Nonviral cell modification

Abstract

This specification provides compositions and methods for improving the non-viral insertion of genes into safe harbor loci of cells such as immune cells. TIFF2026521526000010.tif120128

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 508,059, filed on June 14, 2023, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that is hereby incorporated by reference in its entirety. The XML copy created on June 10, 2024, is named ANB - 220WO_SL.xml and has a size of 115,114 bytes.

Background Art

[0003] In cancer treatment, despite significant research efforts and scientific progress, cancer still continues to impose a major clinical burden. Blood cancers and bone marrow cancers such as multiple myeloma, leukemia, and lymphoma are types of cancers that are frequently diagnosed. Current treatment options for these cancers are not effective for all patients and / or may have substantially harmful side effects. Other types of cancers are also difficult to treat using existing treatment options. Cancer immunotherapy is a promising solution because it is highly specific, can enhance treatment efficacy, and reduce side effects.

[0004] Therapy with genetically modified immune cells is a growing field with the potential to be applied to the treatment of diseases including, but not limited to, cancer. Through modifications in coding genomic regions and / or non - coding genomic regions, researchers have identified, for example, transgenes and insertion sites within cells that enhance cell function, halt cell proliferation, induce cell death, and promote reduction of tumor diameter / volume. The identification of safe harbor sites (SHS) has improved the outcome of genomic modification therapies.

[0005] However, when therapeutic genes are inserted into immune cells via viral insertion, unexpected off-target insertions and decreased gene transfer and editing efficiency can occur. Furthermore, viral vectors present challenges for high-throughput manufacturing processes. Non-viral gene editing methods can address these issues, but they also suffer from low gene transfer efficiency and overall yield of edited cells. Therefore, further methods are needed to improve non-viral gene editing. [Overview of the Initiative]

[0006] In one embodiment, the present invention provides a composition comprising a solution containing an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0007] In one embodiment, the present invention provides a composition comprising a solution containing N-acetyl-L-cysteine ​​(NAC), a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0008] In one embodiment, the present invention provides a composition comprising a solution containing an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0009] In one embodiment, the present invention provides a composition comprising a solution containing a histone deacetylase (HDAC) inhibitor comprising at least one of sodium phenylbutyrate, xynostat, or panobinostat, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0010] In one embodiment, the Specified Composition comprises a solution containing a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), a histone deacetylase (HDAC) inhibitor, and / or an osmotic regulator, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0011] In some embodiments, the solution includes at least the following: i. N-acetyl-L-cysteine ​​(NAC) and osmotic regulators, ii. RNase inhibitors and N-acetyl-L-cysteine ​​(NAC), iii. RNase inhibitors and osmotic regulators, iv. N-acetyl-L-cysteine ​​(NAC) and histone deacetylase (HDAC) inhibitors, v. Osmotic regulators and histone deacetylase (HDAC) inhibitors, vi. RNase inhibitors and histone deacetylase (HDAC) inhibitors, vii. RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), and histone deacetylase (HDAC) inhibitors, viii. N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors. ix. RNase inhibitors, osmotic regulators, and histone deacetylase (HDAC) inhibitors, x. RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), and osmotic regulators, or xi. RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors.

[0012] In some embodiments, the RNase inhibitor is present in the solution at a final concentration of approximately 0.5–2 U / μl.

[0013] In some embodiments, the RNase inhibitor is present in the solution at a final concentration of approximately 1 U / μL.

[0014] In some embodiments, the RNase inhibitor is an RNase A, B, C, T1, or T2 inhibitor.

[0015] In some embodiments, the RNase inhibitor is a mouse, rat, or human RNase inhibitor.

[0016] In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in solution at a final concentration of approximately 1–10 mM.

[0017] In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of approximately 2.5 mM.

[0018] In some embodiments, the osmotic pressure regulator is sorbitol, glycerol, or glycine.

[0019] In some embodiments, the osmotic pressure regulator is sorbitol.

[0020] In some embodiments, sorbitol is present in the solution at a final concentration of about 100–400 mM.

[0021] In some embodiments, sorbitol is present in the solution at a final concentration of about 190 mM or 200 mM.

[0022] In some embodiments, the osmotic pressure regulator is glycerol.

[0023] In some embodiments, glycerol is present in the solution at a final concentration of about 100–400 mM.

[0024] In some embodiments, glycerol is present in the solution at a final concentration of approximately 170 mM.

[0025] In one embodiment, the present invention provides a composition comprising a cell containing at least one heterologous DNA template inserted into a target region of the genome, and a histone deacetylase (HDAC) inhibitor comprising at least one of sodium phenylbutyrate, xynostat, or panobinostat.

[0026] In some embodiments, the histone deacetylase (HDAC) inhibitor is either an HDAC1 inhibitor or an HDAC2 inhibitor.

[0027] In some embodiments, HDAC inhibitors include sodium phenylbutyrate, xinostat, panobinostat, phenylbutyrate, curcumin, mosetinostat, romidespin, SIS17, spritomycin, trichostatin-A, tusidinostat, etinostat, sodium butyrate, valproic acid, CXD101, KT-531, ITF3756, tubastatin A, vorinostat, BML-210, bellinostat, avexinostat, dasinostat, CUDC-101, droxinostat, MC1568, prasinostat, divalproex sodium, PCI-3405, SR-4370, zibinostat, tubasin, AR-42, (-)-parthenolide, and resminostat. These are filepinostat, M344, tasejinarin, sinapic acid, biphenyl-4-sulfonyl chloride, sulforaphane, UF010, suberohydroxamic acid, NKL22, TC-H106, RGFP966, HPOB, RG2833, TMP269, nexturastat A, domatinostat, LMK-235, santacruz amete A, CAY10603, tascinimod, BG45, BRD73954, licorinostat, scriptide, sitalinostat, WT161, TMP195, ACY-738, SKLB-23bb, tinostamstin, TH34, BRD3308, radianine A, isoguanosine, KA2507, ITSA-1, or RNA interference (RNAi) molecules.

[0028] In some embodiments, the HDAC inhibitor is sodium phenylbutyrate, xinostat, or panobinostat.

[0029] In some embodiments, sodium phenylbutyrate is present in the solution at a final concentration of approximately 15.6 μM to 4 mM.

[0030] In some embodiments, sodium phenylbutyrate is present in the solution at a final concentration of about 1 mM, and optionally, the solution contains 1% DMSO.

[0031] In some embodiments, xynostat is present in the solution at a final concentration of approximately 8 nM to 200 nM.

[0032] In some embodiments, xinostat is present in the solution at a final concentration of about 16 nM.

[0033] In some embodiments, panobinostat is present in the solution at a final concentration of approximately 3 nM to 1 μM.

[0034] In some embodiments, panobinostat is present in solution at a final concentration of approximately 37.5 nM.

[0035] In some embodiments, the solution comprises at least one of the following: an RNase inhibitor at a final concentration of approximately 0.5–2 U / μl, NAC at a final concentration of approximately 1–10 mM, sorbitol at a final concentration of approximately 100–400 mM, and / or sodium phenylbutyrate at a final concentration of approximately 15.6 μM–4 mM, xynostat at 8 nM–200 nM, or panobinostat at 3 nM–1 μM.

[0036] In some embodiments, the solution comprises at least one of the following: an RNase inhibitor at a final concentration of about 1 U / μl, NAC at a final concentration of about 2.5 mM, sorbitol at a final concentration of about 200 mM, and / or sodium phenylbutyrate at a final concentration of about 1 mM, 0.016 μM xynostat, or 0.0375 μM panobinostat.

[0037] In some embodiments, the nuclease domain includes a CRISPR-related endonuclease (Cas), optionally a Cas9 nuclease.

[0038] In some embodiments, the size of the DNA template is 300 nucleotides or more.

[0039] In some embodiments, the size of the DNA template is approximately 0.3kb, 0.5kb, 1.0kb, 1.5kb, 2.0kb, 2.5kb, 3.0kb, 3.5kb, 4.0kb, 4.5kb, 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6. 2kb, 6.3kb, 6.4kb, 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8kb, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9k b, 9.0kb, 9.1kb, 9.2kb, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, 10.0kb, 10.1kb, 10.2kb , 10.3kb, 10.4kb, 10.5kb, 10.6kb, 10.7kb, 10.8kb, 10.9kb, 11.0kb, 11.1kb, 11.2kb, 11.3kb, 11.4 DNA templates of sizes kb, 11.5kb, 11.6kb, 11.7kb, 11.8kb, 11.9kb, 12.0kb, 12.1kb, 12.2kb, 12.3kb, 12.4kb, 12.5kb, 12.6kb, 12.7kb, 12.8kb, 12.9kb, 13.0kb or larger, or any size between these sizes.

[0040] In some embodiments, the size of the DNA template is approximately 0.3kb to 13kb, 0.3kb to 0.5kb, 0.3kb to 1kb, 0.3kb to 4kb, 0.3kb to 3kb, 0.3kb to 5kb, 0.3kb to 7kb, 0.3kb to 10kb, 0.5kb to 1kb, 0.5kb to 3kb, 0.5kb to 5kb, 0.5kb to 7kb, 0.5kb to 10kb, 0.5kb to 13kb, 1kb to 3kb, 1kb to 5kb, 1kb to 7kb, 1kb to 10kb b. Approximately 1kb to 13kb, 5kb to 13kb, 5kb to 9kb, 5kb to 8kb, 5kb to 7kb, 5kb to 6kb, 6kb to 13kb, 6kb to 10kb, 6kb to 9kb, 6kb to 8kb, 6kb to 7kb, 7kb to 13kb, 7kb to 10kb, 7kb to 9kb, 7kb to 8kb, 8kb to 13kb, 8kb to 10kb, 8kb to 9kb, 9kb to 13kb, 9kb to 10kb, 10kb to 13kb, or approximately 11kb to 13kb.

[0041] In some embodiments, the composition comprises cells containing a genomic sequence adjacent to the insertion site within the cell's genome.

[0042] In some embodiments, the cells are mammalian cells, human cells, hematopoietic cells, immune cells, primary immune cells, or primary human immune cells.

[0043] In some embodiments, the cells are primary human immune cells.

[0044] In some embodiments, the immune cells are natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, primary T cells, or T cell progenitor cells.

[0045] In some embodiments, the immune cells are primary T cells.

[0046] In some embodiments, the immune cells are primary human T cells.

[0047] In some embodiments, immune cells are undifferentiated.

[0048] In some embodiments, immune cells are CD45RA + and CCR7 + CD45RA + and CCR7 - CD45RA - and CCR7 - , or CD45RA - and CCR7 + That is the case.

[0049] In some embodiments, the cells are virus-free.

[0050] In some embodiments, the cells contain exogenous homologous recombination proteins or DNA repair regulatory proteins.

[0051] In some embodiments, exogenous homologous recombination proteins or DNA repair regulatory proteins are encoded on an episomal plasmid or mRNA molecule.

[0052] In some embodiments, the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

[0053] In some embodiments, the SWSAP1 protein contains the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein contains the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein contains the sequence shown in SEQ ID NO: 127.

[0054] In some embodiments, this involves collecting cells from a patient and introducing a DNA template in vitro or ex vivo.

[0055] In some embodiments, the target region of the cell genome is either the T cell receptor α constant region (TRAC) locus or a genome-safe harbor (GSH).

[0056] In some embodiments, the safe harbor locus is selected from any one of the integration sites designated by GS94, GS88, GS89, GS90, GS91, GS92, GS93, GS95, GS96, GS97, GS98, GS99, GS100, GS101, GS102, GS103, GS104, GS105, GS106, GS107, GS108, GS109, GS110, GS111, GS112, GS113, GS114, GS115, GS116, GS117, GS118, GS119, or GS120.

[0057] In some embodiments, the safe harbor locus is the GS94 integration site.

[0058] In some embodiments, the safe harbor loci are chr11:128340000-128350000, chr10:33130000-33140000, chr10:72290000-72300000, chr11:65425000-65427000 (NEAT1), chr15:92830000-928400 Select from 00, chr16:11220000-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, or chr9:7970000-7980000.

[0059] In some embodiments, the safe harbor locus is a gene selected from APRT, B2M, CAPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28.

[0060] In some embodiments, it includes one or more gRNAs containing any one of SEQ ID NOs: 1 to 120.

[0061] In some embodiments, after at least one sequence is inserted into the safe harbor locus, the cell is CD45RA + and CCR7 + and is.

[0062] In some embodiments, the DNA template is a double-stranded DNA template or a single-stranded DNA template.

[0063] In some embodiments, the DNA template is a linear DNA template or a circular DNA template, and optionally, the circular DNA template is a plasmid.

[0064] In some embodiments, the DNA template contains a heterologous sequence.

[0065] In some embodiments, the DNA template contains a gene.

[0066] In some embodiments, the DNA template contains a priming receptor containing a transcription factor.

[0067] In some embodiments, the DNA template contains a chimeric antigen receptor (CAR).

[0068] In some embodiments, the DNA template includes a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor.

[0069] In some embodiments, the DNA template includes an inductive promoter operably linked to a chimeric antigen receptor.

[0070] In some embodiments, the DNA template further includes a constitutive promoter operably linked to the priming receptor.

[0071] In some embodiments, the DNA template further includes an inductive promoter operably ligated to a chimeric antigen receptor and a constitutive promoter operably ligated to a priming receptor.

[0072] In some embodiments, the DNA template is oriented from 5' to 3': i. Inducible promoter, ii. Chimeric antigen receptor, iii. Constitutive promoters, and iv. Priming receptors Includes.

[0073] In some embodiments, the DNA template is oriented from 5' to 3': i. Constitutive promoter, ii. Priming receptors, iii. Inducible promoters, and iv. Chimeric antigen receptors Includes.

[0074] In some embodiments, the priming receptor is located in the direction from the N-terminus to the C-terminus. i. Extracellular antigen-binding domain having binding affinity to the antigen, ii. A transmembrane domain containing one or more ligand-induced proteolytic cleavage sites, and iii. Intracellular domains containing human or humanized transcription effectors It contains such a component that when an antigen binds to the extracellular antigen-binding domain, cleavage occurs at the ligand-induced proteolytic cleavage site, thereby releasing the intracellular domain.

[0075] In some embodiments, the priming receptor further comprises a near-membrane domain (JMD) or stop transfer sequence (STS) located between the transmembrane domain and the intracellular domain.

[0076] In some embodiments, the transcription factor binds to an inducible promoter to induce CAR expression.

[0077] In some embodiments, the CAR is located from the N-terminus to the C-terminus. i. Extracellular antigen-binding domain having binding affinity to the antigen, ii. Transmembrane domain, iii. Intracellular co-stimulatory domains, and iv. Intracellular activation domain Includes.

[0078] In some embodiments, the priming receptor and CAR bind to different antigens.

[0079] In some embodiments, the priming receptor and the CAR bind to the same antigen.

[0080] In one embodiment, the present specification provides a polypeptide comprising an AcrIIA8 peptide fused to a CDT1 peptide.

[0081] In some embodiments, the polypeptide includes the sequence shown in SEQ ID NO: 127.

[0082] In one embodiment, the present specification provides primary immune cells comprising an exogenous homologous recombination protein or a DNA repair regulatory protein.

[0083] In some embodiments, exogenous homologous recombination proteins or DNA repair regulatory proteins are encoded on an episomal plasmid or mRNA molecule.

[0084] In some embodiments, the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

[0085] In some embodiments, the SWSAP1 protein contains the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein contains the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein contains the sequence shown in SEQ ID NO: 127.

[0086] In some embodiments, the cells are human cells, hematopoietic cells, or primary human immune cells.

[0087] In some embodiments, the immune cells are natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, primary T cells, or T cell progenitor cells.

[0088] In some embodiments, the immune cells are primary T cells.

[0089] In some embodiments, the immune cells are primary human T cells.

[0090] In some embodiments, immune cells are undifferentiated.

[0091] In some embodiments, immune cells are CD45RA + and CCR7 + CD45RA + and CCR7 - CD45RA - and CCR7 - , or CD45RA - and CCR7+ That is the case.

[0092] In some embodiments, the cells are virus-free.

[0093] In some embodiments, the cell includes a DNA template, the 5' and 3' ends of the DNA template containing nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome.

[0094] In some embodiments, the target region of the cell genome is either the T cell receptor α constant region (TRAC) locus or a genome-safe harbor (GSH).

[0095] In some embodiments, the safe harbor locus is selected from any one of the integration sites designated by GS94, GS88, GS89, GS90, GS91, GS92, GS93, GS95, GS96, GS97, GS98, GS99, GS100, GS101, GS102, GS103, GS104, GS105, GS106, GS107, GS108, GS109, GS110, GS111, GS112, GS113, GS114, GS115, GS116, GS117, GS118, GS119, or GS120.

[0096] In some embodiments, the safe harbor locus is the GS94 integration site.

[0097] In some embodiments, the safe harbor loci are chr11:128340000-128350000, chr10:33130000-33140000, chr10:72290000-72300000, chr11:65425000-65427000 (NEAT1), chr15:92830000-928400 Select from 00, chr16:11220000-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, or chr9:7970000-7980000.

[0098] In some embodiments, the safe harbor locus is a gene selected from APRT, B2M, CAPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28.

[0099] In some embodiments, one or more gRNAs include one of sequence numbers 1 to 120.

[0100] In some embodiments, after at least one sequence is inserted into the safe harbor locus, the cells are CD45RA + and CCR7 + That is the case.

[0101] In some embodiments, the DNA template is either a double-stranded DNA template or a single-stranded DNA template.

[0102] In some embodiments, the DNA template is a linear DNA template or a circular DNA template, and optionally, the circular DNA template is a plasmid.

[0103] In some embodiments, the DNA template includes heterologous sequences.

[0104] In some embodiments, the DNA template includes genes.

[0105] In some embodiments, the DNA template comprises a priming receptor containing a transcription factor, a chimeric antigen receptor (CAR), or a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor.

[0106] In some embodiments, the DNA template includes an inductive promoter operably linked to a chimeric antigen receptor.

[0107] In some embodiments, the DNA template further includes a constitutive promoter operably linked to the priming receptor.

[0108] In some embodiments, the DNA template further includes an inductive promoter operably ligated to a chimeric antigen receptor and a constitutive promoter operably ligated to a priming receptor.

[0109] In some embodiments, the DNA template is oriented from 5' to 3': i. Inducible promoter, ii. Chimeric antigen receptor, iii. Constitutive promoters, and iv. Priming receptors Includes.

[0110] In some embodiments, the DNA template is oriented from 5' to 3': i. Constitutive promoter, ii. Priming receptors, iii. Inducible promoters, and iv. Chimeric antigen receptors Includes.

[0111] In some embodiments, the priming receptor is located in the direction from the N-terminus to the C-terminus. i. Extracellular antigen-binding domain having binding affinity to the antigen, ii. A transmembrane domain containing one or more ligand-induced proteolytic cleavage sites, and iii. Intracellular domains containing human or humanized transcription effectors It contains such a component that when an antigen binds to the extracellular antigen-binding domain, cleavage occurs at the ligand-induced proteolytic cleavage site, thereby releasing the intracellular domain.

[0112] In some embodiments, the priming receptor further comprises a near-membrane domain (JMD) or a stop transition sequence (STS) located between the transmembrane domain and the intracellular domain.

[0113] In some embodiments, the transcription factor binds to an inducible promoter to induce CAR expression.

[0114] In some embodiments, the CAR is located from the N-terminus to the C-terminus. i. Extracellular antigen-binding domain having binding affinity to the antigen, ii. Transmembrane domain, iii. Intracellular co-stimulatory domains, and iv. Intracellular activation domain Includes.

[0115] In some embodiments, the priming receptor and CAR bind to different antigens.

[0116] In some embodiments, the priming receptor and the CAR bind to the same antigen.

[0117] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins, ii. Provide a solution containing a ribonucleoprotein complex (RNP) and a DNA template (the RNP contains a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), iii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0118] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide a solution comprising a histone deacetylase (HDAC) inhibitor containing at least one of sodium phenylbutyrate, xinostat, or panobinostat, and cells. ii. The cells are brought into contact with a ribonucleoprotein complex (RNP) and a DNA template (the RNP contains a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), iii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0119] In some embodiments, the method involves contacting edited cells with a solution comprising a histone deacetylase (HDAC) inhibitor, which comprises at least one of sodium phenylbutyrate, xinostat, or panobinostat.

[0120] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide a solution containing an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template (the RNP contains a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome). ii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0121] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide a solution containing N-acetyl-L-cysteine ​​(NAC), a ribonucleoprotein complex (RNP), and a DNA template (the RNP contains a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome). ii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0122] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide a solution containing an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template (the RNP contains a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome). ii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0123] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide a solution comprising a histone deacetylase (HDAC) inhibitor containing at least one of sodium phenylbutyrate, xynostat, or panobinostat, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template containing nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), ii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0124] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins, ii. A solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of the following: an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor (the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), iii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0125] In one embodiment, the present specification provides a method for editing cells, the method being: i. Provide a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of the following: an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor (the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome). ii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0126] In some embodiments, the solution includes at least the following: i. N-acetyl-L-cysteine ​​(NAC) and osmotic regulators, ii. RNase inhibitors and N-acetyl-L-cysteine ​​(NAC), iii. RNase inhibitors and osmotic regulators, iv. N-acetyl-L-cysteine ​​(NAC) and histone deacetylase (HDAC) inhibitors, v. Osmotic regulators and histone deacetylase (HDAC) inhibitors, vi. RNase inhibitors and histone deacetylase (HDAC) inhibitors, vii. RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), and histone deacetylase (HDAC) inhibitors, viii. N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors. ix. RNase inhibitors, osmotic regulators, and histone deacetylase (HDAC) inhibitors, x. RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), and osmotic regulators, or xi. RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors.

[0127] In some embodiments, the RNase inhibitor is present in the solution at a final concentration of approximately 0.5–2 U / μL.

[0128] In some embodiments, the RNase inhibitor is present in the solution at a final concentration of approximately 1 U / μL.

[0129] In some embodiments, the RNase inhibitor is an RNase A, B, C, T1, or T2 inhibitor.

[0130] In some embodiments, the RNase inhibitor is a mouse, rat, or human RNase inhibitor.

[0131] In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in solution at a final concentration of approximately 1–10 mM.

[0132] In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of approximately 2.5 mM.

[0133] In some embodiments, the osmotic pressure regulator is sorbitol, glycerol, or glycine.

[0134] In some embodiments, the osmotic pressure regulator is sorbitol.

[0135] In some embodiments, sorbitol is present in the solution at a final concentration of about 100–400 mM.

[0136] In some embodiments, sorbitol is present in the solution at a final concentration of about 190 mM or 200 mM.

[0137] In some embodiments, the osmotic pressure regulator is glycerol.

[0138] In some embodiments, glycerol is present in the solution at a final concentration of about 100–400 mM.

[0139] In some embodiments, glycerol is present in the solution at a final concentration of approximately 170 mM.

[0140] In some embodiments, the histone deacetylase (HDAC) inhibitor is either an HDAC1 inhibitor or an HDAC2 inhibitor.

[0141] In some embodiments, HDAC inhibitors include sodium phenylbutyrate, xinostat, panobinostat, phenylbutyrate, curcumin, mosetinostat, romidespin, SIS17, spritomycin, trichostatin-A, tusidinostat, etinostat, sodium butyrate, valproic acid, CXD101, KT-531, ITF3756, tubastatin A, vorinostat, BML-210, bellinostat, avexinostat, dasinostat, CUDC-101, droxinostat, MC1568, prasinostat, divalproex sodium, PCI-3405, SR-4370, zibinostat, tubasin, AR-42, and (-)-parthenolide. These are resminostat, fimepinostat, M344, taseginaline, sinapic acid, biphenyl-4-sulfonyl chloride, sulforaphane, UF010, suberohydroxamic acid, NKL22, TC-H106, RGFP966, HPOB, RG2833, TMP269, nextulastat A, domatinostat, LMK-235, Santa Cruz amete A, CAY10603, tascinimod, BG45, BRD73954, licorinostat, scriptide, sitalinostat, WT161, TMP195, ACY-738, SKLB-23bb, tinostamstine, TH34, BRD3308, radianine A, isoguanosine, KA2507, or ITSA-1.

[0142] In some embodiments, the HDAC inhibitor is sodium phenylbutyrate, xinostat, or panobinostat.

[0143] In some embodiments, sodium phenylbutyrate is present in the solution at a final concentration of approximately 15.6 μM to 4 mM.

[0144] In some embodiments, sodium phenylbutyrate is present in the solution at a final concentration of about 1 mM, and optionally, the solution contains 1% DMSO.

[0145] In some embodiments, xynostat is present in the solution at a final concentration of approximately 8 nM to 200 nM.

[0146] In some embodiments, xinostat is present in the solution at a final concentration of about 16 nM.

[0147] In some embodiments, panobinostat is present in the solution at a final concentration of approximately 3 nM to 1 μM.

[0148] In some embodiments, panobinostat is present in solution at a final concentration of approximately 37.5 nM.

[0149] In some embodiments, the solution comprises at least one of the following: an RNase inhibitor at a final concentration of approximately 0.5–2 U / μL, NAC at a final concentration of approximately 1–10 mM, sorbitol at a final concentration of approximately 100–400 mM, and / or sodium phenylbutyrate at a final concentration of approximately 15.6 μM–4 mM, xynostat at 8 nM–200 nM, or panobinostat at 3 nM–1 μM.

[0150] In some embodiments, the solution comprises at least one of the following: an RNase inhibitor at a final concentration of about 1 U / μL, NAC at a final concentration of about 2.5 mM, sorbitol at a final concentration of about 200 mM, and / or sodium phenylbutyrate at a final concentration of about 1 mM, 0.016 μM xynostat, or 0.0375 μM panobinostat.

[0151] In some embodiments, exogenous homologous recombination proteins or DNA repair regulatory proteins are encoded on an episomal plasmid or mRNA molecule.

[0152] In some embodiments, the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

[0153] In some embodiments, the SWSAP1 protein contains the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein contains the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein contains the sequence shown in SEQ ID NO: 127.

[0154] In some embodiments, the method further comprises nonvirally introducing an RNP complex and a DNA template into a cell, where the guide RNA specifically hybridizes to a target region of the cell's genome, and the nuclease domain cleaves the target region to create an insertion site within the cell's genome.

[0155] In some embodiments, nonviral introduction includes electroporation.

[0156] In some embodiments, electroporation includes at least one cycle comprising at least one electrical pulse.

[0157] In some embodiments, at least one cycle includes at least five or more electrical pulses.

[0158] In some embodiments, the electrical pulse is approximately 2300 volts.

[0159] In some embodiments, the duration of the electrical pulse is approximately 3.0 ms.

[0160] In some embodiments, the cycle has a pulse interval of 500 ms.

[0161] In some embodiments, electroporation includes at least one cycle performed using settings of 2300 volts, a pulse duration of 3.0 ms, five pulses, and a pulse interval of 500 ms.

[0162] In some embodiments, exogenous homologous recombination proteins or DNA repair regulatory proteins increase the insertion of DNA templates into the cellular genome compared to cells that do not contain exogenous homologous recombination proteins or DNA repair regulatory proteins.

[0163] In some embodiments, a solution containing at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmoregulator, or a histone deacetylase (HDAC) inhibitor increases the insertion of DNA templates into the cellular genome compared to a control solution that does not contain at least one of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmoregulator, or a histone deacetylase (HDAC) inhibitor.

[0164] In some embodiments, the method includes incubating cells with an HDAC inhibitor solution for about two days before nonvirally introducing the RNP complex and DNA template into the cells.

[0165] In some embodiments, the HDAC inhibitor solution contains sodium phenylbutyrate at a final concentration of approximately 0.1 mM.

[0166] In some embodiments, the insertion of the DNA template into the cell genome increases by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

[0167] In some embodiments, the insertion of the DNA template into the cell genome increases by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

[0168] In some embodiments, the solution enhances the growth of edited cells compared to a control solution that does not contain at least one of the following: an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor.

[0169] In some embodiments, the enlargement of edited cells increases by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

[0170] In some embodiments, the enlargement of the edited cells increases by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

[0171] In some embodiments, the solution increases the yield of edited cells compared to a control solution that does not contain at least one of the following: an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor.

[0172] In some embodiments, the yield of edited cells increases by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

[0173] In some embodiments, the yield of edited cells increases by at least 0.25 times, 0.5 times, 0.75 times, 1 time, 1.25 times, 1.5 times, 1.75 times, 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times, 3.25 times, 3.5 times, 3.75 times, 4 times, 4 times, 4.25 times, 4.5 times, 4.75 times, 5 times, 5.25 times, 5.5 times, 5.75 times, 6 times, 6.25 times, 6.5 times, 6.75 times, or more compared to the control solution.

[0174] In some embodiments, a solution containing at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), or an osmoregulator reduces cell death during the nonviral introduction of RNP complexes and DNA templates into cells compared to a control solution that does not contain at least one of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmoregulator, or a histone deacetylase (HDAC) inhibitor.

[0175] In some embodiments, cell death is reduced by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

[0176] In some embodiments, cell death is reduced by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

[0177] In some embodiments, the nuclease domain includes a CRISPR-related endonuclease (Cas), optionally a Cas9 nuclease.

[0178] In some embodiments, the size of the DNA template is 5 kilobase nucleotides or larger.

[0179] In some embodiments, the size of the DNA template is approximately 0.3kb, 0.5kb, 1.0kb, 1.5kb, 2.0kb, 2.5kb, 3.0kb, 3.5kb, 4.0kb, 4.5kb, 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6. 2kb, 6.3kb, 6.4kb, 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8kb, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9k b, 9.0kb, 9.1kb, 9.2kb, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, 10.0kb, 10.1kb, 10.2kb , 10.3kb, 10.4kb, 10.5kb, 10.6kb, 10.7kb, 10.8kb, 10.9kb, 11.0kb, 11.1kb, 11.2kb, 11.3kb, 11.4 DNA templates of sizes kb, 11.5kb, 11.6kb, 11.7kb, 11.8kb, 11.9kb, 12.0kb, 12.1kb, 12.2kb, 12.3kb, 12.4kb, 12.5kb, 12.6kb, 12.7kb, 12.8kb, 12.9kb, 13.0kb or larger, or any size between these sizes.

[0180] In some embodiments, the size of the DNA template is approximately 0.3kb to 13kb, 0.3kb to 0.5kb, 0.3kb to 1kb, 0.3kb to 4kb, 0.3kb to 3kb, 0.3kb to 5kb, 0.3kb to 7kb, 0.3kb to 10kb, 0.5kb to 1kb, 0.5kb to 3kb, 0.5kb to 5kb, 0.5kb to 7kb, 0.5kb to 10kb, 0.5kb to 13kb, 1kb to 3kb, 1kb to 5kb, 1kb to 7kb, 1kb to 10kb b. Approximately 1kb to 13kb, 5kb to 13kb, 5kb to 9kb, 5kb to 8kb, 5kb to 7kb, 5kb to 6kb, 6kb to 13kb, 6kb to 10kb, 6kb to 9kb, 6kb to 8kb, 6kb to 7kb, 7kb to 13kb, 7kb to 10kb, 7kb to 9kb, 7kb to 8kb, 8kb to 13kb, 8kb to 10kb, 8kb to 9kb, 9kb to 13kb, 9kb to 10kb, 10kb to 13kb, or approximately 11kb to 13kb.

[0181] In some embodiments, the cells are mammalian cells, human cells, hematopoietic cells, immune cells, primary immune cells, or primary human immune cells.

[0182] In some embodiments, the cells are primary human immune cells.

[0183] In some embodiments, the immune cells are natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, primary T cells, or T cell progenitor cells.

[0184] In some embodiments, the immune cells are primary T cells.

[0185] In some embodiments, the immune cells are primary human T cells.

[0186] In some embodiments, immune cells are undifferentiated.

[0187] In some embodiments, immune cells are CD45RA + and CCR7 + CD45RA + and CCR7 - CD45RA - and CCR7 - , or CD45RA - and CCR7 + That is the case.

[0188] In some embodiments, the cells are virus-free.

[0189] In some embodiments, this involves collecting cells from a patient and introducing a DNA template in vitro or ex vivo.

[0190] In some embodiments, the target region of the cell genome is either the T cell receptor α constant region (TRAC) locus or a genome-safe harbor (GSH).

[0191] In some embodiments, the safe harbor locus is selected from any one of the integration sites designated by GS94, GS88, GS89, GS90, GS91, GS92, GS93, GS95, GS96, GS97, GS98, GS99, GS100, GS101, GS102, GS103, GS104, GS105, GS106, GS107, GS108, GS109, GS110, GS111, GS112, GS113, GS114, GS115, GS116, GS117, GS118, GS119, or GS120.

[0192] In some embodiments, the safe harbor locus is the GS94 integration site.

[0193] In some embodiments, the sgRNA target loci are chr11:128340000-128350000, chr10:33130000-33140000, chr10:72290000-72300000, chr11:65425000-65427000 (NEAT1), chr15:92830000-928400 Select from 00, chr16:11220000-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, or chr9:7970000-7980000.

[0194] In some embodiments, the sgRNA target locus is a gene selected from APRT, B2M, CAPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28.

[0195] In some embodiments, one or more gRNAs include one of sequence numbers 1 to 120.

[0196] In some embodiments, after at least one sequence is inserted into the safe harbor locus, the cells are CD45RA + and CCR7 + That is the case.

[0197] In some embodiments, the DNA template is either a double-stranded DNA template or a single-stranded DNA template.

[0198] In some embodiments, the DNA template is a linear DNA template or a circular DNA template, and optionally, the circular DNA template is a plasmid.

[0199] In some embodiments, the DNA template includes heterologous sequences.

[0200] In some embodiments, the DNA template includes genes.

[0201] In some embodiments, the DNA template includes a priming receptor containing a transcription factor.

[0202] In some embodiments, the DNA template includes a chimeric antigen receptor (CAR).

[0203] In some embodiments, the DNA template includes a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor.

[0204] In some embodiments, the DNA template includes an inductive promoter operably linked to a chimeric antigen receptor.

[0205] In some embodiments, the DNA template further includes a constitutive promoter operably linked to the priming receptor.

[0206] In some embodiments, the DNA template further includes an inductive promoter operably ligated to a chimeric antigen receptor and a constitutive promoter operably ligated to a priming receptor.

[0207] In some embodiments, the DNA template is oriented from 5' to 3': i. Inducible promoter, ii. Chimeric antigen receptor, iii. Constitutive promoters, and iv. Priming receptors Includes.

[0208] In some embodiments, the DNA template is oriented from 5' to 3': i. Constitutive promoter, ii. Priming receptors, iii. Inducible promoters, and iv. Contains chimeric antigen receptors.

[0209] In some embodiments, the priming receptor is located in the direction from the N-terminus to the C-terminus. i. Extracellular antigen-binding domain having binding affinity to the antigen, ii. A transmembrane domain containing one or more ligand-induced proteolytic cleavage sites, and iii. Intracellular domains containing human or humanized transcription effectors It contains such a component that when an antigen binds to the extracellular antigen-binding domain, cleavage occurs at the ligand-induced proteolytic cleavage site, thereby releasing the intracellular domain.

[0210] In some embodiments, the priming receptor further comprises a near-membrane domain (JMD) or a stop transition sequence (STS) located between the transmembrane domain and the intracellular domain.

[0211] In some embodiments, the transcription factor binds to an inducible promoter to induce CAR expression.

[0212] In some embodiments, the CAR is located from the N-terminus to the C-terminus. i. Extracellular antigen-binding domain having binding affinity to the antigen, ii. Transmembrane domain, iii. Intracellular co-stimulatory domains, and iv. Intracellular activation domain Includes.

[0213] In some embodiments, the priming receptor and CAR bind to different antigens.

[0214] In some embodiments, the priming receptor and the CAR bind to the same antigen.

[0215] In one embodiment, this specification provides a method for editing immune cells, the method being i. Provide immune cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins, ii. Provide a solution containing a ribonucleoprotein complex (RNP) and a DNA template (the RNP comprising a nuclease domain and guide RNA, the DNA template comprising a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor, and the 5' and 3' ends of the DNA template containing nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), iii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0216] In one embodiment, this specification provides a method for editing immune cells, the method being i. Provide immune cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins, ii. A solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of the following: an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, and / or a histone deacetylase (HDAC) inhibitor (the RNP comprising a nuclease domain and guide RNA, the DNA template comprising a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), iii. This includes editing immune cells by inserting a DNA template into an insertion site within the cell's genome.

[0217] In some embodiments, the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

[0218] In some embodiments, the SWSAP1 protein contains the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein contains the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein contains the sequence shown in SEQ ID NO: 127.

[0219] In one embodiment, this specification provides a method for editing immune cells, the method being i. Provide a solution containing an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprises a nuclease domain and guide RNA, the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), ii. The RNP and DNA template are introduced nonvirally into immune cells (the guide RNA specifically hybridizes to the target region of the cell's genome, and the nuclease domain cleaves the target region to create an insertion site within the cell's genome), iii. This includes editing immune cells by inserting a DNA template into an insertion site within the cell's genome.

[0220] In one embodiment, this specification provides a method for editing immune cells, the method being i. Provide a solution containing N-acetyl-L-cysteine ​​(NAC), ribonucleoprotein complex (RNP), and a DNA template (the RNP contains a nuclease domain and guide RNA, the DNA template contains a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the genome of an immune cell), ii. The RNP and DNA template are introduced nonvirally into immune cells (the guide RNA specifically hybridizes to the target region of the cell's genome, and the nuclease domain cleaves the target region to create an insertion site within the cell's genome), iii. This includes editing immune cells by inserting a DNA template into an insertion site within the cell's genome.

[0221] In one embodiment, this specification provides a method for editing immune cells, the method being i. Provide a solution comprising an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, the DNA template comprising a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the genome of an immune cell), ii. The RNP and DNA template are introduced nonvirally into immune cells (the guide RNA specifically hybridizes to the target region of the cell's genome, and the nuclease domain cleaves the target region to create an insertion site within the cell's genome), iii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0222] In one embodiment, this specification provides a method for editing immune cells, the method being i. Provide a solution comprising a histone deacetylase (HDAC) inhibitor containing at least one of sodium phenylbutyrate, xynostat, or panobinostat, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, the DNA template comprising a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the genome of an immune cell), ii. The RNP and DNA template are introduced into the cell nonvirally (the guide RNA specifically hybridizes to the target region of the cell's genome, and the nuclease domain cleaves the target region to create an insertion site within the cell's genome), iii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0223] In one embodiment, this specification provides a method for editing immune cells, the method being i. A solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of the following: an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, and / or a histone deacetylase (HDAC) inhibitor (the RNP comprising a nuclease domain and guide RNA, the DNA template comprising a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), ii. The RNP and DNA template are introduced nonvirally into immune cells (the guide RNA specifically hybridizes to the target region of the cell's genome, and the nuclease domain cleaves the target region to create an insertion site within the cell's genome), iii. This includes editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0224] In one embodiment, this specification provides a method for treating a subject who has or is at risk of having a disease, the method being: i. To carry out the methods disclosed herein, and ii. This includes administering an effective amount of a composition containing cells or a population thereof to a target.

[0225] In some embodiments, the composition is administered to the subject by injection.

[0226] In some embodiments, the disease is cancer.

[0227] In some embodiments, immune cells are produced by the methods disclosed herein. [Brief explanation of the drawing]

[0228] These and other features, aspects, and advantages of the present invention will be better understood with reference to the following description and the accompanying drawings.

[0229] [Figure 1] A shows the exemplary knock-in rate of transgenes after electroporation in the presence (+) or absence (-) of an RNase inhibitor. B shows the increase in the expansion fold of T cells after electroporation in the presence (+) or absence (-) of an RNase inhibitor. C shows the normalized yield of edited T cells after electroporation in the presence (+) or absence (-) of an RNase inhibitor. [Figure 2] A shows the exemplary knock-in (KI) rate (%) of transgenes in control cells (left bar) and cells treated with an RNase inhibitor (right cells) after electroporation of cells from two different donors using the Xenon MultiShot platform at a clinical scale. B shows the total number of edited cells in control cells (left bar) and cells treated with an RNase inhibitor (right cells) after electroporation of cells from two different donors using the Xenon MultiShot platform at a clinical scale. [Figure 3] The fold change in the yield of edited T cells (upper figure) and the fold change in KI relative to control cells (lower figure) upon electroporation with (+ upper line) and without (- lower line) addition of an RNase inhibitor using the Xenon platform are shown as a function of the exposure time of cells to the payload. Control cells were electroporated without addition of an RNase inhibitor with an exposure time of cells to the payload of 0 minutes. The data shown are from five different donors. Error range = standard error of the mean [Figure 4]A shows the fold change in gene knock-in (KI) in T cells when electroporated with or without addition of RNase inhibitor in five different donor cells using the Xenon platform. B shows the fold change in the yield of edited cells relative to control cells when RNase inhibitor was added in five different donor cells using the Xenon platform. The control cells were electroporated without addition of RNase inhibitor and with a cell and payload exposure time of 0 minutes (dotted line). The data shown are the average of five different donors. Error range = standard error of the mean. [Figure 5] A shows the proportion of stem cell memory T cells (Tscm) and central memory T cells (Tcm) defined by CCR7 and CD45RA expression of T cells electroporated in the presence or absence of RNase inhibitor in cells from 13 different donors using the Xenon platform. B shows the ratio of CD4 T cells to CD8 T cells after electroporation in the presence or absence of RNase inhibitor in cells from the same 13 different donors. The p-value is the value of a two-sided t-test. [Figure 6] A shows the fold change in edited cell yield in cells from five donors after electroporation using the Lonza platform in the presence of 0 mM or 5 mM NAC. B shows the KI rate of the yield of the transgene in cells from five donors after electroporation in the presence of 0 mM (left bar) or 5 mM (right bar) NAC. [Figure 7]A shows the multiplicative change in edited cell yield in cells from three donors after electroporation using the Xenon platform in the presence of 0 mM or 5 mM NAC. B shows the KI rate of transgene yield in cells from three donors after electroporation using the Xenon platform in the presence of 0 mM (left bar) or 5 mM (right bar) NAC. C shows the normalized yield of edited cells in cells from three donors after electroporation using the Xenon platform in the presence of 0 mM (left bar) or 5 mM (right bar) NAC. [Figure 8] A shows the KI rate in cells after electroporation in the presence of 2.5 mM NAC or 5 mM NAC across five different donors. B shows the total number of edited cells in cells after electroporation in the presence of 2.5 mM NAC or 5 mM NAC across five different donors. [Figure 9] A shows the multiplicative change in the yield of edited cells after electroporation using the Xenon platform with 200 mM sorbitol across five different donors. B compares the editing efficiency of the same donors with and without 200 mM sorbitol during electroporation using the Xenon platform. [Figure 10] A shows the correlation of KI efficiency between two donor cell lines electroporated with plasmids transiently expressing the indicated protein. B shows the correlation of KI efficiency between two donor cell lines electroporated with plasmids transiently expressing the indicated protein. Luciferase was used as a control. NLS tags are added where applicable. [Figure 11A] This bar graph shows the KI efficiency (%) between cells from two donors using a 9kb plasmid transiently expressing a GFP reporter. A control plasmid encoding the luciferase reporter gene (luc) was also used. [Figure 11B]A bar graph shows the KI efficiency (%) between cells from two donors using a 9kb plasmid transiently expressing an exemplary myc-tagged CAR. A control plasmid encoding the luciferase reporter gene (luc) was also used. [Figure 12] This shows the multiplicative changes in the knock-in (KI) rates of exemplary transgenes in T cells after electroporation, incubated in culture medium with 0.5 mM, 1 mM, 2 mM, and 4 mM sodium phenylbutyrate in the presence of 1% DMSO. [Figure 13] The knock-in (KI) rates of exemplary transgenes in T cells are shown for two days after pre-incubating with 0.1 mM sodium phenylbutyrate, followed by electroporation and recovery in standard medium, and for T cells recovered in medium containing 1% DMSO and 1 mM sodium phenylbutyrate. [Figure 14] This shows the knock-in (KI) rates of exemplary transgenes in T cells after incubation with 0.016 μM (16 nM) xynostat or 0.0375 μM (37.5 nM) panobinostat following electroporation. [Figure 15] The images show exemplary knock-in (KI) rates of transgenes in T cells after incubation with the compounds shown. [Figure 16A] The graph shows the total number of knock-in (KI) (%) and edited T cells from seven different donors expressing an exemplary transgene(s) after applying four or five electroporation pulses in a medium supplemented with Xenofree Supplement B. Samples with four pulses are shown in the left bar, and samples with five pulses are shown in the right bar. [Figure 16B] The graph shows the knock-in (KI) (%) and total number of edited T cells from five different donors expressing an exemplary transgene(s) after applying four or five electroporation pulses in a culture medium supplemented with Xenofree Supplement A. Samples with four pulses are shown in the left bar, and samples with five pulses are shown in the right bar. [Figure 17]A shows the number of tumor target cells after incubation with edited T cells at an E:T ratio of 1:50. B shows the number of tumor target cells after incubation with edited T cells at an E:T ratio of 1:100. [Figure 18] In T cells derived from five donors, KI (%) and cell expansion (TEC) on days 7 and 9 after electroporation were increased with the 5-pulse protocol compared to the 4-pulse protocol. [Figure 19A] The image shows a nanoplasmid encoding an exemplary transgene (reduced backbone length) and the KI (%) and total number of edited T cells after 5 electroporation pulses (center bar), 4 electroporation pulses (left bar), and 3 electroporation pulses (right bar). [Figure 19B] The graph shows the KI (%) and total number of edited T cells after a standard plasmid encoding an exemplary transgene (total skeleton length), five electroporation pulses (center bar), four electroporation pulses (left bar), and three electroporation pulses (right bar). [Figure 20] A shows the KI (%) and total number of edited T cells after electroporation with 4 or 5 pulses. Cells were incubated in a medium containing xenofree supplement A. B shows the KI (%) and total number of edited T cells after electroporation with 4 or 5 pulses. Cells were incubated in a medium containing xenofree supplement B. [Modes for carrying out the invention]

[0230] Detailed explanation definition Unless otherwise specified, terms used in the claims and specification are defined as set forth below.

[0231] As used herein, the term “gene” refers to the basic unit of heredity, consisting of a segment of DNA arranged along a chromosome that codes for a particular protein or segment of a protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally an intron, and a 3' untranslated region. A gene may further include terminators, enhancers, and / or silencers.

[0232] As used herein, the term “locus” refers to a specific, fixed physical location on a chromosome where a gene or genetic marker is located.

[0233] The term “safe harbor locus” refers to a locus into which a gene or gene element can be incorporated without interrupting the expression or regulation of adjacent genes. These safe harbor loci are also referred to as safe harbor sites (SHS). As used herein, a safe harbor locus refers to an “integration site” or “knock-in site” into which a sequence encoding a transgene, as defined herein, can be inserted. In some embodiments, the insertion occurs with the substitution of a sequence located at the integration site. In some embodiments, the insertion occurs without the substitution of a sequence at the integration site. Examples of intended integration sites are provided in Table 1.

[0234] As used herein, the term “insertion” refers to a nucleotide sequence that has been incorporated (inserted) into a target locus or safe harbor site. “Insertion” can be used to refer to a gene or genetic element that has been incorporated into a target locus or safe harbor site, for example, by homology-directed repair (HDR) CRISPR / Cas9 genome editing or other methods for inserting nucleotide sequences into genomic regions known to those skilled in the art.

[0235] The "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against foreign nucleic acids. The CRISPR / Cas system is found in a wide range of fungal and archaeal organisms. The CRISPR / Cas system includes type I, type II, and type III subtypes. The wild-type type II CRISPR / Cas system utilizes the RNA-directed nuclease Cas9 in a complex with guide RNA and activating RNA to recognize and cleave foreign nucleic acids. Guide RNAs having the activity of both guide RNA and activating RNA are also known in the art. In some cases, such dual-activity guide RNAs are referred to as small guide RNAs (sgRNAs).

[0236] Cas9 homologs are found in a variety of fungi, including but not limited to bacteria of the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and their homologs are described, for example, in Chylinksi, et al., RNA Biol. 2013 May 1;10(5):726-737, Nat.Rev.Microbiol. 2011 June;9(6):467-477, Hou, et al., Proc Natl Acad Sci U S A. 2013 Sep 24;110(39):15644-9, Sampson et al., Nature. 2013 May 9;497(7448):254-7, and Jinek, et al., Science. 2012 Aug 17;337(6096):816-21. The Cas9 nuclease domain can be optimized for efficient activity or enhanced stability in a host cell.

[0237] As used herein, the term "Cas9" refers to an RNA-mediated nuclease (e.g., of bacterial or archaeal origin, or derived therefrom). Exemplary RNA-induced nucleases include, but are not limited to, the aforementioned Cas9 protein and its homologs, and CPF1 (see, for example, Zetsche et al., Cell, Volume 163, Issue 3, pp. 759-771, 22 October 2015). Similarly, as used herein, the terms "Cas9 ribonucleoprotein" complex, etc., refer to a complex between the Cas9 protein and crRNA (e.g., guide RNA or small guide RNA), the Cas9 protein and transactivating crRNA (tracrRNA), the Cas9 protein and small guide RNA, or a combination thereof (e.g., a complex containing the Cas9 protein, tracrRNA, and crRNA guide RNA).

[0238] As used herein, the terms “T lymphocyte” and “T cell” are used interchangeably and refer to cells that have completed maturation in the thymus and can identify specific exogenous antigens in the body. These terms also refer to the major leukocyte types that have various roles in the immune system, including the activation and inactivation of other immune cells. T cells can be any T cells, such as cultured T cells, e.g., primary T cells, or T cells derived from cultured T cell lines, e.g., JuRKAt, SuPT1, etc., or mammalian T cells. Examples of T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subpopulations thereof. T cells can be CD3+ cells. T cells can be CD4 + CD8 + , or CD4 + and CD8 +It can be any type of T cell, CD4+ / CD8+ double-positive T cell, CD4+ helper T cell (e.g., Th1 and Th2 cells), CD8+ T cell (e.g., cytotoxic T cell), peripheral T cell (including but not limited to peripheral blood mononuclear cells (PBMCs) and peripheral blood leukocytes (PBLs)), tumor-infiltrating lymphocytes (TILs), memory T cells, stem memory T cells (Tscm), effector T cells, naive T cells, regulatory T cells, γδ T cells, etc. It can be any T cell at any stage of development. Additional types of helper T cells include Th3 (Treg) cells, Th17 cells, Th9 cells, or Tfh cells. Additional types of memory T cells include cells such as central memory T cells (Tcm cells) and effector memory T cells (Tem cells and TEMRA cells). T cells can also refer to genetically modified T cells, such as T cells that have been modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). T cells can also differentiate from stem cells or progenitor cells.

[0239] "CD4+ T cells" refer to a subset of T cells that express CD4 on their surface and are involved in cellular immune responses. CD4+ T cells are characterized by a post-stimulation secretion profile that may include the secretion of cytokines such as IFN-γ, TNF-α, IL-2, IL-4, and IL-10. "CD4" is a 55kD glycoprotein originally defined as a differentiation antigen on T lymphocytes, but has also been found on other cells, including monocytes / macrophages. The CD4 antigen is a member of the immunoglobulin superfamily and has been suggested as an associative recognition element in MHC (major histocompatibility complex) class II restriction immune responses. On T lymphocytes, the CD4 antigen defines a helper / inducer subset.

[0240] "CD8+ T cells" refer to a subset of T cells that express CD8 on their surface, are MHC class I restricted, and function as cytotoxic T cells. The "CD8" molecule is a differentiation antigen present on thymocytes, as well as on cytotoxic and suppressive T lymphocytes. The CD8 antigen is a member of the immunoglobulin superfamily and is an associative recognition element in major histocompatibility complex class I restriction interactions.

[0241] As used herein, the term “hematopoietic stem cell” refers to a type of stem cell capable of producing blood cells. Hematopoietic stem cells can produce myeloid or lymphoid cells, or a combination thereof. Hematopoietic stem cells are primarily found in the bone marrow, but they can be isolated from peripheral blood or its fractions. Various cell surface markers can be used to identify, sort, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are c-kit + and Lin - It is identified as such. In some cases, human hematopoietic stem cells are CD34 + CD59 + Thy1 / CD90 + CD38 lo / - C-kit / CD117 + ,lin - It is identified as such. In some cases, human hematopoietic stem cells are CD34 - CD59 + Thy1 / CD90 + CD38 lo / - C-kit / CD117 + ,lin - It is identified as such. In some cases, human hematopoietic stem cells are CD133 + CD59 + Thy1 / CD90 + CD38 lo / - C-kit / CD117 + ,lin - It is identified as such. In some cases, mouse hematopoietic stem cells are CD34 lo / - SCA-1 + Thy1 + / lo CD38+ C-kit + ,lin - It is identified as such. In some cases, hematopoietic stem cells are CD150 + CD48 - CD244 - That is the case.

[0242] As used herein, the term “hematopoietic cells” refers to cells derived from hematopoietic stem cells. Hematopoietic cells may be obtained or provided by isolation from an organism, system, organ, or tissue (e.g., blood or a fraction thereof). Alternatively, hematopoietic cells may be obtained or provided by isolating hematopoietic stem cells and differentiating the stem cells. Hematopoietic cells include cells with limited potential for differentiation into further cell types. Such hematopoietic cells include, but are not limited to, pluripotent progenitor cells, lineage-restricted progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells, or megakaryocyte-erythroid progenitor cells. Hematopoietic cells include lymphoid and myeloid cells such as lymphocytes, erythrocytes, granulocytes, monocytes, and platelets.

[0243] As used herein, the term “immune cells” includes all cell types that give rise to immune cells, and immune cells include hematopoietic cells, pluripotent stem cells, and induced pluripotent stem cells (iPSCs). In some embodiments, immune cells are B cells, macrophages, natural killer (NK) cells, induced pluripotent stem cells (iPSCs), human pluripotent stem cells, hematopoietic stem cell progenitor cells (HSPCs), T cells or T cell progenitor cells, or dendritic cells. In some embodiments, the cells are innate immune cells.

[0244] As used herein, the term “primary” in the context of primary cells or primary stem cells refers to cells that have not been transformed or immortalized. Such primary cells may be cultured, subcultured, or subcultured a limited number of times (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times). In some cases, primary cells are adapted to in vitro culture conditions. In some cases, primary cells are isolated from an organism, system, organ, or tissue, selected at will, and used directly, for example, without culture or subculture. In some cases, primary cells are stimulated, activated, or differentiated. For example, primary T cells can be activated by contact with (e.g., by culturing in the presence of) CD3 agonists, CD28 agonists, IL-2, IL-7, IL-15, IFN-γ, or combinations thereof.

[0245] As used herein, the term “ex vivo” generally includes experiments or measurements conducted in or on living tissue, preferably in an artificial environment outside of living tissue, preferably with minimal differences from natural conditions.

[0246] As used herein, the term “construct” refers to a complex of molecules containing macromolecules or polynucleotides. For example, a construct may be a DNA polynucleotide molecule produced by artificial means. In some embodiments, a DNA construct may be propagated via plasmid replication in bacteria.

[0247] As used herein, the term “integration” refers to the process of stably inserting one or more nucleotides of a construct into the cellular genome, i.e., covalently bonding them to a nucleic acid sequence within the cell’s chromosomal DNA. It may also refer to a nucleotide deletion at the site of integration. If there is a deletion at the insertion site, “integration” may further include the deletion of an endogenous sequence or nucleotide substitution with one or more inserted nucleotides.

[0248] As used herein, the term “exogenous” refers to a molecule or activity that is introduced into a host cell and is not native to that cell. This molecule may be introduced, for example, by the introduction of coding nucleic acids into the host genetic material, for example, by integration into the host chromosome, or as non-chromosomal genetic material such as a plasmid. Therefore, when used in relation to the expression of coding nucleic acids, the term refers to the introduction of coding nucleic acids into cells in an expressible form. The term “endogenous” refers to a molecule or activity that is present in the host cell under native, unedited conditions. Similarly, when used in relation to the expression of coding nucleic acids, the term refers to the expression of coding nucleic acids that are contained within the cell and not introduced exogenously.

[0249] The term "heterogeneous" refers to a non-natural nucleic acid or polypeptide sequence or domain adjacent to a sequence, for example, a heterogeneous sequence is not found in nature coupled to the nucleic acid or polypeptide sequences that occur at one or both ends.

[0250] The term "homologous" refers to a nucleic acid or polypeptide sequence or domain that is naturally occurring in an adjacent sequence. For example, homologous sequences are found in nature coupled to nucleic acid or polypeptide sequences that occur at one or both ends.

[0251] As used herein, “polynucleotide donor construct” refers to a nucleotide sequence (e.g., a DNA sequence) that is genetically inserted into a polynucleotide and is exogenous to that polynucleotide. Part or all of a polynucleotide donor construct is transcribed into RNA and optionally translated into a polypeptide. A polynucleotide donor construct may include a prokaryotic sequence, a cDNA derived from eukaryotic mRNA, a genomic DNA sequence derived from eukaryotic (e.g., mammalian) DNA, and a synthetic DNA sequence. For example, a polynucleotide donor construct may be a miRNA, shRNA, a natural polypeptide (i.e., a naturally occurring polypeptide) or a fragment thereof, or a variant polypeptide (e.g., a natural polypeptide having less than 100% sequence identity with the natural polypeptide) or a fragment thereof.

[0252] As used herein, the terms “complementary” or “complementarity” refer to specific base pairings between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), as well as G and C. Guide RNAs described herein may include DNA target sequences that are fully complementary or substantially complementary (e.g., having 1 to 4 mismatches) to a cellular genomic sequence.

[0253] As used herein, the term “transgene” refers to a polynucleotide that has been translocated from one organism to another, either naturally or by one of several genetic modification techniques. It is optionally translated into a polypeptide or a protein. It is optionally translated into a recombinant protein. A “recombinant protein” is a protein encoded by a gene (recombinant DNA or synthetic DNA) that has been cloned in a system that supports gene expression and messenger RNA translation (see expression system). The protein may be a therapeutic agent, for example, a protein that treats a disease or disorder disclosed herein, such as a CAR, priming receptor, or TCR. As used herein, transgene may refer to a polynucleotide that codes for a polypeptide or a protein. Transgene may also refer to a non-protein-coding polynucleotide sequence, such as but not limited to shRNA, siRNA, miRNA, and miR.

[0254] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0255] As used herein, the terms “operably ligated” or “operably ligated” refer to a nucleic acid sequence being bound to a single nucleic acid fragment such that the function of one is influenced by the function of the other. For example, if a promoter can influence the expression of a coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under transcriptional control by the promoter), then the promoter is operably ligated to it. A coding sequence can be operably ligated to a control sequence in both sense-oriented and antisense-oriented orientations.

[0256] As used herein, the term “developing cellular state” refers to a state in which a cell is, for example, inactive, actively expressive, differentiated, or senescent. A developing cellular state may also refer to a progenitor state of a cell (e.g., a T cell progenitor).

[0257] Where used, the term “encodes” refers to a sequence of nucleic acid that codes for a protein, polypeptide, or polynucleotide of interest. The nucleic acid sequence can be either a DNA or RNA molecule. In a preferred embodiment, the molecule is a DNA molecule. In another preferred embodiment, the molecule is an RNA molecule. If present as an RNA molecule, it includes a sequence that instructs the ribosomes of a host cell to begin translation (e.g., a start codon, ATG) and a sequence that instructs the ribosomes to terminate translation (e.g., a stop codon). Between the start codon and the stop codon is an open reading frame (ORF). Such terminology is known to those skilled in the art.

[0258] The term "insertion" refers to the manipulation of nucleotide sequences to introduce non-natural sequences. This is done, for example, by using restriction enzymes and ligases, thereby incorporating a target DNA sequence, usually encoding a gene of interest, into another nucleic acid molecule by digesting both molecules with the appropriate restriction enzymes to create a compatible overlap, and then joining the molecules together using a ligase. Those skilled in the art are very familiar with such operations, and examples can be found in Sambrook et al. (Sambrook, Fritsch, & Maniatis, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Laboratory, 1989), which is incorporated herein by reference in its entirety, including any figures, drawings and tables.

[0259] As used herein, the term “subject” refers to a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cattle, horses, camels, goats, rabbits, pigs, and sheep. In certain embodiments, the subject is a human. In some embodiments, the subject has a disease or condition that can be treated with the modified cells or populations provided herein. In some embodiments, the disease or condition is cancer.

[0260] As used herein, the term “promoter” refers to a nucleotide sequence (e.g., a DNA sequence) that can control the expression of a coding sequence or functional RNA. A promoter sequence consists of proximal and more distal upstream elements, the latter of which are often referred to as enhancers. A promoter may be entirely derived from a native gene, composed of different elements from different promoters found in nature, and / or may include synthetic DNA segments. A promoter may be endogenous to the cell of interest or exogenous to the cell of interest, as intended herein. It is understood by those skilled in the art that different promoters can induce gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. As is known in the art, promoters may be selected according to the strength of the promoter and / or the conditions under which the promoter is active, e.g., constitutive promoters, potent promoters, weak promoters, inducible / repressive promoters, tissue-specific or developmental stage-regulated promoters, cell cycle-dependent promoters, etc.

[0261] The promoter may be an inductive promoter (e.g., a heat shock promoter, a tetracycline-regulating promoter, a steroid-regulating promoter, a metal-regulating promoter, an estrogen receptor-regulating promoter, etc.). The promoter may be a constitutive promoter (e.g., a CMV promoter, a UBC promoter). In some embodiments, the promoter may be a spatially restricted and / or temporally restricted promoter (e.g., a tissue-specific promoter, a cell-type-specific promoter, etc.). See, for example, U.S. Publication No. 20180127786 (the disclosure thereof is incorporated herein by reference in its entirety).

[0262] As intended herein, gene editing may involve knocking in or knocking out a gene (or nucleotide sequence). As used herein, the term “knock-in” refers to the addition of a DNA sequence or fragment thereof to a genome. Such a DNA sequence to be knocked in may include an entire gene, a regulatory sequence associated with the gene, or any of the aforementioned parts or fragments. For example, a polynucleotide donor construct encoding a recombinant protein may be inserted into the genome of a cell containing a mutant gene. In some embodiments, a knock-in strategy involves the replacement of an existing sequence with a provided sequence, e.g., the replacement of a mutant allele with a wild-type copy. On the other hand, the term “knock-out” refers to the removal or expression of a gene. For example, a gene may be knocked out by either the deletion or addition of a nucleotide sequence that leads to the disruption of the leading frame. As another example, a gene may be knocked out by replacing a portion of the gene with an irrelevant (e.g., non-coding) sequence.

[0263] As used herein, the term “non-homologous end junction” or NHEJ refers to a cellular process in which a cleavage end or nick end of a DNA strand is directly ligated without the need for a homologous template nucleic acid. NHEJ can result in the addition, deletion, substitution, or combination thereof of one or more nucleotides at the repair site.

[0264] As used herein, the term “homologous recombination repair” or HDR refers to a cellular process in which a broken or nicked end of a DNA strand is repaired by polymerization from a homologous template nucleic acid. In some embodiments, the original sequence is replaced with the template sequence. In some embodiments, the template sequence is inserted into the genome without replacing an endogenous sequence. The homologous template nucleic acid may be provided by a homologous sequence located elsewhere in the genome (sister chromatid, homologous chromosome, or repeating region on the same or different chromosome). Alternatively, an exogenous template nucleic acid may be introduced to obtain a specific HDR-induced alteration of the sequence at a target site. In this way, a specific mutation may be introduced at the break site.

[0265] As used herein, a single-stranded DNA template or a double-stranded DNA template refers to a DNA oligonucleotide that can be used by cells as a template for HDR. Generally, a single-stranded DNA template or a double-stranded DNA template has at least one region of homology to the target site. In some cases, a single-stranded DNA template or a double-stranded DNA template has two homologous regions adjacent to the region containing the heterologous sequence to be inserted into the target cleavage site.

[0266] The terms “vector” and “plasmid” are used interchangeably and, as used herein, refer to polynucleotide vehicles useful for introducing genetic material into cells. Vectors may be linear or circular. Vectors can be integrated into a target genome in a host cell or replicate independently within a host cell. Vectors may include, for example, origins of replication, multicloning sites, and / or selectable markers. Expression vectors typically contain expression cassettes. Vectors and plasmids include, but are not limited to, integration vectors, prokaryotic plasmids, eukaryotic plasmids, plant synthetic chromosomes, episomes, cosmids, and artificial chromosomes.

[0267] As used herein, the term “introduce” in the context of introducing nucleic acids or nucleic acid-containing complexes, such as the RNP-DNA template complex, refers to the extracellular translocation of the nucleic acid sequence or RNP-DNA template complex into the cell. In some cases, introduction refers to the extracellular translocation of the nucleic acid or complex into the nucleus of a cell. Various methods of such translocation are considered, including but not limited to electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, translocation via cell-permeable peptides, liposome-mediated translocation, and the like.

[0268] As used herein, the term “expression cassette” refers to a recombinant or synthetically produced polynucleotide construct comprising a regulatory sequence operably ligated to a selected polynucleotide in order to facilitate the expression of the selected polynucleotide in a host cell. For example, the regulatory sequence can facilitate the transcription of the selected polynucleotide in a host cell, or the transcription and translation of the selected polynucleotide in a host cell. An expression cassette may be, for example, incorporated into the genome of a host cell or present in an expression vector.

[0269] As used herein, the phrase “subjects requiring it” means subjects who exhibit and / or are diagnosed with one or more symptoms or signs of any of the diseases or disorders described herein.

[0270] "Chemotherapy agents" refer to chemical compounds useful in treating cancer. Examples of chemotherapy agents include "anti-hormone agents" or "endocrine therapy agents" that act to regulate, reduce, block, or inhibit the effects of hormones that can promote cancer growth.

[0271] The term “composition” refers, for example, to a mixture containing the modified cells or proteins intended herein. In some embodiments, the composition may contain additional components such as adjuvants, stabilizers, or excipients. The term “composition” or “pharmaceutical composition” refers to a preparation in which the biological activity of the active ingredients contained herein is effective for the treatment of the subject, and which does not contain additional components that are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0272] The term "improvement" means any therapeutically beneficial outcome in the treatment of a disease condition, such as cancer, including prevention, reduction of severity or progression, remission, or cure.

[0273] The term "in situ" refers to the process that occurs when living cells grow independently of living organisms, such as during tissue culture.

[0274] The term "in vivo" refers to processes that occur within a living organism.

[0275] As used herein, the term “mammal” includes both humans and non-humans, and includes, but is not limited to, humans, non-human primates, dogs, cats, mice, cattle, horses, and pigs.

[0276] The term "percent identity" refers to two or more nucleic acid or polypeptide sequences that, when compared and aligned for maximum correspondence, have a specific percentage of nucleotide or amino acid residues, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art), or by visual inspection. Depending on the application, "percent identity" may exist across regions of the sequences being compared, such as functional domains, or across the full lengths of the two sequences being compared.

[0277] For sequence comparison, typically one sequence serves as the reference sequence against which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are specified as needed, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percentage sequence identity of the test sequence(s) to the reference sequence(s) based on the specified program parameters.

[0278] The optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv.Appl.Math.2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J.Mol.Biol.48:443 (1970), the similarity search method of Pearson & Lipman, Proc.Nat'l.Acad.Sci.USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see Ausubel et al. below for general information).

[0279] One example of a suitable algorithm for determining the percentage of sequence identity and sequence similarity is the BLAST algorithm, described in Altschul et al., J.Mol.Biol.215:403-410 (1990). Software for performing BLAST analysis is available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0280] The term "sufficient amount" means an amount sufficient to produce the desired effect, for example, an amount sufficient to regulate intracellular protein aggregation.

[0281] The term "therapeutic dose" refers to the amount of a substance that is effective in improving the symptoms of a disease. Since prevention can be considered a form of treatment, the therapeutic dose can also be called the "preventive dose."

[0282] As used herein, the term “effective dose” means a sufficient amount of the compound (e.g., the compositions described herein, the cells described herein) to produce a beneficial or desired result. An effective dose may be administered in one or more doses, applications, or prescriptions and is not intended to be limited to a particular formulation or route of administration. As used herein, the term “to treat” includes any effect that results in improvement of a condition, disease, disorder, etc., or improves its symptoms, such as alleviation, reduction, regulation, improvement, or elimination.

[0283] The terms "to regulate" and "to adjust" refer to reducing or inhibiting the listed variables, or alternatively, activating or increasing them.

[0284] The terms “increase” and “activate” refer to increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or more in the listed variables.

[0285] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2x, 3x, 4x, 5x, 10x, 20x, 50x, 100x, or more in the listed variables.

[0286] The term “approximately” indicates and encompasses both the value and the range above and below that value. In certain embodiments, the term “approximately” indicates a specified value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term “approximately” indicates the specified value(s) ± one standard deviation of that value(s).

[0287] When used in this specification and the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context otherwise explicitly indicates.

[0288] Composition for editing cells In some embodiments, compositions are provided herein that comprise a solution comprising cells containing at least one heterologous DNA template inserted into a target region of the genome, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), a histone deacetylase (HDAC) inhibitor, and / or an osmotic regulator. In some embodiments, the solution comprises cells containing at least one heterologous DNA template inserted into a target region of the genome, and N-acetyl-L-cysteine ​​(NAC). In some embodiments, the solution comprises a solution comprising cells containing at least one heterologous DNA template inserted into a target region of the genome, and a histone deacetylase (HDAC) inhibitor. In some embodiments, the solution is an aqueous solution. In some embodiments, at least one of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), and a histone deacetylase (HDAC) inhibitor is present in the solution at the final concentrations described herein.

[0289] In some embodiments, the solution includes a buffer. In some embodiments, the solution includes a cell medium. In some embodiments, the cell medium includes a medium additive. In some embodiments, the cell medium includes a xeno-medium additive. Examples of cell medium additives include, but are not limited to, human serum, fetal bovine serum (FBS), Cell-Vive® T-NK Xeno-Free Serum (BioLegend), Cell-Vive® T-cell CD Serum Substitute (BioLegend), PLT Gold (Sarotrius), Serum Substitute Supplement (Irvine Scientific), Physiologix® XF Serum Replacement (Nucleus Biologics), CTS® Immune Cell Serum Replacement (Thermo Fisher Scientific), and Knock Out Serum Replacement (Thermo Fisher Scientific). In some embodiments, the culture medium additives include Cell-Vive® T-NK Xeno-Free Serum, Cell-Vive® T-cell CD Serum Substitute, PLT Gold, Serum Substitute Supplement, Physiologix® XF Serum Replacement, CTS® Immune Cell Serum Replacement, Knock Out Serum Replacement, fetal bovine serum, or human serum.

[0290] RNase inhibitors In some embodiments, compositions comprising an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template are provided herein, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome. A method for editing cells is also provided, comprising a solution comprising an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template (where the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome). This involves editing cells by inserting DNA templates into insertion sites within the cell's genome. In some embodiments, the RNase inhibitor is present in the solution at a final concentration of about 0.5–2 U / μl. The final concentration of the RNase inhibitor in the solution may be about 0.5 U / μl, 0.75 U / μl, 1 U / μl, 1.25 U / μl, 1.5 U / μl, 1.75 U / μl, or 2 U / μl. The final concentration of the RNase inhibitor in the solution may be about 0.5–2 U / μl, 0.5–1 U / μl, 0.5–0.75 U / μl, 0.75–1 U / μl, 1–1.25 U / μl, 1.25–1.5 U / μl, 1.5–1.75 U / μl, or 1.75–2 U / μl. In some embodiments, the RNase inhibitor is present in the solution at a final concentration of about 1 U / μl. In some embodiments, the RNase inhibitor is a mouse RNase inhibitor. In some embodiments, the RNase inhibitor is an RNase A, B, C, T1, or T2 inhibitor. In some embodiments, the RNase inhibitor is a mouse, rat, or human RNase inhibitor.

[0291] RNase inhibitors are generally commercially available from various vendors such as Thermo Fisher (catalog no. AM2694), Invitrogen (catalog no. EO0381), Promega (catalog no. N2111), New England Biolabs (catalog no. M0314S or M0314L), Takara, and Sigma-Aldrich. In some embodiments, RNase inhibitors specifically inhibit RNase A, B, C, 1, or T1.

[0292] In some embodiments, the RNase inhibitor is added to a solution. In some embodiments, the RNase inhibitor is added to an aqueous solution. In some embodiments, the RNase inhibitor is added to a buffer. In some embodiments, the composition containing the RNase inhibitor may further contain the osmotic regulator and / or N-acetyl-L-cysteine ​​(NAC) described herein. In some embodiments, the composition containing the RNase inhibitor further contains the osmotic regulator. In some embodiments, the composition containing the RNase inhibitor further contains N-acetyl-L-cysteine ​​(NAC). In some embodiments, the composition containing the RNase inhibitor further contains the osmotic regulator and N-acetyl-L-cysteine ​​(NAC).

[0293] Osmotic pressure regulator In some embodiments, compositions are provided herein that comprise a solution comprising an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprise nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome. Methods for editing cells are also provided, comprising providing a solution comprising an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template (where the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprise nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0294] In some embodiments, the osmotic regulator is sorbitol, glycerol, or glycine. In some embodiments, the osmotic regulator is two or more of sorbitol, glycerol, or glycine. In some embodiments, the osmotic regulator is sorbitol. In some embodiments, the osmotic regulator is glycerol. In some embodiments, the osmotic regulator is glycine.

[0295] In some embodiments, the osmoregulator is present in the solution at a final concentration of about 100–400 mM. In some embodiments, the osmoregulator is present in the solution at a final concentration of about 100–150 mM, 150–200 mM, 200–250 mM, 250–300 mM, 300–350 mM, or 350–400 mM. In some embodiments, the osmoregulator is present in the solution at a final concentration of about 100 mM, 125 mM, 150 mM, 175 mM, 190 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, or 400 mM.

[0296] In some embodiments, the osmoregulator is present in the solution at a final concentration of about 0.5% to 1.5%, 0.5% to 1%, 1% to 1.25%, or 1.25% to 1.5%. In some embodiments, the osmoregulator is present in the solution at a final concentration of about 1.25%. In some embodiments, the osmoregulator is present in the solution at a final concentration of less than 2%, 1.9%, 1.8%, 1.7%, 1.6%, or 1.5%.

[0297] In some embodiments, sorbitol is present in the solution at a final concentration of about 100–400 mM. In some embodiments, sorbitol is present in the solution at a final concentration of about 100–400 mM. In some embodiments, sorbitol is present in the solution at a final concentration of about 100–150 mM, 150–200 mM, 200–250 mM, 250–300 mM, 300–350 mM, or 350–400 mM. In some embodiments, sorbitol is present in the solution at a final concentration of about 100 mM, 125 mM, 150 mM, 175 mM, 190 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, or 400 mM. In some embodiments, sorbitol is present in the solution at a final concentration of about 190 mM or 200 mM. In some embodiments, sorbitol is present in the solution at a final concentration of about 200 mM. In some embodiments, sorbitol is present in the solution at a final concentration of about 170 mM.

[0298] In some embodiments, sorbitol is present in the solution at a final concentration of about 0.5% to 1.5%, 0.5% to 1%, 1% to 1.25%, or 1.25% to 1.5%. In some embodiments, sorbitol is present in the solution at a final concentration of about 1.25%. In some embodiments, sorbitol is present in the solution at a final concentration of 2%, 1.9%, 1.8%, 1.7%, 1.6%, or less than 1.5%.

[0299] In some embodiments, glycerol is present in the solution at a final concentration of about 100–400 mM. In some embodiments, glycerol is present in the solution at a final concentration of about 100–400 mM. In some embodiments, glycerol is present in the solution at a final concentration of about 100–150 mM, 150–200 mM, 200–250 mM, 250–300 mM, 300–350 mM, or 350–400 mM. In some embodiments, glycerol is present in the solution at a final concentration of about 100 mM, 125 mM, 150 mM, 175 mM, 190 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, or 400 mM. In some embodiments, glycerol is present in the solution at a final concentration of about 190 mM or 200 mM. In some embodiments, glycerol is present in the solution at a final concentration of about 200 mM. In some embodiments, glycerol is present in the solution at a final concentration of about 170 mM.

[0300] In some embodiments, glycerol is present in the solution at a final concentration of about 0.5%–1.5%, 0.5%–1%, 1%–1.25%, or 1.25%–1.5%. In some embodiments, glycerol is present in the solution at a final concentration of about 1.25%. In some embodiments, glycerol is present in the solution at a final concentration of 2%, 1.9%, 1.8%, 1.7%, 1.6%, or less than 1.5%.

[0301] In some embodiments, glycine is present in the solution at a final concentration of about 100–400 mM. In some embodiments, glycine is present in the solution at a final concentration of about 100–400 mM. In some embodiments, glycine is present in the solution at a final concentration of about 100–150 mM, 150–200 mM, 200–250 mM, 250–300 mM, 300–350 mM, or 350–400 mM. In some embodiments, glycine is present in the solution at a final concentration of about 100 mM, 125 mM, 150 mM, 175 mM, 190 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM, or 400 mM. In some embodiments, glycine is present in the solution at a final concentration of about 190 mM or 200 mM. In some embodiments, glycine is present in the solution at a final concentration of about 200 mM. In some embodiments, glycine is present in the solution at a final concentration of about 170 mM.

[0302] In some embodiments, glycine is present in the solution at a final concentration of about 0.5% to 1.5%, 0.5% to 1%, 1% to 1.25%, or 1.25% to 1.5%. In some embodiments, glycine is present in the solution at a final concentration of about 1.25%. In some embodiments, glycine is present in the solution at a final concentration of 2%, 1.9%, 1.8%, 1.7%, 1.6%, or less than 1.5%.

[0303] In some embodiments, the osmotic regulator is added to a solution. In some embodiments, the osmotic regulator is added to an aqueous solution. In some embodiments, the osmotic regulator is added to a buffer. In some embodiments, the composition containing the osmotic regulator may further contain N-acetyl-L-cysteine ​​(NAC) and / or an RNase inhibitor as described herein. In some embodiments, the composition containing the osmotic regulator (e.g., sorbitol, glycerol, or glycine) further contains N-acetyl-L-cysteine ​​(NAC). In some embodiments, the composition containing the osmotic regulator (e.g., sorbitol, glycerol, or glycine) further contains an RNase inhibitor. In some embodiments, the composition containing the osmotic regulator (e.g., sorbitol, glycerol, or glycine) further contains N-acetyl-L-cysteine ​​(NAC) and an RNase inhibitor.

[0304] Reactive oxygen species (ROS) inhibitors In some embodiments, compositions are provided herein that comprise a solution comprising cells containing at least one heterologous DNA template inserted into a target region of the genome, and a reactive oxygen species (ROS) inhibitor (e.g., N-acetyl-L-cysteine ​​(NAC)). In some embodiments, the solution comprises cells containing at least one heterologous DNA template inserted into a target region of the genome, and N-acetyl-L-cysteine ​​(NAC). In some embodiments, the solution is an aqueous solution. In some embodiments, the reactive oxygen species (ROS) inhibitor (e.g., N-acetyl-L-cysteine ​​(NAC)) is present in the solution at the final concentrations described herein.

[0305] In some embodiments, compositions are provided herein that comprise a solution containing a reactive oxygen species (ROS) inhibitor, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome. Methods for editing cells are also provided, comprising providing a solution containing a reactive oxygen species (ROS) inhibitor, a ribonucleoprotein complex (RNP), and a DNA template (where the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome. In some embodiments, the reactive oxygen species (ROS) inhibitor is a reactive oxygen species (ROS) scavenger.

[0306] Examples of ROS inhibitors include N-acetyl-L-cysteine ​​(NAC), N-acetyl-D-cysteine, quercetin, deferoxamine mesylate, phycocyanobilin, Mito-TEMPO, GSK2795039, diphenyleneiodonium chloride, Tempol, succinylphosphonate trisodium salt, nobiletin, albendazole, imeglimin, N-tert-butyl-α-phenylnitrone, tofogliflozin (hydrate), glutathione, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), and ascorbic acid. Examples of ROS inhibitors include, but are not limited to, tocopherol, α-tocopherol disodium phosphate, tofogliflozin (hydrate), lasidipine, astaxanthin, spiraneoside, moracin O, sodium urate, clobamide, norbergenin, L-theanine, sodium 2-oxopropanate, 3,4-dimethoxycinnamic acid, oleucidine, pelargonidine chloride, randialic acid B, cyclo(L-Phe-L-Pro), decylbiquinone, bixin, sodium thiocyanate, 5-hydroxyoxyndol, and sodium simvastatin hydroxyate. Further ROS inhibitors are commercially available from at least MedChem express (medchemexpress.com / Targets / reactive-oxygen-species / effect / inhibitor.html) and Selleckchem (selleckchem.com / ROS.html).

[0307] In some embodiments, compositions are provided herein that comprise a solution comprising N-acetyl-L-cysteine ​​(NAC), a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprise nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome. Also provided are methods for editing cells, which comprise a solution comprising N-acetyl-L-cysteine ​​(NAC), a ribonucleoprotein complex (RNP), and a DNA template (where the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprise nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0308] In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in solution at a final concentration of about 1–10 mM. In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in solution at a final concentration of about 1–10 mM, 1–2 mM, 2–3 mM, 3–4 mM, 4–5 mM, 5–6 mM, 6–7 mM, 7–8 mM, 8–9 mM, or 9–10 mM. In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in solution at a final concentration of about 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, or 10 mM. In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of about 5 mM. In some embodiments, N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of about 2.5 mM.

[0309] N-acetyl-L-cysteine ​​(NAC) (CAS number 616-91-1), also known as 2-acetamido-3-sulfanylpropanoic acid (IUPAC name), is an antioxidant and mucolytic agent. It is a precursor of the antioxidant glutathione and can increase the cellular pool of free radical scavengers. NAC is commercially available from a variety of vendors, including but not limited to Sigma Aldrich (catalog number A7250), Thermo Fisher (catalog number A15409.36), Santa Cruz Biotechnology (catalog number sc-202232), or Padagis (catalog number NDC0574-0805-30).

[0310] In some embodiments, N-acetyl-L-cysteine ​​(NAC) is added to a solution. In some embodiments, N-acetyl-L-cysteine ​​(NAC) is added to an aqueous solution. In some embodiments, N-acetyl-L-cysteine ​​(NAC) is added to a buffer. In some embodiments, the composition containing N-acetyl-L-cysteine ​​(NAC) may further contain the osmotic regulator and / or RNase inhibitor described herein. In some embodiments, the composition containing N-acetyl-L-cysteine ​​(NAC) further contains an osmotic regulator. In some embodiments, the composition containing N-acetyl-L-cysteine ​​(NAC) further contains an RNase inhibitor. In some embodiments, the composition containing N-acetyl-L-cysteine ​​(NAC) further contains an osmotic regulator and an RNase inhibitor. In some embodiments, the solution contains at least one of an RNase inhibitor at a final concentration of about 0.5 to 2 U / μL, NAC at a final concentration of about 1 to 10 mM, and / or sorbitol at a final concentration of about 100 to 400 mM. In some embodiments, the solution comprises at least one of an RNase inhibitor at a final concentration of about 1 U / μL, NAC at a final concentration of about 5 mM, and / or sorbitol at a final concentration of about 200 mM. In some embodiments, the solution comprises at least one of an RNase inhibitor at a final concentration of about 1 U / μL, NAC at a final concentration of about 2.5 mM, and / or sorbitol at a final concentration of about 200 mM. In some embodiments, the solution comprises at least one of an RNase inhibitor at a final concentration of about 1 U / μL, NAC at a final concentration of about 5 mM, and / or glycerol at a final concentration of about 170 mM. In some embodiments, the solution comprises at least one of an RNase inhibitor at a final concentration of about 1 U / μL, NAC at a final concentration of about 2.5 mM, and / or glycerol at a final concentration of about 170 mM.

[0311] Histone deacetylase (HDAC) inhibitors In some embodiments, compositions comprising a solution containing a histone deacetylase (HDAC) inhibitor, a ribonucleoprotein complex (RNP), and a DNA template are provided herein, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0312] In some embodiments, compositions are provided herein that include a solution comprising cells containing at least one heterologous DNA template inserted into a target region of the genome, and a histone deacetylase (HDAC) inhibitor as described herein. In some embodiments, the solution is an aqueous solution. In some embodiments, the histone deacetylase (HDAC) inhibitor is present in the solution at the final concentrations described herein.

[0313] In some embodiments, compositions are provided herein that comprise a solution comprising a histone deacetylase (HDAC) inhibitor comprising at least one of sodium phenylbutyrate, xynostat, or panobinostat, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprise nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0314] Histone deacetylase (HDAC) inhibitors may be HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and / or HDAC10 inhibitors. In certain embodiments, the HDAC inhibitor is a broad-spectrum HDAC inhibitor. Examples of HDAC inhibitors include sodium phenylbutyrate, xinostat, panobinostat, phenylbutyrate, curcumin, mosetinostat, romidespin, SIS17, spritomycin, trichostatin-A, tusidinostat, etinostat, sodium butyrate, valproic acid, CXD101, KT-531, ITF3756, tubastatin A, vorinostat, BML-210, bellinostat, avexinostat, dasinostat, CUDC-101, droxinostat, MC1568, prasinostat, divalproex sodium, PCI-3405, SR-4370, zibinostat, tubasin, AR-42, (-)-parthenolide, resminostat, fimepinostat. Examples of RNA interference (RNAi) molecules include, but are not limited to, M344, Tasejinarin, Sinapic acid, Biphenyl-4-sulfonyl chloride, Sulforaphane, UF010, Suberohydroxamic acid, NKL22, TC-H106, RGFP966, HPOB, RG2833, TMP269, Nextulastat A, Domatinostat, LMK-235, Santa Cruz Amate A, CAY10603, Tascinimod, BG45, BRD73954, Licorinostat, Scriptide, Sitalinostat, WT161, TMP195, ACY-738, SKLB-23bb, Tinostamstin, TH34, BRD3308, Radianine A, Isoguanosine, KA2507, ITSA-1, or other RNA interference (RNAi) molecules. In some embodiments, the HDAC inhibitor is sodium phenylbutyrate, xinostat, or panobinostat.

[0315] In some embodiments, sodium phenylbutyrate is present in the solution at a final concentration of about 1 mM or about 15.6 μM to 4 mM, and optionally the solution contains 1% DMSO. In some embodiments, sodium phenylbutyrate is present in the solution at a final concentration of about 1 mM or 15.6 μM to 4 mM, and 1% DMSO. In some embodiments, sodium phenylbutyrate is present at concentrations of about 15 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, 550 μM, 600 μM, 650 μM, 700 μM, 750 μM, 800 μM, 850 μM, 900 μM M, 950μM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, or 4mM, or approximately 15.6μM to 4mM, 15μM to 50μ M, 50μM~100μM, 100μM~150μM, 150μM~200μM, 200μM~250μM, 250μM~300μM, 300μM~350 μM, 350μM~400μM, 400μM~450μM, 450μM~500μM, 500μM~550μM, 550μM~600μM, 600μM~ 650μM, 650μM~700μM, 750μM~800μM, 850μM~900μM, 900μM~950μM, 950μM~1000μM, 1~ It is present in the solution at a final concentration of 1.25 mM, 1.25–1.5 mM, 1.5–1.75 mM, 1.75–2 mM, 2–2.25 mM, 2.25–2.5 mM, 2.5–2.75 mM, 2.75–3 mM, 3–3.25 mM, 3.25–3.5 mM, or 3.5–4 mM, or any concentration in between. In some embodiments, sodium phenylbutyrate is present in the solution at a final concentration of about 0.1 mM.

[0316] In some embodiments, xynostat is present in the solution at a final concentration of about 16 nM or about 8 nM to 200 nM. In some embodiments, xynostat is present at about 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 105 nM M, 110nM, 120nM, 125nM, 130nM, 135nM, 140nM, 145nM, 150nM, 155nM, 160nM, 165nM, 170nM, 175nM, 180nM, 185nM, 190nM, 195nM, or 200nM, or approximately 8-10nM, 10-12nM, 10-14nM, 14-16nM, 16-18nM, 18-20nM, 20-25nM , 25~30nM, 30~35nM, 35~40nM, 40~45nM, 45~50nM, 50~55nM, 55~60nM, 60~65nM, 65~70nM, 70~75nM, 75~80 nM, 80~85nM, 85~90nM, 90~95nM, 95~100nM, 100~105nM, 105~110nM, 110~120nM, 120~125nM, 125~130nM, It is present in solution at final concentrations of 130-135 nM, 135-140 nM, 140-145 nM, 145-150 nM, 150-155 nM, 155-160 nM, 160-165 nM, 165-170 nM, 170-175 nM, 175-180 nM, 180-185 nM, 185-190 nM, 190-195 nM, or 195-200 nM, or any concentration in between these.

[0317] In some embodiments, panobinostat is present in the solution at a final concentration of about 37.5 nM or about 3 nM to 1 μM. In some embodiments, panobinostat is present at concentrations of about 3 nM, 5 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 37.5 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM M, 650nM, 700nM, 750nM, 800nM, 850nM, 900nM, 950nM, 1000nM, or approximately 3-5nM, 5-10nM, 10-20nM, 20-30nM, 30-35nM, 35-37.5nM, 35-40nM, 40-45nM, 45-50nM, 55-60nM, 60-65nM, 65-70nM, 70-75nM, 75-80nM, 85-90nM, 90-95nM, 95-100nM, 100- 125nM, 125~150nM, 150~175nM, 175~200nM, 200~225nM, 225~250nM, 250~275nM, 275~200nM, 300~325nM, 325~350nM, 3 50~375nM, 375~300nM, 400~425nM, 425~450nM, 450~475nM, 475~400nM, 500~525nM, 525~550nM, 550~575nM, 575~500nM It is present in solution at final concentrations of 600-625 nM, 625-650 nM, 650-675 nM, 675-600 nM, 700-725 nM, 725-750 nM, 750-775 nM, 775-700 nM, 800-825 nM, 825-850 nM, 850-875 nM, 875-800 nM, 900-925 nM, 925-950 nM, 950-975 nM, or 975-1000 nM, or any concentration in between these.

[0318] In some embodiments, the solution comprises at least one of the following: an RNase inhibitor at a final concentration of approximately 0.5–2 U / μl, NAC at a final concentration of approximately 1–10 mM, sorbitol at a final concentration of approximately 100–400 mM, and / or sodium phenylbutyrate at a final concentration of approximately 15.6 μM–4 mM, xynostat at 8 nM–200 nM, or panobinostat at 3 nM–1 μM.

[0319] combination A composition comprising a solution containing a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), a histone deacetylase (HDAC) inhibitor, and / or an osmoregulator, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome. In some embodiments, the composition comprises at least two, at least three, or all four of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), a histone deacetylase (HDAC) inhibitor, and / or an osmoregulator.

[0320] In some embodiments, the solution contains at least N-acetyl-L-cysteine ​​(NAC) and an osmotic regulator; RNase inhibitor and N-acetyl-L-cysteine ​​(NAC); RNase inhibitor and osmotic regulator; N-acetyl-L-cysteine ​​(NAC) and a histone deacetylase (HDAC) inhibitor; osmotic regulator and a histone deacetylase (HDAC) inhibitor; RNase inhibitor and a histone deacetylase (HDAC) inhibitor; RNase inhibitor, N-acetyl-L-cysteine N-acetyl-L-cysteine ​​(NAC), and histone deacetylase (HDAC) inhibitors; N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors; RNase inhibitors, osmotic regulators, and histone deacetylase (HDAC) inhibitors; RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), and osmotic regulators; or comprising RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors.

[0321] In some embodiments, the solution comprises at least one, two, three, or four of the following: an RNase inhibitor at a final concentration of approximately 0.5–2 U / μl, NAC at a final concentration of approximately 1–10 mM, sorbitol at a final concentration of approximately 100–400 mM, and / or sodium phenylbutyrate at a final concentration of approximately 15.6 μM–4 mM, xynostat at 8 nM–200 nM, or panobinostat at 3 nM–1 μM. In some embodiments, the solution comprises at least one, two, three, or four of the following: an RNase inhibitor at a final concentration of approximately 1 U / μl, NAC at a final concentration of approximately 2.5 mM, sorbitol at a final concentration of approximately 200 mM, and / or sodium phenylbutyrate at a final concentration of approximately 1 mM, xynostat at 0.016 μM, or panobinostat at 0.0375 μM.

[0322] In some embodiments, additional compounds, such as ATR inhibitors like VE-822, PAPR-1 inhibitors like AG14361, Polθ inhibitors like ART-558, CHK inhibitors like AZD7762, and / or DNA-Pk inhibitors like KU0600648 and NU7026, may also be used alone or in combination with the inhibitors and agents described herein.

[0323] In one embodiment, the Specified Composition provides a solution comprising at least one of the following: an ATR inhibitor such as VE-822, a PAPR-1 inhibitor such as AG14361, a Polθ inhibitor such as ART-558, a CHK inhibitor such as AZD7762, and / or a DNA-Pk inhibitor such as KU0600648 or NU7026, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

[0324] In some embodiments, the Specified Inventions provide a solution comprising at least one of the following: an ATR inhibitor such as VE-822, a PAPR-1 inhibitor such as AG14361, a Polθ inhibitor such as ART-558, a CHK inhibitor such as AZD7762, and / or a DNA-Pk inhibitor such as KU0600648 or NU7026, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome); and a method for editing cells comprising inserting the DNA template into the insertion site in the cell's genome.

[0325] Homologous recombination proteins or DNA repair regulatory proteins The cells provided herein may also include exogenous heterologous recombinant proteins or DNA repair regulatory proteins or genes encoding them. Such proteins and / or genes can enhance the efficiency of nonviral modification, resulting in increased expression of target genes (e.g., increased expression of heterologous genes such as priming receptors and / or CARs). In some embodiments, the exogenous homologous recombinant proteins or DNA repair regulatory proteins are SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion proteins.

[0326] The sequences of SWSAP1, dominant-negative KU80, and AcrIIA8_CDT1 fusion proteins are shown below:

[0327] SWSAP1 array:

[0328] MGSGPAAGPPLLLLGTPGSGKTALLFAAALEAAGEGQGPVLFLTRRPLQSMPRGTGTTLDPMRLQKIRFQYPPSTRELFRLLCSAHEAPGPAPSLLLLDGLEEYLAEDPEPQEAAYLIALLLDTAAHFSHRLGPGRDCGLMVALQTQEEAGSGDVLHLALLQRYFPAQCWLQPDAPGPGEHGLRACLEPGGLGPRTEWWVTFRSDGEMMIAPWPTQAGDPSSGKGSSSGGQP (Sequence ID 125)

[0329] Dominant-negative KU80 sequence:

[0330] MGSGQLNAVDALIDSMSLAKKDEKTDTLEDLFPTTKIPNPRFQRLFQCLLHRALHPREPLPPIQQHIWNMLNPPAEVTTKSQIPLSKIKTLFPLIEAKKKDQVTAQEIFQDNHEDGPTAKKLKTEQGGAHFSVSSLAEGSVTSVGSVNPAENFRVLVKQKKASFEEASNQLINHIEQFLDTNETPYFMKSIDCIRAFREEAIKFSEEQRFNNFLKALQEKVEIKQLNHFWEIVVQDGITLITKEEASGSSVTAEEAKKFLAPKDKPSGDTAAVFEEGGDVDDLLDMI (Sequence ID 126)

[0331] AcrIIA8_CDT1 fusion protein sequence:

[0332] MGSGSIFTDMIPAELLINEYKKGQSGAKHDNYVSVGRIMVAIYKNNSFKNTGTVKYQDSTHSGITMSKVFIDGKEYRIDIDTQHYEVQDFDTSGRQTTLILKRIDLYGGSGSGSPSPARPALRAPASATSGSRKRARPPAAPGRDQARPPARRRLRLSVDEVSSPSTPEAPDIPACPSPGQKIKKSTPAAGQPPHLTSAQDQDTI (Sequence ID 127).

[0333] In some embodiments, the SWSAP1 protein contains the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein contains the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein contains the sequence shown in SEQ ID NO: 127.

[0334] Such exogenous heterologous recombinant proteins or DNA repair regulatory proteins can be encoded on episomatic plasmids for transient expression. Any suitable episomatic plasmid can be used to encode exogenous heterologous recombinant proteins or DNA repair regulatory proteins. The episomatic plasmid can be delivered in parallel with the target transgene insertion cassette DNA and Cas9 RNP. In some cases, the episomatic plasmid is non-integrated and non-replicating. Exemplary episomal vectors for gene expression in mammalian cells are described in Van Craenenbroeck K, et al, Eur. J. Biochem. 267:5665-5678(200); and Stavrou, EF, et al. Episomal vectors based on S / MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells. Sci Rep 9, 19765(2019), both of which are incorporated herein by reference in their entirety.

[0335] Exogenous heterologous recombinant proteins or DNA repair regulatory proteins can also be delivered to cells as mRNA or protein using electroporation protocols employing in vitro transcription or protein expression techniques known in the art. Such methods achieve the same goal of delivering DNA repair-promoting elements to cells and are as compatible as electroporation. In the case of mRNA-based delivery, the DNA used for the synthesis of the exogenous heterologous recombinant protein or DNA repair regulatory protein would contain standard elements required for in vitro transcription, such as the T7 promoter and Kozak sequence. The mRNA would be expressed from the DNA and electroporated into target cells using the electroporation protocols described herein.

[0336] For protein-based delivery, exogenous heterologous recombination genes or DNA repair regulatory genes can be cloned into a standard expression vector (e.g., a pET vector) containing the elements necessary for protein expression. This vector can then be used to deliver the protein to target cells.

[0337] In another embodiment, this specification provides a polypeptide comprising an AcrIIA8 peptide fused to a CDT1 peptide. In one embodiment, the polypeptide comprises the sequence shown in SEQ ID NO: 127.

[0338] In one embodiment, a method for editing cells is provided herein, comprising providing cells containing an exogenous homologous recombination protein or a DNA repair regulatory protein, providing a solution comprising a ribonucleoprotein complex (RNP) and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template containing nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0339] Nonviral gene editing methods The terms “gene editing” or “genome editing,” as used herein, refer to a type of genetic manipulation in which DNA is inserted into, replaced in, or removed from a genome using artificially engineered nucleases or “molecular scissors.” This is a useful tool for elucidating the function and effects of sequence-specific genes or proteins, or for modifying cellular behavior (for example, for therapeutic purposes).

[0340] Currently available genome editing tools include zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for inserting genes into safe harbor loci (e.g., the adeno-associated virus integration site 1 (AAVS1) safe harbor locus). The DICE (Dual Integrase Cassette Exchange) system, utilizing phiC31 integrase and Bxb1 integrase, is a tool for targeted insertion. Additionally, clustered, regularly arranged short palindromic repeats / Cas9 (CRISPR / Cas9) techniques can be used for targeted gene insertion.

[0341] Site-directed gene editing approaches may include homology-dependent or homology-independent mechanisms.

[0342] In some embodiments, methods for editing cells are provided herein, the methods comprising a solution comprising an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), This involves editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0343] In some embodiments, methods for editing cells are provided herein, the methods comprising a solution comprising N-acetyl-L-cysteine ​​(NAC), a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the cell's genome), This involves editing cells by inserting DNA templates into insertion sites within the cell's genome.

[0344] In some embodiments, methods for editing cells are provided herein, which include providing a solution comprising an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0345] In some embodiments, methods for editing cells are provided herein, which include providing a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), a histone deacetylase (HDAC) inhibitor, and / or an osmotic regulator (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0346] Cells to be edited can be incubated with at least one of the RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), histone deacetylase (HDAC) inhibitors, and / or osmoregulators disclosed herein, and then these cells can be brought into contact with ribonucleoprotein complexes (RNPs) and DNA templates. These cells can then be edited using the nonviral gene editing method described herein to insert the DNA template into the cell genome. After inserting the DNA template into the cell genome, the edited cells can also be incubated with at least one of the RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), histone deacetylase (HDAC) inhibitors, and / or osmoregulators disclosed herein. The RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), histone deacetylase (HDAC) inhibitors, and / or osmoregulators and the cells can be incubated together in a solution, such as an aqueous solution, before, during, and / or after the nonviral editing process. In some embodiments, the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), histone deacetylase (HDAC) inhibitor, and / or osmoregulator are incubated together with the cells before the editing process. In some embodiments, the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), histone deacetylase (HDAC) inhibitor, and / or osmoregulator are incubated together with the cells during the editing process. In some embodiments, the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), histone deacetylase (HDAC) inhibitor, and / or osmoregulator are incubated together with the cells after the editing process.

[0347] In some embodiments, methods for editing cells are provided herein, which include providing cells comprising an exogenous homologous recombination protein or a DNA repair regulatory protein, providing a solution comprising a ribonucleoprotein complex (RNP) and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0348] In some embodiments, methods for editing cells are provided herein, which include providing a solution comprising a histone deacetylase (HDAC) inhibitor comprising at least one of sodium phenylbutyrate, xynostat, or panobinostat, a ribonucleoprotein complex (RNP), and a DNA template (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0349] In some embodiments, methods for editing cells are provided herein, each method comprising: providing cells with a solution containing a histone deacetylase (HDAC) inhibitor comprising at least one of sodium phenylbutyrate, xynostat, or panobinostat; contacting the cells with a ribonucleoprotein complex (RNP) and a DNA template (the RNP comprising a nuclease domain and guide RNA, the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome); and editing the cells by inserting the DNA template into the insertion site in the cell's genome. In some embodiments, the method further comprises contacting (e.g., incubating) the edited cells with a solution containing a histone deacetylase (HDAC) inhibitor comprising at least one of sodium phenylbutyrate, xynostat, or panobinostat.

[0350] In some embodiments, methods for editing cells are provided herein, which include providing cells comprising an exogenous homologous recombination protein or a DNA repair regulatory protein, and providing a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0351] In some embodiments, methods for editing cells are provided herein, which include providing a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor (the RNP comprising a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template comprising nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome), and editing the cell by inserting the DNA template into the insertion site in the cell's genome.

[0352] In some embodiments, the solution contains at least N-acetyl-L-cysteine ​​(NAC) and an osmotic regulator; RNase inhibitor and N-acetyl-L-cysteine ​​(NAC); RNase inhibitor and osmotic regulator; N-acetyl-L-cysteine ​​(NAC) and a histone deacetylase (HDAC) inhibitor; osmotic regulator and a histone deacetylase (HDAC) inhibitor; RNase inhibitor and a histone deacetylase (HDAC) inhibitor; RNase inhibitor, N-acetyl-L-cysteine N-acetyl-L-cysteine ​​(NAC), and histone deacetylase (HDAC) inhibitors; N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors; RNase inhibitors, osmotic regulators, and histone deacetylase (HDAC) inhibitors; RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), and osmotic regulators, or RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), osmotic regulators, and histone deacetylase (HDAC) inhibitors.

[0353] In some embodiments, the insertion of DNA templates into the cellular genome increases by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution. In some embodiments, the insertion of DNA templates into the cellular genome increases by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4, or more compared to the control solution. In some embodiments, the solution enhances the growth or yield of edited cells compared to a control solution that does not contain at least one of RNase inhibitors, N-acetyl-L-cysteine ​​(NAC), osmotic regulators, or histone deacetylase (HDAC) inhibitors. In some embodiments, the growth or yield of edited cells increases by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution. In some embodiments, the enlargement or yield of edited cells increases by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

[0354] In some embodiments, a solution containing at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor reduces cell death during the nonviral introduction of RNP complexes and DNA templates into cells compared to a control solution that does not contain at least one of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor. In some embodiments, cell death is reduced by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution. In some embodiments, cell death is reduced by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

[0355] In some embodiments, the edited cells are incubated with an HDAC inhibitor during and / or after electroporation. In other embodiments, the cells to be edited are incubated with an HDAC inhibitor solution (e.g., sodium phenylbutyrate) for about 2 days before the RNP complex and DNA template are introduced into the cells nonvirally. In some embodiments, the HDAC inhibitor pre-electroporation solution contains sodium phenylbutyrate. In some embodiments, the HDAC inhibitor pre-electroporation solution contains sodium phenylbutyrate at a final concentration of about 0.01 mM, 0.05 mM, 0.1 mM, 0.10 mM, 0.20 mM, 0.30 mM, 0.40 mM, 0.50 mM, 0.60 mM, 0.70 mM, 0.80 mM, 0.90 mM, or 1 mM.

[0356] In some embodiments, the methods provided herein are methods for editing immune cells, optionally primary immune cells, or primary human immune cells.

[0357] In some embodiments, the cells are mammalian cells, human cells, hematopoietic cells, immune cells, primary immune cells, or primary human immune cells. In some embodiments, the immune cells are autoimmune cells. In some embodiments, the immune cells are alloimmune cells. In some embodiments, the immune cells are natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, primary T cells, or T cell progenitor cells.

[0358] In some embodiments, RNP-DNA templates are introduced into cells nonvirally. Nonviral insertion of RNP-DNA templates can be achieved by chemical or electrical methods. For example, electroporation can be used to introduce the DNA template into cells.

[0359] In some embodiments, nonviral introduction of the RNP-DNA template is performed by electroporation. Any suitable electroporation device can be used, including but not limited to CTS Xenon, Nucleofector, 4D-Nucleofector, Neon NxT, CliniMACS, or MaxCyte Flow. Electroporation devices are commercially available from various vendors such as Invitrogen, BioRad, Thermo Fisher, Lonza, Gibco, Miltenyi Biotec, and Mirus. In some embodiments, the electroporation device is CTS Xenon, Nucleofector, or 4D-Nucleofector.

[0360] In some embodiments, electroporation includes at least one cycle. In some embodiments, electroporation includes multiple cycles (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 cycles, or 4 to 10 cycles). A cycle may be one or more electrical pulses of defined pulse duration and voltage, with a defined time between pulses.

[0361] In some embodiments, one or more electroporation cycles include 1-8, 1-2, 1-4, 1-6, 2-4, 2-8, 4-6, 4-8 electrical pulses, or 1, 2, 3, 4, 5, or 6 electrical pulses. In some embodiments, one or more electroporation cycles include 4 electrical pulses. In some embodiments, one or more electroporation cycles include 5 electrical pulses.

[0362] In some embodiments, one or more cycles or pulses of electroporation are performed independently at 500-2500 volts (e.g., 500 volts, 600 volts, 700 volts, 800 volts, 900 volts, 1000 volts, 1100 volts, 1200 volts, 1300 volts, 1400 volts, 1500 volts, 1600 volts, 1700 volts, 1800 volts, 1900 volts, 2000 volts, 2100 volts, 2200 volts, 2300 volts, 2400 volts, 500-1000 volts, 100-1500 volts, 1500-2000 volts, 2000-2100 volts, 2100-2200 volts, 2200-2300 volts, or 2300-2400 volts, or any number in between). In some embodiments, one or more electroporation cycles are performed at 2300 volts. In some embodiments, one or more pulses are performed at 2300 volts. In some embodiments, one or more pulses in each cycle can be performed at independently different voltages. In some embodiments, one or more pulses in each cycle can all be performed at the same voltage (e.g., 2300 volts). In some embodiments, one or more pulses in each cycle can all be performed at different voltages (e.g., a first pulse at any voltage between 500 and 2500 volts, and a second pulse at any voltage between 500 and 2500 volts, different from the first pulse).

[0363] In some embodiments, one or more pulses of one or more electroporation cycles are performed independently using pulse durations of 1–5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 ms. In some embodiments, one or more pulses of one or more electroporation cycles are performed independently using pulse durations of 1–30 ms, 1 ms, 2 ms, 3 ms, 4 ms, 5 ms, 6 ms, 7 ms, 8 ms, 9 ms, 10 ms, 11 ms, 12 ms, 13 ms, 14 ms, 15 ms, 16 ms, 17 ms, 18 ms, 19 ms, 20 ms, 21 ms, 22 ms, 23 ms, 24 ms, 25 ms, 26 ms, 27 ms, 28 ms, 29 ms, or 30 ms. In certain embodiments, one or more pulses of one or more electroporation cycles are performed independently using a pulse duration of 3 ms.

[0364] In some embodiments, some or all of the electroporation cycles are performed independently using 1-8, 1, 2, 3, 4, 5, 6, 7, or 8 pulses.

[0365] In some embodiments, one or more electroporation cycles are performed independently using pulse intervals of 100-1000, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 100 ms between electrical pulses. In some embodiments, one or more electroporation cycles are performed independently using pulse intervals of 500 ms.

[0366] In some embodiments, electroporation includes one cycle performed using settings of 2200 volts, a pulse duration of 3.0 ms, four pulses, and a pulse interval of 500 ms.

[0367] In some embodiments, electroporation includes one cycle performed using settings of 2300 volts, a pulse duration of 3.0 ms, four pulses, and a pulse interval of 500 ms.

[0368] In some embodiments, electroporation includes one cycle performed using settings of 2300 volts, a pulse duration of 3.0 ms, five pulses, and a pulse interval of 500 ms.

[0369] In some embodiments, the amount of DNA template used in one or more electroporation cycles is approximately 1 μg, 2 μg, 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, or 50 μg of DNA, or approximately 1–10 μg, 10–20 μg, 20–30 μg, 30–40 μg, or 40–50 μg of DNA.

[0370] In some embodiments, the electroporation includes five pulses and a smaller amount of DNA template compared to electroporation with four pulses.

[0371] In some embodiments, electroporation includes an EH115 pulse code.

[0372] Insertion site Methods for genome editing immune cells specifically include methods for editing human T cells, which involve inserting a nucleic acid sequence or construct into a target region within exon 1 of the TCR-α subunit (TRAC) gene in T cells. In some embodiments, the target region is located in exon 1 of the constant domain of the TRAC gene. In other embodiments, the target region is located in exon 1, exon 2, or exon 3, prior to the start of the sequence encoding the TCR-α transmembrane domain. In some embodiments, the nucleic acid sequence or construct encodes a heterologous protein, such as, but not limited to, a priming receptor and / or a chimeric antigen receptor (CAR).

[0373] Genome editing methods for immune cells also include methods for human T cells that involve inserting a nucleic acid sequence or construct into a target region within exon 1 of the TCR-β subunit (TRBC) gene in human T cells. In some embodiments, the target region is located in exon 1 of the TRBC1 or TRBC2 gene.

[0374] Specifically, genome editing methods for immune cells include methods for editing human T cells, which involve inserting nucleic acid sequences or constructs into the target region of a genome-safe harbor (GSH).

[0375] Gene editing therapies include, for example, viral vector integration and site-directed integration. Site-directed integration is a promising alternative to random viral vector integration because it reduces the risk of insertional mutagenesis or insertional carcinogenesis (Kolb et al. Trends Biotechnol. 2005 23:399-406, Porteus et al. Nat Biotechnol. 2005 23:967-973, Paques et al. Curr Gen Ther. 2007 7:49-66). However, site-directed integration continues to face challenges such as low knock-in efficiency, risk of insertional carcinogenesis, instability and / or abnormal expression of adjacent genes or transgenes, and low accessibility (e.g., within 20KB of adjacent genes). These challenges can be addressed in part by identifying and using safe harbor loci or safe harbor sites (SHSs), which are sites where genes or gene elements can be integrated without disrupting the expression or regulation of adjacent genes.

[0376] The most widely used putative human safe harbor site is the AAVS1 site on chromosome 19Q, which was initially identified as a site for recurrent adeno-associated virus insertion. Other potential SHSs have been identified based on homology to sites first identified in other species (e.g., human homology to the tolerant mouse Rosa26 locus), or in an increasing number of human genes that appear intrinsically in some contexts. One of this type of putative SHS is the CCR5 chemokine receptor gene, which, when disrupted, confers resistance to human immunodeficiency virus infection. Additional potential genomic SHSs, as with the original mouse Rosa26 locus, have been identified in humans and other cell types based on viral integration site mapping or gene trapping analysis. The top three SHSs, AAVS1, CCR5, and Rosa26, are located in close proximity to many protein-coding genes and regulatory elements. (See Figure 2 in Sadelain, M., et al. (2012). Safe harbours for the integration of new DNA in the human genome. Nature Reviews Cancer, 12(1), 51-58 (its related disclosure is incorporated herein by reference in its entirety)).

[0377] AAVS1 (also known as the PPP1R12C locus) on human chromosome 19 is a known SHS for hosting transgenes (e.g., DNA transgenes) with expected function. It is located at position 19q13.42. It has an open chromatin structure and is transcriptionally active. The canonical SHS loci of AAVS1 are chr19:55, 625, 241-55, 629, and 351. See Pellenz et al. “New Human Chromosomal Sites with Safe Harbor” “Potential for Targeted Transgene Insertion.” Human gene therapy vol.30,7(2019):814-828 (its relevant disclosure is incorporated herein by reference). Exemplary AAVS1 target gRNAs and target sequences are shown below. ● AAVS1-gRNA sequence: ggggccactagggacaggatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT (Sequence ID 128) ● AAVS1 target sequence: ggggccactagggacaggat (SEQ ID NO: 129)

[0378] Located on chromosome 3 at position 3p21.31, CCR5 encodes the major co-receptor for HIV-1. Disruption of this site in the CCR5 gene has been beneficial in HIV / AIDS treatment, promoting the development of zinc finger nucleases targeting its third exon. The canonical SHS loci of CCR5 are chr3:46, 414, 443-46, 414, 942. See Pellenz et al. “New Human Chromosomal Sites with Safe Harbor” “Potential for Targeted Transgene Insertion.” Human gene therapy vol.30,7(2019):814-828 (its relevant disclosure is incorporated herein by reference).

[0379] The mouse Rosa26 locus is particularly useful for gene modification because it can be targeted with high efficiency and is expressed in most cell types tested. Irion et al. 2007 ("Identification and targeting of the ROSA26 locus in human embryonic stem cells." Nature biotechnology 25.12(2007):1477-1482 (its relevant disclosures are incorporated herein by reference) identified human ROSA26, a human homolog, at chromosome 3 (position 3p25.3). The canonical SHS locus for human Rosa26 (hRosa26) is chr3:9,415,082-9,414,043. See Pellenz et al. "New Human Chromosomal Sites with Safe Harbor Potential for Targeted Transgene Insertion." Human gene therapy vol.30,7(2019):814-828 (its relevant disclosures are incorporated herein by reference).

[0380] For an example of the addition of safe harbor sites, see Pellenz et al. “New Human Chromosomal Sites with “Safe Harbor” “Potential for Targeted Transgene Insertion.” Human gene therapy vol.30,7(2019):814-828 (the relevant disclosure is incorporated herein by reference).

[0381] Table 1 shows additional safe harbor sites and associated sgRNA sequences for targeting these SHSs. [Table 1] TIFF2026521526000003.tif214165TIFF2026521526000004.tif214165TIFF2026521526000005.tif21416 5TIFF2026521526000006.tif223165TIFF2026521526000007.tif219165TIFF2026521526000008.tif36165

[0382] In some embodiments, the safe harbor loci are chr10:33130000-33140000, chr10:72290000-72300000, chr11:128340000-128350000, chr11:65425000-65427000 (NEAT1), chr15:92830000-92840000, chr16:11220000-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-594 It is located at one or more of the sgRNA target loci selected from 60000, chr8:127980000-128000000, chr9:7970000-7980000, APRT, B2M, CAPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, and TRIM28.

[0383] In some embodiments, the target loci are chr10:33130000-33140000, chr10:72290000-72300000, chr11:128340000-128350000, chr11:65425000-65427000 (NEAT1), chr15:92830000-92840000 Selected from chr16:11220000-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, and chr9:7970000-7980000.

[0384] In some embodiments, the target locus is chr11:128340000~128350000 or chr15:92830000~92840000.

[0385] In some embodiments, the target locus is a gene selected from APRT, B2M, CAPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, and TRIM28.

[0386] In some embodiments, the safe harbor locus is the GS94 or GS102 integration site shown in Table 1.

[0387] In some embodiments, the safe harbor loci of this disclosure are useful for insertion of sequences encoding transgenes. In some embodiments, the safe harbor site enables high transgene expression (sufficient to enable transgene functionality or treatment of the disease of interest) and stable expression of the transgene over days, weeks, or months. In some embodiments, knockout of a gene at a safe harbor locus benefits cellular function, or the gene at the safe harbor locus has no known function within the cell. In some embodiments, the safe harbor locus results in stable transgene expression in vitro with or without CD3 / CD28 stimulation, negligible off-target cleavage detected by iGuide-Seq or CRISPR-Seq, fewer off-target cleavage compared to other loci detected by iGuide-Seq or CRISPR-Seq, negligible transgene-independent cytotoxicity, negligible transgene-independent cytokine expression, negligible transgene-independent chimeric antigen receptor expression, negligible deregulation or silencing of nearby genes, and is located outside of cancer-related genes.

[0388] When used, "neighboring genes" can refer to genes located within approximately 100KB, 125KB, 150KB, 175KB, 200KB, 225KB, 250KB, 275KB, 300KB, 325KB, 350KB, 375KB, 400KB, 425KB, 450KB, 475KB, 500KB, 525KB, and 550KB of the safe harbor locus (integration site).

[0389] In some embodiments, this disclosure envisions an insertion comprising one or more transgenes. The transgenes may encode therapeutic proteins, antibodies, peptides, suicide genes, apoptotic genes, or any other gene of interest. Safe harbor loci identified using the methods herein allow for the incorporation of transgenes that result in enhanced therapeutic properties, for example. These enhanced therapeutic properties, as used herein, refer to the therapeutic properties of enhanced cells compared to typical immune cells of the same normal cell type. For example, NK cells with “enhanced therapeutic properties” have enhanced, improved, and / or increased therapeutic outcomes compared to typical, unmodified, and / or naturally occurring NK cells. Therapeutic properties of immune cells may include, but are not limited to, cell transplantation, transport, homing, viability, self-renewal, persistence, immune response regulation and modulation, survival, and cytotoxicity. Therapeutic properties of immune cells are also manifested by antigen-targeting receptor expression, HLA presentation or absence, resistance to the intratumor microenvironment, induction and immunomodulation of bystander immune cells, improved target specificity by reduction, and resistance to treatments such as chemotherapy.

[0390] As used herein, “insertion size” refers to the length of the nucleotide sequence incorporated into (inserted into) the safe harbor site. In some embodiments, the insertion size includes at least about 100, 200, 300, 400, or 500 nucleotides (base pairs). In some embodiments, the insertion size includes about 500 nucleotides (base pairs). In some embodiments, the insertion size includes up to 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 kbp (kilobase pairs) or sizes in between. In some embodiments, the size of the insert is greater than 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 kbp or any size between them. In some embodiments, the size of the insert is in the range of 3 to 15 kbp or any number within that range. In some embodiments, the size of the insert is in the range of 1.5 to 8.3 kbp or any number within that range. In some embodiments, the size of the insert is in the range of 1.5 to 13 kbp or any number within that range. In some embodiments, the size of the insert is at least in the range of 1.5 to 15 kbp or any number within that range. In some embodiments, the size of the insert is in the range of 0.5 to 20 kbp or any number within that range. In some embodiments, the size of the insert is 0.5-10, 0.6-10, 0.7-10, 0.8-10, 0.9-10, 1-10, 2-10, 3-10, 4-10, 5-10, 6-10, 7-10, 8-10, or 9-10 kbp. In some embodiments, the size of the insert is 0.5-11, 0.6-11, 0.7-11, 0.8-11, 0.9-11, 1-11, 2-11, 3-11, 4-11, 5-11, 6-11, 7-11, 8-11, 9-11, or 10-11 kbp.In some embodiments, the size of the insert is 0.5-12, 0.6-12, 0.7-12, 0.8-12, 0.9-12, 1-12, 2-12, 3-12, 4-12, 5-12, 6-12, 7-12, 8-12, 9-12, 10-12, or 11-12kbp. In some embodiments, the size of the insert is 0.5-13, 0.6-13, 0.7-13, 0.8-13, 0.9-13, 1-13, 2-13, 3-13, 4-13, 5-13, 6-13, 7-13, 8-13, 9-13, 10-13, 11-13, or 12-13kbp. In some embodiments, the size of the insert is 0.5-14, 0.6-14, 0.7-14, 0.8-14, 0.9-14, 1-14, 2-14, 3-14, 4-14, 5-14, 6-14, 7-14, 8-14, 9-14, 10-14, 11-14, 12-14, or 13-14kbp. In some embodiments, the size of the insert is 0.5-15, 0.6-15, 0.7-15, 0.8-15, 0.9-15, 1-15, 2-15, 3-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 13-15, or 14-15kbp. In some embodiments, the size of the insert is 0.5-16, 0.6-16, 0.7-16, 0.8-16, 0.9-16, 1-16, 2-16, 3-16, 4-16, 5-16, 6-16, 7-16, 8-16, 9-16, 10-16, 11-16, 12-16, 13-16, 14-16, or 15-16 kbp. In some embodiments, the size of the insert is 0.5-17, 0.6-17, 0.7-17, 0.8-17, 0.9-17, 1-17, 2-17, 3-17, 4-17, 5-17, 6-17, 7-17, 8-17, 9-17, 10-17, 11-17, 12-17, 13-17, or 14-17, 15-17, or 16-17 kbp. In some embodiments, the size of the insert is 0.5-18, 0.6-18, 0.7-18, 0.8-18, 0.9-18, 1-18, 2-18, 3-18, 4-18, 5-18, 6-18, 7-18, 8-18, 9-18, 10-18, 11-18, 12-18, 13-18, 14-18, 15-18, 16-18, or 17-18 kbp.In some embodiments, the size of the insert is 0.5-19, 0.6-19, 0.7-19, 0.8-19, 0.9-19, 1-19, 2-19, 3-19, 4-19, 5-19, 6-19, 7-19, 8-19, 9-19, 10-19, 11-19, 12-19, 13-19, 14-19, 15-19, 16-19, 17-19, or 18-19 kbp. In some embodiments, the size of the insert is 0.5-20, 0.6-20, 0.7-20, 0.8-20, 0.9-20, 1-20, 2-20, 3-20, 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 16-20, 17-20, 18-20, or 19-20 kbp.

[0391] The inserts in this disclosure refer to nucleic acid molecules or polynucleotides inserted into safe harbor sites. In some embodiments, the nucleotide sequence is a DNA molecule, e.g., genomic DNA, or comprises deoxyribonucleotides. In some embodiments, the inserts comprise smaller fragments of DNA, such as plasmids, fosmids, cosmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and / or plastid DNA, mitochondrial DNA, or DNA isolated in the form of any other subgenome segment of DNA. In some embodiments, the inserts are RNA molecules or comprise ribonucleotides. The nucleotides in the inserts are intended to be naturally occurring nucleotides, unnaturally occurring nucleotides, and modified nucleotides. The nucleotides may be chemically or biochemically modified, or may contain unnatural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, and internucleotide modifications. Polynucleotides can be any topological conformation, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, circular conformation, and other three-dimensional conformations as intended in the art.

[0392] The insert may have coding regions and / or non-coding regions. The insert may include non-coding sequences (e.g., regulatory elements, e.g., promoter sequences). In some embodiments, the insert encodes a transcription factor. In some embodiments, the insert encodes an antigen-binding receptor such as a single receptor, a T cell receptor (TCR), a syn-notch, a CAR, or an mAb. In some embodiments, the insert is an RNAi molecule, including but not limited to miRNA, siRNA, or shRNA. In some embodiments, the insert is a human sequence. In some embodiments, the insert is a chimera. In some embodiments, the insert is a multi-gene / multi-module therapeutic cassette. A multi-gene / multi-module therapeutic cassette refers to an insert or cassette having one or more receptors (e.g., synthetic receptors), other exogenous protein-coding sequences, non-coding RNAs, transcriptional regulatory elements, and / or insulating sequences.

[0393] Various cell types are intended to have the safe harbor region as described in this disclosure. Cells containing a safe harbor region and / or cells containing an insert in the safe harbor region as described in this disclosure may be referred to as modified cells. These cells include, but are not limited to, eukaryotic cells, prokaryotic cells, animal cells, plant cells, fungal cells, etc. Optionally, the cells are mammalian cells, e.g., human cells. In some embodiments, the modified cells are stem cells, human cells, primary cells, hematopoietic cells, adaptive immune cells, innate immune cells, T cells, or T cell progenitor cells. Non-limiting examples of immune cells intended in this disclosure include T cells, B cells, natural killer (NK) cells, NKT / iNKT cells, macrophages, myeloid cells, and dendritic cells. Non-limiting examples of stem cells intended in this disclosure include pluripotent stem cells (PSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryo-derived embryonic stem cells (ntES; nuclear transfer ES) obtained by nuclear transfer, male germ cells (GS cells), embryonic germ cells (EG cells), hematopoietic stem / precursor stem cells (HSPCs), somatic cells (adult stem cells), angioblasts, neural stem cells, mesenchymal stem cells, and stem cells of other cells (including osteocytes, chondrocytes, myocytes, cardiomyocytes, neurons, tendinocytes, adipocytes, pancreatic cells, hepatocytes, renal cells, and follicular cells, etc.). In some embodiments, the modified cells are T cells, NK cells, iPSCs, and HSPCs. In some embodiments, the modified cells used in this disclosure are in vitro grown human cell lines (e.g., intentionally immortalized cell lines, cancer cell lines, etc.).

[0394] Methods for incorporating inserts into the safe harbor area may include non-viral delivery techniques.

[0395] In some embodiments, nucleic acid sequences are inserted into the cell genome via nonviral delivery. In nonviral delivery methods, the nucleic acid may be naked DNA or may be in a nonviral plasmid or vector. Nonviral delivery techniques may be site-specific insertion techniques described herein or known to those skilled in the art. Examples of site-specific techniques for insertion into safe harbor loci include, but are not limited to, homology-dependent modification using nucleases and homology-independent targeted insertion using Cas9. In some embodiments, the nonviral delivery method includes electroporation.

[0396] In some embodiments, the insert is incorporated into the safe harbor site by introducing (a) a target nuclease that cleaves the target region of the safe harbor site to create an insertion site, and (b) a nucleic acid sequence (insert) into the modified cell (the insert is incorporated into the insertion site, for example, by HDR). Examples of nonviral delivery techniques that can be used in the methods of this disclosure are provided in U.S. applications 16 / 568,116 and 16 / 622,843, and the relevant disclosures of these are incorporated herein by reference in their entirety.

[0397] Modified cells can retain their undifferentiated state after the insertion of the transgene. In some embodiments, the modified cells are undifferentiated. In some embodiments, after the insertion of the transgene, the modified cells are undifferentiated. In some embodiments, after the insertion of the transgene, the modified cells are CD45RA + and CCR7 + In some embodiments, after the insertion of the transgene, the modified cells are CD45RA + CCR7 + CD27 + That is the case.

[0398] Crispr-Cas gene editing One effective example of gene editing is the Crisp-Cas approach (e.g., Crisp-Cas9). This approach incorporates the use of a guide polynucleotide (e.g., guide ribonucleic acid or gRNA) and a Cas endonuclease (e.g., Cas9 endonuclease).

[0399] As used herein, a polypeptide referred to as “Cas endonuclease” or having “Cas endonuclease activity” refers to a CRISPR-associated (Cas) polypeptide encoded by the Cas gene, where the Cas polypeptide is a target DNA sequence that can be cleaved when operably linked to one or more guide polynucleotides (see, for example, U.S. Patent No. 8,697,359). This definition also includes variants of Cas endonuclease that retain guide polynucleotide-dependent endonuclease activity. The Cas endonuclease used in the donor DNA insertion methods detailed herein is an endonuclease that introduces a double-strand break into DNA at a target site (e.g., within a target locus or a safe harbor site).

[0400] As used herein, the term “guide polynucleotide” refers to a polynucleotide sequence that can be complexed with a Cas endonuclease, enabling the Cas endonuclease to recognize and cleave a DNA target site. A guide polynucleotide may be a single molecule or a double molecule. A guide polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). A guide polynucleotide consisting solely of ribonucleic acid is also referred to as “guide RNA.” In some embodiments, a polynucleotide donor construct is inserted into a safe harbor locus using a guide RNA (gRNA) in combination with a Cas endonuclease (e.g., Cas9 endonuclease).

[0401] A guide polynucleotide comprises a first nucleotide sequence domain (also called a variable targeting domain or VT domain) that is complementary to the nucleotide sequence in the target DNA, and a second nucleotide that interacts with the Cas endonuclease polypeptide. It may be a bimolecule (also called a double-stranded guide polynucleotide) containing a sequence domain (also called a Cas endonuclease recognition domain or CER domain). The CER domain of this bimolecule guide polynucleotide contains two distinct molecules that hybridize along the complementary region. The two distinct molecules may be an RNA sequence, a DNA sequence, and / or an RNA-DNA combination sequence.

[0402] Genome editing using the CRISPR-Cas approach relies on the repair of site-directed DNA double-strand breaks (DSBs) induced by RNA-induced Cas endonucleases (e.g., Cas9 endonuclease). Homologous recombination repair (HDR) of these DSBs enables precise editing of the genome by introducing defined genomic alterations, including base substitutions, sequence insertions, and deletions. Conventional HDR-based CRISPR / Cas9 genome editing involves transfecting cells with Cas9, gRNA, and donor DNA containing homologous arms matching the genomic locus of interest.

[0403] Homologie-Independent Targeted Insertion (HITI) uses a homology-independent strategy based on non-homologous end joining (NHEJ), and this method may be more efficient than HDR. A guide RNA (gRNA) targets the insertion site. For HITI, the donor plasmid lacks homology arms, and DSB repair does not occur via the HDR pathway. The donor polynucleotide construct can be modified to include one or more Cas9 cleavage sites adjacent to the gene or sequence to be inserted. This results in Cas9 cleavage in both the donor plasmid and the genomic target sequence. Both the target and donor have blunt ends, and a linearized donor DNA plasmid is used by the NHEJ pathway, which results in integration into the genomic DSB site. (For example, see Suzuki, K., et al. (2016). In vivo genome editing via CRISPR / Cas9 mediated homology-independent targeted integration. Nature, 540(7631), 144-149 (this related disclosure is incorporated in its entirety herein)).

[0404] Methods for performing gene editing using the CRISPR-Cas approach are known to those skilled in the art. (See, for example, U.S. applications US16 / 312,676, US15 / 303,722, and US15 / 628,533, the disclosures of which are incorporated herein by reference in their entirety). Additionally, the use of endonucleases for inserting transgenes into safe harbor loci is described, for example, in U.S. application 13 / 036,343, the disclosure of which is incorporated herein by reference in its entirety.

[0405] mRNA (or DNA) encoding guide RNA and / or endonucleases can be chemically bound to one or more moieties or complexes that enhance activity, cell distribution, or cellular uptake of oligonucleotides. Non-limiting examples of such moieties include cholesterol moieties, cholic acid, thioethers, thiocholesterol, aliphatic chains (e.g., dodecanediol or undecyl residues), phospholipids, such as dihexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-3-H-phosphonate, polyamine or polyethylene glycol chains, adamantane acetate, palmityl moieties, and lipid moieties such as octadecylamine or hexylamino-carbonyl-t-oxycholesterol moieties. See, for example, U.S. Patent Application No. 15 / 715,068 (these disclosures are incorporated herein by reference in their entirety).

[0406] Nucleic acids and vectors In some embodiments, this disclosure envisions a nucleic acid insert comprising one or more transgenes. The transgenes may encode therapeutic proteins, antibodies, peptides, suicide genes, apoptotic genes, or any other gene of interest. In some embodiments, the transgenes encode priming receptors. In some embodiments, the transgenes encode chimeric antigen receptors. In some embodiments, the insert comprises a priming receptor transgene and a chimeric antigen receptor transgene.

[0407] The inserts may also contain self-cleaving peptides. Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, such as porcine rhinitis virus-1 (P2A) peptide, Thosea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, or foot-and-mouth disease virus (F2A) peptide. Self-cleaving 2A peptides enable the expression of multiple gene products from a single construct. (See, for example, Chang et al. “Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells,” MAbs 7(2): 403-412 (2015)).

[0408] The insert may also contain a WPRE element. The WPRE element is outlined in Higashimoto, T., et al. Gene Ther 14, 1298-1304 (2007) and Zufferey, R., et al. J Virol. 1999 Apr;73(4):2886-92, both of which are incorporated herein by reference.

[0409] In some embodiments, the DNA template further comprises an autoexcisable 2A peptide (P2A).

[0410] In some embodiments, the P2A nucleic acid is located at the 3' end of the DNA template.

[0411] In some embodiments, the DNA template further comprises a woodchuck hepatitis virus posttranslational regulatory element (WPRE).

[0412] In some embodiments, the WPRE is located at the 3' end of the nucleic acid encoding the CAR and at the 5' end of the nucleic acid encoding the priming receptor, or the WPRE is located at the 3' end of the nucleic acid encoding the priming receptor and at the 5' end of the nucleic acid encoding the CAR.

[0413] Priming receptors In certain embodiments of this disclosure, the priming receptor is a Notch protein-based synthetic circuit receptor. Binding of the native Notch receptor to an allogenic ligand, such as a ligand derived from the delta family of proteins, triggers intramembrane proteolysis that cleaves an intracellular fragment of the Notch protein. This intracellular fragment is a transcription regulator that functions only when cleaved from Notch. Cleavage can occur by sequential proteolysis by the ADAM metalloproteinase and gamma-secretase complex. This intracellular fragment enters the cell nucleus and activates intercellular signaling genes. In contrast to the native Notch protein, the synNotch priming receptor replaces the native Notch intracellular fragment with a fragment that activates a CAR-encoding gene upon release from the priming receptor.

[0414] The Notch receptor has a modular domain structure. The external domain of the Notch receptor consists of a series of N-terminal epidermal growth factor (EGF)-like repeats involved in ligand binding. In synthetic Notch receptors or priming receptors, the Notch ligand-binding domain is replaced by a ligand-binding domain that binds to a selected target ligand or antigen. Following the EGF repeats are three LIN-12 / Notch repeat (LNR) modules specific to the Notch receptor, which have been widely reported to be involved in preventing premature receptor activation. The heterodimerization (HD) domain of Notch is split by furin cleavage, so that its N-terminal portion terminates with an extracellular subunit, and its C-terminal half constitutes the beginning of a transmembrane subunit. The receptor has a transmembrane segment and an intracellular domain (ICD) containing transcriptional regulators after the extracellular domain.

[0415] Multiple forms of priming receptors may be used in the methods, cells, and nucleic acids described herein. One type of priming receptor intended for use in the methods and cells described herein comprises a heterogeneous extracellular ligand-binding domain, a linked polypeptide having substantial sequence identity with a Notch receptor containing an NRR, a TMD, and an ICD. The “Fn Notch” receptor comprises a heterogeneous extracellular ligand-binding domain, a linked polypeptide having substantial sequence identity with a Robo receptor (such as mammalian Robo1, Robo2, Robo3, or Robo4), followed by one, two, or three fibronectin repeats ("Fn"), a TMD, and an ICD. The “mini Notch” receptor comprises a heterogeneous extracellular ligand-binding domain, a linked polypeptide having substantial sequence identity with a Notch receptor (lacking an NRR), a TMD, and an ICD. The “minimal Tinker Notch” receptor comprises a heterogeneous extracellular ligand-binding domain, a linked polypeptide (e.g., a synthetic (GGS)n polypeptide sequence) that lacks substantial sequence identity with the Notch receptor, a TMD, and an ICD. The “hinge Notch” receptor comprises a heterogeneous extracellular ligand-binding domain, a hinge sequence containing an oligomerization domain (i.e., a domain that promotes dimerization, trimerization, or higher-order multimerization with synthetic receptors and / or existing host receptors), a TMD, and an ICD. All of these receptor classes are synthetic, recombinant, and do not occur in nature. In some embodiments, the non-naturally occurring receptors disclosed herein bind to a ligand presented on the surface of a target cell, which induces proteolytic cleavage of the receptor and releases transcriptional regulators that modulate custom transcriptional programs in the cell. In some embodiments, the priming receptor does not contain the LIN-12-Notch repeat (LNR) and / or heterodimerization domain (HD) of the Notch receptor.

[0416] priming receptor extracellular domain The priming receptor includes an extracellular domain. In some embodiments, the extracellular domain includes a ligand-binding moiety of the receptor. In some embodiments, the extracellular domain includes an antigen-binding moiety that binds to one or more target antigens. In some embodiments, the antigen-binding moiety includes one or more antigen-binding determinants of the antibody or its functional antigen-binding fragment. In some embodiments, the antigen-binding moiety is selected from the group consisting of antibodies, nanobodies, diabodies, triabodies, or minibodies, F(ab')2 fragments, Fab fragments, single-chain variable fragments (scFv), and single-domain antibodies (sdAb), or functional fragments thereof. In some embodiments, the antigen-binding moiety includes scFv. The antigen-binding moiety may include a naturally occurring amino acid sequence or may be modified, designed, or altered to provide desired and / or improved properties, such as increased binding affinity. An antibody that "selectively binds to" an antigen is an antigen-binding moiety that binds to the antigen with high affinity and does not significantly bind to other unrelated antigens.

[0417] transmembrane domain As described above, the chimeric polypeptide of this disclosure comprises a TMD containing one or more ligand-induced proteolytic cleavage sites.

[0418] In general, suitable TMDs for the chimeric receptors disclosed herein may be any transmembrane domain of a type 1 transmembrane receptor containing at least one gamma-secretase cleavage site. Detailed descriptions of the structure and function of the gamma-secretase complex and its substrate proteins, including amyloid precursor protein (APP) and Notch, can be found, for example, in a recent review by Zhang et al., Frontiers Cell Neurosci (2014). Non-limited preferred TMDs derived from type 1 transmembrane receptors include those derived from CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HLA-A, and IFNAR2, and each TMD contains at least one gamma-secretase cleavage site. Additional TMDs suitable for the compositions and methods described herein include, but are not limited to, transmembrane domains derived from type 1 transmembrane receptors IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBOl, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, YASN, FLT1, CDH5, PKHD1, NECTINl, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd, and PTPRM. In some embodiments, the chimeric polypeptide or Notch receptor TMD of the Disclosure is a TMD derived from a TMD of a member of the calcin-thenin family, such as alkadein alpha and alkadein gamma. In some embodiments, the chimeric polypeptide or Notch receptor TMD of the Disclosure is a TMD known with respect to the Notch receptor. In some embodiments, the chimeric polypeptide or Notch receptor TMD of the Disclosure is a TMD derived from a different Notch receptor.For example, in a human Notch1-based mini-Notch, Notch1 TMD may be replaced with Notch2 TMD, Notch3 TMD, Notch4 TMD, or Notch TMD derived from non-human animals such as Danio rerio, Drosophila melanogaster, Xenopus laevis, or Gallus gallus.

[0419] In some embodiments, the priming receptor includes a Notch cleavage site such as S2 or S3. Additional proteolytic cleavage sites suitable for the compositions and methods disclosed herein include, but are not limited to, metalloproteinase cleavage sites of MMPs selected from collagenase-1, -2, and -3 (MMP-1, -8, and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11), matricin (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP). Another example of a suitable protease cleavage site is a plasminogen activator cleavage site, e.g., a urokinase plasminogen activator (uPA) or tissue plasminogen activator (tPA) cleavage site. Another example of a suitable protease cleavage site is a prolactin cleavage site. Specific examples of uPA and tPA cleavage sequences include sequences containing Yal-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is the tobacco etch virus (TEV) protease cleavage site, e.g., Glu-Asn-Leu-Thr-Gln-Ser (SEQ ID NO: 121), where the protease cleaves between glutamine and serine. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is the enterokinase cleavage site, e.g., Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 122), where the cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is the thrombin cleavage site, e.g., Leu-Val-Pro-Arg (SEQ ID NO: 123).Additional suitable linkers containing protease cleavage sites include sequences cleavable by the following proteases: PreScission® protease (a fusion protein containing human rhinovirus 3C protease and glutathione-S-transferase), thrombin, cathepsin B, Epstein-Barr virus protease, MMP-3 (stromelisin), MMP-7 (matrilysin), MMP-9; thermolysin-like MMP, matrix metalloproteinase 2 (MMP-2), cathepsin L; Tepsin D, matrix metalloproteinase 1 (MMP-1), urokinase-type plasminogen activator, membrane-type 1 matrix metalloproteinase (MT-MMP), stromelycin 3 (or MMP-11), thermolycin, fibroblast collagenase and stromelycin-1, matrix metalloproteinase 13 (collagenase-3), tissue-type plasminogen activator (tPA), human prostate-specific antigen, kallikrein (hK3), neutrophil elastase, and calpain (calcium-activated neutral protease). Non-native proteases (e.g., TEV) can be used as further regulatory mechanisms in host cells expressing the receptor, reducing receptor activation until the protease is expressed or otherwise provided. Additionally, proteases may be tumor-related or disease-related (expressed at significantly higher levels than in normal tissue) and function as independent regulatory mechanisms. For example, some matrix metalloproteinases are highly expressed in certain types of cancer.

[0420] In some embodiments, the amino acid substitution(s) within the TMD include one or more substitutions within the "GV" motif of the TMD. In some embodiments, at least one of such substitution(s) includes a substitution to alanine. Additional sequences and substitutions are described in WO2021061872, which is incorporated herein by reference in its entirety.

[0421] intracellular domain In some embodiments, a priming receptor comprises one or more intracellular domains from or derived from transcription regulators. Transcription regulators activate or repress transcription from homologous promoters. Transcription activators typically bind to nearby transcription promoters and recruit RNA polymerase to directly initiate transcription. Transcription repressors bind to transcription promoters and sterically inhibit transcription initiation by RNA polymerase. Other transcription regulators function as either activators or repressors, depending on where they bind and the cellular conditions. Thus, as used herein, “transcription activating domain” refers to a domain of a transcription factor that interacts with transcriptional regulatory elements and / or transcriptional regulatory proteins (i.e., transcription factors, RNA polymerase, etc.) to increase and / or activate the transcription of one or more genes. Non-limiting examples of transcriptional activation domains include the herpes simplex virus VP16 activation domain, VP64 (a tetrameric derivative of VP16), HIV TAT, NFκB p65 activation domain, p53 activation domains 1 and 2, CREB (cAMP response element binding protein) activation domain, E2A activation domain, NFAT (nuclear factor of activated T cells) activation domain, yeast Gal4, yeast GCN4, yeast HAP1, MLL, RTG3, GLN3, OAF1, PIP2, PDR1, PDR3, PHO4, LEU3 glucocorticoid receptor transcriptional activation domain, B-cell POU homeodomain protein Oct2, plant Ap2, or any other known to those skilled in the art. In some embodiments, the transcriptional regulator is selected from Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-YP64, Gal4-KRAB, and HAP1-VP16. In some embodiments, the transcription regulator is Gal4-VP64. The transcriptional activation domain may contain wild-type or naturally occurring sequences, or it may be a modified, mutated, or derivative version of the original transcriptional activation domain having the desired ability to increase and / or activate the transcription of one or more genes. In some embodiments, the transcription regulator may further include a nuclear localization signal.

[0422] DNA binding domain In some embodiments of the embodiments described herein, the synthetic protein comprises one or more intracellular “DNA-binding domains” (or “DB domains”). Such “DNA-binding domains” refer to sequence-specific DNA-binding domains that bind to specific DNA sequence elements. Thus, as used herein, “sequence-specific DNA-binding domains” refer to protein domain portions that have the ability to selectively bind to DNA having a particular given sequence. Sequence-specific DNA-binding domains may include wild-type or naturally occurring sequences, or they may be modified, variant, or derivative versions of the original domain having the desired ability to bind to a desired sequence. In some embodiments, sequence-specific DNA-binding domains are modified to bind to a desired sequence. Non-limiting examples of proteins having sequence-specific DNA-binding domains that may be used in the synthetic proteins described herein include HNF1a, Gal4, GCN4, reverse tetracycline receptor, THY1, SYN1, NSE / RU5', AGRP, CALB2, CAMK2A, CCK, CHAT, DLX6A, EMX1, their zinc finger proteins or domains, CRISPR / Cas proteins such as Cas9, Cas3, Cas4, Cas5, Cas5e (or CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Ca Examples include s10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu196, as well as TALES.

[0423] In embodiments where a CRISPR / Cas-like protein is used, the CRISPR / Cas-like protein may be a wild-type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild-type or modified CRISPR / Cas protein. The CRISPR / Cas-like protein may be modified to increase its nucleic acid binding affinity and / or specificity, alter its enzymatic activity, and / or change other properties of the protein. For example, the nuclease (i.e., DNase, RNase) domain of the CRISPR / Cas-like protein may be modified, deleted, or inactivated. Alternatively, the CRISPR / Cas-like protein may be cleaved to remove domains not essential to the function of the system described herein. For example, a CRISPR enzyme used as a DNA-binding protein or its domain may be mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR or its domain lacks the ability to cleave nucleic acid sequences containing the DNA-binding domain target site. For example, the D10A mutation, when combined with one or more of the H840A, N854A, or N863A mutations, can produce a Cas9 enzyme that is substantially lacking in DNA cleavage activity.

[0424] membrane-proximal domains The ECD and TMD, or the TMD and ICD, can be linked to each other with linking polypeptides such as a near-membrane domain. The "SynNotch" receptor comprises a heterogeneous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with the Notch receptor JMD (including the NRR), a TMD, and an ICD. The "FnNotch" receptor comprises a heterogeneous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with the Robo receptor (such as mammalian Robo1, Robo2, Robo3, or Robo4), followed by one, two, or three fibronectin repeats ("Fn"), a TMD, and an ICD. The "MiniNotch" receptor comprises a heterogeneous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with the Notch receptor JMD but lacking the NRR (LIN-12-Notch repeat (LNR) module and heterodimerization domain), a TMD, and an ICD. The "minimal linker Notch" receptor has a heterogeneous extracellular ligand-binding domain and a linked polypeptide that lacks substantial sequence identity with the Notch receptor (e.g., synthetic (GGS)). n The "Hinged Notch" receptor comprises a hinge sequence containing a heterogeneous extracellular ligand-binding domain, an oligomerization domain (i.e., a domain that promotes dimerization, trimerization, or higher-order multimerization with synthetic receptors and / or existing host receptors), a TMD, and an ICD.

[0425] In some embodiments, the priming receptor includes a perimembrane domain (JMD) peptide between the extracellular domain and the transmembrane domain. In some embodiments, the priming receptor includes a perimembrane domain (JMD) peptide between the transmembrane domain and the intracellular domain. In some embodiments, the JMD peptide includes an LWF motif. The use of the LWF motif in the receptor construct is described in U.S. Patent No. 10,858,443 (which is incorporated herein by reference in its entirety). In some embodiments, the JMD peptide has substantial sequence identity to the JMDs of Notch1, Notch2, Notch3, and / or Notch4. In some embodiments, the JMD peptide has substantial sequence identity to the JMDs of Notch1, Notch2, Notch3, and / or Notch4, but does not include the LIN-12-Notch repeat (LNR) and / or heterodimerization domain (HD) of the Notch receptor. In some embodiments, the JMD peptide does not have substantial sequence identity to the JMDs of Notch1, Notch2, Notch3, and / or Notch4. In some embodiments, the JMD peptide contains an oligomerizing domain that facilitates the formation of dimers, trimers, or higher-order assemblies of the receptor. Such JMD peptides are described in WO2021061872, which is incorporated herein by reference in whole.

[0426] In the mini-Notch receptor, the linking polypeptide is derived from the Notch JMD sequence after deletion of the NRR and HD domains. The Notch JMD sequence may be derived from Notch1, Notch2, Notch3, or Notch4, and may be derived from non-human homologs such as those from the genera Drosophila, Juncus miyabei, Danio, etc. The remaining 4 to 50 amino acid residues of the Notch sequence can be used as the polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are modified to alter the orientation and / or proximity of the ECD and TMD relative to each other, thereby achieving the desired activity of the chimeric polypeptide, such as the signal transduction level upon ligand induction or in the absence of the ligand.

[0427] In minimal linker Notch receptors, the linked polypeptide has no substantial sequence identity with respect to the Notch JMD sequence (including Notch1, Notch2, Notch3, or Notch4, or Notch JMD sequences derived from their non-human homologs). 4 to 50 amino acid residues can be used as the polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are modified to alter the orientation and / or proximity of the ECD and TMD to each other in order to achieve the desired activity of the chimeric polypeptide of this disclosure. The minimal linker sequence may be designed to include or omit protease cleavage sites, and may include or omit multiple sites for glycosylation sites or other types of post-translational modifications. In some embodiments, the minimal linker does not include protease cleavage sites or glycosylation sites.

[0428] In some embodiments, the priming receptor further comprises a hinge. A hinge linker that may be used in the priming receptor may comprise an oligomerization domain (e.g., a hinge domain) containing one or more polypeptide motifs that promote oligomerization of a chimeric polypeptide via intermolecular disulfide bonds. In these examples, within the chimeric receptors disclosed herein, the hinge domain generally comprises a flexible polypeptide connector region located between the ECD and the TMD. Thus, the hinge domain provides flexibility between the ECD and the TMD and also provides a site for intermolecular disulfide bonds between two or more chimeric polypeptide monomers to form an oligomeric complex. In some embodiments, the hinge domain comprises a motif that promotes dimerization of the chimeric polypeptide disclosed herein. In some embodiments, the hinge domain comprises a motif that promotes trimerization of the chimeric polypeptide disclosed herein (e.g., a hinge domain derived from OX40). Suitable hinge polypeptide sequences for the compositions and methods of this disclosure may be naturally occurring hinge polypeptide sequences (e.g., those derived from naturally occurring immunoglobulins) or may be modified, designed, or altered to provide desired and / or improved properties, such as transcriptional modulating properties. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses such as IgG1 hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or functional variants thereof. In some embodiments, the hinge polypeptide sequence contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence contains one or more CPPC motifs (SEQ ID NO: 124).

[0429] The hinge polypeptide sequence may also be derived from the CD8α hinge domain, CD28 hinge domain, CD152 hinge domain, PD-1 hinge domain, CTLA4 hinge domain, OX40 hinge domain, and functional variants thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from the CD8α hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from the CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from the OX40 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from the IgG4 hinge domain or a functional variant thereof.

[0430] The Fn Notch-linked polypeptide is derived from Robo1 JMD containing a fibronectin repeat (Fn) domain having a short polypeptide sequence between the Fn repeat and the TMD. The Fn Notch-linked polypeptide does not contain a Notch negative regulatory region (NRR) or a Notch HD domain. The Fn-linked polypeptide may contain 1, 2, 3, 4, or 5 Fn repeats. In some embodiments, the chimeric receptor comprises an Fn-linked polypeptide having about 1 to about 5 Fn repeats, about 1 to about 3 Fn repeats, or about 2 to about 3 Fn repeats. The short polypeptide sequence between the Fn repeat and the TMD may be about 2 to about 30 amino acid residues. In some embodiments, the short polypeptide sequence may be about 5 to about 20 amino acids of any sequence. In some embodiments, the short polypeptide sequence may be about 5 to about 20 naturally occurring amino acids of any sequence. In some embodiments, the short polypeptide sequence may be about 5 to about 20 amino acids of any sequence, but has one or fewer proline residues. In some embodiments, the short polypeptide sequence may consist of about 5 to about 20 amino acids, with about 50% or more of the amino acids being glycine. In some embodiments, the short polypeptide sequence may consist of about 5 to about 20 amino acids, selected from glycine, serine, threonine, and alanine. In some embodiments, the length and amino acid composition of the FN-linked polypeptide sequence are modified to alter the orientation and / or proximity of the ECD and TMD to each other in order to achieve the desired activity of the chimeric polypeptide of the Disclosure.

[0431] Stop transition array In some embodiments, priming receptors further include a stop transition sequence (STS) between the transmembrane domain and the intracellular domain. The STS contains a charged lipidophobic sequence. Without being constrained by any particular theory, the STS is thought to function as a membrane anchor, preventing the intracellular domain from crossing the plasma membrane. The use of the STS domain in priming receptors is described in WO2021061872, which is incorporated herein by reference in its entirety. Examples of non-exclusive STS sequences include APLP1, APLP2, APP, TGBR3, CSF1R, CXCL16, CX3CL1, DAG1, DCC, DNER, DSG2, CDH1, GHR, HLA-A, IFNAR2, IGF1R, IL1R1, ERN2, KCNE1, KCNE2, CHL1, LRPl, LRP2, LRP18, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBOl, SORCS3, SORCS1, SORL1, SDC1, S Examples of STS sequences include DC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKTFD1, NECTIN1, KL, IL6R, EFNB1, CD44, CLSTN1, LRP8, PCDHGC3, NRG1, LRP1B, JAG2, EFNB2, DLL1, CLSTN2, EPCAM, ErbB4, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA4, IL1R2, KCNE4, SCN2B, Nradd, PTPRM, Notch1, Notch2, Notch3, and Notch4. In some embodiments, the STS is heterogeneous with respect to the transmembrane domain. In some embodiments, the STS is homologous with respect to the transmembrane domain. STS sequences are described in WO2021061872 (which is incorporated herein in its entirety by reference).

[0432] Chimeric antigen receptor In some embodiments, the chimeric antigen receptor includes an extracellular component containing an antigen-binding domain. The antigen-recognition domain of a receptor such as a CAR can be linked to one or more intracellular signaling components, such as a signaling component that mimics activation via the antigen-receptor complex, e.g., the TCR complex, and / or to a signal mediated by another cell surface receptor. Thus, in some embodiments, the extracellular binding component (e.g., ligand-binding or antigen-binding domain) is linked to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, one of the domains within the receptor, e.g., a transmembrane domain that naturally associates with a CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane proteins and to minimize interaction with other members of the receptor complex.

[0433] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antigen-binding domain as described herein, and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv, VH, or single-domain VH antibody, and the intracellular domain comprises an ITAM. In some embodiments, the intracellular signaling domain comprises a signaling domain of the ζ chain of the CD3ζ(CD3) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0434] In some embodiments, the transmembrane domain includes a transmembrane portion of CD8A or CD28. The extracellular domain and the transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane are linked by a spacer, such as any of those described herein. In some embodiments, the chimeric antigen receptor contains, for example, the intracellular domain of a T cell costimulatory molecule between the transmembrane domain and the intracellular signaling domain. In some embodiments, the T cell costimulatory molecule is CD28 or 41BB.

[0435] Chimeric antigen receptor (CAR) T cells are T cells that have been genetically modified to produce artificial T cell receptors for use in immunotherapy. Chimeric antigen receptors are receptor proteins that have been modified to confer the ability of T cells to target specific proteins. For example, the genetic modification of lymphocytes (e.g., T cells) by incorporating CARs, and the administration of the modified cells to a target, is an example of “adoptive cell therapy.” As used herein, the term “adoptive cell therapy” refers to cell-based immunotherapy for transfusion of autologous or allogeneic lymphocytes called T cells or B cells. In this CAR therapy approach, cells are expanded and cultured ex vivo and genetically modified before transfusion.

[0436] CAR expression allows modified T cells to target and bind to specific proteins, such as tumor antigens. In CAR therapy, T cells are collected from the subject, which may be derived from the subject's own blood or from an autologous T cell from a donor who has not received CAR therapy. Once isolated, the T cells are genetically modified with CAR, expanded ex vivo, and administered to the subject (i.e., the patient), for example, by infusion.

[0437] CARs may be introduced into T cells, for example, using site-specific techniques. Site-specific integration of the transgene (e.g., CAR) may target the transgene to a safe harbor locus or TRAC. Examples of site-specific techniques for integration to safe harbor loci include, but are not limited to, homology-dependent modification using nucleases and homology-independent targeted insertion using Cas9.

[0438] Modified CAR T cells have applications in immuno-oncology. For example, CARs can be selected to target specific tumor antigens. An example of cancer that can be effectively targeted using CAR T cells is hematological malignancies. In some embodiments, CAR T cell therapy can be used to treat solid tumors.

[0439] Chimeric antigen receptor extracellular domain In some embodiments, the extracellular domain includes a ligand-binding moiety of the receptor. In some embodiments, the extracellular domain includes an antigen-binding moiety that binds to one or more target antigens. In some embodiments, the antigen-binding moiety includes one or more antigen-binding determinants of the antibody or its functional antigen-binding fragment. In some embodiments, the antigen-binding moiety is selected from the group consisting of antibodies, nanobodies, diabodies, triabodies, or minibodies, F(ab')2 fragments, Fab fragments, single-chain variable fragments (scFv), and single-domain antibodies (sdAb), or functional fragments thereof. In some embodiments, the antigen-binding moiety includes scFv. The antigen-binding moiety may include a naturally occurring amino acid sequence or may be modified, designed, or altered to provide desired and / or improved properties, such as increased binding affinity.

[0440] CAR transmembrane domain In some embodiments, the transmembrane domains are derived from either natural or synthetic sources. When the source is natural, in some embodiments, the domains are derived from any membrane-bound or transmembrane protein. The transmembrane regions include those derived from the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and / or CD154 (i.e., including at least its transmembrane region(s)). Alternatively, in some embodiments, the transmembrane domains are synthetic. In some embodiments, the synthetic transmembrane domains mainly consist of hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain. In some embodiments, linkage is by linkers, spacers, and / or transmembrane domain(s).

[0441] In some embodiments, the transmembrane domain of the receptor, e.g., CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27-amino acid transmembrane domain of human CD28 (accession number: P10747.1).

[0442] In some embodiments, the CAR includes CD8a or CD28 TMD.

[0443] CAR Hinge In some embodiments, the CAR further includes a spacer, which may be at least a portion of an immunoglobulin constant region or a variant or modified version thereof, e.g., a hinge region, e.g., a CD8A hinge, an IgG4 hinge region, and / or a CH1 / CL and / or FC region, or may include at least a portion thereof. In some embodiments, the constant region or portion is that of human IgG, such as IgG4 or IgG1. In some embodiments, a portion of the constant region functions as a spacer region between an antigen-recognizing component, e.g., scFv, and the transmembrane domain. The spacer may be of a length that results in increased cellular responsiveness after antigen binding compared to the absence of the spacer. In some examples, the spacer is about 12 amino acids long or less. Examples of spacers include those having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and including any integer between any of the endpoints in the listed ranges. In some embodiments, the spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Examples of spacers include CD8a hinges, IgG4 hinges alone, IgG4 hinges linked to CH2 and CH3 domains, or IgG4 hinges linked to the CH3 domain. Examples of spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153 or International Patent Application Publication WO2014031687. In some embodiments, the CAR hinge includes CD8a CD8α, cut-type CD8α, or CD28 hinge domains.

[0444] Some intracellular signaling domains mimic or approximate signaling mediated by native antigen receptors, signaling mediated by such receptors in combination with costimulatory receptors, and / or signaling mediated solely by costimulatory receptors. In some embodiments, short oligo or polypeptide linkers, e.g., those containing glycine and serine, e.g., glycine-serine doublets, or linkers of 2 to 10 amino acids in length, are present, forming a linkage between the transmembrane domain and the cytoplasmic signaling domain of the receptor.

[0445] CAR intracellular domain In some embodiments, upon ligation of a CAR, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of immune cells, e.g., T cells modified to express the receptor. For example, the receptor may induce T cell functions such as cytolytic activity or T helper activity, e.g., secretion of cytokines or other factors. In some embodiments, a cleaved portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used instead of the intact immunostimulatory chain, for example, if it transduces an effector function signal. In some embodiments, the intracellular signaling domain(s) include the cytoplasmic sequence of a T cell receptor (TCR), and, in some embodiments, the sequence of a co-receptor that, in its natural context, acts in coordination with such receptor to initiate signaling after antigen receptor engagement, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same function.

[0446] In some embodiments, the receptor includes a primary cytoplasmic signaling sequence that modulates the primary activation of the TCR complex. The stimulative primary cytoplasmic signaling sequence may contain an immunoreceptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of ITAMs containing a primary cytoplasmic signaling sequence include those derived from TCR or CD3ζ, FCRγ, FCRβ, CD3γ, CD3δ, CD3ε, CDDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ. In some embodiments, the intracellular activation domain includes a CD3ζ domain.

[0447] In some embodiments, the intracellular signaling domain includes a human CD3ζ-stimulated signaling domain or a functional variant thereof, for example, the 112AA cytoplasmic domain of human CD3ζ isoform 3 (accession number: P20963.2) or a CD3ζ signaling domain described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993.

[0448] A receptor, e.g., a CAR, may contain at least one intracellular signaling component. In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain, e.g., a CD3ζ chain, which mediates T cell activation and cytotoxicity. Thus, in some embodiments, the extracellular domain is linked to one or more cellular signaling modules. In some embodiments, the cellular signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., a CAR, further includes a portion of one or more additional molecules, such as FC receptor γ, CD8, CD4, CD25, or CD16. For example, in some embodiments, the CAR includes a chimeric molecule between CD3ζ or FC receptor γ and CD8, CD4, CD25, or CD16.

[0449] In some embodiments, the intracellular domain includes the intracellular costimulatory signaling domain of 41BB, or a functional variant or portion thereof, for example, the 42-amino acid cytoplasmic domain of human 4-1BB (accession number Q07011.1), or a functional variant or portion thereof.

[0450] In some embodiments, the receptor includes one or more, for example, two or more costimulatory domains and an activation domain, for example, a primary activation domain, in its cytoplasmic portion. Exemplary receptors include the intracellular components of CD3ζ, CD28, and 4-1BB. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is 4-1BB.

[0451] In some embodiments, the receptor comprises a signaling domain and / or transmembrane portion of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some embodiments, the same receptor comprises both an activating component and a costimulatory component.

[0452] In certain embodiments, the intracellular signaling domain includes a CD8A transmembrane and signaling domain linked to a CD3 (e.g., CD3ζ) intracellular domain. In some embodiments, the intracellular signaling domain includes a 4-1BB (CD137, TNFRSF9) co-stimulatory domain linked to a CD3ζ intracellular domain. In some embodiments, the CAR includes a 4-1BB co-stimulatory domain.

[0453] In some embodiments, the CAR or other antigen receptor further comprises a marker, such as a cell surface marker, which can be used to confirm the transduction or modification of cells to express a receptor, such as a cleaved version of a cell surface receptor, such as cleaved EGFR (tEGFR). In some embodiments, the marker comprises all or part (e.g., a cleaved form) of CD34, nerve growth factor receptor (NGFR), or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, such as a cleavable linker sequence or a ribosome skip sequence, such as a polynucleotide encoding T2A. See WO2014031687. In some embodiments, the introduction of a construct encoding a CAR and EGFRt separated by a T2A ribosome switch can express two proteins derived from the same construct so that EGFRt can be used as a marker to detect cells expressing such a construct. In some embodiments, the marker and, optionally, the linker sequence may be any, as disclosed in Published Patent Application WO2014031687. For example, the marker may optionally be a cleaved EGFR (tEGFR) linked to a linker sequence such as the T2A ribosome skip sequence.

[0454] In some embodiments, the marker is a molecule not naturally found on T cells or on the surface of T cells, such as a cell surface protein or a portion thereof.

[0455] In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a molecule that is not recognized as "self" by the immune system of the host into which the cell is adopted.

[0456] In some embodiments, the marker does not perform a therapeutic function and / or has no effect other than being used as a marker for genetic modification, for example, to select successfully modified cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that exerts some desired effect in other ways, such as a ligand for cells encountered in vivo, or a co-stimulatory or immune checkpoint molecule to enhance and / or attenuate the cellular response during adoptive transfer and encounter with the ligand.

[0457] CAR may contain one or more naturally occurring amino acids instead of one or more naturally occurring amino acids. Exemplary modified amino acids include aminocyclohexanecarboxylic acid, norleucine, α-amino-N-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, (3-phenylserine (3-hydroxyphenylalanine), phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2, Examples include, but are not limited to, 3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,γ-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0458] For example, in some embodiments, the CAR comprises a single-chain antibody as described herein (e.g., sdAb, containing only the VH region), an antibody or fragment thereof comprising a VH domain and scFv, and a spacer, e.g., a CD8a hinge, a CD8a transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3ζ signaling domain. In some embodiments, the CAR comprises an antibody or fragment comprising sdAb and scFv as described herein, and a spacer, e.g., a CD8A hinge, a CD8A transmembrane domain, a 4-1BB intracellular signaling domain, and a CD3 zeta signaling domain.

[0459] Transgenes expressing priming receptors and CAR systems can be introduced into cells, such as T cells, using site-directed techniques. Site-directed integration of transgenes (e.g., priming receptors and CARs) can target safe harbor loci or TRAC. Examples of site-directed techniques for integration into safe harbor loci include, but are not limited to, homology-dependent modifications using nucleases and homology-independent target insertions using Cas9.

[0460] Modified cells have applications in immuno-oncology. For example, priming receptors and CARs can be selected to target different specific tumor antigens. Examples of cancers that can be effectively targeted using such cells are hematological malignancies or solid tumors. In some embodiments, solid tumors can be treated using immunotherapy.

[0461] In some embodiments, upon ligation of a CAR, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of immune cells, e.g., T cells modified to express the receptor. For example, the receptor may induce T cell functions such as cytolytic activity or T helper activity, e.g., secretion of cytokines or other factors. In some embodiments, a cleaved portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used instead of the intact immunostimulatory chain, for example, if it transduces an effector function signal. In some embodiments, the intracellular signaling domain(s) include the cytoplasmic sequence of a T cell receptor (TCR), and, in some embodiments, the sequence of a co-receptor that, in its natural context, acts in coordination with such receptor to initiate signaling after antigen receptor engagement, and / or any derivative or variant of such molecule, and / or any synthetic sequence having the same function.

[0462] In the context of natural TCRs, complete activation generally requires not only TCR-mediated signaling but also co-stimulatory signals. Therefore, in some embodiments, the receptor also includes components for generating secondary or co-stimulatory signals to promote complete activation. In other embodiments, the receptor does not include components for generating co-stimulatory signals. In some embodiments, additional receptors are expressed within the same cell to provide components for generating secondary or co-stimulatory signals.

[0463] T cell activation is described in some embodiments as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, receptors contain one or both of such signaling components.

[0464] In some embodiments, the receptor comprises a signaling domain and / or transmembrane portion of a costimulatory receptor such as CD28, 4-1BB, OX40, DAP10, and ICOS. In some embodiments, the same receptor comprises both an activating component and a costimulatory component.

[0465] In certain embodiments, the intracellular signaling domain includes a CD28 transmembrane domain and a signaling domain linked to a CD3 (e.g., CD3ζ) intracellular domain. In some embodiments, the intracellular signaling domain includes a chimeric CD28 and CD137 (4-1BB, TNFRSF9) costimulatory domain linked to a CD3ζ intracellular domain.

[0466] In some embodiments, the receptor includes one or more, for example, two or more costimulatory domains and activation domains, for example, a primary activation domain, in its cytoplasmic portion. Exemplary receptors include the intracellular components of CD3ζ, CD28, and 4-1BB.

[0467] In some embodiments, the CAR or other antigen receptor further comprises a marker, such as a cell surface marker, which can be used to confirm the transduction or modification of cells to express a receptor, such as a cleaved version of a cell surface receptor, such as cleaved EGFR (tEGFR). In some embodiments, the marker comprises all or part (e.g., a cleaved form) of CD34, nerve growth factor receptor (NGFR), or epidermal growth factor receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a linker sequence, such as a cleavable linker sequence or a ribosome skip sequence, such as a polynucleotide encoding T2A. See WO2014031687. In some embodiments, the introduction of a construct encoding a CAR and EGFRt separated by a T2A ribosome switch can express two proteins derived from the same construct so that EGFRt can be used as a marker to detect cells expressing such a construct. In some embodiments, the marker and, optionally, the linker sequence may be any, as disclosed in Published Patent Application WO2014031687. For example, the marker may optionally be a cleaved EGFR (tEGFR) linked to a linker sequence such as the T2A ribosome skip sequence.

[0468] In some embodiments, the marker is a molecule not naturally found on T cells or on the surface of T cells, such as a cell surface protein or a portion thereof.

[0469] In some embodiments, the molecule is a non-self molecule, such as a non-self protein, i.e., a molecule that is not recognized as "self" by the immune system of the host into which the cell is adopted.

[0470] In some embodiments, the marker does not perform a therapeutic function and / or has no effect other than being used as a marker for genetic modification, for example, to select successfully modified cells. In other embodiments, the marker may be a therapeutic molecule or a molecule that exerts some desired effect in other ways, such as a ligand for cells encountered in vivo, or a co-stimulatory or immune checkpoint molecule to enhance and / or attenuate the cellular response during adoptive transfer and encounter with the ligand.

[0471] CAR may contain one or more naturally occurring amino acids instead of one or more naturally occurring amino acids. Exemplary modified amino acids include aminocyclohexanecarboxylic acid, norleucine, α-amino-N-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, (3-phenylserine (3-hydroxyphenylalanine), phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2, Examples include, but are not limited to, 3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,γ-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0472] In some cases, CARs are referred to as first, second, and / or third-generation CARs. In some embodiments, first-generation CARs are CARs that provide only CD3 chain-inducing signals when an antigen binds to them. In some embodiments, second-generation CARs provide both such signals and co-stimulatory signals, and include intracellular signaling domains from co-stimulatory receptors such as CD28 or CD137. In some embodiments, third-generation CARs in some aspects include multiple co-stimulatory domains from different co-stimulatory receptors.

[0473] In some embodiments, the chimeric antigen receptor comprises an extracellular component containing the antibody or fragment described herein. In some embodiments, the chimeric antigen receptor comprises an extracellular component containing the antibody or fragment described herein and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv or single-domain VH antibody, and the intracellular domain comprises an ITAM. In some embodiments, the intracellular signaling domain comprises the signaling domain of the ζ chain of the CD3ζ(CD3) chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain linking the extracellular domain and the intracellular signaling domain.

[0474] In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28. The extracellular domain and the transmembrane domain may be linked directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are linked by a spacer, such as any of those described herein. In some embodiments, the chimeric antigen receptor contains, for example, the intracellular domain of a T cell costimulatory molecule between the transmembrane domain and the intracellular signaling domain. In some embodiments, the T cell costimulatory molecule is CD28 or 41BB.

[0475] In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane portion of CD28 or a functional variant thereof, or a transmembrane domain containing such a transmembrane portion, and an intracellular signaling domain comprising a signaling portion of CD28 or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR comprises an antibody, e.g., an antibody fragment, a transmembrane portion of CD28 or a functional variant thereof, or a transmembrane domain containing such a transmembrane portion, and an intracellular signaling domain comprising a signaling portion of 4-1BB or a functional variant thereof and a signaling portion of CD3ζ or a functional variant thereof. In some such embodiments, the receptor further comprises a spacer (e.g., a hinge-only spacer) containing a portion of an Ig molecule (e.g., a human Ig molecule) (e.g., an Ig hinge (e.g., an IgG4 hinge)).

[0476] In some embodiments, the transmembrane domain of the receptor, e.g., CAR, is the transmembrane domain of human CD28 or a variant thereof, e.g., the 27-amino acid transmembrane domain of human CD28 (accession number: P10747.1).

[0477] In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. In some embodiments, the T cell costimulatory molecule is CD28 or 4-1BB.

[0478] In some embodiments, the intracellular signaling domain includes the intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, e.g., its 41-amino acid domain, and / or such domain having an LL-to-GG substitution at positions 186-187 of the native CD28 protein. In some embodiments, the intracellular domain includes the intracellular costimulatory signaling domain of 41BB, or a functional variant or portion thereof, e.g., the 42-amino acid cytoplasmic domain of human 4-1BB (accession number Q07011.1), or a functional variant or portion thereof.

[0479] In some embodiments, the intracellular signaling domain includes a human CD3ζ-stimulated signaling domain or a functional variant thereof, for example, the 112AA cytoplasmic domain of human CD3ζ isoform 3 (accession number: P20963.2) or a CD3ζ signaling domain described in U.S. Patent No. 7,446,190 or U.S. Patent No. 8,911,993.

[0480] In some embodiments, the spacer comprises only the hinge region of IgG, such as only the hinge of IgG4 or IgG1. In other embodiments, the spacer is an Ig hinge linked to the CH2 and / or CH3 domains, e.g., an IgG4 hinge. In some embodiments, the spacer is an Ig hinge linked to the CH2 and CH3 domains, e.g., an IgG4 hinge. In some embodiments, the spacer is an Ig hinge linked to the CH3 domain only, e.g., an IgG4 hinge. In some embodiments, the spacer is a glycine-serine-rich sequence, or other flexible linkers such as known flexible linkers, or comprises them.

[0481] Modified cells This specification also provides modified cells that include at least one DNA template nonvirally inserted into a target region of the cell's genome. In some embodiments, the size of the DNA template is approximately 0.3 kb, 1 kb, 2 kb, 3 kb, 4 kb, 4.5, or 5 kilobase pairs (kb) or larger.

[0482] In some embodiments, the size of the DNA template is approximately 0.3kb, 0.5kb, 1.0kb, 1.5kb, 2.0kb, 2.5kb, 3.0kb, 3.5kb, 4.0kb, 4.5kb, 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6. 2kb, 6.3kb, 6.4kb, 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8kb, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9k b, 9.0kb, 9.1kb, 9.2kb, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, 10.0kb, 10.1kb, 10.2kb , 10.3kb, 10.4kb, 10.5kb, 10.6kb, 10.7kb, 10.8kb, 10.9kb, 11.0kb, 11.1kb, 11.2kb, 11.3kb, 11.4 DNA templates of sizes kb, 11.5kb, 11.6kb, 11.7kb, 11.8kb, 11.9kb, 12.0kb, 12.1kb, 12.2kb, 12.3kb, 12.4kb, 12.5kb, 12.6kb, 12.7kb, 12.8kb, 12.9kb, 13.0kb or larger, or any size between these sizes.

[0483] In some embodiments, the size of the DNA template is approximately 0.3kb to 13kb, 0.3kb to 0.5kb, 0.3kb to 1kb, 0.3kb to 4kb, 0.3kb to 3kb, 0.3kb to 5kb, 0.3kb to 7kb, 0.3kb to 10kb, 0.5kb to 1kb, 0.5kb to 3kb, 0.5kb to 5kb, 0.5kb to 7kb, 0.5kb to 10kb, 0.5kb to 13kb, 1kb to 3kb, 1kb to 5kb, 1kb to 7kb, 1kb to 10kb, 1kb The ranges are approximately b to 13kb, 5kb to 13kb, 5kb to 13kb, 5kb to 9kb, 5kb to 8kb, 5kb to 7kb, 5kb to 6kb, 6kb to 13kb, 6kb to 10kb, 6kb to 9kb, 6kb to 8kb, 6kb to 7kb, 7kb to 13kb, 7kb to 10kb, 7kb to 9kb, 7kb to 8kb, 8kb to 13kb, 8kb to 10kb, 8kb to 9kb, 9kb to 13kb, 9kb to 10kb, 10kb to 13kb, or 11kb to 13kb.

[0484] In some embodiments, the DNA template includes a transgene encoding a protein. The protein may be any protein of interest, such as, but not limited to, a CAR, priming receptor, TCR, or antibody.

[0485] Cells containing inserts at target loci or safe harbor sites as described in this disclosure may be referred to as modified cells. In some embodiments, immune cells are any cells capable of giving rise to pluripotent immune cells. In some embodiments, immune cells may be induced pluripotent stem cells (iPSCs) or human pluripotent stem cells (HSPCs). In some embodiments, immune cells include primary hematopoietic cells or primary hematopoietic stem cells. In some embodiments, modified cells are stem cells, human cells, primary cells, hematopoietic cells, adaptive immune cells, innate immune cells, natural killer (NK) cells, T cells, CD8+ cells, CD4+ cells, or T cell progenitor cells. In some embodiments, immune cells are T cells. In some embodiments, T cells are regulatory T cells, effector T cells, or naive T cells. In some embodiments, T cells are CD8 + These are T cells. In some embodiments, T cells are CD4 + These are T cells. In some embodiments, T cells are CD4 + CD8 + These are T cells.

[0486] In some embodiments, the modified cells are stem cells, human cells, primary cells, hematopoietic cells, adaptive immune cells, innate immune cells, T cells, or T cell progenitor cells. Non-limiting examples of immune cells intended in this disclosure include T cells, B cells, natural killer (NK) cells, NKT / iNKT cells, macrophages, myeloid cells, and dendritic cells. Non-limiting examples of stem cells intended in this disclosure include pluripotent stem cells (PSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), embryo-derived embryonic stem cells (ntES; nuclear transfer ES) obtained by nuclear transfer, male germ cells (GS cells), embryonic germ cells (EG cells), hematopoietic stem / progenitor stem cells (HSPCs), somatic cells (adult stem cells), angioblasts, neural stem cells, mesenchymal stem cells, and stem cells of other cells (including osteocytes, chondrocytes, myocytes, cardiomyocytes, neurons, tendinocytes, adipocytes, pancreatic cells, hepatocytes, renal cells, and follicular cells, etc.). In some embodiments, the modified cells are T cells, NK cells, iPSCs, and HSPCs. In some embodiments, the modified cells used in this disclosure are human cell lines grown in vitro (e.g., intentionally immortalized cell lines, cancer cell lines, etc.).

[0487] Furthermore, a population of cells containing multiple primary immune cells is provided herein. In some embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of the cell genome contains targeted insertions of a heterologous DNA template, the DNA template being at least about 5 kb in size.

[0488] therapeutic use Modified cells, populations thereof, or compositions thereof are administered in effective doses to subjects, generally mammals, generally humans, for therapeutic purposes.

[0489] The modified cells may be administered to the subject by injection (for example, by continuous injection over a period of time) or by other methods of administration known to those skilled in the art.

[0490] The modified cells provided herein can be administered as part of a pharmaceutical composition. In some embodiments, the disclosure provides a composition comprising the guide RNA of the disclosure. The pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and those skilled in the art can select suitable pharmaceutical excipients. Accordingly, the pharmaceutical excipients provided below are illustrative and not limiting. Additional pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009) (which is incorporated in its entirety by reference).

[0491] The modified cells provided herein have been found to have non-pharmaceutical uses, such as not only use in gene therapy, but also in the production of animal models and recombinant cell lines that express the target protein.

[0492] The modified cells of this disclosure may be any cell, generally mammalian cells, generally human cells modified by incorporating a transgene into a safe harbor locus as described herein. In some embodiments, the modified cells are immune cells. In some embodiments, the modified cells are lymphocytes. In some embodiments, the modified cells are T cells or T cell progenitor cells.

[0493] The modified cells, compositions, and methods of this disclosure are useful for therapeutic applications such as CAR T cell therapy and TCR T cell therapy. In some embodiments, insertion of a sequence encoding a transgene within a safe harbor locus maintains TCR expression compared to cases without insertion, enabling transgene expression while maintaining TCR function.

[0494] This specification provides a variety of diseases treated using modified cells, populations thereof, or compositions thereof. Non-limiting examples of such diseases include, but are not limited to, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, cancer, dermatomyositis, diabetes mellitus (type 1), certain juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, certain myocarditis, multiple sclerosis, pemphigus / bullous pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, certain thyroiditis, certain uveitis, vitiligo, polyangiitis (Wegener); and granulomatous autoimmune diseases. Infectious diseases include, but are not limited to, hematopoietic malignancies, including acute and chronic leukemia, lymphoma, multiple myeloma, and myelodysplastic syndromes; tumors and solid tumors of the prostate, breast, lung, colon, uterus, skin, liver, bone, pancreas, ovaries, testes, bladder, kidneys, head, neck, stomach, cervix, rectum, larynx, or esophagus; HIV (human immunodeficiency virus)-related disorders, RSV (respiratory syncytial virus)-related disorders; EBV (Epstein-Barr virus)-related disorders; CMV (cytomegalovirus)-related disorders; and infections including, but not limited to, adenovirus-related disorders and BK polyomavirus-related disorders.

[0495] Cancers that can be treated with the modified cells (e.g., CAR T cells), populations thereof, or compositions thereof of this disclosure include hematological cancers. In some embodiments, the cancers treated using the modified cells (e.g., CAR T cells), populations thereof, or compositions thereof described herein are malignant hematological disorders or leukemias. In some embodiments, the modified cells (e.g., CAR T cells), populations thereof, or compositions thereof described herein are used to treat acute lymphoblastic leukemia (ALL) or diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancers are acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndromes, acute T-lymphoblastic leukemia, or acute promyelocytic leukemia, chronic myelomonocytic leukemia, or chronic myeloid leukemia in the acute transformation phase. Examples of cancers treatable using the modified cells described herein (e.g., CAR T cells) include, but are not limited to, breast cancer, ovarian cancer, esophageal cancer, bladder cancer, or gastric cancer, salivary duct cancer, salivary gland duct cancer, lung adenocarcinoma, or invasive forms of uterine cancer, such as serous endometrial cancer. In some other embodiments, the cancer is brain cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer, pancreatic cancer, rectal cancer, kidney cancer, gastric cancer, testicular cancer, or uterine cancer.In further embodiments, cancer may include squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, neuroblastoma, sarcoma (e.g., angiosarcoma or chondrosarcoma), laryngeal carcinoma, parotid gland carcinoma, biliary tract carcinoma, thyroid carcinoma, acral lentiginous melanoma, actinic keratosis, acute lymphoblastic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous cell carcinoma, anal canal carcinoma, anal carcinoma, rectal carcinoma, astrocytic tumor, greater vestibular adenocarcinoma, basal cell carcinoma, biliary tract carcinoma, bone carcinoma, bone marrow carcinoma, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid plexus papilloma / carcinoma, chronic Lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, gastrointestinal cancer, duodenal cancer, endocrine cancer, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrial adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing's sarcoma, ocular and orbital cancer, female reproductive organ cancer, focal nodular hyperplasia, gallbladder cancer, gastric squamous carcinoma, gastric fundus carcinoma, gastrinoma, glioblastoma, glucagonoma, cardiac cancer, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatobiliary tract cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer, insulino Carcinoma, carcinoma in situ, squamous cell carcinoma in situ, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colorectal cancer, leiomyosarcoma, melanoma derived from lentigo malignant, lymphoma, male reproductive organ cancer, malignant mesothelioma, malignant mesothelioma, medulloblastoma, medullary epithelioma, meningeal carcinoma, mesothelial carcinoma, metastatic cancer, oral cancer, mucoepidermoid carcinoma, multiple myeloma, muscle cancer, nasal cavity cancer, nervous system cancer, neuroepithelial adenocarcinoma, nodular melanoma, non-epithelial skin cancer, non-Hodgkin lymphoma, oat cell carcinoma, oligodendrocyte carcinoma, oral cancer, osteosarcoma, serous papillary adenocarcinoma, penile Cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, paranasal sinus cancer, skin cancer, small cell carcinoma, small intestine cancer, leiomyocyte carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, rhabdomyosarcoma, submesothelial carcinoma, superficial spreading melanoma, T-cell leukemia, tongue cancer, undifferentiated carcinoma, ureteral cancer, urethral cancer, bladder cancer, urinary tract cancer, cervical cancer, endometrial cancer, uveal melanoma, vaginal cancer, verrucous carcinoma, VIP tumor, vulvar cancer, well-differentiated carcinoma, or Wilms' tumor.

[0496] In some embodiments, this disclosure provides a method for treating a subject in need of treatment by administering a composition comprising one of the modified cells described herein to the subject. The terms “treat,” “therapy,” etc., as used herein generally refer to obtaining a desired pharmacological and / or physiological effect. The effect is prophylactic in that it is the complete or partial prevention of a disease, and / or therapeutic in that it is the partial or complete cure of the disease and / or side effects resulting from the disease. As used herein, the term “therapy” encompasses any treatment of a disease in a subject (e.g., a mammal, e.g., a human). Therapy may also refer to administering modified cells provided herein to a subject that is susceptible to a disease but has not yet been diagnosed with the disease, including preventing the onset of the disease, inhibiting the progression of the disease, or mitigating the disease (i.e., regressing the disease). Furthermore, therapy may stabilize or reduce undesirable clinical symptoms in a subject (e.g., a patient). The cells, populations thereof, or compositions thereof provided herein may be administered before, during, or after the onset of a disease or injury.

[0497] In certain embodiments, subjects have a disease, condition, and / or injury that can be treated and / or improved by cell therapy. In some embodiments, subjects requiring cell therapy are subjects having an injury, disease, or condition that thereby necessitates cell therapy (e.g., therapy in which cellular material is administered to the subject). However, the intent is that it is possible to treat, improve, and / or reduce the severity of at least one symptom associated with the injury, disease, or condition. In certain embodiments, subjects requiring cell therapy include, but are not limited to, candidates for bone marrow or stem cell transplantation, subjects who have undergone chemotherapy or radiotherapy, hyperproliferative disorders or cancer (e.g., hematopoietic system), subjects who have or are at risk of developing hyperproliferative disorders or cancer, tumors (e.g., solid tumors), viral infections or subjects who have or are at risk of developing viruses. It is also intended to include subjects who have or are at risk of developing infection-related diseases.

[0498] In some embodiments, the Disclosure provides the compositions of the Disclosure together with instructions for use. The instructions for use may be present in the kit as an accompanying document, on the label of the kit or its component containers, or in digital format (e.g., on a CD-ROM, via an internet link). The kit may contain one or more of the following: genome-targeted nucleic acids, polynucleotides encoding genome-targeted nucleic acids, site-specific polypeptides, and / or polynucleotides encoding site-specific polypeptides. Additional components in the kit, such as buffers (reconstitute buffer, stabilizing buffer, dilution buffer, etc.) and / or one or more control vectors are also intended.

[0499] Combination therapy In some embodiments, the modified cells or compositions thereof of this disclosure are administered together with at least one additional therapeutic agent. Any suitable additional therapeutic agent may be administered together with the modified cells, populations thereof, or compositions thereof provided herein. In some embodiments, the additional therapeutic agent is selected from radioactive materials, ophthalmic agents, cytotoxic agents, chemotherapeutic agents, cell division inhibitors, antihormone agents, immunostimulants, angiogenesis inhibitors, and combinations thereof.

[0500] In some embodiments, the modified cells or compositions thereof are administered together with a steroid. The administration of the steroid can prevent or reduce the risk of the recipient of the modified cells(s) or compositions developing an autoimmune response.

[0501] Additional therapeutic agents may be administered by any preferred means. In some embodiments, the modified cells, populations thereof, or compositions thereof described herein and the additional therapeutic agents are administered in the same pharmaceutical composition, for example, by infusion. In some embodiments, the antibodies and additional therapeutic agents provided herein are contained in different pharmaceutical compositions.

[0502] A pharmaceutical composition may contain one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and those skilled in the art can select a suitable pharmaceutical excipient. Therefore, the pharmaceutical excipients provided below are intended to be illustrative and not limiting. Additional pharmaceutical excipients include, for example, those described in Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009) (which is incorporated in its entirety by reference).

[0503] Various modes of administration of additional therapeutic agents are considered herein. In some embodiments, the additional therapeutic agent is administered by any preferred mode of administration. Generally, but not limited to, modes of administration include intravitreous, subretinal, suprachoroidal, intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, topical, pulmonary, and subcutaneous routes.

[0504] In embodiments in which the modified cells and additional therapeutic agents provided herein are contained in different pharmaceutical compositions, the administration of the modified cells provided herein may be performed before, simultaneously with, and / or after the administration of the additional therapeutic agents.

[0505] Pharmaceutical composition of the present invention Methods for treating cancerous diseases are also included in this disclosure. Such methods include administering a therapeutically effective amount of modified cells, as described herein. Modified cells can be formulated into pharmaceutical compositions. These compositions may include, in addition to one or more of the modified cells, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other materials may depend on the route of administration, e.g., oral, intravenous, transdermal or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0506] Pharmaceutical compositions for oral administration may be in the form of tablets, capsules, powders, or liquids. Tablets may contain a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil, or synthetic oil. They may also contain saline solution, dextrose or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0507] For intravenous, cutaneous, or subcutaneous injection, or injection into the affected area, the active ingredient is in the form of a parenterally acceptable aqueous solution that is pyrogenic, has a suitable pH, isotonicity, and stability. Those skilled in the art can adequately prepare a suitable solution using, for example, an isotonic vehicle such as sodium chloride injection, Ringer's injection, or lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included as needed.

[0508] The dose should preferably be a "therapeutically effective dose" or a "preventively effective dose" (which may be therapeutic in some cases, but prevention can be considered therapeutic), whichever is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. The prescription of treatment, e.g., the determination of dosage, is the responsibility of the general practitioner and other physicians, and should take into account the disorder to be treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0509] Depending on the condition to be treated, the composition can be administered alone, in combination with other treatments, concurrently, or sequentially. [Examples]

[0510] The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantities, temperatures, etc.), a certain degree of experimental error and deviation should naturally be permitted.

[0511] The implementation of this invention will, unless otherwise indicated, utilize conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the scope of the skills of those skilled in the art. Such techniques are adequately described in the literature. For example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3. rd See Ed. (Plenum Press) Vols. A and B (1992).

[0512] Example 1: Increased editing efficiency with RNase inhibitors Materials and methods Newly isolated or freeze-thawed isolated T cells were activated with CD3 / CD28 Dynabeads (Thermo Fisher Scientific, 43500D or 40203D) for 2 days at 37°C in 5% CO2 in complete medium (TexMACS (Miltenyi Biotec, 170-076-306) + 3% PLTGold (Mill Creek Life Sciences, PLTGold 100GMP) + 12.5 ng / mL IL-7 + 12.5 ng / mL IL-15) in a gas-permeable bag (Charter Medical, EXP-3L). The beads were then removed, the cells were enriched, and the buffer was changed to Genome Editing Buffer (Thermo Fisher Scientific, A4998002 or A4998001). Under RNase inhibitor-assisted conditions, an RNase inhibitor (New England Biolabs, M0314L or G8005B-1ML) was added to the cells and incubated for at least 10 minutes before adding the payload. The payload (plasmid DNA and ribonucleoprotein (RNP) consisting of Cas9 protein complexed with GS94-targeting sgRNA) was then added to the cells, and the cells were electroporated using a CTS Xenon Electroporation System (Thermo Fisher Scientific) with either Xenon SingleShot (Thermo Fisher Scientific, A50305) or MultiShot consumable (Thermo Fisher Scientific, A53445). After electroporation, the cells were transferred to a GRex container (Wilson Wolf Manufacturing, 80660M or 81100-CS) containing 1 / 10 the container's filled volume of complete medium. One day after electroporation, the complete medium was added to the full volume. Next, cells were sampled for cell count and flow cytometry, and the knock-in rate was measured 4–7 days after electroporation.

[0513] result The use of RNase inhibitors increased knock-in rates, cell expansion, and normalized yield of edited T cells compared to the absence of RNase inhibitors (Figures 1A–C). Figure 1 summarizes the knock-in rates (%) (edited, Figure 1A), cell expansion (Figure 1B), and yield of edited T cells (Figure 1C) of cells electroporated for insertion of exemplary transgenes using the Xenon platform with and without RNase inhibitors (-) for 15 unique donors. P-values ​​were obtained from one-sided matching t-tests for the 15 unique donors. With the inclusion of RNase inhibitors, each assessed value increased compared to cells electroporated without RNase inhibitors. The addition of RNase inhibitors consistently increased cell editing efficiency (knock-in rates) in all 15 of the 15 donors tested using the Xenon SingleShot platform with an exemplary priming receptor as the transgene insertion (Figure 1A). Cell editing efficiency improved by approximately 26% on average, but in some cases, it reached nearly a 1x improvement. Furthermore, cell proliferation (magnification) was also improved overall with the addition of RNase inhibitors (Figure 1B), and similar or higher yields of edited T cells were obtained in all 15 donors (Figure 1C).

[0514] Figures 2A and 2B show that inclusion of an RNase inhibitor before electroporation (right bar) also improved Xenon MultiShot KI and editing yield in both donors tested, compared to cells incubated without an RNase inhibitor (left bar). Figure 2A shows the percentage of cells with exemplary transgene knock-in after incubation with and without an RNase inhibitor. Figure 2B shows the normalized yield of edited T cells (total number of edited cells divided by the number of electroporated cells) after incubation with and without an RNase inhibitor. Across both donors tested, the addition of an RNase inhibitor resulted in a 30–50% increase in editing efficiency and a 20–110% increase in edited T cell yield.

[0515] Editing efficiency and the yield of edited T cells can be adversely affected by longer exposure times of cells and payloads before electroporation. However, the addition of an RNase inhibitor consistently increased the yield of edited T cells in 5 out of 5 donors tested using a priming receptor as an exemplary transgene insertion. In cells without an RNase inhibitor, both the yield and editing efficiency of edited T cells decreased in 5 out of 5 donors when the cell / payload incubation time before electroporation was between 0 and 60 minutes (Figure 3). When cells / payloads were incubated for 30 minutes before electroporation without an RNase inhibitor, editing efficiency decreased by an average of 30%, and the yield of edited T cells decreased by 50% (Figures 4A and 4B). In contrast, the addition of an RNase inhibitor before electroporation improved both editing efficiency and the yield of edited T cells at all time points (Figures 3, 4A, and 4B). Similar yields of edited T cells were observed at incubation times of 0 minutes without an RNase inhibitor and 10 minutes with an RNase inhibitor (Figures 3 and 4B). Furthermore, at incubation time 0 minutes, the addition of an RNase inhibitor increased the yield of edited T cells by 1.5 times (Figures 4B and 3).

[0516] Therefore, RNase inhibitors consistently increased KI (%) and the yield of edited cells in all donor cells tested. The addition of RNase inhibitors improved the yield of edited cells at all time points (Figures 4A and 4B).

[0517] The addition of RNase inhibitors did not adversely affect the cellular phenotype (Figures 5A and 5B). In six experiments using 13 different donors, the addition of RNase inhibitors did not alter the mean proportion of "Young memory" T cells (stem cell memory T cells / CD45RA+CCR7+ and central memory T cells / CD45RA-CCR7+) (Figure 5A) or the ratio of CD4 T cells to CD8 T cells (Figure 5B). The p-values ​​were obtained from two-sided matched t-tests.

[0518] Therefore, although we do not wish to be bound by theory, the addition of an RNase inhibitor improved gene editing performance compared to all conditions without the RNase inhibitor.

[0519] Example 2: Increased editing efficiency with N-acetyl-L-cysteine ​​(NAC) Materials and methods T cell activation Human primary T cells isolated by CD4 / CD8 were removed from liquid nitrogen and thawed in 1 ml of warmed medium (TexMACS + 10% human AB serum) per 1 ml frozen vial. All cells from the same donor were pooled and transferred to 50 ml tubes containing warmed medium (10 ml per frozen vial). The cells were centrifuged at 400 g for 5 minutes. After centrifugation, 400e6 cells were cultured in 200 ml total hTCM (TexMAC + 3% human AB serum + 12.5 ng / mL IL-7 + 12.5 ng / mL IL-15) in a Grex 100M container containing CTS dynabeads CD3 / CD28 in a 3:1 bead-to-cell ratio. After 2 days, the cells were debeaded and subjected to electroporation.

[0520] Lonza Electroporation T cells were thawed and activated as described above. RNPs were complexed at room temperature for 10 minutes by mixing target sgRNA and Cas9 in a 5:1 ratio. Lonza P3 buffer was added to each RNP complex to prepare an electroporation (EP) reaction mixture. 1 mg / ml of DNA (a GFP-expressing HR repair template) was diluted with Lonza P3 buffer.

[0521] To prepare neutralized NAC, powdered NAC (Sigma, A7250-5G) was dissolved in 50 ml of PBS. The NAC was neutralized by adding 1 ml of 10 M NaOH to 16.3 ml of NAC in a 1:1 molar ratio, resulting in a final stock concentration of 578.035 mM. This solution was filtered sterile. For electroporation, the stock concentration of neutralized NAC was diluted to 50 mM with P3 buffer. A 96-well Lumox plate was used for recovery. 250 μl of hTCM containing and without 5 mM NAC was added to each well and incubated at 37°C. After debedding the cells, 1 e6 cells per reaction were centrifuged at 90 g for 10 minutes, and the supernatant was removed. For NAC conditions, the cell pellet was resuspended in P3 buffer, neutralized NAC, RNP, and DNA. The reaction concentration of NAC was 5 mM. For the control condition without NAC, the cell pellet was resuspended in P3 buffer, RNP, and DNA. Cells were electroporated using a Lonza 384HT equipped with an EH115 pulse code.

[0522] After electroporation, cells were first recovered in the electroporation plate at room temperature for 15 minutes, and then recovered in 96-well Lumox plates in hTCM with and without 5 mM NAC. Cells were divided on day 1 and day 3 post-electroporation and fresh medium with and without 5 mM NAC was added. Five days after electroporation, a portion of the cells were collected for knock-in (KI) efficiency analysis via flow cytometry. Approximately 2e5 cells were collected per sample for flow analysis. Cells were resuspended in BSA staining buffer, CountBright counting beads, and TO-PRO-3 and stained at 4°C for 30 minutes. Cells were washed twice and resuspended in wash buffer volume for analysis. Samples were analyzed by Attune for GFP (indicating knock-in success) and TO-PRO-3 (indicating unviability).

[0523] Large-scale electroporation using Xenon T cells were thawed and activated as described above. After activating the cells for 2 days, they were debeaded before electroporation. 25e6 cells were used per reaction. RNPs were complexed with target sgRNA and Cas9 in a 4:1 ratio at room temperature for 10 minutes. The neutralized NAC stock was diluted to a final concentration of 50 mM in Xenon GE buffer. Grex 6M plates were prepared by adding hTCM to each well and warmed at 37°C. The cells were centrifuged, PBS was added to the cell pellet, and the cells were centrifuged at 400 g for 5 minutes. For the NAC condition, the cell pellet was resuspended in Thermo GE buffer containing RNPs and DNA (GFP-expressing HR repair template) and 5 mM NAC. For the control condition without NAC, the cell pellet was resuspended in Thermo GE buffer containing RNPs and DNA. For each replication, the cell / payload mixture was transferred to a Xenon Single Shot cartridge, and the cells were electroporated in a Thermo Xenon unit at 2300V, 4 pulses, and 3 ms. After electroporation, the cells were allowed to recover in the cartridge at room temperature for 10 minutes. The cells were transferred to a prepared plate containing hTCM with or without 5 mM NAC (final concentration). One day after electroporation, hTCM with or without 5 mM NAC was added to each appropriate well. Three days after electroporation, fresh hTCM with or without 5 mM NAC was added to the cells. Five days after electroporation, the cells were stained and analyzed for KI efficiency using Attune with flow cytometry staining as described above.

[0524] Comparison of NAC Ultrasound Cell editing efficiency and yield using 2.5 mM NAC and 5 mM NAC were also compared. Cells were electroporated using the Xenon protocol described above, and five days after electroporation, electroporation buffer containing either 2.5 mM NAC or 5 mM NAC additive was used.

[0525] result Figure 6A shows the multiplicative change in the total yield of edited cells in NAC-treated samples, normalized to the drug-free control after the Lonza electroporation assay. Cells from five donors treated with 5 mM neutralized NAC during electroporation showed an improved total yield of edited cells (2-6 times improvement) in Lonza small-scale electroporation (Figure 6A). Furthermore, no or minimal change in KI efficiency was observed in NAC-treated samples compared to the control without NAC (Figure 6B). Therefore, the addition of NAC during electroporation increased the yield of edited cells and did not adversely affect the knock-in efficiency of the transgene.

[0526] Figure 7A shows the multiplicative change in the total yield of edited cells in NAC-treated samples, normalized to the drug-free control after the Xenon electroporation assay. Including 5 mM NAC during electroporation doubled the yield of edited cells in three donors. Figure 7C shows the normalized number of edited cells at day 7, normalized to the cell input at day 2. Figure 7B shows the KI rate (%) of the transgene in cells incubated with NAC during electroporation. Compared to the control without NAC, no change or minimal change in KI efficiency was observed in the NAC-treated samples (Figure 7B). Therefore, the addition of NAC during electroporation increased the yield of edited cells and did not adversely affect the knock-in efficiency of the transgene.

[0527] Figure 8A shows the KI rate (%) of target genes after electroporation with 2.5 mM or 5 mM NAC. Figure 8B shows the total number of edited cells after electroporation with 2.5 mM or 5 mM NAC. In the cells of seven donors, no significant difference was observed in the total yield of edited cells with 2.5 mM NAC compared to 5 mM neutralizing NAC buffer. However, cells electroporated in the presence of 2.5 mM neutralizing NAC showed higher KI efficiency in five out of seven donor cells compared to the 5 mM NAC sample.

[0528] An additional assay comparing 5 mM and 2.5 mM NAC in both Cellvive and PLTGold media also showed favorable results under the 2.5 mM NAC condition (data not shown).

[0529] While we do not wish to be bound by theory, applying an electric field during the electroporation process to create cell membrane permeability can induce the production of reactive oxygen species (ROS), potentially leading to damage to the plasmid membrane. If cells are unable to repair the cell membrane, cell death may occur after increased ROS levels, ATP depletion, and calcium influx. Electroporation itself also generates ROS through charge transfer reactions at the electrodes. Therefore, while we do not wish to be bound by theory, cell viability may be increased during electroporation by protecting cells from ROS-mediated cell death caused by cell membrane damage by including ROS scavengers such as N-acetyl-L-cysteine ​​(NAC).

[0530] Example 3: Increased editing efficiency with sorbitol Materials and methods The cells were removed from liquid nitrogen and thawed two days before electroporation. After adding thawing medium (TexMACS + 10% human AB serum) to the cells, the donor cells were pooled and centrifuged at 400g for 4 minutes to form a pellet. The cells were then added to GRex100M in hTCM (TexMACS + 3% human AB serum + 12.5ng / mL IL-7 + 12.5ng / mL IL-15) containing 15ug / mL gentamicin and three times the number of cells as CTS Dynabeads CD3 / CD28.

[0531] Two days later, the cells were debeaded and washed with PBS. The RNPs were complexed at room temperature for 10 minutes by mixing target sgRNA and Cas9 in a 4:1 ratio. The cell pellets were then resuspended in either standard Xenon electroporation (EP) buffer or Xenon electroporation buffer containing 200 mM sterile filtered sorbitol. Next, the plasmid containing the transgene (a GFP-expressing HDR template) was added to the cells in the electroporation buffer containing the RNPs. The mixture was then electroporated in a Xenon electroporator at 2300V, 4 pulses, 3 ms, and 0.5 seconds between pulses. The cells were added to fresh hTCM in a GRex 6-well M plate and returned to the incubator. One day after electroporation, fresh hTCM was added to each sample. Three days after electroporation, the samples were divided and resuspended in fresh hTCM. On day 5, cells were collected for flow cytometry using CountBright counting beads and TO-PRO-3. For GFP and TO-PRO-3 viability staining, the samples were measured using an Attune flow cytometer.

[0532] result Figure 9A shows the multiplicative change in the total number of edited cells normalized to the starting cell number during electroporation in the presence or absence (0mM) of sorbitol. Figure 9B shows the KI editing efficiency (%) in the presence or absence (0mM) of sorbitol. Addition of 200mM sorbitol to the Xenon electroporation buffer increased the yield of edited cells by 2.02 ± 0.79 times in cells from five donors. 200mM sorbitol in the Xenon electroporation buffer improved the yield of edited cells in all donors tested, increasing the editing effect from 27% to 224% (Figure 9A). The effect of sorbitol on the knock-in rate was minimal (Figure 9B). Overall, adding sorbitol to the electroporation buffer improved the yield of viable cells after electroporation, minimized differences in editing efficiency (KI%), and increased the total number of edited cells.

[0533] While we do not wish to be bound by theory, during electroporation, pores are introduced into the cell membrane that allow external ions to enter the cell. Such pores can also enlarge further, becoming irreparable tears. Both external ions and tears can lead to cell rupture and cell death. Therefore, by adjusting the molar osmotic pressure using buffering additives such as sorbitol or glycerol, the pressure outside the membrane increases, and the compressive force stabilizes these pores, while the outward osmotic pressure decreases, preventing cell rupture.

[0534] Example 4: Exogenous DNA repair regulation and / or increased editing efficiency by homologous recombination proteins Materials and methods The success of transgene integration relies primarily on homologous recombination repair (HDR), a high-fidelity DNA repair mechanism. HDR is in kinetic competition with NHEJ, a low-fidelity process not known to enable the insertion of large transgenes. Existing methods to increase KI efficiency include the use of small molecule inhibitors of NHEJ, modification of the DNA molecule carrying the insertion cassette, and modification of CRISPR-Cas proteins to enhance HDR or inhibit NHEJ. However, none of these methods have proven effective when attempting to insert very large (>8kb) expression cassettes into activated primary human T cells.

[0535] To induce HDR via error-prone DNA repair mechanisms such as non-homologous end joining (NHEJ), we designed a plasmid library consisting of genes involved in HDR DNA repair, NHEJ antagonists, and cell cycle regulation. The genes were screened via electroporation for their efficacy in improving gene editing performance, and three genes were identified that, when expressed from separate, non-integrated, non-replicating plasmids (episome plasmids), improved knock-in (KI) of exemplary transgenes (e.g., heterologous proteins, e.g., priming receptors and / or CARs) in T cells. 84 proteins were tested for increased KI efficacy. Luciferase was used as a control. Proteins were tested with or without nuclear localization signal (NLS) tagging. Proteins were transiently expressed from plasmids with the same common skeletal sequence. The plasmids contained a CMV enhancer, a CAG promoter, a hybrid intron, the target gene, and bGH polyA. The plasmid was delivered along with an insertion cassette containing a DNA template and Cas9 RNPs. The plasmid was non-integrated and non-replicating. Modulation of editing efficiency typically relies on small molecule inhibitors, which can have unintended off-target effects. This design emphasizes transient expression peaking within 24 hours, capturing the HDR time window.

[0536] To test plasmids on a 96-well scale, fill each well with 1 × 10⁶ electroporation buffer. 6 Each well contained activated T cells, a Cas9 RNP targeting a safe harbor locus, and plasmids encoding exemplary CAR and GFP markers flanked by 5' and 3' homologous sequences for insertion. The insertion cassette was 9kb long. Plasmids encoding potential HDR-enhancing genes were added to individual wells in array form. Cells were electroporated using an in-house developed system. Cells were transferred to human T cell medium and expanded for 5 days. Editing efficiency in each well was calculated using flow cytometry.

[0537] Plasmids with improved editing efficiency were used in larger-scale trials.

[0538] For larger-scale follow-up studies using a Xenon electroporator, 50 × 10 6 Individual activated T cells were electroporated using the manufacturer's recommended protocol. Cells were administered 20ug of plasmid encoding a Cas9 RNP targeting the safe harbor locus, as well as exemplary CAR and GFP markers flanked by 5' and 3' homologous sequences for insertion. The insertion cassette length was 9kb. Each independent sample also contained 20ug of one plasmid encoding a gene that enhances HDR, selected from a 96-well scale experiment. Cells were transferred to human T cell medium and expanded for 5 days. Editing efficiency in each well was calculated using flow cytometry.

[0539] result This screening method identified three genes—SWSAP1, RAD51 paralog, dominant-negative KU80, and AcrIIA8-CDT1—that improve CAR and GFP marker knock-in (KI) in T cells (Figures 10A and 10B). Figure 10A shows the correlation of KI efficiency between two donor cell lines electroporated with plasmids transiently expressing the indicated proteins. Figure 10B shows the correlation of KI efficiency between two donor cell lines electroporated with plasmids transiently expressing the indicated proteins. Luciferase was used as a control. NLS tags are appended where applicable. Two anti-CRISPR candidate proteins, AcrIIA8 and AcrIIA10, showed improved KI compared to other anti-CRISPRs, AcrII4 and AcrII5. Figure 11A shows a bar graph of KI efficiency (%) between cells from two donors using a 9kb plasmid containing a GFP reporter. Figure 11B shows a bar graph of KI efficiency (%) between cells from two donors using a 9kb plasmid containing an exemplary myc-tagged CAR. In both cases, a control plasmid encoding a luciferase reporter gene (luc) was also used. As shown in both Figures 11A and 11B, the addition of small, non-replicating, non-integrated plasmids encoding i53, dominant-negative (DN) KU80, SWSAP1, or AcrIIA8 improved GFP or exemplary CAR gene knock-in (KI) efficiency.

[0540] It was previously unknown that SWSAP1 expression affects the integration performance of HDR transgenes, and therefore, the improved KI efficacy in cells expressing SWSAP1 was unexpected. Dominant-negative KU80 is a fragment of KU80 that inhibits the formation of the KU70 / KU80 complex involved in NHEJ, and AcrIIA8-CDT1 is a fusion of Cas9 anti-CRISPR (AcrIIA8) and a CDT1 fragment. AcrIIA8-CDT1 acts as a degron specific to the S and G2 cell cycles where HDR occurs. When anti-CRISPR is degraded, it releases Cas9 RNPs, which are cleaved at the appropriate cell cycle stage. While we do not wish to be constrained by theory, these three genes may improve KI efficiency by increasing HDR of target genes by biasing cellular mechanisms toward homologous recombination.

[0541] Example 5: Increased editing efficiency by histone deacetylase inhibition The integration of transgenes by the CRISPR-Cas9 platform is inefficient, with the correct transgene being inserted into only a portion of the input cells. This inefficiency worsens as the size of the gene insertion increases. This leads to problems for several reasons. The majority of cells obtained from the process may not have the inserted gene and may not function as intended, and the absolute number of cells that have the inserted gene and can function as intended may be small. These can cause clinical problems because they require much more cell injection to obtain the intended dose of edited cells, thereby potentially increasing the risk of complications. Furthermore, this can also cause difficulties in producing a sufficient number of edited cells to meet the dose requirements. This embodiment demonstrates how to improve the electroporation (EP)-mediated CRISPR-Cas9 gene editing process by inhibiting histone deacetylases (HDACs) with various compounds to increase knock-in efficiency and the yield of edited cells from the same input. Several different HDAC inhibitors may be effective for these purposes.

[0542] Materials and methods Newly isolated or freeze-thawed isolated T cells were activated for 2 days at 37°C and 5% CO2 in GRex culture flasks (Wilson Wolf, RU81100) containing complete medium (TexMACS (Miltenyi Biotec, 170-076-306) + 3% CellVive (Biolegend, 420502) or human serum (GeminiBio, 100512) + 12.5 ng / mL IL-7 + 12.5 ng / mL IL-15) in a bead:cell ratio of 3:1 or 2:1, using CD3 / CD28 Dynabeads (Thermo Fisher Scientific, 43500D or 40203D), or in complete medium containing 0.1 mM sodium phenylbutyrate. Next, the beads were removed, the cells were pelleted, resuspended in genome editing buffer (Thermo Fisher Scientific, A4998002 or A4998001), then plasmid DNA and ribonucleoprotein (RNP) were added to the cells, and the cells were electroporated using a CTS Xenon Electroporation System (Thermo Fisher Scientific) with a Xenon SingleShot (Thermo Fisher Scientific, A50305). After electroporation, the cells were transferred to a GRex plate (Wilson Wollf, 80660MS) containing complete medium or complete medium containing an HDAC inhibitor (0.5 mM, 1 mM, 2 mM, or 4 mM sodium phenylbutyrate, 0.016 μM xynostat, or 0.0375 μM panobinostat) or other inhibitory compound, at a volume of 1 / 10 of the container's filled volume. One day after electroporation, the complete medium was added to the full volume. Next, cells were sampled for cell count and flow cytometry, and the knock-in rate was measured 4–7 days after electroporation.

[0543] result As shown in Figure 12, incubating T cells after electroporation in media containing 0.5 mM, 1 mM, 2 mM, and 4 mM sodium phenylbutyrate, and 1% DMSO, resulted in increased knock-in (KI) efficiency compared to media without sodium phenylbutyrate. The 1 mM sodium phenylbutyrate condition yielded the best KI (%).

[0544] Sodium phenylbutyrate can also be used as a pretreatment, in which case a concentration of 0.1 mM is added to the culture medium two days before EP. This concentration and treatment duration increased the knock-in (KI%) of the transgene compared to standard recovery without sodium phenylbutyrate (Figure 13). This effect has been shown to be additive to the effect of the recovery treatment, and a better improvement in knock-in can be obtained by combining both pre- and post-treatment with sodium phenylbutyrate than by using either treatment alone.

[0545] As shown in Figure 14, incubation of T cells with 0.016 μM xynostat and 0.0375 μM panobinostat after electroporation also increased knock-in (KI) efficiency compared to medium without either xynostat or panobinostat (Figure 14). The addition of 1% DMSO did not affect the KI (%) of the transgene. Interestingly, novobiosin did not show an improvement in KI (%) in the electroporation-based knock-in method compared to standard electroporation.

[0546] In summary, sodium phenylbutyrate, xynostat, and panobinostat improved CRISPR-edited knock-in of transgenes after electroporation at concentrations that maintained high yields of edited cells (e.g., no loss of T cell output) while producing low toxicity. Furthermore, sodium phenylbutyrate showed an average improvement of 67% in KI (%) efficiency and can be used as both an electroporation pretreatment or posttreatment across multiple electroporation platforms.

[0547] A selection of additional inhibitor compounds also increased the KI (%) of the transgene. For example, ATR inhibitors such as VE-822 can substantially increase knock-in. Other compounds that improved editing efficiency included the PARP-1 inhibitor AG14361, the Polθ inhibitor ART-558, and the CHK inhibitor ADAD7762. DNA-Pk inhibitors such as KU0600648 and NU7026 also showed improved editing efficiency (Figure 15). Furthermore, many compounds previously identified as improving knock-in, or theoretically capable of promoting efficient gene insertion by suppressing non-homologous end joining, did not show improvement.

[0548] Example 6: Increased editing efficiency by increasing the electroporation pulses Materials and methods Development of 5-pulse processes Standard protocol: Freshly isolated or freeze-thawed isolated T cells were activated for 2 days at 37°C and 5% CO2 in complete medium (TexMACS + Xenofree Supplement B or Xenofree Supplement A) with CD3 / CD28 Dynabeads (Thermo Fisher Scientific, 43500D or 40203D). The beads were then removed, and the cells were 100 × 10⁶. 6 The cells were pelleted, RNP and DNA were added, and then resuspended in 170 mM glycerol, 2.5 mM NAC buffer, and gene editing buffer using a final volume of 1 mL of electroporation reaction mixture. The cells were then incubated at room temperature for 10 minutes. Next, 40 ug of plasmid DNA expressing an exemplary transgene (e.g., a heterologous protein such as a priming receptor and / or CAR) and ribonucleoprotein (RNP) were added to the cells. The cells were electroporated at 2300 V for 4 pulses at 3 ms per pulse.

[0549] 5-Pulse Protocol: Freshly isolated or freeze-thawed isolated T cells were activated for 2 days at 37°C and 5% CO2 in complete medium (TexMACS + Xeno-Free Supplement B or Xeno-Free Supplement A) with CD3 / CD28 Dynabeads (Thermo Fisher Scientific, 43500D or 40203D). The beads were then removed, the cells were pelleted, and after adding RNP and DNA, they were resuspended in 170 mM glycerol, 2.5 mM NAC buffer, and gene editing buffer using a final volume of 1 mL of electroporation reaction mixture. The cells were then incubated at room temperature for 10 minutes. Next, 20 ug of plasmid DNA expressing an exemplary transgene (e.g., a heterologous protein such as a priming receptor and / or CAR) and ribonucleoprotein (RNP) were added to the cells. The cells were electroporated at 2300 V for 5 pulses, 3 ms per pulse.

[0550] The DNA insertion cassette in the DNA plasmid was 8.2 kb. Nanoplasmids with a shorter skeleton length compared to the whole plasmid skeleton were also generated and used in the electroporation protocol. RNP without DNA was used as a negative control.

[0551] Functional assay On day 9, edited T cells derived from two donors, generated from either the standard protocol or the 5-pulse protocol, were repeatedly tested at E:T cell ratios of 1:50 or 1:100 using tumor cells expressing analogous ligands of exemplary transgene constructs (e.g., priming receptors and / or CARs). The number of target viable cells after incubation with T cells was assessed.

[0552] Cell expansion assay Cells derived from five different donors were electroporated with an exemplary transgene construct (e.g., CAR) at a number of inputs of 100M using 4 and 5 pulse conditions. Cells were counted on days 7 and 9. Both KI (%) and the total number of edited cells were collected and graphed.

[0553] result Figure 16A shows the total number of knock-in (KI) (%) and edited T cells from seven different donors expressing exemplary transgenes(s). The 4-pulse sample is shown in the left bar, and the 5-pulse sample in the right bar. Figure 16B shows the total number of knock-in (KI) (%) and edited T cells from five different donors expressing exemplary transgenes(s). The 4-pulse sample is shown in the left bar, and the 5-pulse sample in the right bar. The 5-pulse process showed increased editing efficiency compared to the 4-pulse process for all conditions tested with respect to KI (%), and yielded a total number of edited cells equal to or greater than that of the 4-pulse process. Therefore, the 5-pulse process resulted in improved KI (%) and a higher total number of edited cells. Furthermore, the electroporation protocol using 5 pulses (20 ug) required less DNA template compared to the standard protocol using 4 pulses (40 ug). Therefore, the 5-pulse method can improve KI (%) and increase the total number of edited cells while reducing the amount of starting DNA template.

[0554] Functional data of T cells showed comparable antitumor activity across all pulse conditions tested. Figure 17A shows the number of tumor target cells after incubation with edited T cells at an E:T ratio of 1:50. Figure 17B shows the number of tumor target cells after incubation with edited T cells at an E:T ratio of 1:100. T cells from a second donor were also tested and showed the same results (data not shown). In both figures, T cells were electroporated with either 4 or 5 pulses. As shown in Figures 17A and 17B, throughout the 18-day time-course assay, T cells edited under the 5-pulse condition showed similar tumor clearance (e.g., antitumor effect) compared to T cells edited under the 4-pulse condition. Therefore, there was no functional difference between the T cell function under the 4-pulse and 5-pulse conditions, and the additional pulses in the 5-pulse protocol did not adversely affect the function of T cells generated under the 5-pulse condition.

[0555] As shown in Figure 18, in T cells derived from five donors, KI (%) and cell expansion (TEC) on days 7 and 9 after electroporation were increased in the 5-pulse protocol compared to the 4-pulse protocol. The upper line of each bar represents the 5-pulse condition, and the lower line represents the 4-pulse condition.

[0556] KI (%) and the total number of edited cells in the 5-pulse protocol were evaluated using different DNA sizes (nanoplasmids and standard plasmids) compared to the 3-pulse and 4-pulse protocols. As shown in Figures 19A and 19B, the 5-pulse method (center bar) showed increased KI (%) and the total number of edited T cells compared to the 4-pulse method (left bar) and the 3-pulse method (right bar).

[0557] The 5-pulse protocol also increased the transgene KI efficiency of a second exemplary transgene that was inductively expressed. As shown in Figures 20A and 20B, the 5-pulse protocol resulted in a nearly twofold increase in KI in T cells derived from four donors incubated with xenofree medium supplements A and B. Therefore, constitutive expression of the transgene was not required for the KI improvement observed in the 5-pulse process.

[0558] Table 2 shows the number of T cells at the start, intermediate process, and end of an exemplary T cell editing assay. "Intermediate range," "sufficient supply," and "insufficient supply" refer to the number of T cells initially isolated from the donor sample via leukocyte removal (e.g., T cells obtained from Leukopak). Samples in the "insufficient supply" and "intermediate range" categories yield fewer T cells as initiating material compared to samples in the "sufficient supply" category. [Table 2]

[0559] As shown in Table 2, if starting from a lower predicted T cell count ("intermediate range") derived from leukocyte removal, 100 × 10 8 Even when using T cell input, the number of input T cells may be insufficient to perform electroporation. 50 × 10⁶ under 4 pulse conditions 6 Performing electroporation using a T cell input (insufficient supply) is unlikely to yield a sufficient number of edited cells to meet the requirements of an in vivo trial. This limitation can be overcome by using a 5-pulse protocol, which increases the number of edited T cells compared to a 4-pulse protocol. Therefore, the 5-pulse protocol has an improved ability to meet the T cell dose requirements for an in vivo trial.

[0560] Overall, the five-pulse process improved KI(%) and the total number of edited cells in all donors tested. This data was reproducible in multiple assays. Cell function testing showed no significant difference in the performance of electroporated cells at either the 1:50 or 1:100 E:T ratio. ROS is generated during electroporation and leads to increased toxicity associated with the electroporation process. While we do not wish to be bound by theory, the incorporation of NAC, along with other optimizations described herein, improved the ROS scavenger and allowed for additional pulses in the electroporation program. Thus, the fifth electroporation pulse improved KI(%) and the total number of edited cells.

[0561] While the present invention has been particularly shown and described with respect to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made therewith without departing from the spirit and scope of the invention.

[0562] All references, published patents, and patent applications cited herein are incorporated herein by reference in their entirety for any purpose.

Claims

1. A composition comprising a solution containing an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

2. A composition comprising a solution containing N-acetyl-L-cysteine ​​(NAC), a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

3. A composition comprising a solution containing an osmotic pressure regulator, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to an insertion site in the cell's genome.

4. A composition comprising a solution containing a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), a histone deacetylase (HDAC) inhibitor, and / or an osmotic regulator, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

5. The solution is at least a. The N-acetyl-L-cysteine ​​(NAC) and the osmotic pressure regulator, b. The RNase inhibitor and the N-acetyl-L-cysteine ​​(NAC), c. The RNase inhibitor and the osmotic pressure regulator, d. The N-acetyl-L-cysteine ​​(NAC) and the histone deacetylase (HDAC) inhibitors, e. The osmotic pressure regulator and the histone deacetylase (HDAC) inhibitor, f. The RNase inhibitor and the histone deacetylase (HDAC) inhibitor, g. The RNase inhibitor, the N-acetyl-L-cysteine ​​(NAC), and the histone deacetylase (HDAC) inhibitor, h. The N-acetyl-L-cysteine ​​(NAC), the osmotic pressure regulator, and the histone deacetylase (HDAC) inhibitor, i. The RNase inhibitor, the osmotic pressure regulator, and the histone deacetylase (HDAC) inhibitor, j. The RNase inhibitor, the N-acetyl-L-cysteine ​​(NAC), and the osmotic pressure regulator, or k. The RNase inhibitor, the N-acetyl-L-cysteine ​​(NAC), the osmotic pressure regulator, and the histone deacetylase (HDAC) inhibitor The composition according to claim 4, comprising:

6. The composition according to claim 1 or any one of claims 4 to 5, wherein the RNase inhibitor is present in the solution at a final concentration of about 0.5 to 2 U / μl.

7. The composition according to claim 1, or any one of claims 4 to 6, wherein the RNase inhibitor is present in the solution at a final concentration of about 1 U / μl.

8. The composition according to claim 1 or any one of claims 4 to 7, wherein the RNase inhibitor is an RNase A, B, C, T1, or T2 inhibitor.

9. The composition according to claim 1 or any one of claims 4 to 8, wherein the RNase inhibitor is a mouse, rat, or human RNase inhibitor.

10. The composition according to claim 2, or any one of claims 4 to 5, wherein the N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of about 1 to 10 mM.

11. The composition according to any one of claims 2, 4-5, or 10, wherein the N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of about 2.5 mM.

12. The composition according to any one of claims 3 to 5, wherein the osmotic pressure regulator is sorbitol, glycerol, or glycine.

13. The composition according to claim 12, wherein the osmotic pressure regulator is sorbitol.

14. The composition according to claim 12 or 13, wherein the sorbitol is present in the solution at a final concentration of about 100 to 400 mM.

15. The composition according to claims 12 to 14, wherein the sorbitol is present in the solution at a final concentration of about 190 mM or 200 mM.

16. The composition according to claim 12, wherein the osmotic pressure regulator is glycerol.

17. The composition according to claim 12 or 16, wherein the glycerol is present in the solution at a final concentration of about 100 to 400 mM.

18. The composition according to claim 12, 16, or 17, wherein the glycerol is present in the solution at a final concentration of about 170 mM.

19. The composition according to any one of claims 4 to 5, wherein the histone deacetylase (HDAC) inhibitor is an HDAC1 or HDAC2 inhibitor.

20. The aforementioned HDAC inhibitors include sodium phenylbutyrate, xinostat, panobinostat, phenylbutyrate, curcumin, mosetinostat, romidespin, SIS17, spritomycin, trichostatin-A, tusidinostat, etinostat, sodium butyrate, valproic acid, CXD101, KT-531, ITF3756, tubastatin A, vorinostat, BML-210, bellinostat, abexinostat, dasinostat, CUDC-101, droxinostat, MC1568, prasinostat, divalproex sodium, PCI-3405, SR-4370, zibinostat, tubasin, AR-42, (-)-parthenolide, resminostat, fimepinostat, M344, Tasejinarin, Sinapic acid, Biphenyl-4-Sulfonyl chloride, Sulforaphane, UF010, Suberohydroxamic acid, NKL22, TC-H106, RGFP966, HPOB, RG2833, TMP269, Nexturastat A, Domatinostat, LMK-235, Santa Cruz Amate A, CAY10603, Tascinimod The composition according to claim 19, wherein the composition is BG45, BRD73954, licorinostat, scriptide, sitalinostat, WT161, TMP195, ACY-738, SKLB-23bb, tinostamstin, TH34, BRD3308, radianine A, isoguanosine, KA2507, ITSA-1, or an RNA interference (RNAi) molecule.

21. The composition according to claim 20, wherein the HDAC inhibitor is sodium phenylbutyrate, xinostat, or panobinostat.

22. A composition comprising a cell containing at least one heterologous DNA template inserted into a target region of the genome, and a solution comprising at least one histone deacetylase (HDAC) inhibitor, comprising sodium phenylbutyrate, xinostat, or panobinostat.

23. The composition according to any one of claims 20 to 22, wherein the sodium phenylbutyrate is present in the solution at a final concentration of about 15.6 μM to 4 mM.

24. The composition according to claims 20 to 23, wherein the sodium phenylbutyrate is present in the solution at a final concentration of about 1 mM, and optionally the solution contains 1% DMSO.

25. The composition according to any one of claims 20 to 22, wherein the xynostat is present in the solution at a final concentration of about 8 nM to 200 nM.

26. The composition according to any one of claims 20 to 22 or 25, wherein the xynostat is present in the solution at a final concentration of about 16 nM.

27. The composition according to any one of claims 20 to 22, wherein the panobinostat is present in the solution at a final concentration of about 3 nM to 1 μM.

28. The composition according to any one of claims 20 to 22 or 27, wherein the panobinostat is present in the solution at a final concentration of about 37.5 nM.

29. The composition according to any one of claims 4 to 28, wherein the solution comprises at least one of the following: an RNase inhibitor at a final concentration of about 0.5 to 2 U / μl, a NAC at a final concentration of about 1 to 10 mM, sorbitol at a final concentration of about 100 to 400 mM, and / or sodium phenylbutyrate at a final concentration of about 15.6 μM to 4 mM, xynostat at 8 nM to 200 nM, or panobinostat at 3 nM to 1 μM.

30. The composition according to claim 29, wherein the solution comprises at least one of the following: an RNase inhibitor at a final concentration of about 1 U / μl, NAC at a final concentration of about 2.5 mM, sorbitol at a final concentration of about 200 mM, and / or sodium phenylbutyrate at a final concentration of about 1 mM, 0.016 μM xynostat, or 0.0375 μM panobinostat.

31. The composition according to any one of claims 1 to 30, wherein the nuclease domain comprises a CRISPR-related endonuclease (Cas), optionally a Cas9 nuclease.

32. The composition according to any one of claims 1 to 31, wherein the size of the DNA template is 300 nucleotides or more.

33. The sizes of the DNA templates are approximately 0.3kb, 0.5kb, 1.0kb, 1.5kb, 2.0kb, 2.5kb, 3.0kb, 3.5kb, 4.0kb, 4.5kb, 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6.2kb, 6.3kb, and 6.4kb. b, 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8 kb, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9kb, 9.0kb, 9.1kb, 9.2 kb, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, 10.0kb, 10.1kb, 10.2kb, 10.3kb, 10.4kb, 10. 5kb, 10.6kb, 10.7kb, 10.8kb, 10.9kb, 11.0kb, 11.1kb, 11.2kb, 11.3kb, 11.4kb, 11.5kb, 11.6kb, 11 The composition according to any one of claims 1 to 32, wherein the DNA template is 7kb, 11.8kb, 11.9kb, 12.0kb, 12.1kb, 12.2kb, 12.3kb, 12.4kb, 12.5kb, 12.6kb, 12.7kb, 12.8kb, 12.9kb, 13.0kb or larger, or any size between these sizes.

34. The sizes of the DNA templates are approximately 0.3kb to 13kb, 0.3kb to 0.5kb, 0.3kb to 1kb, 0.3kb to 4kb, 0.3kb to 3kb, 0.3kb to 5kb, 0.3kb to 7kb, 0.3kb to 10kb, 0.5kb to 1kb, 0.5kb to 3kb, 0.5kb to 5kb, 0.5kb to 7kb, 0.5kb to 10kb, 0.5kb to 13kb, 1kb to 3kb, 1kb to 5kb, 1kb to 7kb, 1kb to 10kb, 1kb to 13kb, and 5kb. The composition according to any one of claims 1 to 33, wherein b is approximately 13kb, approximately 5kb to approximately 9kb, approximately 5kb to approximately 8kb, approximately 5kb to approximately 7kb, approximately 5kb to approximately 6kb, approximately 6kb to approximately 13kb, approximately 6kb to approximately 10kb, approximately 6kb to approximately 9kb, approximately 6kb to approximately 8kb, approximately 6kb to approximately 7kb, approximately 7kb to approximately 13kb, approximately 7kb to approximately 10kb, approximately 7kb to approximately 9kb, approximately 7kb to approximately 8kb, approximately 8kb to approximately 13kb, approximately 8kb to approximately 10kb, approximately 9kb to approximately 9kb, approximately 9kb to approximately 13kb, approximately 9kb to approximately 10kb, approximately 10kb to approximately 13kb, or approximately 11kb to approximately 13kb.

35. The composition according to any one of claims 1 to 34, further comprising a cell containing a genomic sequence adjacent to the insertion site in the genome of the cell.

36. The composition according to claim 35, wherein the cells are mammalian cells, human cells, hematopoietic cells, immune cells, primary immune cells, or primary human immune cells.

37. The composition according to claim 36, wherein the cells are primary human immune cells.

38. The composition according to any one of claims 36 to 37, wherein the immune cells are natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, primary T cells, or T cell progenitor cells.

39. The composition according to any one of claims 36 to 38, wherein the immune cells are primary T cells.

40. The composition according to any one of claims 36 to 39, wherein the immune cells are primary human T cells.

41. The composition according to any one of claims 36 to 40, wherein the immune cells are undifferentiated.

42. The aforementioned immune cells are CD45RA ＋ and CCR7 ＋ CD45RA ＋ and CCR7 － CD45RA － and CCR7 － , or CD45RA － and CCR7 ＋ The composition according to any one of claims 36 to 41.

43. The composition according to any one of claims 35 to 42, wherein the cells are virus-free.

44. The composition according to any one of claims 35 to 43, wherein the cells comprise an exogenous homologous recombination protein or a DNA repair regulatory protein.

45. The composition according to claim 44, wherein the exogenous homologous recombination protein or DNA repair regulatory protein is encoded on an episomal plasmid or mRNA molecule.

46. The composition according to claim 44 or 45, wherein the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

47. The composition according to claim 46, wherein the SWSAP1 protein comprises the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein comprises the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein comprises the sequence shown in SEQ ID NO:

127.

48. The composition according to any one of claims 35 to 47, further comprising collecting the cells from a patient and introducing the DNA template in vitro or ex vivo.

49. The composition according to any one of claims 1 to 48, wherein the target region of the cell genome is a T cell receptor α constant region (TRAC) gene locus or a genome-safe harbor (GSH).

50. The composition according to claim 49, wherein the safe harbor locus is selected from any one of the integration sites designated by GS94, GS88, GS89, GS90, GS91, GS92, GS93, GS95, GS96, GS97, GS98, GS99, GS100, GS101, GS102, GS103, GS104, GS105, GS106, GS107, GS108, GS109, GS110, GS111, GS112, GS113, GS114, GS115, GS116, GS117, GS118, GS119, or GS120.

51. The composition according to claim 50, wherein the safe harbor gene locus is the GS94 integration site.

52. The aforementioned safe harbor loci are chr11:128340000-128350000, chr10:33130000-33140000, chr10:72290000-72300000, chr11:65425000-65427000 (NEAT1), chr15:92830000-92840000, chr16:11220000 A composition according to any one of claims 1 to 51, selected from -11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, or chr9:7970000-7980000.

53. The composition according to any one of claims 1 to 52, wherein the safe harbor locus is a gene selected from APRT, B2M, CAMPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28.

54. The composition according to any one of claims 1 to 53, comprising one or more gRNAs, each containing one of sequence numbers 1 to 120.

55. After the at least one sequence is inserted into the safe harbor locus, the cell is CD45RA ＋ and CCR7 ＋ The composition according to any one of claims 1 to 54, which is such.

56. The composition according to any one of claims 1 to 55, wherein the DNA template is a double-stranded DNA template or a single-stranded DNA template.

57. The composition according to any one of claims 1 to 56, wherein the DNA template is a linear DNA template or a circular DNA template, and optionally the circular DNA template is a plasmid.

58. The composition according to any one of claims 1 to 57, wherein the DNA template includes heterologous sequences.

59. The composition according to any one of claims 1 to 58, wherein the DNA template comprises a gene.

60. The composition according to any one of claims 1 to 59, wherein the DNA template comprises a priming receptor containing a transcription factor.

61. The composition according to any one of claims 1 to 60, wherein the DNA template comprises a chimeric antigen receptor (CAR).

62. The composition according to any one of claims 1 to 61, wherein the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor.

63. The composition according to any one of claims 1 to 62, wherein the DNA template comprises an inducible promoter operably linked to the chimeric antigen receptor.

64. The composition according to any one of claims 1 to 63, wherein the DNA template further comprises a constitutive promoter operably linked to the priming receptor.

65. The composition according to any one of claims 1 to 64, wherein the DNA template further comprises an inductive promoter operably linked to the chimeric antigen receptor and a constitutive promoter operably linked to the priming receptor.

66. The aforementioned DNA template is positioned from the 5' to the 3' direction. a. The inductive promoter, b. The aforementioned chimeric antigen receptor, c. The aforementioned constitutive promoter, and d. The priming receptor A composition according to any one of claims 1 to 65, comprising:

67. The aforementioned DNA template is positioned from the 5' to the 3' direction. a. The aforementioned constitutive promoter, b. The priming receptor, c. The inductive promoter, and d. The chimeric antigen receptor A composition according to any one of claims 1 to 66, comprising:

68. The priming receptor is located from the N-terminus towards the C-terminus. a. Extracellular antigen-binding domain having binding affinity to the antigen, b. A transmembrane domain containing one or more ligand-induced proteolytic cleavage sites, and c. Intracellular domains containing human or humanized transcription effectors The composition according to any one of claims 60 to 67, wherein when an antigen binds to the extracellular antigen-binding domain, cleavage occurs at the ligand-induced proteolytic cleavage site, thereby releasing the intracellular domain.

69. The composition according to claim 68, wherein the priming receptor further comprises a near-membrane domain (JMD) or a stop transfer sequence (STS) located between the transmembrane domain and the intracellular domain.

70. The composition according to any one of claims 60 to 69, wherein the transcription factor binds to the inducible promoter and induces the expression of the CAR.

71. The aforementioned CAR extends from the N-terminus to the C-terminus. a. Extracellular antigen-binding domain having binding affinity to the antigen, b. Transmembrane domain, c. Intracellular co-stimulatory domains, and d. Intracellular activation domain A composition according to any one of claims 61 to 70, comprising:

72. The composition according to any one of claims 60 to 71, wherein the priming receptor and the CAR bind to different antigens.

73. The composition according to any one of claims 60 to 71, wherein the priming receptor and the CAR bind to the same antigen.

74. A polypeptide containing an AcrIIA8 peptide fused to a CDT1 peptide.

75. The polypeptide according to claim 74, wherein the polypeptide comprises the sequence shown in sequence number 127.

76. Primary immune cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins.

77. The primary immune cell according to claim 76, wherein the exogenous homologous recombination protein or DNA repair regulatory protein is encoded on an episomal plasmid or mRNA molecule.

78. The primary immune cell according to claim 76 or 77, wherein the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

79. The primary immune cell according to claim 78, wherein the SWSAP1 protein contains the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein contains the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein contains the sequence shown in SEQ ID NO:

127.

80. The primary immune cell according to any one of claims 76 to 79, wherein the cell is a human cell, a hematopoietic cell, or a primary human immune cell.

81. The primary immune cell according to claim 80, wherein the immune cell is a natural killer (NK) cell, a T cell, a CD8+ T cell, a CD4+ T cell, a primary T cell, or a T cell progenitor cell.

82. The primary immune cells according to claims 80 to 81, wherein the immune cells are primary T cells.

83. The primary immune cell according to any one of claims 80 to 82, wherein the immune cell is a primary human T cell.

84. The primary immune cells according to any one of claims 80 to 83, wherein the immune cells are undifferentiated.

85. The aforementioned immune cells are CD45RA ＋ and CCR7 ＋ CD45RA ＋ and CCR7 － CD45RA － and CCR7 － , or CD45RA － and CCR7 ＋ The primary immune cell according to any one of claims 80 to 84.

86. The primary immune cells according to any one of claims 76 to 85, wherein the cells are virus-free.

87. The primary immune cell according to any one of claims 76 to 86, wherein the cell includes a DNA template, and the 5' and 3' ends of the DNA template include nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome.

88. The primary immune cell according to claim 87, wherein the target region of the cell's genome is a T cell receptor α constant region (TRAC) gene locus or a genome-safe harbor (GSH).

89. The primary immune cell according to claim 88, wherein the safe harbor locus is selected from any one of the integration sites designated by GS94, GS88, GS89, GS90, GS91, GS92, GS93, GS95, GS96, GS97, GS98, GS99, GS100, GS101, GS102, GS103, GS104, GS105, GS106, GS107, GS108, GS109, GS110, GS111, GS112, GS113, GS114, GS115, GS116, GS117, GS118, GS119, or GS120.

90. The primary immune cell according to claim 89, wherein the safe harbor locus is the GS94 integration site.

91. The aforementioned safe harbor loci are chr11:128340000-128350000, chr10:33130000-33140000, chr10:72290000-72300000, chr11:65425000-65427000 (NEAT1), chr15:92830000-92840000, chr16:1122000 Primary immune cells according to claims 88 to 90, selected from 0-11230000, chr2:87460000-87470000, chr3:186510000-186520000, chr3:59450000-59460000, chr8:127980000-128000000, or chr9:7970000-7980000.

92. The primary immune cell according to claim 88, wherein the safe harbor locus is a gene selected from APRT, B2M, CAMPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28.

93. A primary immune cell according to any one of claims 76 to 92, comprising one or more gRNAs including one of sequence numbers 1 to 120.

94. After the insertion of the at least one sequence into the safe harbor locus, the cell becomes CD45RA ＋ and CCR7 ＋ The primary immune cell according to any one of claims 76 to 93.

95. The primary immune cell according to any one of claims 87 to 94, wherein the DNA template is a double-stranded DNA template or a single-stranded DNA template.

96. The primary immune cell according to any one of claims 87 to 95, wherein the DNA template is a linear DNA template or a circular DNA template, and optionally the circular DNA template is a plasmid.

97. The primary immune cell according to any one of claims 87 to 96, wherein the DNA template includes heterologous sequences.

98. The primary immune cell according to any one of claims 87 to 97, wherein the DNA template includes a gene.

99. The primary immune cell according to any one of claims 87 to 98, wherein the DNA template comprises a priming receptor containing a transcription factor, a chimeric antigen receptor (CAR), or a priming receptor containing a chimeric antigen receptor (CAR) and a transcription factor.

100. The primary immune cell according to any one of claims 87 to 99, wherein the DNA template comprises an inducible promoter operably linked to the chimeric antigen receptor.

101. The primary immune cell according to any one of claims 87 to 100, wherein the DNA template further comprises a constitutive promoter operably linked to the priming receptor.

102. The primary immune cell according to any one of claims 87 to 101, wherein the DNA template further comprises an inductive promoter operably linked to the chimeric antigen receptor and a constitutive promoter operably linked to the priming receptor.

103. The aforementioned DNA template is positioned from the 5' to the 3' direction. a. The inductive promoter, b. The aforementioned chimeric antigen receptor, c. The aforementioned constitutive promoter, and d. The priming receptor Primary immune cells according to any one of claims 87 to 102, including the above.

104. The aforementioned DNA template is positioned from the 5' to the 3' direction. a. The aforementioned constitutive promoter, b. The priming receptor, c. The inductive promoter, and d. The chimeric antigen receptor Primary immune cells according to any one of claims 87 to 102, including the above.

105. The priming receptor is located from the N-terminus towards the C-terminus. a. Extracellular antigen-binding domain having binding affinity to the antigen, b. A transmembrane domain containing one or more ligand-induced proteolytic cleavage sites, and c. Intracellular domains containing human or humanized transcription effectors A primary immune cell according to any one of claims 99 to 104, wherein when an antigen binds to the extracellular antigen-binding domain, cleavage occurs at the ligand-induced proteolytic cleavage site, thereby releasing the intracellular domain.

106. The primary immune cell according to claim 105, wherein the priming receptor further comprises a near-membrane domain (JMD) or a stop transition sequence (STS) located between the transmembrane domain and the intracellular domain.

107. The primary immune cell according to any one of claims 99 to 106, wherein the transcription factor binds to the inducible promoter to induce the expression of the CAR.

108. The aforementioned CAR extends from the N-terminus to the C-terminus. a. Extracellular antigen-binding domain having binding affinity to the antigen, b. Transmembrane domain, c. Intracellular co-stimulatory domains, and d. Intracellular activation domain Primary immune cells according to any one of claims 99 to 107, including the above.

109. The primary immune cell according to any one of claims 99 to 108, wherein the priming receptor and the CAR bind to different antigens.

110. The primary immune cell according to any one of claims 99 to 108, wherein the priming receptor and the CAR bind to the same antigen.

111. A method of cell editing, a. To provide cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins. b. To provide a solution comprising a ribonucleoprotein complex (RNP) and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and c. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

112. A method of cell editing, a. To provide a solution comprising an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and b. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

113. A method of cell editing, a. To provide a solution comprising N-acetyl-L-cysteine ​​(NAC), ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and b. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

114. A method of cell editing, a. To provide a solution comprising an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and b. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

115. A method of cell editing, a. To provide a solution comprising a histone deacetylase (HDAC) inhibitor containing at least one of sodium phenylbutyrate, xinostat, or panobinostat, and cells. b. Contacting the cells with a ribonucleoprotein complex (RNP) and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and c. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

116. The method according to claim 115, comprising contacting the edited cells with a solution comprising a histone deacetylase (HDAC) inhibitor comprising at least one of sodium phenylbutyrate, xinostat, or panobinostat.

117. A method of cell editing, a. To provide cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins. b. To provide a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and c. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

118. A method of cell editing, a. To provide a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and b. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

119. The solution is at least a. The N-acetyl-L-cysteine ​​(NAC) and the osmotic pressure regulator, b. The RNase inhibitor and the N-acetyl-L-cysteine ​​(NAC), c. The RNase inhibitor and the osmotic pressure regulator, d. The N-acetyl-L-cysteine ​​(NAC) and the histone deacetylase (HDAC) inhibitors, e. The osmotic pressure regulator and the histone deacetylase (HDAC) inhibitor, f. The RNase inhibitor and the histone deacetylase (HDAC) inhibitor, g. The RNase inhibitor, the N-acetyl-L-cysteine ​​(NAC), and the histone deacetylase (HDAC) inhibitor, h. The N-acetyl-L-cysteine ​​(NAC), the osmotic pressure regulator, and the histone deacetylase (HDAC) inhibitor, i. The RNase inhibitor, the osmotic pressure regulator, and the histone deacetylase (HDAC) inhibitor, j. The RNase inhibitor, the N-acetyl-L-cysteine ​​(NAC), and the osmotic pressure regulator, or k. The RNase inhibitor, the N-acetyl-L-cysteine ​​(NAC), the osmotic pressure regulator, and the histone deacetylase (HDAC) inhibitor The method according to claim 117 or 118, including the method described in claim 117 or 118.

120. The method according to claim 112 or any one of claims 117 to 119, wherein the RNase inhibitor is present in the solution at a final concentration of about 0.5 to 2 U / μL.

121. The method according to any one of claims 112 or 117 to 120, wherein the RNase inhibitor is present in the solution at a final concentration of about 1 U / μL.

122. The method according to any one of claims 112 or 117 to 121, wherein the RNase inhibitor is an RNase A, B, C, T1, or T2 inhibitor.

123. The method according to claim 112 or any one of claims 117 to 122, wherein the RNase inhibitor is a mouse, rat, or human RNase inhibitor.

124. The method according to any one of claims 113, 117, 118, or 119, wherein the N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of about 1 to 10 mM.

125. The method according to any one of claims 113, 117, 118, 119, or 124, wherein the N-acetyl-L-cysteine ​​(NAC) is present in the solution at a final concentration of about 2.5 mM.

126. The method according to any one of claims 114 to 119, wherein the osmotic pressure regulator is sorbitol, glycerol, or glycine.

127. The method according to claim 126, wherein the osmotic pressure regulator is sorbitol.

128. The method according to claim 126 or 127, wherein the sorbitol is present in the solution at a final concentration of about 100 to 400 mM.

129. The method according to claims 126 to 128, wherein the sorbitol is present in the solution at a final concentration of about 190 mM or 200 mM.

130. The method according to claim 126, wherein the osmotic pressure regulator is glycerol.

131. The method according to claim 126 or 130, wherein the glycerol is present in the solution at a final concentration of about 100 to 400 mM.

132. The method according to claim 126, 130, or 131, wherein the glycerol is present in the solution at a final concentration of about 170 mM.

133. The method according to any one of claims 117 to 119, wherein the histone deacetylase (HDAC) inhibitor is an HDAC1 or HDAC2 inhibitor.

134. The aforementioned HDAC inhibitors include sodium phenylbutyrate, xinostat, panobinostat, phenylbutyrate, curcumin, mosetinostat, romidespin, SIS17, spritomycin, trichostatin-A, tusidinostat, etinostat, sodium butyrate, valproic acid, CXD101, KT-531, ITF3756, tubastatin A, vorinostat, BML-210, bellinostat, abexinostat, dasinostat, CUDC-101, droxinostat, MC1568, prasinostat, divalproex sodium, PCI-3405, SR-4370, zibinostat, tubasin, AR-42, (-)-parthenolide, resminostat, fimepinostat, M 344, Tasejinarin, Sinapic acid, Biphenyl-4-Sulfonyl chloride, Sulforaphane, UF010, Suberohydroxamic acid, NKL22, TC-H106, RGFP966, HPOB, RG2833, TMP269, Nexturastat A, Domatinostat, LMK-235, Santa Cruz Amate A, CAY10603, Tascinimod, B The method according to any one of claims 117 to 119 or 133, wherein the substance is G45, BRD73954, licorinostat, scriptide, sitalinostat, WT161, TMP195, ACY-738, SKLB-23bb, tinostamstin, TH34, BRD3308, radianine A, isoguanosine, KA2507, or ITSA-1.

135. The method according to claim 134, wherein the HDAC inhibitor is sodium phenylbutyrate, xinostat, or panobinostat.

136. The method according to any one of claims 115 to 116, 134, or 135, wherein the sodium phenylbutyrate is present in the solution at a final concentration of about 15.6 μM to 4 mM.

137. The method according to any one of claims 115 to 116 or 134 to 136, wherein the sodium phenylbutyrate is present in the solution at a final concentration of about 1 mM, and optionally the solution contains 1% DMSO.

138. The method according to any one of claims 115 to 116 or 134 to 136, wherein the xynostat is present in the solution at a final concentration of about 8 nM to 200 nM.

139. The method according to any one of claims 115 to 116, 134, or 135, wherein the xynostat is present in the solution at a final concentration of about 16 nM.

140. The method according to any one of claims 115 to 116, 134 to 136, wherein the panobinostat is present in the solution at a final concentration of about 3 nM to 1 μM.

141. The method according to any one of claims 115 to 116, 134, or 135, wherein the panobinostat is present in the solution at a final concentration of about 37.5 nM.

142. The method according to any one of claims 112 to 129, wherein the solution comprises at least one of the following: an RNase inhibitor at a final concentration of about 0.5 to 2 U / μL, a NAC at a final concentration of about 1 to 10 mM, a sorbitol at a final concentration of about 100 to 400 mM, and / or sodium phenylbutyrate at a final concentration of about 15.6 μM to 4 mM, xynostat at 8 nM to 200 nM, or panobinostat at 3 nM to 1 μM.

143. The method according to claim 142, wherein the solution comprises at least one of the following: an RNase inhibitor at a final concentration of about 1 U / μL, NAC at a final concentration of about 2.5 mM, sorbitol at a final concentration of about 200 mM, and / or sodium phenylbutyrate at a final concentration of about 1 mM, 0.016 μM xynostat, or 0.0375 μM panobinostat.

144. The method according to claims 111, 117, or 119-143, wherein the exogenous homologous recombination protein or DNA repair regulatory protein is encoded on an episomal plasmid or mRNA molecule.

145. The method according to claim 111, 117, or 119-144, wherein the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

146. The method according to claim 145, wherein the SWSAP1 protein comprises the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein comprises the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein comprises the sequence shown in SEQ ID NO:

127.

147. The method according to any one of claims 111 to 146, further comprising nonvirally introducing the RNP complex and the DNA template into the cell, wherein the guide RNA specifically hybridizes to a target region of the cell's genome, and the nuclease domain cleaves the target region to create the insertion site in the cell's genome.

148. The method according to any one of claims 111 to 147, wherein the nonviral introduction includes electroporation.

149. The method according to claim 148, wherein the electroporation comprises at least one cycle comprising at least one electrical pulse.

150. The method according to claim 149, wherein the at least one cycle comprises at least five or more electrical pulses.

151. The method according to claim 149 or 150, wherein the electrical pulse is approximately 2300 volts.

152. The method according to any one of claims 149 to 151, wherein the electrical pulse has a duration of about 3.0 ms.

153. The method according to any one of claims 149 to 152, wherein the cycle has a pulse interval of 500 ms.

154. The method according to any one of claims 148 to 153, wherein the electroporation comprises at least one cycle performed using the settings of 2300 volts, a pulse duration of 3.0 ms, five pulses, and a pulse interval of 500 ms.

155. The method according to any one of claims 111 to 154, wherein the exogenous homologous recombination protein or DNA repair regulatory protein increases the insertion of the DNA template into the genome of the cell compared to cells that do not contain the exogenous homologous recombination protein or DNA repair regulatory protein.

156. The method according to any one of claims 112 to 155, wherein the solution comprising at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor increases the insertion of the DNA template into the genome of the cells compared to a control solution not comprising at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor.

157. The method according to any one of claims 115 to 156, comprising incubating the cells with an HDAC inhibitor solution for about two days before nonvirally introducing the RNP complex and DNA template into the cells.

158. The method according to claim 157, wherein the HDAC inhibitor solution contains sodium phenylbutyrate at a final concentration of approximately 0.1 mM.

159. The method according to any one of claims 156 to 158, wherein the insertion of the DNA template into the genome of the cells is increased by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

160. The method according to any one of claims 156 to 158, wherein the insertion of the DNA template into the genome of the cells is increased by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

161. The method according to any one of claims 112 to 160, wherein the solution enhances the growth of the edited cells compared to a control solution that does not contain at least one of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor.

162. The method according to claim 161, wherein the magnification of the edited cells increases by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

163. The method according to claim 161, wherein the magnification of the edited cells is increased by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

164. The method according to any one of claims 112 to 162, wherein the solution increases the yield of edited cells compared to a control solution that does not contain at least one of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, or a histone deacetylase (HDAC) inhibitor.

165. The method according to claim 164, wherein the yield of the edited cells increases by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

166. The method according to claim 164, wherein the yield of the edited cells is increased by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 6, 6.25, 6.5, 6.75, or more compared to the control solution.

167. The method according to any one of claims 147 to 160, wherein the solution comprising at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), or an osmoregulator reduces cell death during the nonviral introduction of the RNP complex and DNA template into the cells, compared to a control solution not comprising at least one of the RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmoregulator, or a histone deacetylase (HDAC) inhibitor.

168. The method according to claim 167, wherein the death of the cells is reduced by 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 300%, or more compared to the control solution.

169. The method according to claim 167, wherein the death of the cells is reduced by at least 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times or more compared to the control solution.

170. The method according to any one of claims 111 to 169, wherein the nuclease domain comprises a CRISPR-related endonuclease (Cas), optionally a Cas9 nuclease.

171. The method according to any one of claims 111 to 170, wherein the size of the DNA template is 5 kilobase nucleotides or more.

172. The sizes of the DNA templates are approximately 0.3kb, 0.5kb, 1.0kb, 1.5kb, 2.0kb, 2.5kb, 3.0kb, 3.5kb, 4.0kb, 4.5kb, 5.0kb, 5.1kb, 5.2kb, 5.3kb, 5.4kb, 5.5kb, 5.6kb, 5.7kb, 5.8kb, 5.9kb, 6.0kb, 6.1kb, 6.2kb, 6.3kb, and 6.4kb. , 6.5kb, 6.6kb, 6.7kb, 6.8kb, 6.9kb, 7.0kb, 7.1kb, 7.2kb, 7.3kb, 7.4kb, 7.5kb, 7.6kb, 7.7kb, 7.8k b, 7.9kb, 8.0kb, 8.1kb, 8.2kb, 8.3kb, 8.4kb, 8.5kb, 8.6kb, 8.7kb, 8.8kb, 8.9kb, 9.0kb, 9.1kb, 9.2k b, 9.3kb, 9.4kb, 9.5kb, 9.6kb, 9.7kb, 9.8kb, 9.9kb, 10.0kb, 10.1kb, 10.2kb, 10.3kb, 10.4kb, 10.5 kb, 10.6kb, 10.7kb, 10.8kb, 10.9kb, 11.0kb, 11.1kb, 11.2kb, 11.3kb, 11.4kb, 11.5kb, 11.6kb, 11.7 The method according to any one of claims 111 to 171, wherein the DNA template is of a size of 13.0kb, 11.8kb, 11.9kb, 12.0kb, 12.1kb, 12.2kb, 12.3kb, 12.4kb, 12.5kb, 12.6kb, 12.7kb, 12.8kb, 12.9kb, 13.0kb or larger, or any size between these sizes.

173. The sizes of the DNA templates are approximately 0.3kb to 13kb, 0.3kb to 0.5kb, 0.3kb to 1kb, 0.3kb to 4kb, 0.3kb to 3kb, 0.3kb to 5kb, 0.3kb to 7kb, 0.3kb to 10kb, 0.5kb to 1kb, 0.5kb to 3kb, 0.5kb to 5kb, 0.5kb to 7kb, 0.5kb to 10kb, 0.5kb to 13kb, 1kb to 3kb, 1kb to 5kb, 1kb to 7kb, 1kb to 10kb, 1kb to 13kb, and 5kb. The method according to any one of claims 111 to 172, wherein the bit depth is approximately 13kb, 5kb to 9kb, 5kb to 8kb, 5kb to 7kb, 5kb to 6kb, 6kb to 13kb, 6kb to 10kb, 6kb to 9kb, 6kb to 8kb, 6kb to 7kb, 7kb to 13kb, 7kb to 10kb, 7kb to 9kb, 7kb to 8kb, 8kb to 13kb, 8kb to 10kb, 8kb to 9kb, 9kb to 13kb, 9kb to 10kb, 10kb to 13kb, or approximately 11kb to 13kb.

174. The method according to any one of claims 111 to 173, wherein the cells are mammalian cells, human cells, hematopoietic cells, immune cells, primary immune cells, or primary human immune cells.

175. The method according to claim 174, wherein the cells are primary human immune cells.

176. The method according to any one of claims 174 to 175, wherein the immune cells are natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, primary T cells, or T cell progenitor cells.

177. The method according to any one of claims 174 to 176, wherein the immune cells are primary T cells.

178. The method according to any one of claims 174 to 177, wherein the immune cells are primary human T cells.

179. The method according to any one of claims 174 to 178, wherein the immune cells are undifferentiated.

180. The aforementioned immune cells are CD45RA ＋ and CCR7 ＋ CD45RA ＋ and CCR7 － CD45RA － and CCR7 － , or CD45RA － and CCR7 ＋ The method according to any one of claims 174 to 179.

181. The method according to any one of claims 111 to 180, wherein the cells are virus-free.

182. The method according to any one of claims 111 to 181, further comprising collecting the cells from a patient and introducing the DNA template in vitro or ex vivo.

183. The method according to any one of claims 111 to 182, wherein the target region of the cell genome is a T cell receptor α constant region (TRAC) locus or a genome-safe harbor (GSH).

184. The method according to claim 183, wherein the safe harbor locus is selected from any one of the integration sites designated by GS94, GS88, GS89, GS90, GS91, GS92, GS93, GS95, GS96, GS97, GS98, GS99, GS100, GS101, GS102, GS103, GS104, GS105, GS106, GS107, GS108, GS109, GS110, GS111, GS112, GS113, GS114, GS115, GS116, GS117, GS118, GS119, or GS120.

185. The method according to claim 184, wherein the safe harbor locus is the GS94 integration site.

186. The aforementioned sgRNA target gene loci are chr11:128340000-128350000, chr10:33130000-33140000, chr10:72290000-72300000, chr11:65425000-65427000 (NEAT1), chr15:92830000-92840000, chr16:11220000- The method according to any one of claims 111 to 186, selected from 11230000, chr2: 87460000-87470000, chr3: 186510000-186520000, chr3: 59450000-59460000, chr8: 127980000-128000000, or chr9: 7970000-7980000.

187. The method according to any one of claims 111 to 186, wherein the sgRNA target locus is a gene selected from APRT, B2M, CAMPNS1, CBLB, CD2, CD3E, CD3G, CD5, EDF1, FTL, PTEN, PTPN2, PTPN6, PTPRC, PTPRCAP, RPS23, RTRAF, SERF2, SLC38A1, SMAD2, SOCS1, SRP14, SRSF9, SUB1, TET2, TIGIT, TRAC, or TRIM28.

188. The method according to any one of claims 111 to 187, wherein the one or more gRNAs include one of sequence numbers 1 to 120.

189. After the insertion of the at least one sequence into the safe harbor locus, the cell becomes CD45RA ＋ and CCR7 ＋ The method according to any one of claims 111 to 188.

190. The method according to any one of claims 111 to 189, wherein the DNA template is a double-stranded DNA template or a single-stranded DNA template.

191. The method according to any one of claims 111 to 190, wherein the DNA template is a linear DNA template or a circular DNA template, and optionally the circular DNA template is a plasmid.

192. The method according to any one of claims 111 to 191, wherein the DNA template includes heterologous sequences.

193. The method according to any one of claims 111 to 192, wherein the DNA template includes a gene.

194. The method according to any one of claims 111 to 193, wherein the DNA template comprises a priming receptor containing a transcription factor.

195. The method according to any one of claims 111 to 193, wherein the DNA template comprises a chimeric antigen receptor (CAR).

196. The method according to any one of claims 111 to 195, wherein the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor containing a transcription factor.

197. The method according to any one of claims 111 to 196, wherein the DNA template includes an inducible promoter operably linked to the chimeric antigen receptor.

198. The method according to any one of claims 111 to 197, wherein the DNA template further comprises a constitutive promoter operably linked to the priming receptor.

199. The method according to any one of claims 111 to 198, wherein the DNA template further comprises an inductive promoter operably linked to the chimeric antigen receptor and a constitutive promoter operably linked to the priming receptor.

200. The aforementioned DNA template is positioned from the 5' to the 3' direction. a. The inductive promoter, b. The aforementioned chimeric antigen receptor, c. The aforementioned constitutive promoter, and d. The priming receptor The method according to any one of claims 111 to 199, including the method described in any one of claims 111 to 199.

201. The aforementioned DNA template is positioned from the 5' to the 3' direction. a. The aforementioned constitutive promoter, b. The priming receptor, c. The inductive promoter, and d. The chimeric antigen receptor The method according to any one of claims 111 to 199, including the method described in any one of claims 111 to 199.

202. The priming receptor is located from the N-terminus towards the C-terminus. a. Extracellular antigen-binding domain having binding affinity to the antigen, b. A transmembrane domain containing one or more ligand-induced proteolytic cleavage sites, and c. Intracellular domains containing human or humanized transcription effectors The method according to any one of claims 111 to 201, comprising, wherein when the antigen binds to the extracellular antigen-binding domain, cleavage occurs at the ligand-induced proteolytic cleavage site, thereby releasing the intracellular domain.

203. The method according to claim 202, wherein the priming receptor further comprises a near-membrane domain (JMD) or a stop transition sequence (STS) located between the transmembrane domain and the intracellular domain.

204. The method according to any one of claims 111 to 203, wherein the transcription factor binds to the inducible promoter to induce the expression of the CAR.

205. The aforementioned CAR extends from the N-terminus to the C-terminus. a. Extracellular antigen-binding domain having binding affinity to the antigen, b. Transmembrane domain, c. Intracellular co-stimulatory domains, and d. Intracellular activation domain The method according to any one of claims 111 to 204, including the method described in any one of claims 111 to 204.

206. The method according to any one of claims 111 to 205, wherein the priming receptor and the CAR bind to different antigens.

207. The method according to any one of claims 111 to 206, wherein the priming receptor and the CAR bind to the same antigen.

208. A method for editing immune cells, a. To provide immune cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins. b. To provide a solution comprising a ribonucleoprotein complex (RNP) and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and c. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

209. A method for editing immune cells, a. To provide immune cells containing exogenous homologous recombination proteins or DNA repair regulatory proteins. b. To provide a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, and / or a histone deacetylase (HDAC) inhibitor, wherein the RNP comprises a nuclease domain and guide RNA, the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and c. Editing the immune cells by inserting the DNA template into the insertion site within the genome of the cells. The method, including the method described above.

210. The method according to claim 208 or 209, wherein the exogenous homologous recombination protein or DNA repair regulatory protein is SWSAP1, dominant-negative KU80, or AcrIIA8-CDT1 fusion protein.

211. The method according to claims 208 to 210, wherein the SWSAP1 protein comprises the sequence shown in SEQ ID NO: 125, the dominant-negative KU80 protein comprises the sequence shown in SEQ ID NO: 126, or the AcrIIA8-CDT1 fusion protein comprises the sequence shown in SEQ ID NO:

127.

212. A method for editing immune cells, a. To provide a solution comprising an RNase inhibitor, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome. b. Nonvirally introducing the RNP and DNA template into the immune cell, wherein the guide RNA specifically hybridizes to a target region of the cell's genome, and the nuclease domain cleaves the target region to create the insertion site within the cell's genome, and c. Editing the immune cells by inserting the DNA template into the insertion site within the genome of the cells. The method, including the method described above.

213. A method for editing immune cells, a. To provide a solution comprising N-acetyl-L-cysteine ​​(NAC), ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the genome of the immune cell. b. Nonvirally introducing the RNP and DNA template into the immune cell, wherein the guide RNA specifically hybridizes to a target region of the cell's genome, and the nuclease domain cleaves the target region to create the insertion site within the cell's genome, and c. Editing the immune cells by inserting the DNA template into the insertion site within the genome of the cells. The method, including the method described above.

214. A method for editing immune cells, a. To provide a solution comprising an osmotic regulator, a ribonucleoprotein complex (RNP), and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to the genomic sequence adjacent to the insertion site in the genome of the immune cell. b. Nonvirally introducing the RNP and DNA template into the immune cell, wherein the guide RNA specifically hybridizes to a target region of the cell's genome, and the nuclease domain cleaves the target region to create the insertion site within the cell's genome, and c. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

215. A method for editing immune cells, a. To provide a solution comprising a histone deacetylase (HDAC) inhibitor containing at least one of sodium phenylbutyrate, xinostat, or panobinostat, and cells. b. Contacting the cells with a ribonucleoprotein complex (RNP) and a DNA template, wherein the RNP comprises a nuclease domain and guide RNA, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome, and c. Introducing the RNP and DNA template into the cell nonvirally, wherein the guide RNA specifically hybridizes to a target region of the cell's genome, and the nuclease domain cleaves the target region to create the insertion site in the cell's genome, and d. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

216. A method for editing immune cells, a. To provide a solution comprising a ribonucleoprotein complex (RNP), a DNA template, and at least one of an RNase inhibitor, N-acetyl-L-cysteine ​​(NAC), an osmotic regulator, and / or a histone deacetylase (HDAC) inhibitor, wherein the RNP comprises a nuclease domain and guide RNA, the DNA template comprises a chimeric antigen receptor (CAR) and a priming receptor comprising a transcription factor, and the 5' and 3' ends of the DNA template contain nucleotide sequences homologous to a genomic sequence adjacent to the insertion site in the cell's genome. b. Nonvirally introducing the RNP and DNA template into the immune cell, wherein the guide RNA specifically hybridizes to a target region of the cell's genome, and the nuclease domain cleaves the target region to create the insertion site within the cell's genome, and c. Editing the cell by inserting the DNA template into the insertion site within the cell's genome. The method, including the method described above.

217. A method for treating a subject who has or is at risk of having a disease, a. To carry out the method described in any one of claims 111 to 216, and b. Administering an effective amount of composition containing the cells or population thereof to the subject. The method, including the method described above.

218. The method according to claim 217, wherein the composition is administered to the subject by injection.

219. The method according to claim 217 or 218, wherein the disease is cancer.

220. Immune cells produced by the method according to any one of claims 111 to 216.

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