Non-viral circular single-stranded DNA systems and uses thereof

Abstract

Disclosed herein are systems for LNP-mediated delivery of circular single-stranded DNA (cssDNA). The systems can be used to facilitate integration of the cssDNA into a cellular genome for a variety of purposes, including treatment of human diseases.

Description

NON- VIRAL CIRCULAR SINGLE-STRANDED DNA SYSTEMSAND USES THEREOFFIELD OF THE DISCLOSURE

[0001] This disclosure relates to compositions and systems for LNP-mediated delivery of circular single-stranded DNA (cssDNA) to a cell and methods of introducing, modifying or editing nucleic acids using such systems.SEQUENCE LISTING

[0002] The present application contains a Sequence Listing which has been submitted electronically in XML format. Said XML copy, created on March 3, 2025, is named “01373- 0003-00PCT.xml’' and is 1,082,703 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] Nucleic acid-based medicines have become an increasingly important treatment modality for various medical indications, including in the fields of cancer immunotherapy and genetic disease. However, there remains an unmet need for non-viral approaches for gene delivery using DNA without eliciting undesired immune response effects. Such non-viral approaches would provide for new therapeutics that integrate a gene into the genome of a cell . The herein described compositions, systems, and methods comprising a recombinase enzyme are useful in a variety of diagnostic and therapeutic settings, such as in the treatment of human disease and disorders.SUMMARY

[0004] In some embodiments, the disclosure provides a genome editing system comprising: a) an mRNA encoding a large serine recombinase (LSR); and b) a circular single-stranded DNA (cssDNA) comprising a donor attachment site (AttD) and a sequence of interest; wherein the genome editing system is capable of integrating the sequence of interest into a cellular genome. In some embodiments, the disclosure provides a composition comprising (a) an mRNA encoding a large serine recombinase (LSR). (b) a cssDNA compnsing a donor attachment site (AttD) and a sequence of interest, (c) a lipid nanoparticle, and optionally (d) anadditional component, wherein the composition is capable of integrating the sequence of interest into a cellular genome.

[0005] In certain embodiments herein, an additional component beyond the cssDNA and the mRNA and the lipid nanoparticle as described herein is optional and not required. Furthermore, in certain embodiments, it is not necessary for a cell to be engineered to incorporate an installed attachment site (also referred to as a “landing pad") to which the sequence of interest is to be integrated.

[0006] In some embodiments, a genome editing system comprises a lipid nanoparticle (LNP). In some embodiments, the ratio of the cssDNA to mRNA in the genome editing system is 3: 1 to 1 :3. In some embodiments, the ratio of the cssDNA to mRNA in the genome editing system is 2: 1 to 1 :2. In some embodiments, the ratio of the mRNA to the cssDNA in the genome editing system is 1 : 1.

[0007] In some instances, the genome editing system further comprises a lipid-based formulation comprising (a) and (b). In some embodiments, the genome editing system further comprises a first lipid-based formulation comprising (a) and a second lipid-based formulation comprising (b).

[0008] In some aspects, the endotoxin level of (b) is less than 0. lEU / pg, less than 0.05EU / pg, O.OlEU / pg, or 0.005EU / pg.

[0009] In some embodiments, the sequence of interest in the genome editing system comprises a gene or a portion of a gene. In some instances, the sequence of interest comprises a therapeutic protein.

[0010] In some embodiments, the sequence of interest encodes a CAR. In some embodiments, the cssDNA has reduced immunogenicity in an organism as compared to a composition or genome editing system comprising dsDNA encoding the sequence of interest. For example, in some embodiments, reduced immunogenicity may be shown by lower levels of inflammatory cytokines, such as IL-6, IFN-beta, IFN-alpha, and / or MCP-1, and / or by no significant changes in body weight in a subject administered with the system compared to a system comprising dsDNA encoding the sequence of interest.

[0011] In some embodiments, the genome editing system disclosed herein comprises an mRNA encoding an LSR, wherein the LSR is a genome targeting LSR. Thus, in some embodiments, the LSR is not a landing pad LSR.

[0012] In some embodiments, the genome editing system comprises a lipid-based formulation. In some instances, the lipid-based formulation comprises a cationic lipid, helper lipid, cholesterol, and a PEG lipid. In some instances, the lipid-based formulation furthercomprises a polypeptide. In some embodiments, the polypeptide comprises atargeting-moiety. The targeting moiety may comprise, for example, an antibody or antibody fragment.

[0013] In some embodiments, the lipid-based formulation is a lipid nanoparticle (LNP). In some aspects, the LNP has a size of 50-100 nm.

[0014] In some embodiments, the genome editing system comprises a first lipid-based formulation comprising an mRNA encoding a large serine recombinase (LSR); and a second lipid-based formulation comprising a circular single-stranded DNA (cssDNA) comprising a donor attachment site (AttD) and a sequence of interest. In some embodiments, the first and second lipid-based formulations are nearly identical in composition and size.

[0015] In some embodiments, a first lipid-based formulation comprises an mRNA encoding a large serine recombinase (LSR). and a second lipid-based formulation comprising a circular single-stranded DNA (cssDNA) comprising a donor attachment site (AttD) and a sequence of interest. In some embodiments, the first and second lipid-based formulation are not identical in composition and size.

[0016] In some embodiments, the AttD site comprises at least 26. 34, 36, 39, 48, or 52 nucleotides, wherein the nucleotides comprise the dinucleotide core and an even number of nucleotides directly adjacent on either side of the dinucleotide core. In some embodiments, the AttD comprises an AttP site. In some embodiments, the cellular genome is a human genome. In some embodiments, the cellular genome does not comprise an installed attachment site.

[0017] Also provided herein are methods for generating an engineered cell, comprising contacting a cell with any one of the compositions or genome editing systems disclosed herein. In some embodiments, methods are provided for introducing a sequence of interest in a cell, comprising contacting the cell with any one of the compositions, or genome editing systems disclosed herein. In some embodiments, an engineered cell is provided, wherein the engineered cell comprises the integrated sequence of interest of any one of the compositions, or genome editing systems disclosed herein. In some embodiments, methods for treating disease in a subject are disclosed, comprising administering to a subject any one of the compositions or genome editing systems or engineered cells disclosed herein. In some embodiments, the disease is cancer.

[0018] In some embodiments, the cell for expression of the sequence of interest in the genome editing systems, methods, or engineered cells disclosed herein is a dividing cell. In some embodiments, the cell is a non-dividing cell. In some embodiments, the cell is a T cell. In some embodiments, the sequence of interest in the composition, genome editing system, or engineered cell comprises a CAR. In some embodiments, it is not necessary for a cell to beengineered to incorporate any installed attachment site (also referred to as a “landing pad’') to which the sequence of interest is to be integrated.

[0019] Also provided is a method for integration of a sequence of interest into a cellular genome, comprising contacting a cell with the composition or genome editing system described herein such that the sequence of interest is integrated into the cellular genome. In some instances, the cell is mammalian. In some instances, the cell is human. In some embodiments, the cell is a T cell, natural killer cell (NK cell), non-human embryonic stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell (HSC), liver cell, muscle cell, monocyte, B cell, neuron, astrocyte, or microglial cell. In some embodiments, the cell is a T cell. In some embodiments, the sequence of interest comprises a CAR. In some embodiments, the contacting of the cell is in vivo. In some embodiments, the contacting of the cell is ex vivo. In some embodiments, a Cas or a guide RNA is not used in the contacting of the cell. In some embodiments, the method results in the generation of a CAR-T cell. In some embodiments, it is not necessary7for a cell to be engineered to incorporate any installed attachment site (also referred to as a “landing pad”) to which the sequence of interest is to be integrated.

[0020] Accordingly, certain embodiments herein include the following, for example:

[0021] Embodiment 1: A genome editing system comprising: a) an mRNA encoding a large serine recombinase (LSR); and b) a circular single-stranded DNA (cssDNA) comprising a donor attachment site (AttD) and a sequence of interest; wherein the genome editing system is capable of integrating the sequence of interest into a cellular genome.

[0022] Embodiment 2: The genome editing system of embodiment 1, wherein the genome editing system further comprises a lipid-based formulation comprising (a) and (b).

[0023] Embodiment 3: The genome editing system of embodiment 1, wherein the genome editing system further comprises a first lipid-based formulation comprising (a) and a second lipid-based formulation comprising (b).

[0024] Embodiment 4: The genome editing system of embodiments 2 or 3, wherein the lipid-based formulation is an LNP.

[0025] Embodiment 5: The genome editing system of any one of embodiments 1-4, wherein the ratio of b) to a) in the genome editing system is 3: 1 to 1:3 or wherein the ratio of b) to a) in the genome editing system is 2: 1 to 1 :2.

[0026] Embodiment 6: The genome editing system of any one of embodiments 1-5. wherein the ratio of b) to a) in the genome editing system is 1 : 1.

[0027] Embodiment 7: The genome editing system of any one of embodiments 1-6, wherein the endotoxin level of (b) is less than 0.1 EU / pg, less than 0.05 EU / pg, 0.01 EU / pg, or 0.005 EU / pg.

[0028] Embodiment 8: The genome editing system of any one of embodiments 1-7, wherein the sequence of interest comprises a gene or a portion of a gene.

[0029] Embodiment 9: The genome editing system of any one of embodiments 1-8. wherein the sequence of interest comprises a therapeutic protein.

[0030] Embodiment 10: The genome editing system of any one of embodiments 1-9, wherein the sequence of interest comprises a CAR.

[0031] Embodiment 11: The genome editing system of any one of embodiments 1-10, wherein the LSR is a genome targeting LSR.

[0032] Embodiment 12: The genome editing system of any one of embodiments 1-11, wherein the LSR is not a landing pad LSR.

[0033] Embodiment 13: The genome editing system of any one of embodiments 1-12, comprising a lipid-based formulation, comprising a lipid-based formulation comprising a cationic lipid, helper lipid, cholesterol, and a PEG lipid.

[0034] Embodiment 14: The genome editing system of embodiment 13, wherein the lipid- based formulation further comprises a polypeptide.

[0035] Embodiment 15: The genome editing system of any one of embodiments 2-14, wherein the lipid-based formulation comprises an LNP having a size of 50-100 nm.

[0036] Embodiment 16: The genome editing system any one of embodiments 1 -15, comprising a first lipid-based formulation comprising a) and a second lipid-based formulation comprising b). wherein the first and second lipid-based formulations are nearly identical in composition and size.

[0037] Embodiment 17: The genome editing system any one of embodiments 1-16, comprising a first lipid-based formulation comprising a) and a second lipid-based formulation comprising b), wherein the first and second lipid-based formulation are not identical in composition and size.

[0038] Embodiment 18: The genome editing system of any one of embodiments 1-17, wherein the AttD site comprises at least 26, 34, 36, 39, 48, or 52 nucleotides, wherein the nucleotides comprise the dinucleotide core and an even number of nucleotides directly adjacent on either side of the dinucleotide core.

[0039] Embodiment 19: The genome editing system of any one of embodiments 1-18, wherein the AttD site comprises an AttP site.

[0040] Embodiment 20: The genome editing system of any one of embodiments 1-19, wherein the cellular genome is a human genome.

[0041] Embodiment 21 : The genome editing system of any one of embodiments 1-20, wherein the cellular genome does not comprise an installed attachment site.

[0042] Embodiment 22: A method for integration of a sequence of interest into a cellular genome, comprising contacting a cell with the genome editing system of any one of embodiments 1-21 such that the sequence of interest is integrated into the cellular genome.

[0043] Embodiment 23: A method for generating an engineered cell, comprising contacting a cell with the genome editing system of any one of the embodiments 1-21.

[0044] Embodiment 24: A method for introducing a sequence of interest in a cell, comprising contacting a cell with the genome editing system of any one of the embodiments 1-21.

[0045] Embodiment 25: The method of any one of embodiments 22-24, wherein the cell is mammalian, optionally wherein the cell is human.

[0046] Embodiment 26: The method of embodiment 25. wherein the cell is a T cell, natural killer cell (NK cell), non-human embryonic stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell (HSC), liver cell, muscle cell, monocyte, B cell, neuron, astrocyte, or microglial cell.

[0047] Embodiment 27: The method of embodiment 26. wherein the cell is a T cell.

[0048] Embodiment 28: The method of any one of embodiments 22-27, wherein the contacting of the cell is in vivo.

[0049] Embodiment 29: The method of any one of embodiments 22-27, wherein the contacting of the cell is ex vivo.

[0050] Embodiment 30: The method of any one of embodiments 22-29, wherein a Cas or a guide RNA is not used in the contacting of the cell.

[0051] Embodiment 31: The method of any one of embodiments 22-30, wherein the method does not comprise modifying the cell to insert an attachment site for the sequence of interest into the cellular genome.

[0052] Embodiment 32: An engineered cell comprising the genome editing system of anyone of the embodiments 1-21.

[0053] Embodiment 33: The engineered cell of embodiment 32, wherein the cell is a human cell.

[0054] Embodiment 34: The engineered cell of embodiment 32 or 33, wherein the cell is a T cell, natural killer cell (NK cell), non-human embryonic stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell (HSC), liver cell, muscle cell, monocyte, B cell, neuron, astrocyte, or microglial cell.

[0055] Embodiment 35: A method for treating a disease in a subject, comprising administering to the subject the genome editing system of any one of the embodiments 1- 21. or the engineered cell of any one of embodiments 32-34.BRIEF DESCRIPTION OF THE FIGURES

[0056] FIGS 1A-1B show cssDNA constructs used in experiments with the LSR Cp36 mRNA. FIG 1A shows a schematic for cssDNA constructs “cssDNA5’?and "cssDN A6"; FIG IB is agarose gel electrophoresis showing purification of constructs at the expected sizes.

[0057] FIGS 2A and 2B show reduced immunogenicity of cssDNA as compared to a dsDNA nanoplasmid control in THPl-Dual cells for cssDNA5 (FIG 2A) and cssDNA6 (FIG 2B)

[0058] FIGS 3A-3B show the percent integration by ddPCR using cssDNA constructs and the LSR lxFlag-Cp36 mRNA by reverse transfection with Lipofectamine 3000 in HEK293FT cells. FIG 3A shows the percent integration over a dose range; FIG 3B shows a comparison of the integration rates observed between cssDNA and dsDNA with dsDNA as the denominator.

[0059] FIG 4 shows the percent integration at the left and right sides of the integration site by ddPCR with cssDNA constructs and the LSR lxFlag-Cp36 mRNA by reverse transfection with Lipofectamine 3000 in HEK293FT cells.

[0060] FIG 5 shows the percent integration by ddPCR with cssDNA and the LSR IxFlag- Cp36 mRNA by electroporation in primary T cells.

[0061] FIGS 6A-6B show cssDNA constructs used in experiments with the LSR BxBl. FIG 6A shows the construct design for “cssDNA8’‘; FIG 6B is agarose gel electrophoresis of cssDNA showing purification at the expected size.

[0062] FIGS 7A-7B shows percent integration at the left and right sides by ddPCR with cssDNA and reverse transfection with Lipofectamine 3000 (FIG 7A) and Lipofectamine MessengerMAX (FIG 7B) in HEK293FT cells expressing BxBl and with an installed Bxbl attachment site.

[0063] FIG 8 shows percent integration at the left and right sides by ddPCR with cssDNA and LNPs in HEK293FT cells expressing BxBl and with an installed Bxbl attachment site.

[0064] FIG 9 shows the percent integration by ddPCR with cssDNA6 and lxFlag-Cp36 plasmid compared to cssDNA6 and lxFlag-Cp36-2A-RAD51 plasmid in HEK293FT cells.

[0065] FIG 10 shows structures of certain known ionizable lipids.

[0066] FIG 11 shows cssDNA reduces cGAS / STING pathway-associated immunogenicity relative to double stranded DNA or cGAMP in primary human T cells. Cells were harvested 1 hour after electroporation and analyzed by immunoblotting for levels of pTBKl, pIRF3, and pSTING.

[0067] FIG 12 shows LSR-mediated genome integration of a cssDNA donor template with an mRNA LSR variant as measured by ddPCR in HEK293FT cells 48 hours post-transfection.

[0068] FIGS 13A-B show electroporation of cssDNA and a variant LSR as mRNA results in CAR integration in primary human T cells. Cells were evaluated 7 days after electroporation for CAR expression by flow cytometry (FIG 13A) and for CAR integration by ddPCR at four genome integration sites (FIG 13B).

[0069] FIG 14 shows LNP delivery of cssDNA and a variant LSR as mRNA results in CAR positive expression in human primary’ T cells by flow cytometry on day 7 as compared to nanoplasmid.

[0070] FIGS 15A-D show delivery' of cssDNA and a variant LSR as mRNA with an oligo (FIG 15A) or with an oligo containing locked nucleic acids (FIG 15C) and integration in HEK293FT cells by ddPCR following transfection with Lipo3000 in FIG 15B and FIG 15D, respectively.

[0071] FIGS 16A-B show the efficiency of expression of a CD19-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 CD19-CAR transgene by flow cytometry (FIG 16A) or integration by ddPCR (FIG 16B). CD3-targeted LNPs encapsulated either LSR mRNA only or cssDNA donor template only, or co-encapsulated both LSR mRNA and donor template cssDNA. FIG 16A shows the percent of CAR+ T cells. FIG 16B show s the % integration in T cells at four genomic sites (GS10, GS11, GS12, and GS13).

[0072] FIGS 17A-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 CD19-CAR transgene. FIG 17A and FIG 17B 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 cytometryshowed significantly reduced B cells (FIG 17A) and no significant change in T cells (FIG 17B). 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 17C) or encapsulating cssDNA only (FIG 17D). 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 17E) or as % integration for each individual mouse dosed with CD3-targeted LNPs with LSR mRNA and / or cssDNA donor template (as indicated) (FIG 17F).

[0073] FIGS 18A-C 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 cssDNA encoding the CD 19 CAR (solid line circles) compared to LNPs encapsulating mRNA or DNA only (hashed lines) (FIG 18A). 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 18B). CD19-CAR T cells were detected in blood 10 days after LNP dosing by flow cytometry (FIG 18C).

[0074] FIGS 19A-E show cssDNA reduces cGAS pathway-associated immunogenicity in vivo in wildtype mice injected with cssDNA encapsulated in LNPs as compared to injection with nanoplasmid or plasmid. Reduced immunogenicity was evidenced by reductions in IL-6 (FIG 19A), IFN-a (FIG 19B), IFN-p (FIG 19C), and MCP-1 (FIG 19D), as well as no statistically significant change in body weight (FIG 19E).

[0075] FIGS 20A-B show- testing of five ratios of cssDNA to LSR mRNA in coencapsulated LNP formulations in primary T cells. FIG 20A shows CAR expression and FIG 20B shows percent integration at the GS 10 integration site four days after dosing with LNP.

[0076] FIGS 21 A-E show cssDNA produced by an enzymatic conversion method is active as a template for LSR-mediated integration in primary T cells. FIG 21A is a schematic illustrating the process of converting double-stranded DNA into cssDNA and FIG 21B is a schematic of the starting plasmid used. FIG 21C shows gel electrophoresis of the reaction products. FIG 21D is a chromatogram showing elution of cssDNA as measured by OD260m. FIG 2 IE shows percent integration of cssDNA in primary T cells from two donors.DETAILED DESCRIPTION

[0077] Disclosed herein are non- viral compositions and systems for delivery and integration of a gene to a cell. The inventors have surprisingly determined that cssDNA delivered in anLNP can be used to successfully integrate a sequence of interest into the cellular genome using an LSR and cssDNA comprising an AttD and a sequence of interest, and further that such integration can be achieved in primary T cells without eliciting an immune response.

[0078] The disclosure provides compositions and gene editing systems comprising cssDNA and an LSR in LNP formulations that are capable of inserting a sequence of interest into a cellular genome without installation of an attachment site in the cellular genome. Such compositions and genome editing systems disclosed herein result in expression of the sequence of interest in the cell.

[0079] The disclosure further provides methods for integrating a sequence of interest (e.g., a gene) from the cssDNA in a genome of a subject, treating a disease by integrating a sequence of interest (e.g., a gene) from the cssDNA in a genome of a subject, or generating an engineered cell, or a cell that expresses a new protein or has a new function through the introduction of a sequence of interest (e.g., a gene) from the cssDNA, that may be administered to a subject, thereby treating a disease or disorder using the genome editing systems disclosed herein.I. DEFINITIONS

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.IL COMPOSITIONS AND GENOME EDITING SYSTEMS

[0086] Disclosed herein are compositions and genome editing systems comprising cssDNA having a sequence of interest for: integration of the sequence of interest into a cell (e.g., with an LSR as disclosed herein) for expression of a transgene or silencing of target genes (e.g., with a sequence of interest encoding an miRNA or shRNA). In some embodiments, the sequence of interest encodes a chimeric antigen receptor (CAR). In particular, disclosed herein are compositions, systems, and methods for delivering in vivo an LNP comprising a genome editing system for integrating a sequence of interest into a cell. As shown in the Examples herein, for instance, where the sequence of interest on the cssDNA encodes a CAR, administration of an LSR mRNA and a cssDNA co-encapsulated in an LNP formulation surprisingly supported CAR-T levels sufficient for treating disease. In some embodiments herein, compositions, genome editing systems, or LNPs herein show reduced immunogenicity in treated subjects compared to a dsDNA composition, genome editing system, or LNP that encodes the sequence of interest.

[0087] In some embodiments, compositions are provided comprising (a) an mRNA encoding an LSR; b) cssDNA comprising an AttD and a sequence of interest, (c) a lipid nanoparticle, and optionally (d) an additional component.

[0088] In some embodiments, the composition comprises a) an mRNA encoding an LSR; b) cssDNA comprising an AttD and a sequence of interest, and (c) a lipid nanoparticle. In some embodiments, the sequence of interest encodes a therapeutically relevant protein. In some embodiments, the sequence of interest encodes a CAR. In some embodiments, the cssDNA in the composition has reduced immunogenicity in a subject as compared to a composition comprising dsDNA that encodes the sequence of interest. This reduced immunogenicity may be shown by lower levels of inflammatory cytokines, such as IL-6, IFN-beta, IFN-alpha, and / or MCP-1, and / or by no significant changes in body weight in subjects compared to those treated with a composition comprising dsDNA that encodes the sequence of interest. In some embodiments, no additional component (d) is present. For example, in some embodiments, (a), (b), and (c) are present but (d) is not present. In some embodiments, the ratio of b) to a) is 3: 1 to 1 :3. In some embodiments, the ratio of b) to a) is 2: 1 to 1 :2. In some embodiments, the ratio of b) to a) is 1 : 1.

[0089] In some embodiments, the genome editing system comprises a nucleic acid encoding a large serine recombinase (LSR), and cssDNA comprising a donor attachment site (AttD) anda sequence of interest to a cell, wherein the sequence of interest is integrated into the cellular genome. In some embodiments, the nucleic acid encoding the LSR is an mRNA.

[0090] In some instances, composition or the genome editing system comprises a single lipid-based formulation comprising the mRNA encoding an LSR and a cssDNA comprising an AttD and a sequence of interest (z.e., co-encapsulation). In some embodiments, the genome editing system comprises two lipid-based formulations, wherein one lipid-based formulation comprises the mRNA encoding an LSR, and the other lipid-based formulation comprises the cssDNA comprising an AttD and a sequence of interest (z.e., separate encapsulation). In some embodiments, the lipid-based formulation is an LNP. In some embodiments, the ratio of b) to a) in the genome editing system is 3: 1 to 1 :3. In some embodiments, the ratio of cssDNA to mRNA is 2: 1 to 1 :2. In some embodiments, the ratio of cssDNA to mRNA in the genome editing system is 1 : 1.

[0091] In some embodiments, the genome editing system comprises an LSR, a cssDNA comprising an AttD and a sequence of interest, and an additional component. In some embodiments, the additional component enhances nuclear import of the cssDNA into the nucleus of the cell. For example, in some embodiments, the additional component is a singlestranded 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 cssDNA by increasing the formation of double-stranded DNA from the cssDNA in the cell. In some embodiments, the additional component is a nucleic acid (e.g., an oligo) that increases the formation of double-strand DNA from the cssDNA in the cell. In some embodiments, the additional component is an oligo. In some embodiments, the additional component is an antisense oligo. In some embodiments, the additional component is an oligo comprising a modified nucleic acid, optionally a locked form of DNA.A. LARGE SERINE RECOMBINASE COMPONENT

[0092] The present disclosure provides compositions and genome editing systems for expression and delivery of mRNA encoding large serine recombinases (LSRs) and cssDNA comprising an AttD site and a sequence of interest to a cell for integration of the sequence of interest. As used herein, LSR refers to a protein having an amino acid sequence of a naturally- occurring LSR, as well as variants of such LSRs, including sequence modifications, mutants, deletions, conjugates, chemical modifications, LSR-fusion proteins, truncations and the like. In some embodiments, the cssDNA and the mRNA encoding the LSR are sufficient for expression of the sequence of interest in the cell, such that an additional component is not required. In some embodiments, the LSR is a “genome targeting LSR” (i.e., an LSR that canintegrate DNA primarily at an integration site in the genome without any installed attachment site). Thus, in some embodiments, the LSR is not a "‘landing pad LSR” (i.e., an LSR that requires that the cell be modified to insert an attachment site as the primary site for integration into the cellular genome).

[0093] 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, <DpC31, RDF, ©BT1, RI. R2, R3. R4. R5. TP901-1. Al 18. ®FC1, ©Cl. MR1 1. TGI, ©370.1, W , BL3, SPBc. K38, or variants thereof. See e.g., Durrant et al.. Nature Biotechnology 41, 488-499 (2023); Yamall et al., 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.

[0094] 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.

[0095] 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), Nanodropspectrophotometer (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.

[0096] In some embodiments, the LSR is capable of integrating a sequence from a nucleic acid (e.g., cssDNA) into a 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.

[0097] 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).

[0098] In some embodiments, the genome editing system comprises 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 cell-penetrating 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 domaincomprises 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.

[0099] 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.

[0100] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO: 2024 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: 2024.

[0101] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO:2029 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: 2029.

[0102] In some embodiments, the LSR comprises the amino acid sequence of SEQ ID NO:2030 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: 2030.

[0103] 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 polyprimidine tract, or a combination or variant thereof.

[0104] The LSRs of the genome editing systems disclosed herein can be prepared using suitable methods known in the art. In some embodiments, the mRNA is prepared by in vitro transcription.B. CIRCULAR SINGLE-STRANDED DNA COMPONENT

[0105] The cssDNA of the compositions and genome editing systems 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.

[0106] In some embodiments, the cssDNA does not comprise a sequence from an fl ori. In some embodiments, the cssDNA comprises a sequence from an fl ori. In some embodiments, cssDNA does not comprise an antibiotic resistance gene. In some embodiments, cssDNA comprises an antibiotic resistance gene. In some embodiments, the cssDNA does not comprise a sequence from the bacterial replication ori. In some embodiments, the cssDNA comprises a sequence from the bacterial replication ori. In some embodiments, the cssDNA does not comprise a packaging signal. In some embodiments, the cssDNA comprises a packaging signal. In some embodiment, sequences phagemid sequences are removed from the cssDNA.

[0107] In some embodiments, the cssDNA is purified to reduce the endotoxin level resulting from preparation in a bacterial cell. In some embodiments, the endotoxin level of the cssDNA is less than 0.1 EU / pg, 0.05 EU / pg, 0.01 EU / pg, or 0.005 EU / pg. In some embodiments, the endotoxin level of the cssDNA is less than 0.1 EU / pg. In some embodiments, the endotoxin level of the cssDNA is less than 0.05 EU / pg. In some embodiments, the endotoxin level of the cssDNA is less than 0.01 EU / pg. In some embodiments, the endotoxin level of the cssDNA isless than 0.005 EU / pg. The endotoxin level of the cssDNA disclosed herein can be reduced using commercially available kits and filtration methods. In some embodiments, the Norgen Biotek Endotoxin Removal Kit is used to optimally reduce the endotoxin level of the cssDNA.

[0108] In some embodiments, the cssDNA is produced using an enzymatic conversion method starting from dsDNA. See e.g., Tang et al., Nature Communications 14: 6665 (2023). The dsDNA starting material may be a plasmid or nanoplasmid. In some embodiments, the cssDNA for use herein is produced from a dsDNA plasmid or nanoplasmid containing BbvCI sites. The dsDNA is nicked and then degraded with an exonuclease, resulting in cssDNA that may be purified using column purification methods.

[0109] In some embodiments, the cssDNA 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.

[0110] In some embodiments, the cssDNA of the disclosed compositions and genome editing systems comprises about 0.3 to 20 kb. In some embodiments, the cssDNA 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 cssDNA 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.

[0111] In some embodiments, the cssDNA comprises about 5 to 13kb. In some embodiments, the cssDNA comprises about 5 to lOkb. In some embodiments, the cssDNA comprises about 5 to 9kb. In some embodiments, the cssDNA comprises about 5 to 8kb. In some embodiments, the cssDNA comprises about 5 to 7kb. In some embodiments, the cssDNA comprises about 5 to 6kb.1. AttD site

[0112] In some embodiments, the cssDNA component of the compositions and genome editing system comprises an AttD site. LSRs of the disclosed compositions and 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 cssDNAvia its associated AttD site “pairing” with a cognate AttA site in the cellular genome. In the compositions and genome editing systems encompassed herein, the cssDNA comprises an AttD site that will function with the LSR to integrate a sequence of interest in the cellular genome.

[0113] In some embodiments, the cssDNA comprises more than one AttD site (i.e., an “array" of AttDs). In some embodiments, the cssDNA comprises two AttDs. In some embodiments, the cssDNA comprises three AttDs. In some embodiments, the cssDNA comprises four AttDs. In some embodiments, the cssDNA comprises five AttDs. In some embodiments, the cssDNA comprises six AttDs. In some embodiments, the cssDNA comprises seven AttDs. In some embodiments, the cssDNA comprises eight AttDs. In some embodiments, the cssDNA comprises nine AttDs. In some embodiments, the cssDNA comprises ten AttDs.

[0114] 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(l l):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(l l):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: 2025 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 nucleotide sequence of SEQ ID NO: 2025.

[0115] 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 site comprises 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.

[0116] 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.2. Sequence of interest

[0117] In some embodiments, the cssDNA component of the composition or genome editing system comprises a sequence that encodes one or more sequences of interest for integrating into a cellular genome. In some embodiments, the sequence of interest is a noncoding 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 (i.e., a portion of a gene encoding a particular domain of a protein, or an open reading frame encoding a protein or protein domain derived from a gene, an exon from a gene or series of exons from a gene, or a cDNA sequence encoding a protein, wherein the portion of the gene is associated with a function). 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 encodes 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. In some embodiments the sequence of interest comprises a sequence that encodes a therapeutic protein (i.e., a protein that may be used as a therapeutic agent to treat, manage or prevent a disease). In some embodiments the sequence ofinterest encodes a therapeutic RNA. 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 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 encodes an miRNA. In some embodiments, the sequence encodes an shRNA. In some embodiments, the sequence encodes a circRNA.

[0118] 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.

[0119] 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 clotting factor. In some embodiments, the sequence of interest encodes a cytokine. In some embodiments, the sequence of interest encodes a hormone. In some embodiments, the sequence of interest encodes an enzyme. In some embodiments, the sequence of interest encodes an antibody, or a portion thereof. 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.

[0120] In some cases, the term “transgene” is used herein to refer to the sequence of interest for integration in the cellular genome.

[0121] In some embodiments, the cssDNA comprises a sequence of interest and further comprises a promoter for expression of the sequence of interest. In some embodiments, thepromoter 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 [3 actin (CBA). CMV early enhancer / chicken 0 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-l-antitrypsin promoter.

[0122] In some embodiments, the sequence of interest of the cssDNA comprises about 0.3 to 20 kb. In some embodiments, the sequence of interest of the cssDNA 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 cssDNA 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 1 1 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.

[0123] In some embodiments, the cssDNA comprises about 5 to 13kb. In some embodiments, the cssDNA comprises about 5 to lOkb. In some embodiments, the cssDNA comprises about 5 to 9kb. In some embodiments, the cssDNA comprises about 5 to 8kb. In some embodiments, the cssDNA comprises about 5 to 7kb. In some embodiments, the cssDNA comprises about 5 to 6kb.C. LIPID-BASED FORMULATIONS

[0124] In some embodiments, the composition or genome editing system comprises a lipid- based formulation (e.g., a lipid nanoparticle) comprising 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 (see Figure 10).

[0125] 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.

[0126] 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, 0-sitosterol, Sitosterol, or DC cholesterol.

[0127] 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 Cl 8- PEG. In some embodiments, the PEG molecular weight can vary between 500 Da-10000 Da. In some embodiments, the PEG molecular weight is between 500 Da-4000 Da. In some embodiments, the PEG molecular weight is betw een 2000 Da-4000 Da. In some embodiments, the PEG molecular w eight is between 2000 Da-3500 Da.

[0128] 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-based formulation 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. In some embodiments, the lipid- based formulation comprises between about 35-50% ionizable lipid, about 15-25% helper lipid, about 23-40% cholesterol, and about 1-5% PEG lipid, wherein the total percentage equals 100%. In some embodiments, the lipid-based formulation comprises between about 45-50%ionizable lipid, about 20-22% helper lipid, about 23-28% cholesterol, and about 2-3% PEG lipid, wherein the total percentage equals 100%. In some embodiments, the lipid-based formulation comprises 50% ionizable lipid, 22% helper lipid, 25% cholesterol, and about 3% PEG lipid, wherein the total percentage equals 100%. In some embodiments, the PEG lipid % in the lipid-based formulation comprises a modified PEG lipid (i.e., a PEG lipid molecule that has been chemically modified). In some embodiments, the PEG lipid % in the lipid-based formulation comprises an unmodified PEG lipid and a modified PEG lipid.

[0129] 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.

[0130] 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.

[0131] In some embodiments, the lipid-based formulation further comprises a polypeptide. Iln some embodiments, the lipid-based formulation comprises a targeting moiety. In some embodiments, the targeting moiety is a polypeptide (e.g., an antibody or antibody fragment). In some embodiments, the lipid-based formulation further comprises a small molecule, peptide, oligo (e.g., siRNA or ASO) encapsulated in the formulation. In some embodiments, the targeting moiety is a small molecule.

[0132] In some embodiments, the targeting moiety is an antibody or antibody fragment. In some embodiments, the targeting moiety is a bispecific antibody or bispecific antibody fragment. In some embodiments, the targeting moiety is a Fab. In some embodiments, the targeting moiety7is an scFv. In some embodiments, the targeting moiety is a nanobody. In some embodiments, the targeting moiety is an antibody or antibody fragment that binds to CD3, CD4. CD5, or CD8. In some embodiments, the targeting moiety is / V-acetylgalactosamine (GalNAc) targeting ligand. In some embodiments, the lipid-based formulation comprises a GalNAc lipid. In some embodiments, the targeting moiety targets the lipid-based formulation to T cells. In some embodiments, the targeting moiety targets the lipid-based formulation to liver cells.D. ADDITIONAL COMPONENT

[0133] In some embodiments, the composition, or genome editing system further comprises an additional component that enhances integration of the cssDNA. In other embodiments, no such additional component is included. In some embodiments, where present, an additional component enhances integration of the cssDNA by increasing the import of the cssDNA into the nucleus of the cell. In some embodiments, an additional component enhances integration of the cssDNA by increasing the import of the cssDNA into the nucleus of the cell from an LNP. In some embodiments, an additional component enhances integration of the cssDNA by increasing the formation of double-stranded DNA from the cssDNA in the cell.

[0134] Such enhancements of integration may be measured, for e.g., 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 (z. e. , 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.

[0135] In some embodiments, the composition or genome editing system further comprises a nucleic acid encoding a single-stranded DNA-binding protein. In some embodiments, the mRNA encodes the single-stranded 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, the single-stranded DNA binding protein is a separate nucleic acid from the mRNA LSR. In some embodiments, the single-stranded DNA binding protein is a separate mRNA. In some embodiments, the single-stranded DNA binding protein is a separate DNA. In some embodiments, the single-stranded DNA binding protein is 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).

[0136] In some embodiments, the composition or genome editing system further comprises a cis sequence element that e.g., enhances nuclear import of the cssDNA. In some embodiments, the cssDNA encodes the cis sequence element. In some embodiments, the cis sequence element is a separate nucleic acid from the cssDNA. 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 HMGB 1.

[0137] In some embodiments, the composition or genome editing system further comprises a nucleic acid that increases double-strand formation of the cssDNA in the cell. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is a modified DNA. In some embodiments, the nucleic acid is an oligo. In some embodiments, the nucleic acid is a locked form of DNA. In some embodiments, nucleic acid has a 3’ hydroxyl group. In some embodiments, the nucleic acid is 40 nucleotides. In some embodiments, the nucleic acid is less than 40 nucleotides. In some embodiments, the nucleic acid is less than 39. 38. 37. 36. 35, 34, 33, 32, 31, 30, 25, or 20 nucleotides. In some embodiments, the nucleic acid is between 40 and 90 nucleotides. In some embodiments, the nucleic acid is between 40 and 80 nucleotides. In some embodiments, the nucleic is between 40 and 70 nucleotides. In some embodiments, the nucleic acid is between 40 and 60 nucleotides. In some embodiments, the nucleic acid is between 40 and 50 nucleotides. In some embodiments, the nucleic acid is an antisense oligo. In some embodiments, the nucleic acid is an sense oligo.

[0138] In some embodiments, the additional component is an oligo comprising a modified nucleic acid. In some embodiments, the additional component is an oligo comprising a locked form of DNA. In some embodiments, an oligo hybridizes to the cssDNA and comprises the locked form of DNA (also called locked nucleic acid or LNA). In some embodiments, the modified nucleic acid is an LNA. In some embodiments, the modified nucleic acid is selected from any one of: phosphothioate, 2’0-Methyl, 2'-Floro, 2’, 2’methoxyethyl (MOE), phosphorodiamidate Morpholino oligomer (PMO), cEt (constrained ethyl bridged nucleic acid), ENA (ethylene-bridged nucleic acid), PNA (peptide-nucleic acid), tcDNA (tricyclo DNA).

[0139] In some embodiments, the oligo hybridizes to at least a portion of the AttD site in the cssDNA. In some embodiments, the LNA is located on the oligo at least 5, 10, 15, 20, or 25 nucleotides away from the dinucleotide core of the AttD site.III. NUCLEIC ACID MODIFICATIONS

[0140] In some embodiments, the mRNA and / or the cssDNA in the genome editing system is modified. Any DNA or mRNA modification known to those of skill in the art is encompassed. In some embodiments, a modified mRNA or DNA is synthesized with a non- canonical nucleoside or nucleotide. Modifications such as those listed herein can be combined to provide modified mRNA or DNA comprising nucleosides and nucleotides that can have two, three, four, or more modifications. In some embodiments, every base of a mRNA or DNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In some embodiments, modified mRNA 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.

[0141] In some embodiments, the mRNA or DNA comprises 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 mRNA or DNA are modified nucleosides or nucleotides.

[0142] In some embodiments, the mRNA modification comprises a uridine modification. In some embodiments, the mRNA modification comprises 1 -methyl-pseudo uridine. In some embodiments, the mRNA modification comprises pseudouridine. In some embodiments, the mRNA modification comprises a replacement of each uridine for 1-methyl-pseudouridine. In some embodiments, the mRNA modification comprises a replacement of each uridine for pseudouridine.

[0143] Some embodiments encompass a phosphate backbone modification. Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyd 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 moi eties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, 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.

[0144] Some embodiments encompass a sugar modification. For example, the 2' hydroxyl group (OH) 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 methoxy ethyl 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, heteroarylamino, 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, cycloalkyd, ary 1, 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

[0145] In some aspects, methods are provided for generating an engineered cell, comprising contacting a cell with any one of the compositions, or genome editing systems disclosed herein. In some embodiments, methods are provided for introducing a sequence of interest in a cell, comprising contacting the cell with any7one of the compositions, or genome editing systems disclosed herein. In some embodiments, methods for treating disease in a subject are disclosed, comprising administering to a subject any one of the compositions, or genome editing systems, or engineered cells disclosed herein. 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 sequence of interest is a CAR. In some embodiments, the cell is a T cell.

[0146] In some aspects, the present disclosure provides a method for integration of a sequence of interest into a cellular genome comprising contacting a cell with any one or moreof the disclosed compositions or genome editing systems such that the sequence of interest is integrated into the cellular genome. In some embodiments, the method occurs in vivo. In some embodiments, the method occurs in vitro. In some embodiments, the method occurs ex vivo.

[0147] In some embodiments, a method of editing chromosomal locations in a human cell comprises contacting the human cell with any one or more of the disclosed compositions or genome editing systems such that the recombination activity of the LSR results in a sequence of interest of the composition or genome editing system 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.

[0148] In some aspects, the present disclosure provides a method of integrating a sequence of interest into the GS10 integration site in an immune cell using a composition or genome editing system, such that the protein encoded by the sequence of interest is expressed by the cell. The GS10 integration site is chrl0:69637352-69637354 (Hg38 genome). In some embodiments, the immune cell is a T cell. In other embodiments, the immune cell is a B cell, NK cell, lymphocyte, or myeloid cell. In some embodiments, the protein encoded by the sequence of interest is a therapeutic protein. In some embodiments, the protein encoded by the sequence of interest is an antigen receptor, for example a CAR. In some embodiments, the composition or genome editing system comprises (a) an mRNA encoding an LSR and (b) a cssDNA comprising an AttD and a sequence of interest (for example, encoding a therapeutic protein, antigen receptor, or CAR). In some embodiments, the composition or genome editing system further comprises a lipid-based formulation comprising (a) and (b). In some embodiments, the lipid-based formulation comprises an LNP. In some embodiments, the LSR comprises at least 90%, 95%, 99%, or 100% identity to the amino acid sequence of SEQ ID NOs: 2024, 2029, or 2030. In some embodiments, the cssDNA comprises SEQ ID NO: 2025. In some embodiments, the ratio of b) to a) in the LNP is 3: 1 to 1 :3. In some embodiments, the ratio of b) to a) in the LNP is 2: 1 to 1 :2. In some embodiments, the ratio of b) to a) in the LNP is 1: 1.

[0149] In some embodiments, the composition or gene editing system after introduction into the cell is less immunogenic than a double-stranded DNA or plasmid control. For example, in some embodiments, the gene editing system has reduced levels of phosphorylated proteinspTBKl, pTIRF3, and / or pSTING following integration into primary T cells compared to a double-stranded DNA control. In some embodiments, the gene editing system results in reduced levels of IL6, IFNa, IFNb, and / or TNFa in vivo compared to a double-stranded DNA control.

[0150] In some embodiments, the methods disclosed herein result in the generation of a CAR-T cell. In some embodiments, wherein a Cas or a guide RNA is not used in the contacting of the cell.

[0151] The present disclosure provides a method of treating a disease in a subject using the compositions or genome editing systems disclosed herein. In some embodiments, the disease is cancer. 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.

[0152] In some embodiments, the method comprises administering an effective amount of a composition or genome editing system to a subject, wherein the sequence of interest of the composition or genome editing system integrates into the subject’s genome, thereby treating the subject’s disease.

[0153] In some embodiments, methods are provided for contacting a subject with a composition or genome editing system comprising (a) an mRNA encoding an LSR, (b) a cssDNA comprising an AttD and sequence of interest, wherein (a) and (b) are comprised in a lipid-based formulation comprising an LNP, wherein the sequence of interest encodes a CAR, thereby generating CAR+ cells in the subject. In some embodiments, the cells are immune cells such as T cells. In some embodiments, the cssDNA has reduced immunogenicity in the subject as compared to dsDNA, such that the level of CAR+ cells is sufficient to treat cancer in the subject. In some embodiments, reduced immunogenicity may be shown by lower levels of inflammatory cytokines, such as IL6, IFN-alpha, IFN-beta, or MCP-1 in the subject compared to those for a subject administered a composition or genome editing system comprising dsDNA encodes the sequence of interest. In some embodiments, reduced immunogenicity may be shown by a lack of significant change in body weight in the subject, and / or a lack of a significant increase in one or more of an inflammatory cytokine, such as IL6, IFN-alpha, IFN- beta, or MCP-1 in the subject following administration of the composition or system. In some embodiments, the cells are T cells and the levels of CAR-T cells in the subject are sufficient to deplete B cells in the subject. In some embodiments, the cells are T cells and the levels of CAR-T cells are sufficient to reduce tumor burden in the subject. In some embodiments, the LSR comprises at least 90%, 95%, 99%, or 100% identity to the amino acid sequence of SEQ ID NOs: 2024, 2029, or 2030. In some embodiments, the cssDNA comprises SEQ ID NO: 2025. In some embodiments, the ratio of b) to a) in the LNP is 3: 1 to 1 :3. In some embodiments, the ratio of b) to a) is 2: 1 to 1 :2. In some embodiments, the ratio of b) to a) in the LNP is 1: 1.

[0154] In some aspects, the present disclosure provides methods for treating a disease in a subject by administering any one or more of the disclosed compositions or genome editing systems to a patient a need thereof, thereby generating an engineered cell within the patient. In some aspects, the present disclosure provides methods for treating a disease in a subject by generating an engineered cell using any one or more of the disclosed compositions or genome editing 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 cells with an effective amount of any one or more of the disclosed compositions or genome editing systems to yield engineered cells and (ii) administering the engineered cells to the subject, thereby treating the disease. In some embodiments, the subject is a human. In some embodiments, the disease is caused by a genetic mutation.

[0155] Any suitable cell type may be used in the embodiments disclosed herein. In some embodiments, the cell or genome is a T cell, a natural killer cell, an induced pluripotent stem cell, a hematopoietic stem cell, a liver cell, a muscle cell, a B cell, a fibroblast, a neuron, an astrocyte, or a microglial cell.

[0156] In some embodiments, the cell is a T cell. In some embodiments, the cell is a natural killer cell. In some embodiments, the cell is an induced pluripotent stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments, the cell is a liver cell. In some embodiments, the cell is a muscle cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a fibroblast. In some embodiments, the cell is a neuron. In some embodiments, the cell is an astrocyte. In some embodiments, the cell is a microglial cell.

[0157] The number of administrations of treatment to a subject may vary. Introducing the engineered cells 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 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.

[0158] The present disclosure provides a composition or genome editing system for use in treating a disease in a subject, wherein the use comprises administering an effective amount ofthe composition or genome editing system to the subject or contacting isolated cells with an effective amount of the composition genome editing system to yield engineered cells and administering the engineered cells to the subject. In some embodiments, the disease is cancer. In some embodiments, the disease is caused by a genetic mutation. In some embodiments, the use comprises administering an effective amount of the composition or genome editing system to the subject. In some embodiments, the use comprises contacting isolated cells with an effective amount of the composition or genome editing system to yield engineered cells and administering the engineered cells to the subject.

[0159] In some embodiments, the engineered cells disclosed herein are CAR-T cells. In some embodiments, the levels of CAR-T cells administered to the subject are sufficient to deplete B cells in the subject. In some embodiments, the levels of CAR-T cells are sufficient to reduce tumor burden in the subject.

[0160] The present disclosure provides uses of a composition or genome editing system 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 is for treating cancer.V. PHARMACEUTICAL COMPOSITIONS

[0161] The compositions and genome editing systems of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers, diluents, or vehicles. 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 subject 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

[0162] 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 present disclosure 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 nonlimitingexample, 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.

[0163] 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, embodiments, 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.

[0164] 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.

[0165] The abbreviation, “e.g..” is derived from the Latin exempli gratia, and is used herein to indicate anon-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.”

[0166] 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.

[0167] 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.

[0168] Section headings, materials, methods, and examples are illustrative only and not intended to be limiting.EXAMPLESExample 1: Preparation of circular single-stranded DNA

[0169] Circular single-stranded DNA constructs were prepared for evaluation as compared to a double-stranded DNA plasmid. Two cssDNA constructs were prepared for use with the Cp36 LSR: 1) “cssDNA5” (SEQ ID NO: 4000) encoded 5’ to 3’ - Cp36 corresponding AttDsite (SEQ ID NO: 2001), EFla, mCherry. bGHpA, fl ori, ColEl, AmpR; 2) “cssDNAO” (SEQ ID NO: 4001) encoded 5’ to 3’ - Cp36 corresponding AttD site (SEQ ID NO: 2001), EFla, mCherry, bGHpA, fl ori. See Figure 1A.

[0170] For experiments with Bxbl, the following cssDNA construct ‘'cssDNA8” (SEQ ID NO: 4002) was prepared encoding 5‘ to 3’ - Bxbl corresponding AttD site (SEQ ID NO: 2003), mCherry, bGHpA, fl ori. See Figure 6A.

[0171] The cssDNA constructs were prepared as follows. A phagemid plasmid encoding either cssDNA5, cssDNA6, or cssDNA8 and helper plasmid were co-transformed in the E. coll XLl-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 shakerincubator 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-incubator 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 w ere 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 w as gently removed and the phage-PEG / NaCl pellets were redispersed in 20 mL lx TBS buffer using vortexing. 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, MgCh, 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 w as 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 further purified using Norgen Biotek Endotoxin Removal Kit (Mini) to reducethe endotoxin level of cssDNA. The concentration of cssDNA was measured using Nanodrop and the endotoxin level of cssDNA was measured using Endosafe nexgen-PTS.

[0172] Agarose gel electrophoresis of cssDNA showing the purified cssDNA constructs at the expected sizes is shown in Figure IB (for Cp36) and Figure 6B (for BxBl).Example 2; Reduced immunogenicity with cssDNA

[0173] The immunogenicity of cssDNA was compared to dsDNA in THPl-Dual™ Cells (thpd-nfis, InvivoGen). The cssDNA constructs from Example 1 were used. The dsDNA construct was a nanoplasmid (SEQ ID NO: 4003) encoding 5’ to 3’: BxblAttA, PaOlAttD, Si74AttD and Cp36AttD, ApoE, hAAT, hEPO, 2A, Firefly. Nanoplasmid was synthesized from Genscript (<0.01 EU / pg).

[0174] The endotoxin level of the cssDNAs was reduced using Norgen Biotek Endotoxin Removal Kit. After the removal process, the endotoxin levels of cssDNA5 and cssDNA6 were 0.049 EU / pg and 0.042 EU / pg, respectively. The endotoxin level of cssDNA8 was 0.016 EU / pg.

[0175] THPl-Dual™ Cells were seeded at 50,000 cells / well in a round-bottom 96-well plate in 100 pL of media consisting of RPMI 1640 (Gibco), 9% FBS (Gibco). and lx Pen-Strep (Gibco). After one hour, 2 pg of cssDNA or dsDNA was added to 100 pL LyoVec (lyec-1, InvivoGen) and briefly pipette mixed. The solution was incubated at room temperature for 15 min before being dosed to the cells. After overnight incubation, 20 pL of the supernatant in each well was pipetted into anew white 96-well plate. Luciferase dye (rep-qlc41gl, InvivoGen) was diluted 1 :20 with water, and 50 pL of the diluted luciferase solution was added to each well. The mixture was immediately measured with a luminometer (SpectraMax i3x) with a 0.1 second reading time. Measurements are plotted as luminescence fold over background, where background refers to non-treated cell conditions.

[0176] Figures 2A and 2B show the luciferase fold over background with increasing dose for cssDNA5 and cssDNA6, respectively, and show that immunogenicity of the cssDNAs was lower than the dsDNA.Example 3: Integration of cssDNA in HEK293FT cells with LSR Cp36

[0177] Genome integration of cssDNA was evaluated in HEK293FT cells over a range of doses using cssDNA constructs from Example 1. The dsDNA construct was dsDNA plasmid (“pAF999’‘; SEQ ID NO: 4004) encoding 5’ to 3’: Cp36 corresponding AttD site (SEQ ID NO: 2001), EFla, PuroR, 2A, mCherry, bGHpA, pUC, AmpR. The LSR used for testing was lxFlag-Cp36 (lxFlag-Cp36 mRNA (SEQ ID NO: 2000)). See Durrant et al., NatureBiotechnology 41, 488-499 (2023); WO2023081762. The mRNA encoding lxFlag-Cp36 was purchased from TriLink technologies.

[0178] The lxFlag-Cp36 mRNA and DNA (either cssDNA or dsDNA), were delivered to HEK293FT cells by reverse transfection in a 96-well plate on the first day. 100 ng mRNA / well was prepared with Lipofectamine MessengerMAX according to the manufacture’s protocol and different doses of DNA (either cssDNA or dsDNA) were prepared with Lipofectamine 3000. mRNA mixture and DNA mixture were then added to each well. 30,000 (Figure 3) or 60,000 (Figure 4) cells were seeded in one well of 96-well plate. After 48 hours incubation, cells were harvested, and the total DNA was extracted for ddPCR using Quick-DNA 96 kit (Zymo Research, D3010).

[0179] Percent integration was measured by ddPCR. ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (Bio-Rad) according to the manufacturer’s protocol. Each test was prepared in a 22 pL mixture including 5.5 pL of 4x ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad), forward / reverse primers (900 nM), FAM or HEX labelled PrimeTime™ qPCR probes (250 nM), 4 units of Hindlll-HF restriction enzyme (NEB) and 2.2 pL of DNA templates (20-50 ng). The mixture was used for droplet generation by the Automated Droplet Generator. The generated droplet was amplified by PTC Tempo Deepwell Thermal Cycler with an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s, and annealing-extension at 60 °C for 60 s. Enzyme inactivation was performed at 98 °C for 10 min and an infinite incubation at 4 °C. After amplification, the droplet was subjected to quantification by the QX600 Droplet Reader (Bio-Rad). Data analysis was performed with the QX Manager. The integration rate was defined as the concentration of integration target divided by that of a reference gene. For the top integration site, the test was performed with both the target and reference primer / probe in duplicate. A gBlock was also generated to test the sensitivity of the primer / probe design.

[0180] Primers and probe were designed using PrimerQuest (IDT) by default setting and ordered from IDT. dsDNA and ssDNA templates used the same primers and probe when evaluating integration at the left side but a different forward primer for integration on the right side. A gBlocks Gene Fragment (dsDNA from IDT) of each expected template sequence was ordered to validate the primer / probe design and measure the sensitivity of the assay. Primers and probes sequences are shown in Table 1 below.Table 1: ddPCR reagents for Cp36

[0181] Results for percent integration with Cp36 are shown in Figure 3A. The integration rate of cssDNA to dsDNA was calculated for three doses (100, 500. and 1000 ng) and is shown in Figure 3B with cssDNA reaching -60-70% of the integration rate of dsDNA.

[0182] In a separate experiment with Cp36, increasing doses of cssDNA were evaluated. As above, lxFlag-Cp36 mRNA and DNA (either cssDNA or dsDNA) were delivered to cells by reverse transfection with Lipofectamine on day 1. After 48 hours, total DNA was extracted and integration rates were assessed by ddPCR for the left and right sides. Results for percent integration with cssDNA for the left and right sides are show n in Figure 4.Example 4; Integration of cssDNA in primary T cells by electroporation with LSR Cp36

[0183] Genome integration of cssDNA was evaluated in primary T cells over a range of cssDNA doses using the cssDNA6 construct from Example 1. The dsDNA construct was dsDNA plasmid (“pAF999”; SEQ ID NO: 4004) encoding 5’ to 3’: Cp36 corresponding AttD site (SEQ ID NO: 2001), EFla, PuroR, 2A, mCherry, bGHpA, pUC, AmpR. The LSR used for testing was lxFlag-Cp36 (lxFlag-Cp36 mRNA (SEQ ID NO: 2000)). The mRNA encoding lxFlag-Cp36 was purchased from Trilink technologies.

[0184] Twenty million frozen human primary T cells were thawed and seeded in a T75 tissue culture flask in 20 mL of X-Vivo-15 (Lonza BP04-774Q) medium consisting of 2% human serum (Valley Biomedical), CD3 / CD28 Dynabeads at a 3: 1 bead to cell ratio (Gibco, 11132D), and supplemented with 100 lU / mL IL-2 (STEMCELL Technologies) every 2-3 days. After 48-72 hours of activation, T cells were harvested and counted.

[0185] The harvested cells we concentrated by centrifugation at 500 g for 5 min. The cell supernatant was removed, and the cells were resuspended in P3 Primary Cell Solution (Lonza, V4XP-3032) supplemented with Supplement 1 (Lonza, V4XP-3032). Each well of a 12-well nucleocuvette strip (Lonza, V4XP-3032) was loaded with 19 pL of P3 solution containing 200,000 cells and along with 1 pL containing DNA and / or RNA as indicated in Figure 5. The cuvette was placed in the 4D-Nucleofector (Lonza, AAF-1003X) and cells were transfected with program EO-115.

[0186] After electroporation, cells were immediately seeded in a 96-well plate round bottom plate (Coming, 351177) at 50,000 cells / well and diluted in media to 150 pL. After 48-72 hours of activation, cells were harvested for ddPCR.

[0187] ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (Bio-Rad) according to the manufacturer’s protocol. Each test was prepared in a 22 pL mixture including 5.5 pL of 4x ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad), forward / reverse primers (900 nM), FAM or HEX labelled PrimeTime™ qPCR probes (250 nM), 4 units of Hindlll-HF restriction enzyme (NEB) and 2.2 pL of DNA templates (20-50 ng). The mixture was used for droplet generation by the Automated Droplet Generator. The generated droplet was amplified by PTC Tempo Deepwell Thermal Cycler with an initial denaturation at 95 °C for 10 min, follow ed by 40 cycles of denaturation at 94 °C for 30 s, and annealing-extension at 60 °C for 60 s. Enzyme inactivation was performed at 98 °C for 10 min and an infinite incubation at 4 °C. After amplification, the droplet was subjected to quantification by the QX600 Droplet Reader (Bio-Rad). Data analysis was performed with the QX Manager. The integration rate was defined as the concentration of integration target divided by that of the RPPH reference gene. For each integration site, the test was performed with both the target and reference primer / probe in duplicate. A gBlock was also generated to test the sensitivity of the primer / probe design.

[0188] Results for percent integration with Cp36 are shown in Figure 5.Example 5; Integration of cssDNA in HEK293FT cells expressing LSR BxBl

[0189] Genome integration of cssDNA was evaluated in HEK293FT cells over a range of doses using either Lipofectamine, Messenger Max, or LNP, and the cssDNA8 construct from Example 1. The dsDNA construct was dsDNA plasmid (‘;pAF755”; SEQ ID NO: 4005) encoding 5’ to 3’: Bxbl corresponding AttD site (SEQ ID NO: 2003), mCherry, bGHpA, pUC, AmpR. The LSR used for testing was Bxbl (SEQ ID NO: 2002). Bxbl was expressed from Bxbl landing pad HEK293FT cells, which were prepared using lentiviral transduction as follows. Lentivirus w as generated via transfection of HEK293T cells with a packaging plasmidencoding necessary' proteins for assembly of mature virions, pseudotyping plasmid encoding Vesicular Stomatitis Virus G protein (VSV-G), and a transfer plasmid (“pJT301”, SEQ ID NO: 4006) comprising modified 5’ and 3’ long terminal repeats (LTRs) and an expression cassette comprising an EFla promoter, Bxbl AttA acceptor site, and an open reading frame encoding Bxbl enzyme, P2A peptide, and EGFP reporter. The region of transfer plasmid pJT301 comprising 5’ to 3’ LTR, i.e. the “‘Bxbl landing pad”, was integrated into the cell genome of HEK293FT cells to generate Bxbl landing pad cells.

[0190] The DNA (either cssDNA or dsDNA) were delivered to HEK293FT cells by reverse transfection in a 96-well plate on the first day. Different doses of DNA (either cssDNA or dsDNA) were prepared with Lipofectamine 3000 (Figure 7A) or Lipofectamine MessengerMAX (Figure 7B) and then DNA mixture was added to each well. 60,000 cells were seeded in one well of 96-well plate. After 48 hours incubation, cells were harvested, and the total DNA was extracted for ddPCR using Quick-DNA 96 kit (Zymo Research, D3010).

[0191] Percent integration was measured by ddPCR. ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (Bio-Rad) according to the manufacturer's protocol. Each test was prepared in a 22 pL mixture including 5.5 pL of 4x ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad), forward / reverse primers (900 nM), FAM or HEX labelled PrimeTime™ qPCR probes (250 nM), 4 units of Hindlll-HF restriction enzy me (NEB) and 2.2 pL of DNA templates (20-50 ng). The mixture was used for droplet generation by the Automated Droplet Generator. The generated droplet was amplified by PTC Tempo Deepwell Thermal Cycler with an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s, and annealing-extension at 60 °C for 60 s. Enzyme inactivation was performed at 98 °C for 10 min and an infinite incubation at 4 °C. After amplification, the droplet was subjected to quantification by the QX600 Droplet Reader (Bio-Rad). Data analysis was performed with the QX Manager. The integration rate was defined as the concentration of integration target divided by7that of a reference gene. For the top integration site, the test was performed with both the target and reference primer / probe in duplicate. A gBlock was also generated to test the sensitivity’ of the primer / probe design.

[0192] Primers and probe were designed using PrimerQuest (IDT) by default setting and ordered from IDT. dsDNA and ssDNA templates used the same primers and probe when evaluating integration at the left side but a different forward primer for integration on the right side. To calculate the integration percentage, the integrated DNA was compared to the number of available landing pad sites measured by an orthogonal set of primers and probe. A gBlocks Gene Fragment (dsDNA from IDT) of each expected template sequence was ordered to validatethe primer / probe design and measure the sensitivity of the assay. Primers and probes sequences are shown in Table 2 below.Table 2: ddPCR reagents for Bxbl

[0193] Results for percent integration with Bxbl for the left and right sides using Lipofectamine 3000 transfection and Lipofectamine MessengerMAX are shown in Figure 7A and 7B, respectively.

[0194] Lipid nanoparticle (LNP) formulations were prepared as follows. Five lipids (an ionizable lipid, a helper lipid (DSPC (BP-25623)), cholesterol (BP-26125), (l-sitosterol (BP- 40650), and a PEG lipid (DMG-PEG 2000 (BP-25496), all purchased from Broadpharm) were dissolved in ethanol at a ratio of 50:23: 10: 15:2. The lipid mixture was combined with acid aqueous buffer containing DNA at a volumetric (aqueous: ethanol) ratio of 3: 1 using a microfluidic mixer (Ignite, Percision Nanosystems). 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. Formulations were stored at -80 °C until further use.

[0195] Different doses of LNP-DNA (either cssDNA or dsDNA) were added to a 96-well plate with supplement of ApoE (final concentration 3 ng / pl), then the Bxbl-LP HEK 293FT cells were added with 60,000 cells / well in a 96-well plate. After 48 hours incubation, cells were harvested, and the total DNA was extracted for ddPCR using Quick-DNA 96 kit (Zymo Research, D3010). Results for percent integration with Bxbl for the left and right sides using LNP are shown in Figure 8.Example 6; Integration of cssDNA with single-stranded DNA binding protein

[0196] Genome integration of cssDNA was evaluated in HEK293FT cells with the addition of the single-stranded DNA binding protein RAD51. The construct cssDNA6 (Example 1) and the LSR lxFlag-Cp36 plasmid were used in the experiment. cssDNA6 and lxFlag-Cp36 plasmid were compared to cssDNA6 and lxFlag-Cp36-2A-RAD51 plasmid. The LSR IxFlag- Cp36 and RAD51 proteins were expressed using EFla promoter in the plasmids. The sequence for RAD51 is provided as SEQ ID NO: 4007.

[0197] The cssDNA and LSR DNA were delivered to HEK293FT cells by electroporation in a 96-well plate on the first day. 1000 ng cssDNA6 / well or 250 ng pAF999Avell was electroporated with 250 ng lxFlag-Cp36 or lxFlag-Cp36-2A-RAD51 plasmid / well according to the manufacturer’s protocol. The electroporation was performed using the 4D-Nucleofector (Lonza, AAF-1003X) with the program DS-150.

[0198] ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (Bio-Rad) according to the manufacturer's protocol. Each test was prepared in a 22 pL mixture including 5.5 pL of 4x ddPCR™ Supermix for Probes (No dUTP) (Bio-Rad), forward / reverse primers (900 nM), FAM or HEX labelled PrimeTime™ qPCR probes (250 nM), 4 units of Hindlll-HF restriction enzyme (NEB) and 2.2 pL of DNA templates (20-50 ng). The mixture was used for droplet generation by the Automated Droplet Generator. The generated droplet was amplified by PTC Tempo Deepwell Thermal Cycler with an initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 94 °C for 30 s, and annealing-extension at 60 °C for 60 s. Enzyme inactivation was performed at 98 °C for 10 min and an infinite incubation at 4 °C. After amplification, the droplet was subjected to quantification by the QX600 Droplet Reader (Bio-Rad). Data analysis was performed with the QX Manager. The integration rate was defined as the concentration of integration target divided by that of the RPPH reference gene. For each integration site, the test was performed with both the target and reference primer / probe in duplicate. A gBlock was also generated to test the sensitivity of the primer / probe design.

[0199] ddPCR primers and probe (SEQ ID NOs: 2004, 2005, 2006). The results for the percent integration by ddPCR are shown in Figure 9.Example 7; cssDNA reduces cGAS pathway-associated immunogenicity in human primary T cells and in vivo

[0200] The immunogenicity profile of cssDNA constructs was evaluated in human primary T cells. The construct cssDNA6 (Example 1) was prepared as in Example 1.

[0201] Human peripheral blood pan-T cells (StemCell) were stimulated by Dynabead Human T-Activator CD3 / CD28 (ThermoFisher) at a ratio of 75uL of Dynabeads per le6 T cells and lOOIU / mL IL-2, lOng / mL IL-7, and lOng / mL IL-15 (StemCell) in X-Vivo 15 media (Lonza) supplemented with 2% human AB serum (Valley Biomedical). After 3 days, the Dynabeads were magnetically removed and the T cells were electroporated in P3 buffer using the EO-115 program on the 4D-Nucleofector X Unit (Lonza). Electroporation was conducted with 2e6 activated T cells and (i) no additive, (ii) 4ug cGAMP per le6 cells, (iii) 4ug circular single stranded DNA (cssDNA6) per le6 cells, or (iv) 4ug nanoplasmid double stranded DNA per le6 cells. One hour after electroporation, the cells were harvested and lysed using RIPA buffer (ThermoFisher) supplemented with HALT protease and phosphatase inhibitor (ThermoFisher). Protein lysates were quantified using Pierce BCA Protein Assay Kits (ThermoFisher) and 20ug of protein from each lysate was loaded onto the Western Blot. The Western Blot membrane was then stained for GAPDH and the phosphorylated proteins pTBKl , pIRF3, pSTING (all antibodies at 1 : 1000) (Cell Signaling Technology, Inc ).

[0202] The cssDNA sample showed reduced levels of pTBKl, pTIRF3. and pSTING as compared to cGAMP and nanoplasmid DNA samples (Figure 11).

[0203] Immunogenicity was further evaluated in vivo in C57BL / 6j wildtype mice with LNP delivery7of cssDNA6 (SEQ ID NO: 4001). Mice were injected through the tail vein with Img / kg LNP (25 ug LNP for a 25g mice) containing cssDNA or nanoplasmid or plasmid. After 4 hours, 100 pl blood was collected retro-orbitally and serum was processed for cytokine detection. LEGENDplex™ Mouse Anti-Virus Response Panel (Biolegend, CAT# 740621 ) was used to test a panel of cytokine levels. Body weight was recorded daily up to 7 days post LNP injection.

[0204] Mice that were injected with cssDNA had a reduction in immune response as evidenced by reduced cytokine levels (Figures 19A-19D) and no statistically significant change in body weight (Figure 19E). As compared to nanoplasmid or plasmid, cssDNA demonstrated a reduction in IL-6 (Figure 19A), IFN-a (Figure 19B), IFN-b (Figure 19C), and MCP-1 (Figure 19D)Example 8; Integration of a cssDNA template with an LSR variant in HEK293FT cells

[0205] Integration of cssDNA with an LSR variant (SEQ ID NO: 2024) (a genome targeting LSR) was evaluated in HEK293FT cells by transient transfection. The following cssDNA construct “cssDNAW’ (SEQ ID NO: 4008) was prepared as in Example 1 and encoded from 5?to 3?- LSR variant corresponding AttD site (SEQ ID NO: 2025)-CMV-hEPO-2A-eGFP- bGHpA-flori. The LSR variant construct was 3xMyc-NLS-Flag- LSR as mRNA.

[0206] One day prior to transfection, HEK293FT cells (Thermo Fisher Scientific) were seeded at 20,000 cells / mL in a 96-well plate. The cssDNA was transfected using Lipofectamine 3000 (Thermo Fisher Scientific) at indicated doses following manufacturer's protocol, and 250ng of 3xMyc-NLS-Flag-LSR mRNA was transfected per well using MessengerMAX (Thermo Fisher Scientific) following manufacturer's protocol.

[0207] Two days post-transfection, genomic DNA was harvested using the Quick-DNA 96 kit (Zymo) and ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (BioRad) to quantify integration events at the genome integration site referred to as “GS 10”. 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 enzy me (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 enzy me 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. The primers for the GS10 site were SEQ ID NO: 2026 (forward primer), SEQ ID NO: 2027 (reverse primer), and SEQ ID NO: 2028 (probe FAM).

[0208] The percent integration with the variant LSR increased at the GS10 site with increasing amount of cssDNA (Figure 12).Example 9; cssDNA can be used as an LSR template to integrate a CAR in human primary T cells

[0209] cssDNA was evaluated for use as an LSR template to integrate a CAR sequence in human primary T cells with an LSR variant (SEQ ID NO: 2024). The following cssDNA construct “cssDNA19’?(SEQ ID NO: 4010) was prepared as in Example 1 and encoded from 5’ to 3’ - LSR variant corresponding AttD site (SEQ ID NO: 2025)-EFla-CAR-bGHpA-flori. The LSR variant construct was 3xMyc-NLS-Flag- LSR as mRNA.

[0210] Human peripheral blood pan-T cells (StemCell) were stimulated by Dynabead Human T-Activator CD3 / CD28 (ThermoFisher) at a ratio of 75uL of Dynabeads per le6 T cells and lOOIU / mL IL-2, lOng / mL IL-7, and lOng / mL IL-15 (StemCell) in X-Vivo 15 media (Lonza) supplemented with 2% human AB serum (Valley Biomedical). After 1 day, the Dynabeads were magnetically removed and the T cells were electroporated in P3 buffer using the EO-115 program on the 4D-Nucleofector X Unit (Lonza). Electroporation was comprised of 2e6 activated T cells and (i) 6ug LSR mRNA per le6 cells, (ii) 2ug nanoplasmid doublestranded DNA encoding the CAR per 1 e6 cells, (iii) 2ug cssDNA encoding the CAR and 6ug LSR mRNA each per le6 cells, or (iv) 4ug cssDNA encoding the CAR and 6ug LSR mRNA each per le6 cells. After electroporation, the cells were put back onto CD3 / CD28 Dynabeads at the same ratio as before. Cytokines were replenished every other day at the same concentrations. Seven days after electroporation, cells were harvested and split into two populations for flow cytometry using FITC-Labeled Monoclonal Anti-FMC63 Antibody (Aero Biosystems) and genomic DNA analysis via ddPCR. Genomic DNA was harvested using the Quick-DNA 96 kit (Zymo) and ddPCR was performed with QX600 AutoDG Droplet Digital PCR System (Bio-Rad) to quantify integration events at GS10. Each test was prepared using 4x ddPCR™ Multiplex Mix for Probes (Bio-Rad); forward / reverse primers (900 nM); FAM, HEX, Cy5, or Cy5.5 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.

[0211] Significant CAR expression was observed in primary human T cells by flow cytometry (Figure 13A). CAR integration was demonstrated with the variant LSR at the GS 10 site by ddPCR (Figure 13B).Example 10: cssDNA and LSR delivered in LNPs integrates a CAR in human primary T cells

[0212] CAR integration was further evaluated in human primary T cells with delivery of cssDNA and a variant LSR mRNA (SEQ ID NO: 2024) delivery in LNPs. The construct cssDNA18 (SEQ ID NO: 4009) was prepared as in Example 1. The LSR variant construct was 3xMyc-NLS-Flag- LSR as mRNA.

[0213] LNPs were prepared with an ionizable lipid, a helper lipid (DSPC (BP-25623)), cholesterol (BP-26125), a PEG lipid (DMG-PEG 2000 (BP-25496)), and a modified PEG lipid and were dissolved in ethanol at a ratio of 50:25:22:2.5:0.5. The lipid mixture was combined with acid aqueous buffer containing mRNA and / or cssDNA at a volumetric (aqueous: ethanol) ratio of 3 : 1 using a microfluidic mixer (Ignite, Precision Nanosystems). LNPs were conj ugated with an anti-CD3 Fab fragment (comprising SEQ ID NOs: 2031 and 2032 for heavy and light chain variable regions, respectively). Formulations were dialyzed against 50 mM Tris (pH 7.5),75 mM NaCl, and 10% sucrose in dialysis cassettes for at least 18 hr. The concentration of the nucleic acid within the LNPs was determined with RiboGreen (R1149L ThermoFisher). Formulations were stored at -80°C until further use.

[0214] Cryopreserved T cells were obtained from Stemcell Technologies. T cells were thawed activated in CTS OpTmizer T cell expansion media (Gibco) supplemented with 2% human AB serum (Fisher), lOOIU / mL IL-2, lOng / mL IL-7, lOng / mL IL- 15 (Stemcell Technologies) and 1 : 100 dilution of TransAct (Miltenyi). The cells were seeded at 100.000 cells / well in a 96-well plate and incubated overnight. The following day, T cells were treated with 5 pg / mL of LNPs encapsulating either cssDNA or nanoplasmid and co-delivered with either 1 pg / mL, 3 pg / mL or 5 pg / mL of LSR mRNA LNP. All LNP treatments were codelivered with 4 pg / mL ApoE. Controls included cssDNA LNP only, nanoplasmid LNP only and media only groups.

[0215] CAR positive T cells were detected by flow' cytometry on day 7 as shown in Figure 14 and increased with greater doses of the LSR.Example 11: cssDNA integration with oligos in HEK293FT cells

[0216] cssDNA integration was tested with the addition of oligos of various lengths and with locked nucleic acids (LNAs) at various locations on the oligo was tested in HEK293FT cells with Lipo3000 transfection. The construct cssDNAlO (SEQ ID NO: 4008) was prepared as in Example 1. The LSR variant construct was 3xMyc-NLS-Flag-LSR as mRNA with LSR variant (SEQ ID NO: 2024).

[0217] Supplementation of antisense oligos resulted in enhanced integration of cssDNA as measured by percent integration at the GS10 site (FIG 15A-B). Locked nucleic acids further enhanced integration (FIG 15C-D). While enhanced integration was observed by ddPCR. no difference in expression of the GFP transgene was observed as compared to cssDNA without an oligo.Example 12: In vitro CAR-T generation with cssDNA encoding a CAR in LNPs

[0218] CAR-T cells were generated in vitro by incubation of primary human T cells w ith CD3-targeted LNPs containing either LSR mRNA alone, cssDNA encoding CD 19 CAR alone, or co-encapsulated LSR mRNA and cssDNA encoding CD19 CAR (cssDNA19) (SEQ ID NO: 5000). The LSR variant construct was 3xMyc-NLS-Flag-LSR as mRNA with LSR variant (SEQ ID NO: 2024). LNPs were prepared as in Example 11 with an anti-CD3 Fab fragment (comprising SEQ ID NOs: 2031 and 2032 for heavy and light chain variable regions, respectively). The cssDNA was prepared as in Example 1.

[0219] 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 rnL. 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 16A) and by ddPCR for integration (FIG 16B).

[0220] 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 (TreeStar).

[0221] 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 GS10 site. lOOng gDNA was used for each reaction. Each test was prepared using 2x ddPCR™ Supermix for Probes (No dUTP) (BioRad), 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 13; In vivo CAR-T generation in NSG mice with CD3-targeted LNPs coencapsulating LSR mRNA and cssDNA encoding a CAR

[0222] 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 CD19 CAR transgene. The construct cssDNA19 (SEQ ID NO: 5000) was prepared as described above. The LSR variant construct was 3xMyc-NLS-Flag-LSR as mRNA with LSR variant (SEQ ID NO: 2024).

[0223] CD3-targeted LNPs were prepared and validated for mediating CAR expression and integration in vitro using primary T cells as described above in Example 11.

[0224] Human CD34+ stem cell engrafted NSG mice were dosed with CD3-LNPs through the tail vein and the dose for each group as listed in Table 3. Mice in group 3 received LNPs co-encapsulating cssDNA and mRNA at a ratio of 1 : 1.Table 3: LNP contents in treatment groups and doses for in vivo study

[0225] 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 12. Other tissues including splenic leukocytes, bone marrow-derived leukocytes, and lymph node-derived leukocytes 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 CD19 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 C AR-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.

[0226] Terminal tissues evaluated by flow cytometry at day 8 after dosing showed significantly reduced B cells (FIG 17A) and no significant change in T cells (FIG 17B).

[0227] Direct evidence of in vivo CAR-T cell generation was found in T cells in the spleen of one mouse dosed with LNP co-encapsulating LSR + cssDNA. CAR expression by flow cytometry was measured in splenic T cells of a mouse treated with LNP co-encapsulating LSR + cssDNA (FIG 17C) and a mouse treated with LNP containing cssDNA only (FIG 17D).

[0228] LSR-mediated CAR integration was demonstrated with ddPCR in terminal tissues as an average of all mice within a group (FIG 17E) and for each individual mouse dosed with LNP co-encapsulating LSR + cssDNA (FIG 17F). 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.Example 14: In vivo anti-tumor activity with integrated CD 19 CAR-T cells

[0229] Anti -tumor activity of CD 19 CAR-T cells from CD3-targeted LNPs coencapsulating mRNA encoding a variant LSR and cssDNA template encoding a CD 19 CAR transgene was evaluated in a tumor regression mouse model, NSG-MHCI / II-DKO mice. Two genome targeting LSR variants were tested, “LSR- A’" (SEQ ID NO: 2024) and “LSR-B” (SEQ ID NO: 2029) using the same AttD (SEQ ID NO: 2025) and integrating at the GS10 site. PBMCs were purchased from iXCells Biotechnologies USA, Inc. CD3-targeted LNPs were prepared with a CD3 Fab as described above.

[0230] 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 cssDNA encoding CD19- CAR; and 2) CD3-targeted LNPs co-encapsulating LSR mRNA and mScarlet mRNA (“LSR mRNA only”). A control group for episomal expression receiving CD3-targeted LNPs encapsulating nanoplasmid DNA encoding CD19-CAR only was dosed with 0.5 mg / kg (“DNA 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.

[0231] Tumor burden was assessed by IVIS imaging on Day -1, 4, 7, 10, 14, 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% isofl wane 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 Awa In Vivo Imaging software (Spectral Instruments) following the manufacturer’s instruction. Total flux (p / s) was used to indicate the intensity of luciferase signal.

[0232] Tumor reduction was observed with LNPs co-encapsulating LSR mRNA and the CD19-CAR transgene (FIG 18A). CD3-targeted LNPs were well-tolerated with modest body weight loss in all groups followed by recovery.

[0233] 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 (FIG 18B). 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 cssDNA encoding the CD19-CAR transgene (FIG 18C). 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 measwing the percent of CD20+ cells in the CD45+ cell population by flow cytometry.

[0234] The above results demonstrated functional CAR integration and tumor reduction in NSG mice with CD3-targered LNPs co-encapsulating LSR mRNA and cssDNA encoding a CD19-CAR transgene.Example 15: LNP formulation ratios of cssDNA:RNA in prim ary T cells

[0235] The ratio of cssDNA to mRNA was evaluated by testing five LNP formulations. The five formulations were: LNP-1 (ratio 1 : 1 of cssDNA:mRNA); LNP-2 (ratio 2: 1 of cssDNA:mRNA); LNP-3 (ratio 1:2 of cssDNA: mRNA); LNP-4 (ratio 3: 1 of cssDNA mRNA); LNP-5 (ratio 1:3 of cssDNA:mRNA). LNP-6 was a control without mRNA.

[0236] Five lipids (an ionizable lipid, a helper lipid (DSPC (BP-25623)), cholesterol (BP- 26125), a PEG lipid (DMG-PEG 2000 (BP-25496))), and a modified PEG lipid, were dissolved in ethanol at a ratio of 50:25:22:2.5:0.5. The lipid mixture was combined with acid aqueous buffer containing mRNA and / or cssDNA at a volumetric (aqueous: ethanol) ratio of 3: 1 using a microfluidic mixer (Ignite, Precision Nanosystems). The LNPs were then incubated and labeled with a T-cell targeting ligand. Formulations were dialyzed against 50 mM Tris (pH7.5), 75 mM NaCl. and 10% sucrose in dialysis cassettes for at least 18 hr. The concentration of the nucleic acid within the LNPs was determined with RiboGreen (R11491, ThermoFisher). Formulations were stored at -80°C until further use.

[0237] Primary T cells (Stemcell Tech, 70024. 1) were thawed and incubated overnight. The next day, 100k cells were placed in a flat-bottom 96 well plate. LNPs were directly administered to each well. The cells were incubated for four days and then evaluated by flow cytometry for % CAR + cells (FIG 20 A) and by ddPCR for percent integration at the GS10 integration site (FIG 20B).Example 16: cssDNA produced by enzymatic conversion is active for LSR-mediated integration

[0238] Production of cssDNA was evaluated using an enzymatic conversion process, which does not use a phage system (FIG 21A). A double-stranded plasmid was used as starting material, containing 7 Bbvl sites all on the same strand (FIG 21B). Enzymes were purchased from New England Biolabs, as well as the lOx reaction buffer (CutSmart). Reactions were setup with 40 ng / ul plasmid DNA, 0.04 units / pl BbvCI, 0.2 units / pl Nt.BbvCL 1 unit / pl T7 exo and lx cutsmart buffer in 50 pl, and incubated at 37°C for approximately 16 hours. Reactions were analyzed by gel electrophoresis on a 1% agarose TAE gel cast with SybrGold stain (FIG 21C).

[0239] cssDNA DNA was purified by anion exchange chromatograph. Reactions were diluted 1 :33 in Equilibration Buffer (100 mM Tris HC1 pH 8.0, 300 mM Guanidine HC1, 100 mM NaCl) and loaded onto a DEAE CIMmultus Monolithic column with 6pm pore. The column was washed with Equilibration Buffer and then cssDNA was collected by rising the column with Elution buffer (100 mM Tris HC1 pH 8.0, 300 mM Guanidine HC1, 1,000 mM NaCl). All operations were carried out on an Akta Avant FPLC instrument. A chromatogram showing purified cssDNA is shown in FIG 21D.

[0240] Purified cssDNA, or the parent dsDNA plasmid, was electroporated into primary human T cells (two donors tested) together with an LSR variant (SEQ ID NO: 2030) (a genome targeting LSR) as above. The AttD was SEQ ID NO: 2025. Integration of the DNA payload (either cssDNA or plasmid dsDNA) at the GS10 integration site was measured by ddPCR (FIG 21E)Sequence Listing

Claims

CLAIMS1 . A genome editing system comprising: a. an mRNA encoding a large serine recombinase (LSR); and b. a circular single-stranded DNA (cssDNA) comprising a donor attachment site (AttD) and a sequence of interest; wherein the genome editing system is capable of integrating the sequence of interest into a cellular genome.

2. The genome editing system of claim 1, wherein the genome editing system further comprises a lipid-based formulation comprising (a) and (b).

3. The genome editing system of claim 1, wherein the genome editing system further comprises a first lipid-based formulation comprising (a) and a second lipid-based formulation comprising (b).

4. The genome editing system of claims 2 or 3. wherein the lipid-based formulation is an LNP.

5. The genome editing system of any one of claims 1-4, wherein the ratio of b) to a) in the genome editing system is 3: 1 to 1 :3 or wherein the ratio of b) to a) in the genome editing system is 2: 1 to 1 :2.

6. The genome editing system of any one of claims 1 -5, wherein the ratio of b) to a) in the genome editing system is 1: 1.

7. The genome editing system of any one of claims 1-6, wherein the endotoxin level of (b) is less than 0.1 EU / pg, less than 0.05 EU / pg, 0.01 EU / pg, or 0.005 EU / pg.

8. The genome editing system of any one of claims 1-7, wherein the sequence of interest comprises a gene or a portion of a gene.

9. The genome editing system of any one of claims 1-8, wherein the sequence of interest comprises a therapeutic protein.

10. The genome editing system of any one of claims 1-9, wherein the sequence of interest comprises a CAR.

11. The genome editing system of any one of claims 1-10, wherein the LSR is a genome targeting LSR.

12. The genome editing system of any one of claims 1-11, wherein the LSR is not a landing pad LSR.

13. The genome editing system of any one of claims 1-12, comprising a lipid-based formulation, comprising a lipid-based formulation comprising a cationic lipid, helper lipid, cholesterol, and a PEG lipid.

14. The genome editing system of claim 13, wherein the lipid-based formulation further comprises a polypeptide.

15. The genome editing system of any one of claims 2-14, wherein the lipid-based formulation comprises an LNP having a size of 50-100 nm.

16. The genome editing system any one of claims 1-15, comprising a first lipid-based formulation comprising a) and a second lipid-based formulation comprising b), wherein the first and second lipid-based formulations are nearly identical in composition and size.

17. The genome editing system any one of claims 1-16, comprising a first lipid-based formulation comprising a) and a second lipid-based formulation comprising b), wherein the first and second lipid-based formulation are not identical in composition and size.

18. The genome editing system of any one of claims 1-17, wherein the AttD site comprises at least 26, 34. 36, 39, 48, or 52 nucleotides, wherein the nucleotides comprise the dinucleotide core and an even number of nucleotides directly adj acent on either side of the dinucleotide core.

19. The genome editing system of any one of claims 1-18, wherein the AttD site comprises an AttP site.

20. The genome editing system of any one of claims 1-19, wherein the cellular genome is a human genome.

21. The genome editing system of any one of claims 1-20, wherein the cellular genome does not comprise an installed attachment site.

22. A method for integration of a sequence of interest into a cellular genome, comprising contacting a cell with the genome editing system of any one of claims 1-21 such that the sequence of interest is integrated into the cellular genome.

23. A method for generating an engineered cell, comprising contacting a cell with the genome editing system of any one of the claims 1-21.

24. A method for introducing a sequence of interest in a cell, comprising contacting a cell with the genome editing system of any one of the claims 1-21.

25. The method of any one of claims 22-24, wherein the cell is mammalian, optionally wherein the cell is human.

26. The method of claim 25, wherein the cell is a T cell, natural killer cell (NK cell), nonhuman embryonic stem cell, induced pluripotent stem cell (iPSC), hematopoietic stemcell (HSC), liver cell, muscle cell, monocyte, B cell, neuron, astrocyte, or microglial cell.

27. The method of claim 26, wherein the cell is a T cell.

28. The method of any one of claims 22-27, wherein the contacting of the cell is in vivo.

29. The method of any one of claims 22-27, wherein the contacting of the cell is ex vivo.

30. The method of any one of claims 22-29, wherein a Cas or a guide RNA is not used in the contacting of the cell.

31. The method of any one of claims 22-30, wherein the method does not comprise modifying the cell to insert an attachment site for the sequence of interest into the cellular genome.

32. An engineered cell comprising the genome editing system of any one of the claims 1- 21.

33. The engineered cell of claim 32, wherein the cell is a human cell.

34. The engineered cell of claim 32 or 33, wherein the cell is a T cell, natural killer cell (NK cell), non-human embryonic stem cell, induced pluripotent stem cell (1PSC), hematopoietic stem cell (HSC), liver cell, muscle cell, monocyte, B cell, neuron, astrocyte, or microglial cell.

35. A method for treating a disease in a subject, comprising administering to the subject the genome editing system of any one of the claims 1-21, or the engineered cell of any one of claims 32-34.

Images