some embodiments, the medicament is for treating a disease caused by a genetic mutation. In some embodiments, the medicament comprises engineered immune cells, such as engineered T cells. In some embodiments, the medicament is for use in treating a B cell cancer. In some embodiments, the subject is human.V. PHARMACEUTICAL COMPOSITIONS

[0172] The lipid-based formulations herein can be made into delivery systems in some embodiments by combination with appropriate pharmaceutically acceptable carriers, excipients, diluents, or vehicles. Thus, a delivery system may in such cases comprise a pharmaceutical composition. Pharmaceutically acceptable vehicles may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. In some embodiments, the disclosed genome editing system is administered to a subj ect via intravenous, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, or intraocular administration. The active agent may be systemic after administration or may be localized by the use of regional administration or targeted delivery mechanisms.General Considerations

[0173] At various places in the present disclosure, substituents, or properties of compounds of the present disclosure are disclosed in groups or in ranges. It is intended that the presentAttorney Docket No: 01373-0004-00PCTdisclosure comprise each and every individual or sub-combination of the members of such groups and ranges, and that such groups or ranges include the endpoints. By way of nonlimiting example, if a group or range is from about 1 to about 10, then the group or range includes both the value of about 1 and the value of about 10.

[0174] Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that comprise "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The present disclosure can include embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure can include embodiments in which more than one, or the entire group members, are present in, employed in, or otherwise relevant to a given product or process.

[0175] The term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the terms “consisting of’ and “consisting essentially of’ are also encompassed and disclosed.

[0176] The abbreviation, “e.g.,” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.,” is synonymous with the term “for example.” The abbreviation, “i.e.,” is derived from the Latin id est, and is used herein to indicate a non-limiting rewording or clarification. Thus, the abbreviation “i.e.,” is synonymous with the term “that is.”

[0177] Any embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Any embodiment of the agents, methods, and / or compositions of the present disclosure can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0178] The present specification will control in instances where publications, patent applications, patents, and other references mentioned herein are incorporated by reference and are in conflict with the present specification.

[0179] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting.EXAMPLESExample 1: CD3-targeted LNPs deliver mRNA to primary human T cells

[0180] CD3 -targeted LNPs were prepared with mRNA encoding mCherry and evaluated for reporter expression in primary human T cells. LNPs were prepared with either an anti-CD3Attorney Docket No: 01373-0004-00PCTIgG antibody, or anti-CD3 Fab antibody, or unconjugated (non-targeting (nT)). The anti-CD3 antibody heavy chain and light chain variable region sequences used herein are SEQ ID NO: 6 and SEQ ID NO: 8, respectively.

[0181] LNP formulations were prepared as follows. Six lipids (an ionizable lipid, cholesterol, a helper lipid (DSPC), a PEG lipid (DMG-PEG 2000), a PEG-conjugation lipid (DSPE-PEG-TCO), and a lipid dye (Atto647N-DOPE), were dissolved in ethanol at a ratio of 50:24.95:22:2.5:0.5:0.05. The lipid mixture was combined with acid aqueous buffer containing mRNA at a volumetric (aqueous: ethanol) ratio of 3:1 using a microfluidic mixer (Ignite, Percision Nanosystems). CD3-targeting antibodies containing a methyltetrazine modification were then conjugated to the LNPs. Formulations were dialyzed against 50 mM Tris (pH 7.5), 75 mM sodium chloride, and 10% sucrose in dialysis cassettes for at least 18 hours. Formulations were concentrated using amicon spin filters (Millipore Sigma, UFC910008) to remove unbound antibodies. Formulations were stored at -80 °C until further use.

[0182] Cryopreserved human PBMCs were obtained from Stemcell Technologies. PBMCs were thawed in X-Vivol5 media (Lonza) supplemented with 2% human AB serum (Fisher), lOOIU / mL IL-2, lOng / mL IL-7, lOng / mL IL- 15 (Stemcell Technologies) with or without Dynabead Human T-Activator CD3 / CD28 at a ratio of 75uL of Dynabeads per le6 PBMCs (Gibco). The cells were seeded at 200,0000 cells / well in a 12-well plate and incubated overnight. The following day, PBMCs were treated with 1.2 pg / e6 cells of LNPs encapsulating mCherry mRNA. LNPs were either non-targeting or were conjugated with CD3-targeting moieties (anti-CD3 IgG or anti-CD3 Fab fragment). All LNP treatments were carried out in the absence of ApoE. 48 hours after LNP addition, PBMCs were harvested for flow cytometry and were stained with PBMC lineage panel fluorophore conjugated antibodies against CD3 (BV711; Tcells); CD19 (BV605; B cells); CD14 (BV421; Monocytes); CDllc (PercP Cy5.5; Monocytes) and CD16 (PECy7; NK cells) along with LiveDead Aqua, mCherry, or T cell panel using fluorophore conjugated antibodies against CD3 (APC Cy7; T lineage); CD4 (BV711; T lineage); CD8 (PerCP Cy5.5; T lineage) along with LiveDead Aqua and mCherry. Cells were analyzed on a Thermo fisher Attune Flow cytometer. Expression of mCherry in different live cell lineages and in T cells was analyzed using FlowJo 10.10.0 software (BD).

[0183] For lineage gating, single cells were identified, and live cells were gated on Live / Dead Aqua negative population. Subsequently CD3+CD19- were identified as T cells and CD3-CD19+ cells were identified as B cells. Further, CD3-CD19- population of cells was further gated on CD14+CDllc+ population which was identified as Monocytes. CD14-CD11c- population was further gated on CD 16 and CD 16+ population was identified as NKAttorney Docket No: 01373-0004-00PCTcells. All subpopulations were subsequently gated on mCherry to identify mCherry+ fraction. For determining activation status of T cells, live cell population was gates as described above. Subsequently, CD3+CD4+ and CD3+CD8+ were identified as helper T cells and cytotoxic T cells separately. Each of these populations were gated on identifying the CD25+CD69+ population as the early activation stage cells and the CD25+HLADR+ cells as the late activation stage cells. All subpopulations were subsequently gated on mCherry to identify mCherry+ fraction.

[0184] Both types of anti-CD3 LNPs delivered mRNA to a high proportion of T cells within PBMCs as measured by % mCherry expression by flow cytometry (FIG 1A). Delivery was higher to T cells than macrophages and other cell types (FIG IB).Example 2: CD3-targeted LNPs deliver co-encapsulated mRNA and DNA payloads in humanized NSG mice

[0185] Human CD34+ HSC-engrafted NSG mice were dosed with CD3-targeted LNPs coencapsulating mRNA encoding mScarlet reporter protein and nanoplasmid DNA encoding a GFP reporter transgene.

[0186] CD3 -targeted LNPs were prepared as above. The lipid mixture was combined with acid aqueous buffer containing mScarlet mRNA and GFP nanoplasmid DNA (RNA: DNA=1:1) at a volumetric (aqueous: ethanol) ratio of 3:1 using a microfluidic mixer (Ignite, Percision Nanosystems). After formulation, the CD3-targeting ligand was added to the preformed LNPs. Formulations were dialyzed against 50 mM Tris (pH 7.5), 75 mM sodium chloride, and 10% sucrose in dialysis cassettes for at least 18 h. Unbound targeting ligand was removed through centrifugation with an Amicon filter (UFC9100, Sigma Aldrich). Formulations were stored at -80 °C until further use.

[0187] Mice were dosed at 2 mg / kg of total nucleic acids through tail vein, and 50 ul blood was collected retro-orbitally under anesthesia on Day -1 (for pre-dose baseline), Day 3, 5, 7, 10, 14 and 18 to monitor the human T cells numbers as well as mScarlet and GFP expression.

[0188] To analyze cell number and reporter expression in whole blood, a no-wash flow cytometry protocol was used. First, 10 pL whole blood from each mouse sample was reverse-pipetted into each well of a 96-well U-bottom plate (Fisher, Waltham, MA). To each well, 25 pL of a mixture of fluorescent antibodies directed against human CD45 (PerCP / Cyanine5.5 anti-human CD45, BioLegend, 982312), human CD3 (APC / Cyanine7 anti-human CD3, BioLegend, 300426), human CD 19 (Brilliant Violet 605 anti-human CD 19, BioLegend, 302244), human CD4 (Brilliant Violet 510 anti-human CD4, BioLegend, 300546), and human CD8 (PE / Cyanin7 anti-human CD8, BioLegend, 344712), diluted 1:50 in buffer (Stain BufferAttorney Docket No: 01373-0004-00PCT(FBS), BD Biosciences, 554657), was added. The 96-well plate was incubated for 30 minutes at room temperature protected from light. After the incubation, 165 pL lx RBC Lysis Buffer (diluted in DI water from lOx, BioLegend, 420302) was added to each well. The plate was incubated for 15 minutes at room temperature protected from light before immediately acquiring the samples on a flow cytometer (Attune NxT configured with Blue, Red, Violet, and Yellow lasers). 150 pL of each sample was collected volumetrically by the flow cytometer and analyzed for spectral emission corresponding to the antibodies and reporters used.

[0189] Data were exported as FCS 3.0 files and visualized in FlowJo 10 software (TreeStar, Ashland, OR). Live cells were located by forward scatter and side scatter signal. Single cells were gated by plotting forward scatter height by forward scatter area. For all analyses, a gate was first drawn around the human immune cell population as marked by CD45 positivity. Further gates within this population were made to enumerate T cells (CD3+) and B cells (CD19+). Within the T cell population, helper T cells (CD4+) were distinguished from cytotoxic T cells (CD8+). To measure expression of mRNA and DNA expression, the mScarlet and GFP signals within the CD3+ population were measured respectively by the yellow laser and blue laser.

[0190] Delivery of nucleic acid by CD3-targeted LNPs to human T cells was evaluated by flow cytometry and measured as a percentage of T cells expressing mRNA (mScarlet fluorescence) or nuclear DNA (GFP fluorescence) at time points of peak expression level after LNP dosing (day 3 for mRNA, day 5 for DNA (FIG 2)).Example 3: In vitro CAR-T generation with cssDNA encoding a CAR

[0191] CAR-T cells were generated in vitro by incubation of primary human T cells with CD3 -targeted LNPs containing either LSR mRNA alone, cssDNA encoding CD 19 CAR alone, or co-encapsulated LSR mRNA and cssDNA encoding CD 19 CAR. The AttD was SEQ ID NO: 1. The LSR variant construct was 3xMyc-NLS-Flag-LSR as mRNA with LSR variant, “LSR-A”, (SEQ ID NO: 2). LNPs were conjugated with an anti-CD3 Fab fragment.

[0192] The cssDNA was prepared as follows. A phagemid plasmid encoding cssDNA19 and helper plasmid were co-transformed in the E. coli XL 1 -Blue competent cells. The cells were spread on LB / agar plates having 100 pg / mL carbenicillin and 50 pg / mL kanamycin and grown at 37 °C overnight. The single colonies were inoculated in 3 mL of 2xYT media with carbenicillin, kanamycin, and MgSO4 in 14 mL Falcon round-bottom tubes and grown at 37 °C and 220 r.p.m. in a New Brunswick Innova 44 shaker-incubator overnight. The 0.5 mL of grown cell cultures were transferred to 250 mL of 2xYT media with carbenicillin, kanamycin, and MgSO4 in 1000 mL Erlenmeyer flasks and grown at 37 °C and 220 r.p.m. in the shaker-Attorney Docket No: 01373-0004-00PCTincubator overnight. The cultures were transferred to 250 mL PPCO centrifuge bottles and centrifuged at 6,000 g and 4 °C for 30 min in a Sorvall LYNX 4000 Superspeed centrifuge. The supernatant containing phage was collected in 500 mL Pyrex media storage bottles and E. coli cell pellets were discarded. To purify the phage, 72 mL 50% (w / v) PEG8000 and 36 mL 5M NaCl were added in the phage solution. The phage-PEG / NaCl mixtures were incubated at 4 °C for 24 h and then centrifuged at 15,000 g for 30 min. After discarding the supernatant, the mixtures were centrifuged at 15,000 g for 10 min. The supernatant was gently removed and the phage-PEG / NaCl pellets were redispersed in 20 mL lx TBS buffer using vortex. The redispersed phage solution was centrifuged at 15,000 g and 4 °C for 20 min and the supernatant was transferred to new tubes. To remove extra DNA or RNA in the phage solution, MgC12, DNase I, and RNase A were added in the solution. The solution was incubated at 37 °C and 220 r.p.m. for 2 h in the shaker-incubator. To inactivate DNase I, the solution was incubated at 70 °C for 10 min. To lyse the phage particles and extract cssDNA, 20 mL of M2 Buffer (2% Triton X-100, 1 M Guanidine-HCl, 20 mM MOPS, pH 6.5) was added and incubated at 80 °C for 1 h. The solution was cooled to room temperature and centrifuged at 15,000 g for 10 min. The supernatant was transferred to new 50 mL endotoxin-free centrifuge tubes. To remove endotoxin from the solution, 2.5 mL Qiagen Buffer ER was added and incubated on ice for 30 min. And then, the cssDNA was purified using Qiagen-tip 500, Buffer QC, and Buffer QN in the Qiagen Endofree Plasmid Maxi Kit. The purified cssDNA was purified one more time using Norgen Biotek Endotoxin Removal Kit (Mini) to reduce the endotoxin level of cssDNA. The concentration of cssDNA was measured using Nanodrop and the endotoxin level of cssDNA was measured using Endosafe nexgen-PTS.

[0193] CAR-T cells were prepared and analyzed as follows. Cryopreserved human T cells were obtained from Stemcell Technologies. T cells were thawed and resuspended in X-Vivol5 media (Lonza) supplemented with 2% human AB serum (Fisher), 100 lU / mL IL-2, 10 ng / mL IL-7, 10 ng / mL IL-15 (Stemcell Technologies) at 1,000,000 live cells per mL. The T cells were seeded at 100,000 cells per well in a 96-well flat-bottom plate in duplicate or triplicate and treated with CD3 -targeted LNP at the concentrations indicated in each graph. Every 2-3 days, T cells were supplemented with additional cytokines. On day 6, T cells were analyzed by flow cytometry for CAR expression (FIG 3A) and by ddPCR for integration (FIG 3B).

[0194] For analysis of CAR expression by flow cytometry, T cells were stained with a fluorophore-conjugated antibody against the anti-CD19 CAR and were analyzed on a Thermo fisher Attune NxT Flow cytometer. Data was analyzed using FlowJo 10.10.0 software (Tree Star).Attorney Docket No: 01373-0004-00PCT

[0195] For integration analysis by ddPCR, the cells were lysed with lOOul genomic lysis buffer from Quick-DNA™ 96 Kit (Zymo research, D3012), according to the manufacturer’s instructions, and ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (Bio-Rad) to quantify integration events at the genome integration site referred to as “GS10”. lOOng gDNA was used for each reaction. Each test was prepared using 2x ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad), forward / reverse primers (900 nM), FAM or HEX labeled PrimeTime™ qPCR probes (250 nM), Hindlll-HF restriction enzyme (NEB) and 20-50 ng of DNA templates. Reaction mixture droplets were generated with the Automated Droplet Generator, then DNA amplification in the generated droplets was performed with the PTC Tempo DeepWell Thermal Cycler (Bio-Rad) followed by enzyme inactivation. The droplets were then quantified using a QX600 Droplet Reader (Bio-Rad). Data analysis was performed with QX Manager (Bio-Rad) to count positive and negative droplets for each probe, and integration events were calculated relative to RPPH1 as genome copy number reference.Example 4: In vivo CAR-T generation in NSG mice with CD3-targeted LNPs coencapsulating LSR mRNA and cssDNA encoding a CAR

[0196] In vivo generation of CAR T cells was demonstrated by IV delivery of CD3-targeted LNPs co-encapsulating mRNA encoding a variant LSR and cssDNA donor template encoding a CD 19 CAR transgene. The AttD was SEQ ID NO: 1. The LSR variant construct was 3xMyc-NLS-Flag-LSR as mRNA with LSR variant, “LSR-A”, (SEQ ID NO: 2).

[0197] CD3 -targeted LNPs were prepared and validated for mediating CAR expression and integration in vitro using primary T cells as described above in Example 3.

[0198] Human CD34+ stem cell engrafted NSG mice were dosed with CD3-taregeted LNPs through the tail vein and the dose for each group as listed in Table 1.Table 1: LNP contents in treatment groups and doses for in vivo study

[0199] Animals were sacrificed on day 8 after dosing. Several tissues including whole blood, spleen, bone marrow, and mesenteric lymph node were collected and dissociated. Whole blood was stained and measured by flow cytometry as described in Example 3. Other tissues including splenic leukocytes, bone marrow-derived leukocytes, and lymph node-derivedAttorney Docket No: 01373-0004-00PCTleukocytes were prepared for staining by pipetting 200 pL of each dissociated sample per well of a 96-well U-bottom plate. For bone marrow-derived leukocytes, samples were first passed through a mesh filter to remove particulate debris before being added to the well. The plate was centrifuged at 500 x g for 5 minutes at ambient temperature to pellet the cells. Supernatants were discarded in a biohazard container. All cell pellets were resuspended in 50 pL of a mixture of human and mouse Fc receptor blockers (Human TruStain FcX, BioLegend, 422302, and TruStain FcX anti-mouse CD16 / 32, BioLegend, 101320) diluted 1:20 in buffer. The plate was incubated for 20 minutes at 4°C protected from light. Without washing, 25 pL of antibody mixture was added to each well. To measure expression of the CAR on the surface of T cells, an antibody specific for the CD 19 CAR was added to the antibody mixture at 1:50 dilution prior to addition to the plate. Tissue-derived cells were incubated for 20 minutes at 4°C protected from light. After incubation, 150 pL buffer was added to each well and the plate was centrifuged as before. Supernatants were discarded in a biohazard container, and the wash step was repeated a second time using 200 pL buffer. After discarding the supernatant from the second wash, all tissue-derived cells were resuspended in 200 pL buffer and acquired on the flow cytometer as described. In each sample type, the frequency of CAR-T cells was measured by first gating on CD3+events in the CD45+population of single live cells, and then plotting forward scatter against the CAR+ signal.

[0200] Terminal tissues evaluated by flow cytometry at day 8 after dosing showed significantly reduced B cells (FIG 4A) and no significant change in T cells (FIG 4B).

[0201] Direct evidence of in vivo CAR T generation was found in T cells in the spleen of one mouse dosed with LNPs co-encapsulating LSR + cssDNA. CAR expression by flow cytometry was measured in splenic T cells of a mouse treated with LNPs co-encapsulating LSR + cssDNA (FIG 4C) and a mouse treated with LNP containing cssDNA only (FIG 4D).

[0202] LSR-mediated CAR integration was demonstrated with ddPCR in terminal tissues as an average of all mice within a group (FIG 4E) and for each individual mouse dosed with LNPs co-encapsulating LSR + cssDNA (FIG 4F). To prepare genomic DNA from the samples, 100 pl whole blood was treated with red blood cell lysis buffer for 10 min at room temperature, centrifuged at 1000 rpm for 5 min, supernatant was discarded, and the cell pellets were used for genomic DNA extraction. Other samples including spleen, bone marrow and mesenteric lymph node were processed to single cell suspension and le6 cells were transferred to each well of a 96-well plate. Cells were lysed with lOOpl genomic lysis buffer from Quick-DNA™ 96 Kit (Zymo research, D3012), according to the manufacturer’s instructions. lOOng genomic DNA was used for each reaction and ddPCR was performed as above.Attorney Docket No: 01373-0004-00PCTExample 5: In vivo anti-tumor activity with integrated CD19 CAR T cells

[0203] Anti-tumor activity of CD19 CAR T cells from CD3-targeted LNPs coencapsulating mRNA encoding a variant LSR and nanoplasmid donor template or circular single-stranded “cssDNA” donor template encoding a CD 19 CAR transgene was evaluated in a tumor regression mouse model, NSG-MHCI / II-DKO mice. Two LSR variants were tested, “LSR-A” (SEQ ID NO: 2) and “LSR-B” (SEQ ID NO: 3) using the same AttD (SEQ ID NO: 1) and targeting the same genomic site. PBMCs were purchased from iXCells Biotechnologies USA, Inc. CD3-targeted LNPs were prepared with a CD3 Fab as described above.

[0204] On day -3, mice were intravenously injected with 2.5xl0e5 tumor cells (Nalm6-luciferase). On day 0, mice were intravenously injected with 20e6 PBMCs followed by intravenous dosing of LNPs. Mice were dosed with 1 mg / kg LNPs per mouse for the following groups: 1) CD3-targeted LNPs co-encapsulating LSR mRNA and nanoplasmid or cssDNA encoding CD19-CAR; and 2) CD3-targeted LNPs co-encapsulating LSR mRNA and mScarlet mRNA (“LSR mRNA only” or “mRNA only”). A control group for episomal expression receiving CD3 -targeted LNPs encapsulating nanoplasmid encoding CD19-CAR only was dosed with 0.5 mg / kg (“DNA only” or “dsDNA only”). Additional controls included a tumor growth baseline with T cells control that received IV injections of tumor cells and PBMCs but did not receive LNPs and another control arm of tumor free baseline for IVIS imaging that did not receive IV injections of any of the tumor cells, PBMCs, or LNPs.

[0205] Tumor burden was assessed by IVIS imaging on Day -1, 4, 7, 10, 14, and 19. 26, and 33. Mice were injected IP (150 mg / kg) with 15 mg / mL D-luciferin solution in D-PBS and maintained on 2.5% isoflurane via nose cones attached to the internal anesthesia manifold. Mice were placed on the heated (37 °C) shelf of the imaging chamber of the AMI HTX Spectral Imaging (Spectral Instruments) system for ventral image acquisition. The bioluminescence signal was quantitated using Aura In Vivo Imaging software (Spectral Instruments) following the manufacturer’s instruction. Total flux (p / s) was used to indicate the intensity of luciferase signal.

[0206] Tumor reduction was observed with LNPs co-encapsulating LSR mRNA and the CD19-CAR transgene delivered with nanoplasmid (FIG 5A) or with cssDNA (FIG 6A). CD3 -targeted LNPs were well-tolerated with modest body weight loss in all groups followed by recovery.

[0207] Correlative assessments showed integration of CD19-CAR and generation of CAR T cells in the blood. Integration was assessed by ddPCR as above and measured as a sum of the top 8 integration sites. Data are shown for individual mice at days 4 and 26 for nanoplasmidAttorney Docket No: 01373-0004-00PCT(FIG 5B) and cssDNA (FIG 6B). CAR+ T cells (CD3+ T cells) were detected by flow cytometry as above at the indicated time points after dosing of LNPs in mice that received LNPs co-encapsulating LSR mRNA and nanoplasmid DNA encoding the CD19-CAR transgene (FIG 5C) or cssDNA (FIG 6C). No CAR+ T cells were detected in the episomal expression control. B cells from the PBMCs were assessed for depletion 10 days after LNP dosing by measuring the percent of CD20+ cells in the CD45+ cell population by flow cytometry. Normal B cells were depleted from PBMCs in mice that received LNPs coencapsulating LSR mRNA and nanoplasmid DNA encoding the CD19-CAR transgene (FIG 5D)

[0208] The above results demonstrated functional CAR integration and tumor reduction in NSG mice with CD3-targered LNPs co-encapsulating LSR mRNA and dsDNA or cssDNA encoding a CD19-CAR transgene.Example 6: CD20 CAR Study in Mice

[0209] NSG-MHC I / II double knockout (DKO) mice (7-9 weeks old, Strain #025216) were inoculated systemically with 0.25 * 106Raji cells on Day -7. Tumor growth was assessed by IVIS imaging on Day -2, and mice were randomized into treatment groups. On Day -1, each mouse received 20 * 106healthy human PBMCs via tail vein injection. On Day 0, mice were treated with either PBS or T-cell targeting LNPs. After treatment on Day 0, IVIS, a non-invasive imaging technique, was conducted periodically throughout the study to monitor changes in tumor burden over time. Blood samples were collected over time to track circulating CAR-T cells. The study concluded on Day 28, with terminal collection of cardiac blood, spleen, and bone marrow for CAR expression analysis.Circular single-stranded DNA (cssDNA) as an CD20 CAR donor template

[0210] Mice received either PBS (no LNP) or T-cell targeted LNPs comprising circular single-stranded DNA (cssDNA) encoding a CD20 CAR and mRNA expressing a functional LSR or a catalytically inactive LSR (“Dead LSR” as an episomal CAR control). Both LNP groups were dose-matched and contained mRNA and cssDNA. IVIS imaging showed significant tumor regression after 7 days of LNP administration, with low tumor burden maintained through study completion on Day 25 (FIG. 7).Nanoplasmid double-stranded DNA (dsDNA) as an CD20 CAR donor template

[0211] The mice were treated with PBS (no LNP) or T-cell targeted LNPs comprising dsDNA encoding CD20 CAR and mRNA expressing either a functional LSR or a catalytic inactive LSR (“Dead LSR” as an episomal expressed CAR control). Both LNP groups were dose-matched and contained mRNA and dsDNA. IVIS imaging showed significant tumorAttorney Docket No: 01373-0004-00PCTregression after 7 days of LNP administration, with low tumor burden maintained through study completion on Day 25 (FIG. 8).Example 7: CD20 CAR Study in Naive Cynomolgus Monkeys (NHPs)

[0212] Naive cynomolgus monkeys (NHP) were employed in this study. Prior to treatment, all animals were screened for anti-PEG antibodies (IgM and IgG), and only antibody-negative animals were selected. On Day 0, NHPs received CD3-LNPs via a 2-hour infusion. Blood samples were collected periodically to monitor B and T cell populations in peripheral circulation.

[0213] Messenger RNA (mRNA) expressing LSR having a high enzymatic activity and cssDNA encoding a CD20 CAR were encapsulated in T-cell targeting LNPs comprising ionizable lipid, DSPC, cholesterol, DMG-PEG and DSPE-PEG-CD3binder. Shown in FIG. 9 are fold-changes in B cell numbers taken from blood samples collected at Day 14 post LNP infusion. B cells were gated by CD20+ cells. B cell numbers were measured and then normalized to the pre-dose baseline. As shown in FIG. 9, dose-dependent B cell depletion was observed for both total LNP doses (mg / kg) and estimated anti-CD3 Fab concentration per LNP (z.e., anti-CD3 Fab densities per LNP in “Low” (0.25 mol%) to “High” (0.5 mol%). The PBS control group showed a relatively stable B cell number in the blood.Attorney Docket No: 01373-0004-00PCTSequence Listing

Claims

Attorney Docket No: 01373-0004-00PCTCLAIMS1. A lipid-based formulation for nucleic acid delivery to an immune cell, the formulation comprising:a. an mRNA encoding a large serine recombinase (LSR);b. a DNA comprising a donor attachment site (AttD) and a sequence of interest; and c. a targeting moiety for the immune cell;wherein, the formulation comprises an activation factor for the immune cell or a nucleic acid sequence encoding an activation factor for the immune cell, optionally wherein (i) the targeting moiety is an activation factor, (ii) the activation factor is encoded by the mRNA of (a) encoding the LSR, or (iii) the formulation comprises an additional mRNA encoding the activation factor; andwherein the formulation is capable of integrating the sequence of interest into the genome of the immune cell, optionally wherein the immune cell is a human immune cell; andoptionally wherein the formulation comprises a lipid nanoparticle (LNP).

2. The lipid-based formulation of claim 1, wherein the immune cell is a T cell.

3. The lipid-based formulation of claim 1 or 2, wherein the T cell is a human T cell.

4. The lipid-based formulation of any one of the preceding claims, wherein the sequence of interest encodes a chimeric antigen receptor (CAR).

5. The lipid-based formulation of any one of claims 1-4, wherein the targeting moiety is an activation factor for the immune cell.

6. The lipid-based formulation of any one of claims 1-4, wherein the targeting moiety is not an activation factor for the immune cell.

7. The lipid-based formulation of any one of claims 1-6, wherein the formulation comprises an LNP, and wherein activation factor is on the surface of LNPs in the formulation.

8. The lipid-based formulation of any one of claims 1-4, wherein the activation factor is encoded by an additional mRNA in the formulation.Attorney Docket No: 01373-0004-00PCT9. The lipid-based formulation of any one of claims 1-4, wherein the activation factor is encoded by the mRNA encoding the LSR.

10. The lipid-based formulation of any one of claims 1-9, wherein the activation factor is antigen-dependent.

11. The lipid-based formulation of claim 10, wherein the activation factor is a CAR.

12. The lipid-based formulation of claim 11, wherein the activation factor is a CD19 CAR.

13. The lipid-based formulation of any one of claims 1-9, wherein the activation factor is antigen independent.

14. The lipid-based formulation of claim 13, wherein the activation factor is selected from any one of: IL-12B, BATF, LTBR, JUNB, constitutively active beta-catenin, TCF7, FOXO1, IRF4, CARD11-PIK3R3, CD79A / CD40, ITK-SYK, FYN-TRAF3IP2, KHDRBS1-LCK, SIN3A-FOXO1, RNMT, RAS, a BRAF mutant such as BRAF-G469A, a PLCG1 mutant such as PLCG1-D1165H or PLCG1-R48W or PLCG1-E1164K or PLCG1-S520F, a RASGRP1 mutant such as RASGRP1-M2611, a CARD 11 mutant such as CARD11-Y361C or CARD11-S615F or CARD11-D357N, TNFRSF1B-T3771, CD28 antibody, CD80, soluble CD58, SLC7A5, SLC1A5, SLC7A1, SLC38A1, SLC38A2, GLUT1 (SLCA1), GLUT3 (SLCA3), MCT1 (SLC16A1), MCT4 (SLC16A3), phospholipase Cyl), IP3, IP3R, P2X7, CD40, CD86, 0X40, 4-1BBL, AHCY, CDK1, CDK2, AKR1C4, ATF6B, ITM2A, AHNAK, or FOXQ1, or wherein the activation factor is a chimeric molecule comprising a T cell activating domain and a dimer or multimerization motif.

15. The lipid-based formulation of any one of claims 1-14, wherein the DNA is circular single-stranded DNA (cssDNA).

16. The lipid-based formulation of any one of claims 1-14, wherein the DNA is double stranded DNA.

17. The lipid-based formulation of any one of claims 1-16, wherein the targeting moiety targets CD3 (e.g., on the surface of T cells).Attorney Docket No: 01373-0004-00PCT18. The lipid-based formulation of claim 17, wherein the targeting moiety is an anti-CD3 antibody or antigen binding domain.

19. The lipid-based formulation of any one of claims 1-18, wherein the sequence of interest encodes a CAR that binds to B cells or malignant B cells, optionally wherein the B cells or malignant B cells are human B cells or human malignant B cells.

20. The lipid-based formulation of any one of claims 1-19, wherein the sequence of interest binds a CAR that binds to plasma cells or malignant plasma cells, optionally wherein the plasma cells or malignant plasma cells are human plasma cells or human malignant plasma cells.

21. The lipid-based formulation of claim 19 or 20, wherein the sequence of interest encodes a CD 19 CAR or a CD20 CAR.

22. The lipid-based formulation of any one of claims 1-21, wherein the formulation comprises and LNP, and wherein LNP comprises a cationic lipid, helper lipid, cholesterol, and a PEG lipid.

23. The lipid-based formulation of claim 22, wherein the targeting moiety is covalently attached to at least one lipid of the LNP, optionally to a PEG-lipid.

24. The lipid-based formulation of any one of claims 1-23, wherein the mRNA encodes a nuclear localization signal (NLS).

25. The lipid-based formulation of any one of claims 1-24, wherein the DNA of (b) is modified.

26. The lipid-based formulation of any one of claims 1-25, wherein the mRNA of (a) is modified.

27. A method for integration of a chimeric antigen receptor (CAR) into a cellular genome of a T cell, comprising contacting the T cell with the lipid-based formulation of any one of claims 1-26, wherein the sequence of interest encodes the CAR, such that the CAR is integrated into the cellular genome of the T cell.Attorney Docket No: 01373-0004-00PCT28. A method for introducing a nucleic acid encoding a chimeric antigen receptor (CAR) into a T cell, comprising contacting the T cell with the lipid-based formulation of any one of claims 1-26, optionally wherein the T cell is a human T cell.

29. An engineered T cell expressing the chimeric antigen receptor (CAR) encoded by the DNA of the lipid-based formulation of any one of claims 1-26, optionally wherein the engineered T cell is a human T cell.

30. An engineered T cell modified by contact with the lipid-based formulation of any one of claims 1-26.

31. A method for treating a disease in a subject, comprising administering to the subject the lipid-based formulation of any one of claims 1-26 or the engineered T cell of claim 29 or 30.

32. The lipid-based formulation of any one of claims 1-26 for use in treating a disease in a subject.

33. Use of the lipid-based formulation of any one of claims 1-26 in the preparation of a medicament for treating a disease in a subject.

34. The method, lipid-based formulation, or use of any one of claims 31-33, wherein the disease is a B cell cancer.

35. The method, lipid-based formulation, or use of claim 34, wherein the B cell cancer is a plasma cell neoplasm, a plasmacytoma, Hodgkin lymphoma, non-Hodgkin lymphoma, acute lymphocytic leukemia (ALL), Hairy cell leukemia, multiple myeloma, B cell lymphoma, Burkitt lymphoma, chronic lymphocytic leukemia / small lymphoplasmacytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma (DLBL), follicular lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, or marginal zone lymphoma.

36. The method, lipid-based formulation, or use of any one of claims 31-35, wherein the administration of the lipid-based formulation or engineered T cell results in depletion of B cells in the subject.

37. The method, lipid-based formulation, or use of any one of claims 31-36, wherein the subject is a human.

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