Modified cells and therapeutic methods

Abstract

The present invention provides a genetically modified composition for treating cancer, and a method for preparing and using a genetically modified composition in the treatment of cancer. [Solution] The compositions and methods disclosed herein can be used to identify cancer-specific T cell receptors (TCRs) that recognize unique immunogenic mutations in a patient's cancer and to treat any type of cancer in the patient. Insertion of these transgenes encoding cancer-specific TCRs into T cells, using non-viral methods (e.g., CRISPR, TALEN, transposon-based ZEN, meganuclease, or Mega-TAL), is a novel technique that opens up new opportunities to extend immunotherapy to many cancer types.

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Application No. 62 / 199,905 filed on 31 July 2015; No. 62 / 232,983 filed on 25 September 2015; No. 62 / 286,206 filed on 22 January 2016; No. 62 / 295,670 filed on 16 February 2016; No. 62 / 330,464 filed on 2 May 2016; and No. 62 / 360,245 filed on 8 July 2016, all of which are incorporated herein by reference in whole. [Background technology]

[0002] background Despite the remarkable progress in cancer treatment over the past 50 years, many tumor types still exist that are resistant to chemotherapy, radiation therapy, or biotherapy, especially in advanced stages, and cannot be treated surgically. In recent years, molecular targets on tumors have been used in Remarkable advances in the genetic engineering of lymphocytes recognized in vivo have resulted in a remarkable number of cases of targeted tumor remission. However, these successes have been largely limited to hematopoietic malignancies, and broader application to solid tumors is limited due to the lack of identifiable molecules expressed by cells within specific tumors, and the lack of molecules that can be used to specifically bind to tumor targets in order to mediate tumor destruction. Some recent advances have focused on identifying tumor-specific mutations that, in some cases, induce antitumor T cell responses. For example, these endogenous mutations can be identified using whole-exome sequencing (Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer," Science, vol. 344: pp. 641-644 (2014)). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer," Science, Vol. 344: pp. 641-644 (2014). [Overview of the project] [Means for solving the problem]

[0004] The compositions and methods disclosed herein can be used to identify cancer-specific T cell receptors (TCRs) that recognize unique immunogenic mutations in a patient's cancer and to treat any type of cancer in the patient. Insertion of these transgenes encoding cancer-specific TCRs into T cells, using non-viral methods (e.g., CRISPR, TALEN, transposon-based ZEN, meganuclease, or Mega-TAL), is a novel technique that opens up new opportunities to extend immunotherapy to many cancer types. Embedding by reference All publications, patents, and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually referenced. In the event of any conflict between the terminology herein and the terminology of any incorporated reference, the terminology herein shall prevail.

[0005] Summary of the Invention This specification discloses a modified cell comprising disruption of at least one gene and at least one non-viral integrated T cell receptor (TCR) sequence, wherein the gene can be disrupted by the non-viral integrated TCR sequence. This gene may be a checkpoint gene, for example, an immune checkpoint gene. This gene may include adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-associated (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), and hepatitis A liver. This could be inflammatory virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5). In some cases, this gene could be PD-1.

[0006] Manipulated cells may contain a single TCR sequence. A TCR sequence may contain a manipulated TCR sequence. A TCR sequence may contain two or more chains. Two or more chains may contain at least one alpha chain. Two or more chains may contain at least one beta chain. A TCR sequence may contain an extracellular domain, a transmembrane domain, and an intracellular domain. A TCR sequence may produce a functional TCR. A TCR sequence may recognize an antigen. A TCR sequence may recognize an antigen in the context of major histocompatibility complex (MHC). The MHC may be class I. The MHC may be HLA-A02. The MHC may be class II. A TCR may bind to a mutation. Mutations to which a TCR binds can be identified by whole exome sequencing. A TCR may bind to cancer cells.

[0007] Manipulated cells may be primary cells. Manipulated cells may be immune cells. Manipulated cells may be T cells, stem cells, or progenitor cells. Manipulated cells may be hematopoietic progenitor cells. Manipulated cells may be human cells. Manipulated cells can be selected. Manipulated cells can be enlarged ex vivo. Manipulated cells can be enlarged in vivo. Manipulated cells may be CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), or IL-7Rα(+). Manipulated cells may be autologous cells for a target requiring them. Manipulated cells may be non-autologous cells for a target requiring them. Manipulated cells may be Good Manufacturing Practice (GMP) compliant reagents. Manipulated cells may be part of a combination therapy to treat cancer, infection, autoimmune disorders, or graft-versus-host disease (GVHD) in a target requiring them.

[0008] This specification also discloses a method for producing manipulated cells, comprising the steps of: a) introducing one or more polynucleic acids containing at least one exogenous T cell receptor (TCR) sequence, sandwiched between recombinant arms, into cells non-virally; and b) contacting at least one exogenous TCR sequence with a double-strand break region containing a gene. The recombinant arms may be complementary to the gene portion. The gene may include adenosine A2a receptor, CD276, V-set domain-containing T cell activation inhibitor 1, B lymphocyte and T lymphocyte-related, cytotoxic T lymphocyte-related protein 4, indoleamine 2,3-dioxygenase 1, and KIR3DL1 (killer cell immunoglobulin). It may be a ulin-like receptor, three domains, long cytoplasmic tail 1), lymphocyte activating gene 3, programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2, V-domain immunoglobulin suppressor for T cell activation, or natural killer cell receptor 2B4. In some cases, the gene may be PD-1. In some cases, the gene may be a checkpoint gene. In some cases, the checkpoint gene may be an immune checkpoint gene.

[0009] Double-strand break regions can be repaired by insertion of at least one exogenous TCR sequence. Insertion of at least one exogenous TCR sequence may involve disruption of at least one gene. Insertion of at least one exogenous TCR sequence can be assisted by a homologous recombination (HR) enhancer. The enhancer may be derived from a viral protein. The enhancer may be E1B55K, E4orf6, Scr7, or L755507. In some cases, the enhancer may be a chemical inhibitor. In some cases, the enhancer may inhibit ligase IV. In some cases, the enhancer may facilitate TCR sequence insertion. The insertion may involve homologous repair.

[0010] In some cases, double-strand break regions can be created by CRISPR, TALEN, transposon-based ZEN, meganuclease, or Mega-TAL. In some cases, double-strand break regions can be created by CRISPR. In some cases, CRISPR can be multiplexed. In some cases, multiplexing can be carried out by adding at least two guide RNAs. The TCR sequence can be inserted near the double-strand break region.

[0011] In some cases, the polynucleic acid may be RNA. In some cases, the RNA may be mRNA. In some cases, the cell may be brought into contact with reverse transcriptase (RT). In some cases, the cell may be brought into contact with a primer complementary to the polynucleic acid. In some cases, RT transcribes mRNA to a first ssDNA template. In some cases, RT transcribes the first ssDNA template to a second dsDNA template. In some cases, transcription may be performed in situ. The ssDNA or dsDNA may contain at least one exogenous TCR sequence. In some cases, the presence of RT can be determined using the primer sequence. The forward primer for the reverse transcriptase (RT) reporter may be AAC GTG CTG GTT GTT GTG CTG (SEQ ID NO: 180). In other cases, the reverse primer for the reverse transcriptase (RT) reporter may be used. The reverse primer for the RT reporter may be AAA GTG GTG GTA GAA TAG GCT C (SEQ ID NO: 181).

[0012] In some cases, nonviral delivery may include electroporation or nucleofection. Polynucleic acids can be co-delivered with at least one modifier that modifies the cellular response to the polynucleic acid. At least one modifier may reduce cytotoxicity. The modifier may include the pancaspase inhibitor Z-VAD-FMK or BX795. The present invention may include primary cells. Primary cells may be immune cells. Immune cells may be T cells, stem cells, or progenitor cells. The method may include progenitor cells. In some cases, the progenitor cells are hematopoietic progenitor cells. In some cases, the cells are human cells. The method may be compliant with "Good Manufacturing Practice" (GMP) standards for pharmaceuticals and quasi-drugs.

[0013] Depending on the circumstances, the procedure may include administering a therapeutically effective amount of a pharmaceutical composition containing manipulated cells to a subject in need. The pharmaceutical composition may be administered intravenously. The pharmaceutical composition may be administered topically. Depending on the circumstances, the method may further include a step of administering one or more further treatments. One or more further treatments may involve transplantation. This may include: One or more further treatments may include immunotherapy. In some cases, the manipulated cells may be autologous cells for the target. In some cases, the manipulated cells may be allogeneic cells for the target.

[0014] This specification also refers to adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-associated (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), and VISTA (V-set domain of T cell activation). Also disclosed are polynucleic acids comprising at least one exogenous T cell receptor (TCR) sequence flanked by at least two recombinant arms having a sequence complementary to a genomic sequence that may be a main immunoglobulin suppressor, natural killer cell receptor 2B4 (CD244), CISH (cytokine-induced SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS site (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5).

[0015] Polynucleotide sequences may be complementary to genomic sequences, which may be partial sequences. In some cases, the binding of a recombinant arm to a sequence complementary to the genomic sequence inserts an exogenous TCR sequence. In some cases, the binding of a recombinant arm to a sequence complementary to the genomic sequence repairs double-strand breaks. In some cases, the genomic sequence contains coding sequences. In some cases, the genomic sequence contains non-coding sequences. In some cases, the genomic sequence contains one or more genes. Insertion of an exogenous TCR sequence may disrupt one or more genes. In some cases, the genomic sequence may be PD-1.

[0016] In some cases, the polynucleic acid may be a plasmid vector. The plasmid vector may contain a promoter. In some cases, the promoter may be a constitutive promoter. In some cases, the promoter may be an inductive promoter. The promoter may be CMV, U6, MND, or EF1a. In some cases, the promoter may be flanked by an exogenous TCR sequence. In some cases, the plasmid vector may further contain a splicing acceptor. In some cases, the splicing acceptor may be flanked by an exogenous TCR sequence. The promoter sequence may be a PKG promoter or an MND promoter. The MND promoter may be a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer.

[0017] In some cases, the plasmid vector further contains an "ATG" sequence. The "ATG" sequence may be adjacent to a TCR sequence. In some cases, the TCR sequence encodes a fusion protein. In some cases, the TCR sequence may be a TCR sequence within a multicistronic vector. In some cases, the polynucleic acid contains an exogenous promoter, an endogenous promoter via splicing, and / or an endogenous promoter via in-frame translation.

[0018] In some cases, plasmids can be modified. Modifications may include demethylation, addition of CpG methylation, removal of bacterial methylation, and addition of mammalian methylation. The TCR sequence may be a manipulated TCR sequence. In some cases, polynucleic acids can be designed for delivery to cells by nonviral methods. In some cases, polynucleic acids may be Good Manufacturing Practice (GMP) compliant reagents.

[0019] This specification also discloses a method for facilitating homology-guided repair (HDR), comprising the steps of: a) introducing mRNA, reverse transcriptase (RT), enhancer, and primer into a cell non-virally; b) reverse transcribing the mRNA into one or more copies of a polynucleic acid; and c) facilitating HDR between the cellular genome and the polynucleic acid. The method may optionally include a step of inducing a double-strand break. The double-strand break may optionally be induced by CRISPR, TALEN, transposon-based ZEN, meganuclease, and Mega-TAL. The double-strand break may optionally be induced by CRISPR. The HDR in c) may repair the double-strand break. The CRISPR may optionally be multiplexed with at least two guide RNAs. The polynucleic acid may optionally be DNA. The polynucleic acid may optionally be cDNA. The polynucleic acid may optionally be single-stranded.

[0020] In some cases, RT transcribes mRNA into a first ssDNA template. In some cases, the polynucleic acid may be double-stranded. In some cases, RT transcribes mRNA into a second dsDNA template in situ. The mRNA or polynucleic acid may contain at least one TCR sequence. In some cases, the TCR sequence contains at least two flanking recombination arms having sequences complementary to a genomic region. In some cases, the TCR sequence can be used within the HDR of c). In some cases, the TCR sequence can be used within the HDR of c), further including the binding of recombination arms to a complementary region of the cellular genome. In some cases, the TCR sequence can be used within the HDR of c), further including the binding of recombination arms to a complementary region of the cellular genome, and further including the insertion of the TCR sequence. In some cases, the HDR between the cellular genome and the polynucleic acid disrupts one or more genes. One or more genes may include immune checkpoint genes. Depending on the case, one or more genes may be involved in the following: adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-associated (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD). -1) Includes hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5). In some cases, one or more genes include PD-1. In some cases, one or more genes include TCR.

[0021] In some cases, HDR between the cellular genome and polynucleic acid can be supported by one or more homologous recombination (HR) enhancers. One or more enhancers may include viral proteins. In some cases, one or more enhancers may include E1B55K, E4orf6, Scr7, and / or L755507. In some cases, the enhancer may include a chemical inhibitor. In some cases, the enhancer may inhibit ligase IV. In some cases, the enhancer may facilitate the insertion of polynucleic acid into the cellular genome. The enhancer may prevent non-homologous end joining (NHEJ). In some cases, the polynucleic acid may be inserted at or near a double-strand break. In some cases, mRNA, reverse transcriptase, primers, HR enhancer, and CRISPR are brought into contact with the cell. In some cases, polynucleic acid, CRISPR, and HR enhancer are brought into contact with the cell. The cell may be a primary cell. The cell may be an immune cell. The cell may be a T cell, stem cell, or progenitor cell. In some cases, the cell is a T cell. Depending on the case, the cells are progenitor cells. Depending on the case, the cells are hematopoietic progenitor cells. The cells may be human cells. Depending on the case, the T cells may be autologous cells. Depending on the case, the T cells may be non-autologous cells. Depending on the case, the method may be compliant with "Good Manufacturing Practice" (GMP) standards for pharmaceuticals and quasi-drugs.

[0022] This specification also discloses methods for reducing cytotoxicity to exogenously modified polynucleic acids, comprising the step of modifying one or more cellular responses to polynucleic acids. One or more cellular responses may include cytoplasmic DNA sensing pathways. Optionally, the step of modifying one or more cellular responses may involve DAI (DNA-dependent activator of IFN regulators), IFN-inducible protein 16 (IFI16), DEAD-box polypeptide 41 (DDX41), AIM2 (absent in melanoma 2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), STING (IFN gene stimulator), TANK-binding kinase (TBK1), interleukin 1β (IL-1β), MRE11 (meiotic recombination 11), Trex1, caspase 1 (aspartate-specific cysteine ​​protease), 3' repair exonuclease, DAI (DNA-dependent activator of This includes modifying IRF, IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), MRE11 (meiotic recombination 11) homolog A, and / or IFN regulator (IRF) 3 or 7. Depending on the compound, one or more may alter one or more cellular responses. One or more compounds may include inhibitors. One or more compounds may include activators. Depending on the compound, one or more may include the pancaspase inhibitor Z-VAD-FMK, and / or Z-VAD-FMK.

[0023] Depending on the circumstances, one or more compounds may be modified. One or more compounds may prevent apoptosis and pyroptosis of cells. Depending on the circumstances, one or more compounds may inhibit the cleavage of pro-IL-1β and pro-IL-18 by caspase 1. Depending on the circumstances, one or more compounds may modify ASC (apoptosis-associated The activity of a speck-like protein containing a CARD may be modulated. One or more compounds may modulate the cGAS-STING pathway. One or more compounds may prevent the expression of type I interferon. Depending on the circumstances, one or more compounds may comprise two or more compounds. Depending on the circumstances, the compounds may be compliant with "Good Manufacturing Practices" (GMP) for pharmaceuticals and quasi-drugs.

[0024] In some cases, the compound may be brought into contact with the cells before the cells are brought into contact with one or more exogenous modified polynucleic acids. In some cases, the method may further include the step of bringing the cells into contact with one or more homologous recombination (HR) enhancers.

[0025] In some cases, the method may further include a step of selecting cells. In some cases, the method may further include a step of expanding cells. In some cases, the method may result in GMP-compliant cell therapy.

[0026] This specification discloses a method for genome manipulation comprising: a) contacting cells with one or more signaling-modifying compounds; and b) contacting cells with a polynucleic acid comprising at least one antigen receptor sequence flanked by at least two recombinant arms complementary to at least one genomic region. Optionally, one or more signaling-modifying compounds alter cytoplasmic DNA sensing pathways. Optionally, one or more signaling-modifying compounds include DAI (DNA-dependent activator of IFN regulators), IFN-inducible protein 16 (IFI16), and DEAD-box polynucleic acid. Peptide 41 (DDX41), AIM2 (absent in melanoma 2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), STING (IFN gene stimulator), TANK-binding kinase (TBK1), interleukin 1β (IL-1β), MRE11 (meiotic recombination 11), Trex1, caspase 1 (aspartate-specific cysteine ​​protease), 3' repair exonuclease, DAI (DNA-dependent activator of IRF), IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), MRE11 (meiotic recombination 11) homolog A, and / or IFN regulator (IRF) 3 or 7 are modified. Depending on the case, one or more signal transduction modifying compounds include inhibitors. Depending on the case, one or more signal transduction modifying compounds include activators. One or more signal transduction-modifying compounds may include the pancaspase inhibitor Z-VAD-FMK, and / or Z-VAD-FMK.

[0027] Depending on the circumstances, one or more signaling-modifying compounds may be modified. One or more signaling-modifying compounds may prevent apoptosis and pyroptosis of cells. Depending on the circumstances, one or more signaling-modifying compounds may inhibit the cleavage of pro-IL-1β and pro-IL-18 by caspase 1. One or more signaling-modifying compounds may modulate the activity of ASC (apoptosis-associated speck-like protein containing a CARD). Depending on the circumstances, one or more signaling-modifying compounds may modulate the cGAS-STING pathway. One or more signaling-modifying compounds may prevent the expression of type I interferon. One or more signaling-modifying compounds may comprise two or more compounds. Depending on the circumstances, one or more signaling-modifying compounds may be brought into contact with cells before contacting the cells with one or more exogenous modified polynucleic acids. Depending on the circumstances, the method may further comprise the step of bringing cells into contact with one or more homologous recombination (HR) enhancers. Depending on the circumstances, the cells are primary cells. Depending on the circumstances, the cells are immune cells. Depending on the case, the cells may be T cells, stem cells, or progenitor cells. The present invention may include progenitor cells. The cells may be hematopoietic progenitor cells. The cells may be human cells.

[0028] Also disclosed herein are unmethylated polynucleic acids comprising at least one manipulated antigen receptor flanked by at least two recombinant arms complementary to at least one genomic region. The polynucleic acid may be modified. Modifications may include demethylation, addition of CpG methylation, removal of bacterial methylation, and / or addition of mammalian methylation. The polynucleic acid may be capable of undergoing homologous recombination. The recombinant arms may bind to complementary genomic regions. The antigen receptor may include a TCR or a chimeric antigen receptor (CAR).

[0029] Also disclosed herein are mammalian methylated polynucleic acids comprising at least one manipulated antigen receptor. The polynucleic acid may be further modified. Modifications may include demethylation, addition of CpG methylation, removal of bacterial methylation, and / or addition of mammalian methylation. The polynucleic acid may be capable of undergoing homologous recombination. The mammalian methylated polynucleic acid may further comprise a recombinant arm that binds to at least one complementary genomic region. The recombinant arm may bind to the complementary genomic region. The mammalian methylated polynucleic acid may comprise an antigen receptor comprising a TCR or a chimeric antigen receptor (CAR).

[0030] This specification may also disclose compositions for reducing cytotoxicity, comprising a caspase modulator and a cGAS-STING pathway modulator. Caspase modulators can alter the cytoplasmic DNA sensing pathway. cGAS-STING pathway modulators can alter the cytoplasmic DNA sensing pathway. The cytoplasmic DNA sensing pathway may include caspase 1. Caspase modulators can be caspase inhibitors. Caspase modulators can inhibit the cleavage of pro-IL-1β and pro-IL-18 by caspase 1.

[0031] The cytoplasmic DNA sensing pathway may include DAI (DNA-dependent activator of IFN regulators), IFN-inducible protein 16 (IFI16), DEAD-box polypeptide 41 (DDX41), AIM2 (absent in melanoma 2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), STING (IFN gene stimulator), TANK-binding kinase (TBK1), interleukin 1β (IL-1β), MRE11 (meiotic recombination 11), Trex1, caspase 1 (aspartate-specific cysteine ​​protease), 3' repair exonuclease, DAI (DNA-dependent activator of IRF), IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), MRE11 (meiotic recombination 11) homolog A, and / or IFN regulator (IRF) 3 or 7. cGAS-STING pathway modulators may be cGAS-STING pathway inhibitors. cGAS-STING pathway inhibitors may include the pancaspase inhibitor Z-VAD-FMK, and / or Z-VAD-FMK. The compositions may prevent apoptosis and pyroptosis of cells. The compositions may prevent the expression of type I interferons. In some cases, compositions containing modified caspase modulators may reduce cytotoxicity. In some cases, compositions containing modified cGAS-STING pathway modulators may reduce cytotoxicity. Modifications may include deuteration, lipidization, glycosylation, alkylation, PEGylation, oxidation, phosphorylation, sulfation, amidation, biotinylation, citrullination, isomerization, ubiquitination, protonation, conjugation of small molecules, reduction, dephosphorylation, nitrosylation, and / or proteolysis. In some cases, modifications can improve the activity of modified caspase modulators and modified cGAS-STING pathway modulators.Activity can be increased by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 500%, 750%, or 1000%, or more, or by these ratios, compared to an unmodified caspase modulator or an unmodified cGAS-STING pathway modulator. Activity can be increased by at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 500%, 750%, or 1000%, or more, or by at least these ratios, compared to an unmodified caspase modulator or an unmodified cGAS-STING pathway modulator. Activity can be increased by at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 500%, 750%, or 1000%, and up to 100%, or at least by these ratios, compared to an unmodified caspase modulator or an unmodified cGAS-STING pathway modulator. The composition may be introduced into cells. The composition may prevent intracellular toxicity. The cells may be further exposed to polynucleic acid.

[0032] This specification describes a method for producing manipulated cells, comprising the steps of: introducing into cells a guide polynucleotide containing a spacer region complementary to a target nucleic acid within a cellular genome region; a nuclease guided by the guide polynucleotide; and a polynucleotide encoding an exogenous T cell receptor; site-specifically cleaving the target nucleic acid within the cell with the nuclease guided by the guide polynucleotide; and inserting the polynucleotide encoding the exogenous T cell receptor into the cellular genome region at the cleavage site. A method comprising a step is disclosed. The nuclease may be Cas9. The guide polynucleotide may be a single guide polynucleotide. The guide polynucleotide may be RNA. The target nucleic acid may be DNA. The spacer region may be between 10 and 30 nucleotides in length. The nuclease may induce a double-strand break within the target nucleic acid.

[0033] In some cases, guide polynucleotides can be introduced into cells by electroporation. Guide nucleic acids can be introduced into cells by nucleofection. Nucleases can also be introduced into cells by delivery vectors. Polynucleotides encoding exogenous T cell receptors may further contain promoter sequences. Exogenous T cell receptors can be inserted by homologous recombination. By guiding polynucleotides and nucleases, a nuclear protein complex can be formed.

[0034] It is within the scope of the present invention that a genomic nucleic acid sequence that is replaced by a polynucleotide encoding an exogenous T cell receptor can be removed by cleaving a target nucleic acid. The polynucleotide encoding the exogenous T cell receptor may further comprise a first recombinant arm and a second recombinant arm. The first recombinant arm may comprise a first sequence identical to a first portion of the target nucleic acid, and the second recombinant arm may comprise a second sequence identical to a second portion of the target nucleic acid. Optionally, the first recombinant arm may comprise a first sequence identical to a first portion adjacent to the target nucleic acid, and the second recombinant arm may comprise a second sequence identical to a second portion adjacent to the target nucleic acid. The target nucleic acid may be located within a gene. The genes include adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), and hepatitis A virus. The following can be selected: T cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5). The gene may be PD-1. The gene may be a checkpoint gene. The checkpoint gene may be an immune checkpoint gene.

[0035] In some cases, insertion of an exogenous TCR sequence at a cleavage site can result in gene disruption. The target nucleic acid may be located within an intergenic region. Exogenous T cell receptors can be expressed intracellularly. Manipulated cells can be introduced into organisms. Manipulated cells can be enlarged ex vivo.

[0036] The ability to suppress non-homologous end joining (NHEJ) intracellularly is within the scope of the present invention. Suppression of intracellular NHEJ may include inhibition of ligase IV. Suppression of intracellular NHEJ may also include the introduction of a homologous recombination (HR) enhancer. The enhancer may be derived from a viral protein. The enhancer may be E1B55K, E4orf6, Scr7, or L755507. Suppression of intracellular NHEJ may facilitate the insertion of a polynucleotide encoding an exogenous TCR by homologous recombination at the cleavage site.

[0037] This disclosure may further include a step of introducing a modifying agent into cells to reduce cytotoxicity. The modifying agent may be the pancaspase inhibitor Z-VADFMK and / or BX795. The cells may be T cells. The cells may be mammalian cells. It may be a primary cell. Primary cells may be immune cells. The cell may be a stem cell or a progenitor cell. In some cases, the cell is a progenitor cell. Progenitor cells may be hematopoietic progenitor cells. The cell may be a human cell.

[0038] This specification may also disclose compositions comprising manipulated cells. The manipulated cells can be administered to a subject in a therapeutically effective dose. Administration of the manipulated cells may result in a therapeutic outcome in the subject that is modulated by an exogenous TCR.

[0039] This specification may disclose engineered cells comprising at least one exogenous receptor sequence that may be adjacent to a protospacer-adjacent motif sequence of genomic DNA. The protospacer-adjacent motif sequence (PAM) may be recognized by a CRISPR endonuclease. The endonuclease may be a Cas protein. Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, C A selection can be made from a list including mr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, their homologs, or modified forms thereof. In some cases, the CRISPR endonuclease may be Cas9. The Cas9 of the present invention may recognize a PAM sequence that may be 5'NGG3'.

[0040] This specification may disclose at least one exogenous receptor capable of disrupting at least one gene. The gene may be a checkpoint gene. Checkpoint genes include adenosine A2a receptor (ADORA), CD276, V-set domain-containing T-cell activating inhibitor 1 (VTCN1), B-lymphocyte and T-lymphocyte-associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activator gene 3 (LAG3), programmed cell death 1 (PD-1), and hepatitis A liver. The following can be selected: inflammatory virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5).

[0041] In some cases, the gene may contain a protospacer. The protospacer can be disrupted by insertion of an exogenous receptor sequence. In some cases, at least one exogenous receptor sequence may be an immune receptor sequence. The immune receptor sequence may be selected from a list including T cell receptor (TCR) sequences, B cell receptor (BCR) sequences, or chimeric antigen receptor (CAR) sequences. The TCR sequence may contain two or more chains. The two or more chains may contain at least one alpha chain in this invention. The two or more chains may also contain at least one beta chain. The TCR sequence may contain an extracellular domain, a transmembrane domain, and an intracellular domain. The TCR sequence may produce a functional TCR. The TCR sequence may recognize an antigen. The TCR sequence may recognize an antigen in the context of a major histocompatibility complex (MHC). In some cases, the MHC may be class I. In some cases, the MHC may be HLA-A02. In other cases, the MHC may be class II.

[0042] This specification may disclose exogenous receptors capable of binding to mutations. Mutations can be identified by whole-exome sequencing. Exogenous receptor sequences can bind to cancer cells. The cells of the present invention may optionally be primary cells. Primary cells may be immune cells. The cells may be T cells, stem cells, or progenitor cells. The cells may be progenitor cells. Progenitor cells may be hematopoietic progenitor cells. The cells of the present invention may be human cells. The cells may be selected. The cells may be enlarged ex vivo. The cells may be enlarged in vivo. The cells may also be CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), IL-7Rα(+), or combinations thereof.

[0043] The cells of the present invention may be autologous cells for a target requiring them. The cells may also be non-autologous cells for a target requiring them. The cells may be Good Manufacturing Practice (GMP) compliant reagents. The cells may be part of a combination therapy to treat cancer, infection, autoimmune disorders, or graft-versus-host disease (GVHD) in a target requiring them. In some cases, the cells of the present invention may be administered to a target requiring them as monotherapy.

[0044] This specification describes a composition comprising at least one guide RNA that binds to an endogenous CISH (cytokine-inducible SH2-containing) gene, and an adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), and lymphocyte-activating gene 3 (LAG3). A composition may be disclosed that includes a secondary guide RNA that binds to an endogenous gene selected from the group consisting of programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), and chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5).

[0045] This specification may disclose manipulated cells involving disruption of the endogenous CISH (cytokine-induced SH2-containing) gene sequence and at least one secondary disruption within the endogenous genes. The endogenous genes include adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (P D-1) can be selected from the group consisting of hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), and chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5).

[0046] Depending on the circumstances, the cells of the present invention may further contain exogenous receptors. The exogenous receptors can be selected from the group including T cell receptors (TCRs), chimeric antigen receptors (CARs), or B cell receptors (BCRs). The exogenous receptors may bind to mutations. Mutations can be identified by whole-exome sequencing. The exogenous receptors may bind to cancer cells. The manipulated cells may be primary cells. Primary cells may be immune cells. Cells may be T cells, stem cells, or progenitor cells. Cells may be progenitor cells. Progenitor cells may be hematopoietic progenitor cells. The cells of the present invention may be human cells.

[0047] This specification discloses a target polynucleic acid comprising: lymphocytes derived from a human subject; a polynucleic acid targeting polynucleic acid engineered to hybridize to a specific region of a target gene within the lymphocyte genome; a nuclease capable of associating with the polynucleic acid targeting polynucleic acid to form a nuclear protein complex, wherein the nuclear protein complex may be capable of causing targeted double-strand breaks in the target gene within the lymphocyte genome; and a target polynucleic acid which may be genomic DNA containing double-strand breaks within the target gene, wherein the double-strand breaks within the target gene result in the disruption of the target gene function, and the disruption of the target gene function occurs with at least 60% efficiency when the nuclear protein complex can be brought into contact with a population of primary lymphocytes. This further discloses a genetically modified immune cell population of lymphocytes with altered target gene function, which may be capable of producing a clonal population of lymphocytes suitable for administration to humans in need.

[0048] This specification discloses a method for efficient disruption of checkpoint inhibitors within T cells, comprising the steps of: contacting T cells with a Cas9 nuclease and guide RNA, wherein the guide RNA contains a region of 17-22 nucleotides substantially complementary to a region in a target gene; cleaving a target gene, the target gene being PD-1, wherein a knockout event occurs in at least 30% of primary T cells when a population of primary T cells is contacted with the Cas9 nuclease and guide RNA; and disrupting checkpoint inhibitors within T cells.

[0049] This specification discloses a method for treating a subject in need thereof, comprising the steps of: recovering lymphocytes from a human; ex vivo genetically modifying lymphocytes by contacting them with a ribonuclease capable of knocking out the function of the PD-1 protein by inducing double-strand breaks in a specific target region of genomic DNA within the lymphocytes, wherein the target region of genomic DNA within the lymphocytes is located within the PD-1 gene, and the double-strand break occurs in a target region of genomic DNA that is 3' to the target DNA region and contains a protospacer adjacent motif, and is capable of hybridizing to at least 15 nucleotides of the ribonuclease; expanding the population of genetically modified lymphocytes having PD-1 protein knockout to produce a population of PD-1 knockout T cells; and administering the population of PD-1 knockout T cells to a subject, wherein the PD-1 knockout T cells are suitable for administration to a patient.

[0050] In some embodiments, the disclosure presents a method for producing genetically modified cells, comprising the step of obtaining one or more cells from a subject. In some embodiments, the method comprises the step of introducing a first nucleic acid into one or more cells. In some embodiments, the method comprises a first nucleic acid, the first nucleic acid comprising a first transgene encoding at least one anti-DNA sensing protein. In some embodiments, the method comprises at least one DNA sensing pathway, and disrupts at least one DNA sensing pathway in one or more cells by at least one anti-DNA sensing protein. In some embodiments, the method comprises the step of introducing a second nucleic acid into one or more cells. In some embodiments, the method comprises a second nucleic acid, the second nucleic acid comprising a second transgene encoding an engineered T cell receptor (TCR). In some embodiments, the method comprises at least one endogenous immune checkpoint gene, and disrupts at least one endogenous immune checkpoint gene in one or more cells by insertion of the second transgene. In some embodiments, the method disrupts at least one DNA sensing pathway. The method includes disruption of at least one DNA sensing pathway, which reduces the cytotoxicity induced by the second trans gene, thereby maintaining or increasing the viability of one or more cells. In some embodiments, the method includes one or more cells, one or more of which are immune cells. In some embodiments, the method includes one or more cells, one or more of which are T cells, naive T cells, CD4+ cells, CD8+ cells, stem cells, induced pluripotent stem cells, progenitor cells, hematopoietic cells, primary cells, or any combination thereof. In some embodiments, the method includes a first nucleic acid, the first nucleic acid being DNA, RNA, or a hybrid thereof. In some embodiments, the method includes a first nucleic acid, the first nucleic acid being single-stranded or double-stranded. In some embodiments, the method includes a second nucleic acid, the second nucleic acid being DNA, RNA, or a hybrid thereof. In some embodiments, the method includes a second nucleic acid, the second nucleic acid being single-stranded or double-stranded. In some embodiments, the method includes a step of introducing a first nucleic acid, the step of introducing the first nucleic acid including nonviral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method includes viral gene transfer, the viral gene transfer including adeno-associated virus. In some embodiments, the method includes at least one DNA sensing pathway, the at least one DNA sensing protein including 3' repair exonuclease 1 (TREX1), deadbox helicase 41 (DDX41), DAI (DNA-dependent activator of IFN-regulatory factor), Z-type DNA-binding protein 1 (ZBP1), interferon-gamma-inducible protein 16 (IFI16), LRRFIP1 (leucine-rich repeat (In FLII) interacting protein).1) DEAH box helicase 9 (DHX9), DEAH box helicase 36 (DHX36), Ku70 (Lupus Ku autoantigen protein p70), XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6), STING (stimulator of interferon gene), transmembrane protein 173 (TMEM173), TRIM32 (tripartite motif containing 32), TRIM56 (tripartite motif containing 56), β-catenin (CTNNB1), MyD88 (myeloid differentiation primary response 88), AIM2 (absent in melanoma 2), ASC (apoptosis-associated speck-like protein containing a CARD), procaspase 1 (pro-CASP1), caspase 1 (CASP1), prointerleukin 1 beta (pro-IL-1β), prointerleukin 18 (pro-IL-18), interleukin 1 beta (IL-1β), interleukin 18 (IL-18), interferon regulator 1 (IRF1), interferon regulator 3 (IRF3), interferon regulator 7 (IRF7), ISRE7 (interferon-stimulated response element 7), ISRE1 / 7 (interferon-stimulated response element 1 / 7), NF-κB (nuclear factor kappa B), RNA polymerase III (RNA Pol III), melanoma differentiation-related protein 5 (MDA-5), LGP2 (Laboratory of Genetics and Physiology 2), retinoic acid-inducible gene 1 (RIG-I), IPS-1 (mitochondrial antiviral-signaling protein), TNF receptor-related factor 3 (TRAF3), TANK (TRAF family member associated NFKB activator), NAP1 (nucleosome assembly protein 1), TANK-binding kinase 1 (TBK 1) Selected from the group consisting of Atg9a (autophagy-related 9A), tumor necrosis factor alpha (TNF-α), interferon lambda 1 (IFNλ1), phosphorylated forms of these proteins, or any combination or derivative thereof. In some embodiments, the method comprises disruption of at least one DNA sensing pathway, wherein the disruption of at least one DNA sensing pathway comprises at least partial inhibition of at least one DNA sensing protein by an anti-DNA sensing protein. In some embodiments, the method comprises disruption of at least one DNA sensing pathway, wherein the disruption of at least one DNA sensing pathway comprises activation of at least one DNA sensing protein by an anti-DNA sensing protein. In some embodiments, the method comprises at least one anti-DNA sensing protein, which is selected from the group consisting of c-FLiP, HCMV pUL83, DENV NS2B~NS3, HPV18 E7, hAd5 E1A, HSV1 ICP0, VACV B13, VACV C16, TREX1, HCoV-NL63, SARS-CoV, HBV Pol, PEDV, and any combination or derivative thereof. In some embodiments, the method comprises at least one endogenous immune checkpoint gene, which is PD-1. In some embodiments, the method comprises at least one endogenous immune checkpoint gene, the at least one endogenous immune checkpoint gene being an adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activating inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-associated (BTLA), indoleamine 2,3-dioxygenase 1 (IDO1), or KIR3DL1 (killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail).1) Lymphocyte activator gene 3 (LAG3), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS site (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD160), TIGIT (T-cell immunoreceptor with Ig and ITIM domains), CD96 molecule (CD96), CRTAM (cytotoxic and regulatory T-cell (molecule), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), FADD (Fas associated via death domain), FAS (Fas cell surface death Transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI oncogene (SKI), SKI-like oncogene (SKIL), TGIF1 (TGFB-induced factor homeobox 1), programmed cell death 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), interleukin-10 receptor subunit alpha (IL10RA), interleukin-10 receptor subunit beta (IL10RB), HMOX2 (heme oxygenase2) Interleukin 6 receptor (IL6R), IL6ST (interleukin 6 signal transducer), c-src tyrosine kinase (CSK), PAG1 (phosphoprotein membrane anchor with glycosphingoprotein ngolipid microdomains 1), SIT1 (signaling threshold regulating transmembrane adapter 1), FOXP3 (forkhead box P3), PR domain 1 (PRDM1), BATF (basic leucine zipper transcription factor, Selected from the group consisting of ATF-like proteins, soluble guanylate cyclase 1 alpha-2 (GUCY1A2), soluble guanylate cyclase 1 alpha-3 (GUCY1A3), soluble guanylate cyclase 1 beta-2 (GUCY1B2), the prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, or soluble guanylate cyclase 1 beta-3 (GUCY1B3), T cell receptor alpha locus (TRA), T cell receptor beta locus (TRB), egl-9 family hypoxia-inducible factor 1 (EGLN1), egl-9 family hypoxia-inducible factor 2 (EGLN2), egl-9 family hypoxia-inducible factor 3 (EGLN3), PPP1R12C (protein phosphatase 1 regulatory subunit 12C), and any combination or derivative thereof. In some embodiments, the method comprises at least one endogenous immune checkpoint gene, the at least one endogenous immune checkpoint gene comprising a double-strand break. In some embodiments, the method comprises a double-strand break, and the creation of the double-strand break comprises CRISPR. In some embodiments, the method comprises a double-strand break, and the creation of the double-strand break comprises CRISPR, TALEN, transposon-based ZEN, meganuclease, or Mega-TAL. In some embodiments, the method comprises a double-strand break, which is repaired by insertion of a second trans gene encoding the operational TCR. In some embodiments, the method comprises a second nucleic acid, the second nucleic acid comprising recombinant arms, in this case flanking the second trans gene encoding the operational TCR with the recombinant arms. In some embodiments, the method comprises recombinant arms, the recombinant arms at least partially complementary to at least a portion of the at least one endogenous immune checkpoint gene.In some embodiments of the methods of this disclosure, increased isogeneity between the recombinant arm and at least one endogenous immune checkpoint gene corresponds to increased efficiency of insertion of a second transgene. In some embodiments, the method includes insertion of a second transgene, and the efficiency of insertion of the second transgene is measured using fluorescence-activated cell sorting. In some embodiments, the method includes a step of introducing a second nucleic acid, the step of introducing the second nucleic acid includes nonviral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method includes insertion of a second transgene, and the insertion of a second transgene encoding an operational TCR includes homology-guided repair (HDR). In some embodiments, the method includes insertion of a second transgene, and the insertion of the second transgene is assisted by a homologous recombination (HR) enhancer. In some embodiments, the method includes an enhancer, the enhancer is derived from a viral protein. In some embodiments, the method comprises an HR enhancer, which is selected from the group consisting of E4orf6, E1b55K, E1b55K-H354, E1b55K-H373A, Scr7, L755507, or any combination thereof. In some embodiments, the method comprises an HR enhancer, which is a chemical inhibitor. In some embodiments, the method comprises an HR enhancer, which inhibits ligase IV. In some embodiments, the method comprises a reduction in cytotoxicity, which includes at least one of the following, or any combination thereof: DNA cleavage, cell death, apoptosis, nuclear condensation, cell lysis, necrosis, alteration of cell motility, alteration of cell rigidity, alteration of cytoplasmic protein expression, alteration of membrane protein expression, swelling, loss of membrane integrity, cessation of metabolic activity, decrease in metabolic activity, increase in metabolic activity, increase in reactive oxygen species, cytoplasmic contraction.In some embodiments, the method includes measuring viability, which is determined by at least one of the following: fluorescence-activated cell sorting, trypan blue exclusion, CD4+ cell surface marker, CD8+ cell surface marker, telomere length, or these. Measurements are taken using any combination. In some embodiments, the method includes a subject, which is a human subject.

[0051] In some embodiments, the disclosure presents a method for producing a therapeutically effective composition comprising one or more cells. In some embodiments, the method includes measuring the viability of one or more cells after gene editing. In some embodiments, the method comprises gene editing, which includes the step of introducing a first nucleic acid into one or more cells. In some embodiments, the method comprises a first nucleic acid, which comprises a first trans gene encoding at least one anti-DNA sensing protein. In some embodiments, the method comprises at least one DNA sensing pathway, which disrupts at least one DNA sensing pathway in one or more cells with at least one anti-DNA sensing protein. In some embodiments, the method comprises gene editing, which includes the step of introducing a second nucleic acid into one or more cells. In some embodiments, the method comprises a second nucleic acid, which comprises a second trans gene encoding an engineered T cell receptor (TCR). In some embodiments, the method comprises at least one endogenous immune checkpoint gene, which is disrupted in one or more cells by insertion of a second trans gene. In some embodiments, the method comprises disruption of at least one DNA sensing pathway, which reduces the cytotoxic effect induced by the second trans gene, thereby maintaining or increasing the viability of one or more cells. In some embodiments, the method comprises a step of measuring the efficiency of gene editing in one or more cells. In some embodiments, the method comprises a step of calculating the amount of one or more cells required to produce a therapeutic response when administered to a subject. In some embodiments, the method comprises a step of calculating the amount of cells required to produce a therapeutic response, and the calculation of the amount comprises measuring viability and measuring efficiency. In some embodiments, the method comprises a step of contacting the calculated amount of one or more cells with at least one excipient.In some embodiments, the method includes measuring viability, the measurement of viability including at least one of fluorescence-activated cell sorting, trypan blue exclusion, CD4+ cell surface marker, CD8+ cell surface marker, telomere length, or any combination thereof. In some embodiments, the method includes one or more cells, one or more of which are immune cells. In some embodiments, the method includes one or more cells, one or more of which are T cells, naive T cells, CD4+ cells, CD8+ cells, stem cells, induced pluripotent stem cells, progenitor cells, hematopoietic cells, primary cells, or any combination thereof. In some embodiments, the method includes a first nucleic acid, the first nucleic acid being DNA, RNA, or a hybrid thereof. In some embodiments, the method includes a first nucleic acid, the first nucleic acid being single-stranded or double-stranded. In some embodiments, the method includes a second nucleic acid, the second nucleic acid being DNA, RNA, or a hybrid thereof. In some embodiments, the method includes a second nucleic acid, the second nucleic acid being single-stranded or double-stranded. In some embodiments, the method includes a step of introducing a first nucleic acid, the step of introducing the first nucleic acid including nonviral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method includes viral gene transfer, the viral gene transfer including adeno-associated virus. In some embodiments, the method includes at least one DNA sensing pathway, the at least one DNA sensing protein being 3' repair exonuclease 1 (TREX1), deadbox helicase 41 (DDX41), DAI (DNA-dependent activator of IFN-regulatory factor), Z-type DNA-binding protein 1 (ZBP1), interferon-gamma-inducible protein 16 (IFI16), LRRFIP1 (leucine-rich repeat (In FLII) interacting protein 1), DEA. These include H-box helicase 9 (DHX9), DEAH-box helicase 36 (DHX36), Ku70 (Lupus Ku autoantigen protein p70), XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6), STING (stimulator of interferon gene), transmembrane protein 173 (TMEM173), TRIM32 (tripartite motif containing 32), TRIM56 (tripartite motif containing 56), β-catenin (CTNNB1), MyD88 (myeloid differentiation primary response 88), AIM2 (absent in melanoma 2), and ASC (apoptosis-associated speck-like protein containing a CARD), procaspase 1 (pro-CASP1), caspase 1 (CASP1), pro-interleukin 1 beta (pro-IL-1β), pro-interleukin 18 (pro-IL-18), interleukin 1 beta (IL-1β), interleukin 18 (IL-18), interferon regulator 1 (IRF1), interferon regulator 3 (IRF3), interferon regulator 7 (IRF7), ISRE7 (interferon-stimulated response element 7), ISRE1 / 7 (interferon-stimulated response element 1 / 7), NF-κB (nuclear factor kappa B), RNA polymerase III (RNA Pol III), melanoma differentiation-related protein 5 (MDA-5), LGP2 (Laboratory of Genetics and Physiology 2), retinoic acid-inducible gene 1 (RIG-I), IPS-1 (mitochondrial antiviral-signaling TNF receptor-related factor 3 (TRAF3), TANK (TRAF family member associated NFKB)The group consists of an activator, NAP1 (nucleosome assembly protein 1), TANK-binding kinase 1 (TBK1), Atg9a (autophagy-related 9A), tumor necrosis factor alpha (TNF-α), interferon lambda 1 (IFNλ1), phosphorylated forms of these proteins, or any combination or derivative thereof. In some embodiments, the method comprises disruption of at least one DNA sensing pathway, wherein the disruption of at least one DNA sensing pathway comprises at least partial inhibition of at least one DNA sensing protein by an anti-DNA sensing protein. In some embodiments, the method comprises disruption of at least one DNA sensing pathway, wherein the disruption of at least one DNA sensing pathway comprises activation of at least one DNA sensing protein by an anti-DNA sensing protein. In some embodiments, the method comprises at least one anti-DNA sensing protein, where the at least one anti-DNA sensing protein is c-FLiP, HCMV pUL83, DENV NS2B~NS3, HPV18 E7, hAd5 E1A, HSV1 ICP0, VACV B13, VACV C16, TREX1, HCoV-NL63, SARS-CoV, HBVThe group consists of Pol, PEDV, and any combination or derivative thereof. In some embodiments, the method comprises at least one endogenous immune checkpoint gene, the at least one endogenous immune checkpoint gene being PD-1. In some embodiments, the method comprises at least one endogenous immune checkpoint gene, the at least one endogenous immune checkpoint gene being adenosine A2a receptor (ADORA), CD276, V-set domain-containing T-cell activation inhibitor 1 (VTCN1), B-lymphocyte and T-lymphocyte-associated (BTLA), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T-cell activation), natural killer cell receptor 2B4 (CD244), CIS H (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS site (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD160), TIGIT (T-cell immunoreceptor with Ig and ITIM domains), CD96 molecule (CD96), CRTAM (cytotoxic and regulatory T-cell (molecule), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), FADD (Fas associated via death domain), FAS (Fas cell surface death , Transforming Growth Factor Beta Receptor II (TGFBRII), Transforming Growth Factor Beta Receptor I (TGFBR1), SMAD Family Member 2 (SMAD2), SMAD Family Member 3 (SMAD3), SMAD Family Member 4 (SMAD4), SKI Oncogene (SKI), SKI-like Oncogene (SKIL), TGIF1 (TGFB-induced factor homeobox 1), Programmed Cell Death 1 (PD-1), Cytotoxic T Lymphocyte-Associated Protein 4 (CTLA4), Interleukin 10 Receptor Subunit Alpha (IL10RA), Interleukin 10 Receptor Subunit Beta (IL10RB), HMOX2 (heme oxygenase 2), Interleukin 6 Receptor (IL6R), IL6ST (interleukin 6 signal transducer), c-src tyrosine kinase (CSK), PAG1 (phosphoprotein membrane anchor withglycosphingolipid microdomains 1), SIT1 (signaling threshold regulating transmembrane adapter 1), FOXP3 (forkhead box P3), PR domain 1 (PRDM1), BATF (basic leucine zipper transcription factor, ATF-like), soluble guanylate cyclase 1 alpha-2 (GUCY1A2), soluble guanylate cyclase 1 alpha-3 (GUCY1A3), soluble guanylate cyclase 1 beta-2 (GUCY1B2), the protein prolyl hydroxylase domain (PHD1, PHD2, PHD3) family, or soluble guanylate cyclase 1 beta-3 (GUCY1B3), T cell receptor alpha locus (TRA), T cell receptor beta locus (TRB), egl-9 family hypoxia-inducible factor 1 (EGLN1), egl-9 family hypoxia-inducible factor 2 (EGLN2), egl-9 family hypoxia-inducible factor 3 (EGLN3), PPP1R12C (protein phosphatase 1 regulatory subunit12C), and selected from the group consisting of any combination or derivative thereof. In some embodiments, the method comprises at least one endogenous immune checkpoint gene, the at least one endogenous immune checkpoint gene comprising a double-strand break. In some embodiments, the method comprises a double-strand break, and the creation of the double-strand break comprises CRISPR. In some embodiments, the method comprises a double-strand break, and the creation of the double-strand break comprises CRISPR, TALEN, transposon-based ZEN, meganuclease, or Mega-TAL. In some embodiments, the method comprises a double-strand break, and the double-strand break is repaired by insertion of a second trans gene encoding a operative TCR. In some embodiments, the method comprises a second nucleic acid, the second nucleic acid comprising recombinant arms, in this case flanking the second trans gene encoding the operative TCR with the recombinant arms. In some embodiments, the method comprises recombinant arms, the recombinant arms being at least partially complementary to at least a portion of the at least one endogenous immune checkpoint gene. Some embodiments of the methods of the present disclosure In this configuration, increased isogeneity between the recombinant arm and at least one endogenous immune checkpoint gene corresponds to increased efficiency of insertion of the second trans gene. In some embodiments, the method includes insertion of the second trans gene, and the efficiency of gene editing corresponds to the efficiency of insertion of the second trans gene. In some embodiments, the method includes a step of measuring the efficiency of gene editing, and the step of measuring the efficiency of gene editing includes at least one of fluorescence-activated cell sorting, real-time PCR, or Droplet Digital PCR. In some embodiments, the method includes a step of introducing a second nucleic acid, and the step of introducing the second nucleic acid includes nonviral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method includes insertion of the second trans gene, and the insertion of the second trans gene encoding the operational TCR includes homology-guided repair (HDR). In some embodiments, the method includes the insertion of a second transgene, with the insertion of the second transgene being assisted by a homologous recombination (HR) enhancer. In some embodiments, the method includes an enhancer, which is derived from a viral protein. In some embodiments, the method includes an HR enhancer, which is selected from the group consisting of E4orf6, E1b55K, E1b55K-H354, E1b55K-H373A, Scr7, L755507, or any combination thereof. In some embodiments, the method includes an HR enhancer, which is a chemical inhibitor. In some embodiments, the method includes an HR enhancer, which inhibits ligase IV.In some embodiments, the method comprises reducing cytotoxicity, which includes at least one of the following, or any combination thereof: DNA cleavage, cell death, apoptosis, nuclear condensation, cell lysis, necrosis, alteration of cell motility, alteration of cell rigidity, alteration of cytoplasmic protein expression, alteration of membrane protein expression, swelling, loss of membrane integrity, cessation of metabolic activity, decrease in metabolic activity, increase in metabolic activity, increase in reactive oxygen species, cytoplasmic contraction. In some embodiments, the method comprises an amount of one or more cells necessary to produce a therapeutic response, and when administered to a subject, the amount of one or more cells necessary to produce a therapeutic response is approximately 5 × 10⁶ cells. 10 Includes a number of cells. In some embodiments, the method includes an amount of one or more cells necessary to produce a therapeutic response, and when administered to a subject, the amount of one or more cells necessary to produce a therapeutic response is at least about 5 × 10 cells. 7The method includes a number of cells. In some embodiments, the method includes one or more cells, which are living cells. In some embodiments, the method includes a second trans gene, which is inserted into at least one endogenous immune checkpoint gene in one or more cells. In some embodiments, the method includes a subject, which is a human subject. In some embodiments, the method includes a therapeutic response, which includes preventing, reducing, or eliminating cancer in the subject. In some embodiments, the method includes cancer, which is bladder cancer, bone cancer, brain tumor, breast cancer, esophageal cancer, gastrointestinal cancer, hematopoietic malignancy, leukemia, liver cancer, lung cancer, lymphoma, myeloma, ovarian cancer, prostate cancer, sarcoma, stomach cancer, or thyroid cancer. In some embodiments, the method comprises at least one excipient, which is selected from the group consisting of acetate, acid, alcohol, alginate, ammonium, cell culture medium, cellulose, chitosan, collagen, dextran, dextrose, ester, ethanol, gelatin, glucose, glycerol, lactose, mannitol, mannose, mercury compounds, mineral oil, phenol, phosphate, polyacrylic acid, polyethylene glycol (PEG), Ringer's solution, physiological saline, sorbitol, starch, sucrose, vegetable oil, water, petroleum, or combinations thereof. In some embodiments, the method comprises the step of administering to a subject an amount of manipulated cells necessary to produce a therapeutic response in the subject.

[0052] Novel features of the present invention are expressed in detail in the appended claims. Refer to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are used, and the accompanying drawings. This will lead to a better understanding of the features and advantages of the present invention. The present invention provides, for example, the following items: (Item 1) a. Lymphocytes derived from human subjects; b. A polynucleic acid targeting polynucleic acid, which has been engineered to hybridize to a specific region of a target gene within the genome of the lymphocyte; c. A nuclease capable of associating with a polynucleic acid that targets the aforementioned polynucleic acid to form a nuclear protein complex, wherein the nuclear protein complex is a nuclease capable of causing targeted double-strand breaks of the target gene in the genome of the lymphocyte; d. A target polynucleic acid which is genomic DNA containing a double-strand break within the target gene, wherein the double-strand break within the target gene results in the disruption of the target gene function, and when the nuclear protein complex is brought into contact with a population of primary lymphocytes, the disruption of the target gene function occurs with an efficiency of at least 60%, Expanding on this, it is possible to create a clonal population of lymphocytes in which the function of the target gene has been altered, and the clonal population of lymphocytes is a genetically modified immune cell suitable for administration to humans who need it. (Item 2) The endogenous CISH (cytokine-inducible SH2-containing) gene is sequence-dependent and involves at least one further disruption within the endogenous gene, wherein the endogenous gene is related to adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), and KIR3DL1 (killer cell immunoglobulin-like receptor, 3-domain). Modified primary cells selected from the group consisting of: long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS1), and chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5). (Item 3) a) at least one exogenous T cell receptor (TCR); b) Disruption of at least one genome of programmed death ligand 1 (PD-1); c) comprising genomic disruption of at least one TCR alpha (TCRA) chain gene and a TCR beta (TCRB) chain gene, Manipulated cells in which the TCR has been introduced using a lentiviral vector and the genome disruption has been performed using a CRISPR endonuclease system. (Item 4) a. At least one exogenous T cell receptor (TCR) sequence; b. At least one nucleic acid that targets a nucleic acid nuclease complex, i. A targeting nucleic acid comprising at least one sequence substantially complementary to a target genome sequence; and ii. Cells containing nucleic acids, including exogenous endonucleases. (Item 5) At least one exogenous T cell receptor (TCR) sequence; At least one complex, a. At least one manipulated polynucleic acid having a sequence complementary to at least one genomic sequence; and b. Manipulated cells comprising at least one complex containing at least one exogenous endonuclease. (Item 6) a. At least one exogenous T cell receptor (TCR); b. Disruption of at least one genome of programmed death ligand 1 (PD-1); c. comprising at least one genomic disruption of at least one endogenous gene, Manipulated cells in which the TCR has been introduced using a lentiviral vector and the genome disruption has been performed using a CRISPR system. (Item 7) A modified cell comprising the disruption of at least one gene and at least one non-viral integrated T cell receptor (TCR) sequence, wherein the gene is disrupted by the non-viral integrated TCR sequence. (Item 8) Cells described in any of the preceding items, which have been manipulated using a CRISPR nuclease. (Item 9) Cells described in any of the preceding items, which have been manipulated using an AAV vector. (Item 10) Manipulated cells as described in any one of the preceding items, further comprising exogenous receptors. (Item 11) A modified cell as described in any one of the preceding items, wherein the exogenous receptor is selected from the group including T cell receptors (TCRs), chimeric antigen receptors (CARs), or B cell receptors (BCRs). (Item 12) A pharmacological composition containing the cells described in any of the preceding items. (Item 13) Cells described in any of the preceding items that can expand more than 40 times in 12 days. (Item 14) A TIL, which is a cell described in any of the preceding items. (Item 15) A cell that is an immune cell, as described in any of the preceding entries. (Item 16) Human cells, as described in any of the preceding entries. (Item 17) A modified cell as described in any one of the preceding items, wherein the exogenous receptor is selected from the group including T cell receptors (TCRs), chimeric antigen receptors (CARs), or B cell receptors (BCRs). (Item 18) A method for treating a patient in need thereof, comprising the step of administering cells described in any of the preceding items. (Item 19) At least one guide RNA that binds to the endogenous CISH (cytokine-inducible SH2-containing) gene, and adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-associated (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1) Lymphocyte activator gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS site (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene). A composition comprising a secondary guide RNA that binds to an endogenous gene selected from (CCR5). (Item 20) A modified cell as described in any one of the preceding items, wherein the exogenous receptor is selected from the group including T cell receptors (TCRs), chimeric antigen receptors (CARs), or B cell receptors (BCRs). (Item 21) A method for efficient disruption of checkpoint inhibitors in T cells, A step of contacting aT cells with Cas9 nuclease and guide RNA, wherein the guide RNA contains a region of 17-22 nucleotides that is substantially complementary to a region in the target gene; b. A step of cleaving the target gene, wherein the target gene is PD-1, and when a population of primary T cells is brought into contact with Cas9 nuclease and guide RNA, a knockout event occurs in at least 30% of the primary T cells; c. The step of destroying the checkpoint inhibitor in the T cell. A method that includes this. (Item 22) A method for treating an object that requires it, a. The step of collecting lymphocytes from humans; b. A step of genetically modifying lymphocytes in ex vivo by contacting them with a ribonuclease capable of knocking out the function of the PD-1 protein by inducing double-strand breaks within a specific target region of genomic DNA within the lymphocytes, wherein the target region of the genomic DNA within the lymphocytes is located within the PD-1 gene, and the double-strand break occurs within a target region of genomic DNA that is 3' to the region of the target DNA capable of hybridizing to at least 15 nucleotides of the ribonuclease, and 5' to the region of the target DNA containing a protospacer adjacent motif; c. A step of expanding a population of genetically modified lymphocytes having PD-1 protein knockout to create a population of PD-1 knockout T cells; d. A step of administering the population of PD-1 knockout T cells to the subject, wherein the PD-1 knockout T cells are suitable for administration to the patient. A method that includes this. (Item 23) The method according to item 21, wherein the population of genetically modified lymphocytes is expanded for at least 12 days, and the population of genetically modified lymphocytes increases by at least 40 times. (Item 24) The method according to item 22, wherein the population of genetically modified lymphocytes increases by at least 100 times. (Item 25) A method for producing manipulated cells, a. The step of introducing one or more polynucleic acids, each containing at least one exogenous T cell receptor (TCR) sequence, into cells via a nonviral method, with recombinant arms; b. The step of bringing at least one exogenous TCR sequence into contact with a double-strand break region containing a gene. A method that includes this. (Item 26) The method according to item 24, wherein the recombinant arm is complementary to the portion of the gene. (Item 27) The aforementioned gene is adenosine A2a receptor (ADORA), CD276, V set domey This product contains T-cell activating inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related proteins (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activating gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), and VIST. The method described in item 24, which is A (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS site (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5). (Item 28) The method according to item 24, wherein the double-strand break region is repaired by insertion of the at least one exogenous TCR sequence. (Item 29) The method according to item 24, wherein the insertion of the at least one exogenous TCR sequence comprises the disruption of the at least one gene. (Item 30) The method according to item 28, wherein the insertion of at least one exogenous TCR sequence is supported by a homologous recombination (HR) enhancer. (Item 31) The method of item 24, wherein the insertion includes a restoration guided by homology. (Item 32) The method according to any one of the preceding items, wherein the double-strand break is induced by a nuclease. (Item 33) The method according to item 31, wherein the nuclease is selected from the group consisting of Cas9, Argonaute, Cpf1, CRISPR, TALEN, transposase, ZEN, meganuclease, or Mega-TAL. (Item 34) The method according to any one of items 36 to 52, wherein the double-strand break region is created by CRISPR. (Item 35) The method described in item 32, which involves multiplexing the nuclease. (Item 36) The method according to item 34, wherein the multiplexing is carried out by adding at least two guide polynucleic acids. (Item 37) The aforementioned nonviral introduction is a method of any of the preceding items, including electroporation or nucleofection. (Item 38) The method according to any one of the preceding items, wherein the polynucleic acid is co-delivered with at least one modifier that alters the cellular response to the polynucleic acid. (Item 39) A method for facilitating homology-driven restoration (HDR), a. A step of introducing mRNA, reverse transcriptase (RT), enhancer, and primer into cells using a non-viral method; b. The step of reverse transcribing the mRNA into one or more copies of a polynucleic acid; c. A step to facilitate HDR between the genome of the cell and the polynucleic acid. A method that includes this. (Item 40) A method for reducing cytotoxicity of cells to one or more exogenously modified polynucleic acids, comprising the step of modifying one or more cellular responses to the polynucleic acids by contacting the cells with the one or more exogenously modified polynucleic acids, wherein the one or more cellular responses include a cytoplasmic DNA sensing pathway. (Item 41) The step of modifying one or more cellular responses includes DAI (DNA-dependent activator of IFN regulators), IFN-inducible protein 16 (IFI16), DEAD-box polypeptide 41 (DDX41), and AIM2 (absent in melanoma). 2) The method according to item 39, comprising modifying DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), STING (IFN gene stimulator), TANK-binding kinase (TBK1), interleukin-1β (IL-1β), MRE11 (meiotic recombination 11), Trex1, caspase-1 (aspartate-specific cysteine ​​protease), 3' repair exonuclease, DAI (DNA-dependent activator of IRF), IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), MRE11 (meiotic recombination 11) homolog A, and / or IFN regulator (IRF) 3 or 7. (Item 42) a. The step of bringing cells into contact with one or more signal transduction modifying compounds; b. A method for genome manipulation, comprising the step of contacting the cells with a polynucleic acid comprising at least one antigen receptor sequence sandwiched between at least two recombinant arms complementary to at least one genomic region. (Item 43) The method according to item 39, wherein the one or more signal transduction modifying compounds alter the cytoplasmic DNA sensing pathway. (Item 44) The one or more signal transduction modifying compounds mentioned above include DAI (DNA-dependent activator of IFN regulators), IFN-inducible protein 16 (IFI16), DEAD-box polypeptide 41 (DDX41), AIM2 (absent in melanoma 2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), STING (IFN gene stimulator), TANK-binding kinase (TBK1), interleukin 1β (IL-1β), MRE11 (meiotic recombination 11), Trex1, caspase 1 (aspartate-specific cysteine ​​protease), 3' repair exonuclease, and DAI (DNA-dependent activator of The method described in item 39, which modifies IRF, IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), MRE11 (meiotic recombination 11) homolog A, and / or IFN regulator (IRF) 3 or 7. (Item 45) A method for creating manipulated cells, a. To cells, i. A guide polynucleotide comprising a spacer region complementary to the target nucleic acid within the genomic region of the cell; ii. Nucleases guided by the aforementioned guide polynucleotides; and iii. Polynucleotides encoding exogenous T cell receptors Steps to implement; b. The step of site-specifically cleaving the target nucleic acid within the cell by the nuclease guided by the guide polynucleotide; c. The step of inserting the polynucleotide encoding the exogenous T cell receptor into the genomic region of the cell at the cleavage site. A method that includes this. (Item 46) The method according to item 44, wherein the gene is PD-1. (Item 47) The method according to item 44, wherein insertion of the exogenous TCR sequence at the cleavage site results in the disruption of the gene. (Item 48) The method according to item 44, further comprising the step of expressing the exogenous T cell receptor within the cell. (Item 49) The method according to any one of the preceding items, further comprising the step of introducing the manipulated cells into an organism. (Item 50) The method according to any one of the preceding items, further comprising the step of expanding the manipulated cells ex vivo. (Item 51) A method for producing manipulated cells, a. The step of introducing at least one exogenous T cell receptor (TCR) into at least one genome of the cell by a virus; b. A step of disrupting the genome by disrupting at least one endogenous gene; c. A step of genome disruption of at least one immune checkpoint gene and Includes, The method by which the genome disruption is adjacent to a protospacer-adjacent motif (PAM) sequence of the cell. (Item 52) A method for producing manipulated cells, a. The step of introducing at least one polynucleic acid encoding at least one exogenous T cell receptor (TCR) sequence by a virus; b. A step of genomically disrupting at least one gene with at least one endonuclease or a functional portion thereof. A method that includes this. (Item 53) A method for producing manipulated cells, a) The step of introducing at least one polynucleic acid encoding at least one exogenous T cell receptor (TCR) sequence; b) The step of introducing at least one guide RNA (gRNA) containing at least one modification; c) A step of introducing at least one endonuclease and Includes; The gRNA comprises at least one sequence that is complementary to at least one endogenous genome. method. [Brief explanation of the drawing]

[0053] [Figure 1] Figure 1 is a diagram showing an overview of some of the methods disclosed herein.

[0054] [Figure 2] Figure 2 shows several exemplary transposon constructs for TCR transgene integration and TCR expression.

[0055] [Figure 3] Figure 3 illustrates the transcription of mRNA in vitro and its use as a template for producing homologous recombination (HR) substrates within any cell type (e.g., primary cells, cell lines, etc.). For in vitro transcription of the viral cassette, T7, T3, or other transcription start sequences can be placed upstream of the 5'LTR region of the viral genome. Yields can be improved by using mRNA encoding both the sense and antisense strands of the viral vector.

[0056] [Figure 4] Figure 4 shows the structures of four plasmids, including the Cas9 nuclease plasmid, HPRT gRNA plasmid, Amaxa EGFPmax plasmid, and HPRT target vector.

[0057] [Figure 5] Figure 5 shows an exemplary HPRT target vector with a 0.5kb targeting arm.

[0058] [Figure 6]Figure 6 illustrates three potential TCR trans gene knock-in designs targeting an exemplary gene (e.g., the HPRT gene): (1) exogenous promoter: transcription of the TCR trans gene ("TCR") by an exogenous promoter ("promoter"); (2) in-frame splice acceptor (SA) transcription: transcription of the TCR trans gene by an endogenous promoter via splicing (indicated by the arrow); and (3) in-frame fusion translation: transcription of the TCR trans gene by an endogenous promoter via in-frame translation. All three exemplary designs can knock out gene function. For example, when knocking out the HPRT gene or the PD-1 gene by insertion of a TCR trans gene, 6-thioguanine selection can be used as a selection assay.

[0059] [Figure 7] Figure 7 illustrates that co-transfection of Cas9 + gRNA + target plasmid resulted in good transfection efficiency within the bulk population.

[0060] [Figure 8] Figure 8 shows the results of EGFP FACS analysis for CD3+ T cells, supporting the findings.

[0061] [Figure 9] Figure 9 shows two types of T cell receptors.

[0062] [Figure 10] Figure 10 shows the achievement of T cell transfection efficiency using two platforms.

[0063] [Figure 11] Figure 11 shows the efficient transfection when the number of T cells is scaled up, for example, when the number of T cells is increased.

[0064] [Figure 12] Figure 12 shows the percentage of gene modification caused by CRISPR gRNA at potential target sites.

[0065] [Figure 13] Figure 13 illustrates CRISPR-induced double-segment brain blocks (DSBs) in stimulated T cells.

[0066] [Figure 14] Figure 14 shows the optimization of RNA delivery.

[0067] [Figure 15] Figure 15 illustrates double-strand breaks at the target site. Gene targeting successfully induced double-strand breaks in T cells activated with anti-CD3 and anti-CD28 before introducing the targeted CRISPR-Cas system. For illustrative purposes, the system was validated using the immune checkpoint genes PD-1, CCR5, and CTLA4.

[0068] [Figure 16] Figure 16 shows the integration of the TCR in CCR5. It is an exemplary design of a plasmid targeting vector for CCR5 with a 1kb recombination arm. To target other target genes using homologous recombination, a 3kb trans gene for TCR expression can be inserted into a similar vector with a recombination arm to a different gene. Successful TCR integration in the gene can be confirmed by PCR analysis using primers outside the recombination arm.

[0069] [Figure 17] Figure 17 illustrates the integration of the TCR into the CCR5 gene within stimulated T cells. The positive PCR result confirms successful homologous recombination in the CCR5 gene 72 hours after transfection.

[0070] [Figure 18] Figure 18 shows the death of T cells in response to plasmid DNA transfection.

[0071] [Figure 19] Figure 19 is a schematic diagram of innate immunosensing pathways of cytoplasmic DNA present in different cell types, including but not limited to T cells. T cells express both pathways to detect foreign DNA. Cytotoxicity can arise from the activation of these pathways during genomic manipulation.

[0072] [Figure 20] Figure 20 illustrates how the inhibitor shown in Figure 19 blocks apoptosis and pyroptosis.

[0073] [Figure 21] Figure 21 shows a schematic diagram of typical plasmid modifications. Standard plasmids contain bacterial methylation, which can trigger the innate immune sensing system. Removing bacterial methylation can reduce the toxicity caused by standard plasmids. Alternatively, bacterial methylation can be removed and mammalian methylation added so that the vector resembles "self-DNA". Modifications may also include the use of synthetic single-stranded DNA.

[0074] [Figure 22] Figure 22 shows a representative functionally modified TCR antigen receptor. This modified TCR is highly reactive to melanoma cell lines expressing MART-1. The TCRα chain and TCRβ chain are linked by a 2A ribosome skipping peptide following the furin cleavage site.

[0075] [Figure 23]Figures 23A and 23B show the expression of PD-1, CTLA-4, PD-1 and CTLA-2, or CCR5, PD-1 and CTLA-4 at day 6 after transfection with guide RNA. Representative guides are PD-1 (P2, P6, P2 / 6), CTLA-4 (C2, C3, C2 / 3), or CCR5 (CC2). A shows the percentage expression of inhibitory receptors. B shows the expression of inhibitory receptors normalized against control guide RNA.

[0076] [Figure 24] Figures 24A and 24B show the expression of CTLA-4 in primary human T cells after electroporation with CRISPR and guides #2 and #3, which are CTLA-4-specific guide RNAs, compared to an unstained, unguided control. Figure B shows the expression of PD-1 in primary human T cells after electroporation with CRISPR and guides #2 and #6, which are PD-1-specific guide RNAs, compared to an unstained, unguided control.

[0077] [Figure 25] Figure 25 shows the FACS results for CTLA-4 and PD-1 expression in primary human T cells after electroporation with CRISPR and multiplexed CTLA-4 guide RNA and PD-1 guide RNA.

[0078] [Figure 26]Figures 26A and 26B show the dual knockout percentages in primary human T cells after CRISPR treatment. A shows the CTLA-4 knockout percentages in T cells treated with CTLA-4 guides #2, #3, #2 and #3, PD-1 guide #2 and CTLA-4 guide #2, PD-1 guide #6 and CTLA-4 guide #3, compared to Zap alone, Cas9 alone and all guide RNA controls. B shows the PD-1 knockout percentages in T cells treated with PD-1 guide #2, PD-1 guide #6, PD-1 guides #2 and #6, PD-1 guide #2 and CTLA-4 guide #2, PD-1 guide #6 and CTLA-4 guide #3, compared to Zap alone, Cas9 alone and all guide RNA controls.

[0079] [Figure 27] Figure 27 shows the survival rates of T cells after electroporation with guide RNAs specific to CRISPR and CTLA-4, PD-1, or combinations thereof.

[0080] [Figure 28] Figure 28 shows the results of the CEL-I assay, illustrating cleavage by PD-1 guide RNA #2, #6, #2, and #6 under the conditions of introducing only PD-1 guide RNA, introducing PD-1 guide RNA and CTLA-4 guide RNA, introducing CCR5 guide RNA, PD-1 guide RNA, and CTLA-4 guide RNA, under the control condition of using Zap alone, or under the control condition of using gRNA alone.

[0081] [Figure 29]Figure 29 shows the results of the CEL-I assay, illustrating cleavage by CTLA-4 guide RNA #2, #3, #2 and #3 under the conditions of introducing only CTLA-4 guide RNA, introducing PD-1 guide RNA and CTLA-4 guide RNA, introducing CCR5 guide RNA, PD-1 guide RNA, and CTLA-4 guide RNA, under the control condition of using Zap alone, or under the control condition of using gRNA alone.

[0082] [Figure 30] Figure 30 shows the results of the CEL-I assay, comparing the cleavage by CCR5 guide RNA #2 under conditions where CCR5 guide RNA is introduced, and under conditions where CCR5 guide RNA, PD-1 guide RNA, or CTLA-4 guide RNA is introduced, with control conditions using Zap alone, Cas9 alone, or guide RNA alone.

[0083] [Figure 31] Figure 31 shows TCR alpha knockout in primary human T cells using optimized CRISPR guide RNA with 2'O-methylRNA modification at doses of 5 micrograms and 10 micrograms, as measured by CD3 expression in FACS.

[0084] [Figure 32] Figure 32 illustrates the method for measuring T cell viability and phenotype after treatment with CRISPR and CTLA-4 guide RNA. Phenotype was measured by quantifying the frequency of treated cells exhibiting a normal FSC / SSC profile, standardized against the frequency of a control obtained by electroporation alone. Viability was also measured by excluding viability dyes from cells within an FSC / SSC-gated population. T cell phenotype was measured by CD3 and CD62L.

[0085] [Figure 33]Figure 33 shows a method for measuring T cell viability and phenotype after treatment with CRISPR, guide RNA to PD-1, and guide RNA to PD-1 and CTLA-4. Phenotype was measured by quantifying the frequency of treated cells exhibiting a normal FSC / SSC profile, standardized against the frequency of a control obtained by electroporation alone. Viability was also measured by excluding viability dyes from cells within an FSC / SSC-gated population. T cell phenotype was measured by CD3 and CD62L.

[0086] [Figure 34] Figure 34 shows the results of a T7E1 assay to detect CRISPR gene editing in primary human T cells and Jurkat controls four days after transfection with PD-1 guide RNA or CTKA-4 guide RNA. NN represents a control without T7E1 nuclease.

[0087] [Figure 35] Figure 35 shows the results of TIDE (tracking of indels by decomposition) analysis. The gene editing efficiency percentages are shown for PD-1 guide RNA and CTLA-4 guide RNA.

[0088] [Figure 36] Figure 36 shows the results of TIDE (tracking of indels by decomposition) analysis for single guide transfection. The percentage of sequences with deletions or insertions is shown for primary human T cells transfected with PD-1 guide RNA or CTLA-1 guide RNA and CRISPR.

[0089] [Figure 37] Figure 37 shows the deletion of the PD-1 sequence by dual targeting.

[0090] [Figure 38] Figure 38 shows the sequencing results of PCR products for PD-1 sequence deletions by dual targeting. Samples 6 and 14 are shown as fusions of two gRNA sequences with an intervening 135 bp excised.

[0091] [Figure 39] Figure 39 shows the deletion of the CTLA-4 sequence by dual targeting. Deletions between the two guide RNA sequences are also present in the sequencing of CTLA-4 targeted by dual guides (samples 9 and 14). The T7E1 assay confirms the deletion by PCR.

[0092] [Figure 40] Figures 40A and 40B show FACS analysis of human T cell viability (A) 6 days after CRISPR transfection and human T cell transfection efficiency (GFP-positive %).

[0093] [Figure 41] Figure 41 shows a FACS analysis of CTLA-4 expression in stained human T cells transfected with anti-CTLA-4 CRISPR guide RNA. PE is anti-human CD152(CTLA-4).

[0094] [Figure 42] Figures 42A and 42B show CTLA-4 FACS analysis of CTLA-4-positive human T cells after transfection with anti-CTLA-4 guide RNA and CRISPR. Figure B shows the CTLA-4 knockout efficiency in human T cells compared to pulsed control after transfection with anti-CTLA-4 guide RNA and CRISPR.

[0095] [Figure 43] Figure 43 shows a minicircle DNA containing the manipulated TCR.

[0096] [Figure 44] Figure 44 is a diagram illustrating modified sgRNAs for CISH, PD-1, CTLA4, and AAVS1.

[0097] [Figure 45] Figure 45 illustrates the FACS results for PD-1 KO at 14 days post-transfection with CRISPR and anti-PD-1 guide RNA. PerCP-Cy5.5 is mouse anti-human CD279 (PD-1).

[0098] [Figure 46] Figures 46A and 46B show, A. the percentage of PD-1 expression after transfection with an anti-PD-1 CRISPR system, and B. the percentage of PD-1 knockout efficiency compared to a control using Cas9 alone.

[0099] [Figure 47] Figure 47 shows FACS analysis of FSC / SSC subsets of human T cells transfected with the CRISPR system, along with anti-PD-1 guide #2, anti-PD-1 guide #6, anti-PD1 guides #2 and #6, or anti-PD-1 guides #2 and #6 and anti-CTLA-4 guides #2 and #3.

[0100] [Figure 48] Figure 48 shows FACS analysis of human T cells 6 days after transfection with CRISPR and anti-CTLA-4 guide RNA. The PE is mouse anti-human CD152 (CTLA-4).

[0101] [Figure 49]Figure 49 shows FACS analysis of human T cells and control Jurkat cells on day 1 after transfection with CRISPR, anti-PD-1 guide RNA, and anti-CTLA-4 guide RNA. The viability and transfection efficiency of human T cells are shown compared to transfected Jurkat cells.

[0102] [Figure 50] Figure 50 illustrates quantitative data obtained from FACS analysis of CTLA-4 stained human T cells transfected with CRISPR and anti-CTLA-4 guide RNA. It shows data for CTLA-4 expression percentage and knockout percentage 6 days post-transfection.

[0103] [Figure 51] Figure 51 shows FACS analysis of PD-1 stained human T cells transfected with CRISPR and anti-PD-1 guide RNA. Data on PD-1 expression (anti-human CD279 PerCP-Cy5.5) at 14 days post-transfection are shown.

[0104] [Figure 52] Figure 52 shows the percentage of PD-1 expression and PD-1 knockout in human T cells transfected with CRISPR and anti-PD-1 guide RNA, compared to a control mediated by Cas9 alone.

[0105] [Figure 53] Figure 53 shows the cell count and viability of human T cells transfected with CRISPR, anti-CTLA-4, and anti-PD-1 guide RNA on day 14.

[0106] [Figure 54]Figure 54 shows FACS data for human T cells 14 days after electroporation using CRISPR, as well as anti-PD-1 guide #2 alone, anti-PD-1 guides #2 and #6, or anti-CTLA-4 guide #3 alone. Manipulated T cells were restimulated for 48 hours, and CTLA-4 and PD-1 expression was evaluated and compared with control cells electroporated without guide RNA.

[0107] [Figure 55] Figure 55 shows FACS data for human T cells 14 days after electroporation with CRISPR, as well as anti-CTLA-4 guides #2 and #3, anti-PD-1 guide #2 and anti-CTLA-4 guide #3, or anti-PD-1 guides #2 and #6, anti-CTLA-4 guides #3 and #2. Manipulated T cells were restimulated for 48 hours, and CTLA-4 and PD-1 expression was evaluated and compared with control cells electroporated without guide RNA.

[0108] [Figure 56] Figure 56 illustrates the results of a Surveyor assay for CRISPR-mediated gene modification at the CISH locus in primary human T cells.

[0109] [Figure 57] Figures 57A, 57B, and 57C show the following: A. is a schematic diagram of the T cell receptor (TCR); B. is a schematic diagram of the chimeric antigen receptor; and C. is a schematic diagram of the B cell receptor (BCR).

[0110] [Figure 58] Figure 58 shows that the mutagenesis load of somatic cells varies among tumor types. Theoretically, the generation and presentation of tumor-specific neoantigens are directly proportional to the mutagenesis load.

[0111] [Figure 59]Figure 59 shows pseudouridine-5'-triphosphate modification and 5-methylcytidine-5'-triphosphate modification that can be applied to nucleic acids.

[0112] [Figure 60] Figure 60 shows a comparison of TIDE and density measurement data for 293T cells transfected with CRISPR and CISH gRNA1, 3, 4, 5, or 6.

[0113] [Figure 61] Figure 61 illustrates a series of experiments involving density measurements of 293T cells transfected with CRISPR and CISH gRNA1, 3, 4, 5, or 6.

[0114] [Figure 62] Figures 62A and 62B show the results of dual TIDE analyses A and B for CISH gRNA 1.

[0115] [Figure 63] Figures 63A and 63B show the results of dual TIDE analyses A and B for CISH gRNA 3.

[0116] [Figure 64] Figures 64A and 64B show the results of dual TIDE analyses A and B for CISH gRNA 4.

[0117] [Figure 65] Figures 65A and 65B show the results of dual TIDE analyses A and B for CISH gRNA 5.

[0118] [Figure 66] Figures 66A and 66B show the results of dual TIDE analyses A and B for CISH gRNA 6.

[0119] [Figure 67] Figure 67 shows a Western blot illustrating the loss of CISH protein after CRISPR knockout in primary T cells.

[0120] [Figure 68] Figures 68A, 68B, and 68C show DNA viability by cell number, illustrating the DNA viability at A. 1 day, B. 2 days, and C. 3 days after transfection of single-stranded or double-stranded DNA. The ss / dsDNA of M13 is 7.25 kb. pUC57 is 2.7 kb. The GFP plasmid is 6.04 kb.

[0121] [Figure 69] Figure 69 shows the mechanistic pathways that can be modulated during or after the preparation of manipulated cells.

[0122] [Figure 70]Figures 70A and 70B show the cell counts after transfection with the CRISPR system (15ug Cas9, 10ug gRNA) on day 3 and day 7, respectively. Sample 1: Untreated. Sample 2: Pulsed only. Sample 3: GFP mRNA. Sample 4: Pulsed only Cas9. Sample 5: Pulsed only 5 microgram minicircle donor. Sample 6: Pulsed only 20 microgram minicircle donor. Sample 7: Plasmid donor (5 micrograms). Sample 8: Plasmid donor (20 micrograms). Sample 9: +guide PD1-2 / +Cas9 / -donor. Sample 10: +guide PD1-6 / +Cas9 / -donor. Sample 11: +guide CTLA4-2 / +Cas9 / -donor. Sample 12: +guide CTLA4-3 / +Cas9 / -donor. Sample 13: Donor of PD1-2 / 5ug. Sample 14: Donor of PD1 (double) / 5ug. Sample 15: Donor of CTLA4-3 / 5ug. Sample 16: Donor of CTLA4 (double) / 5ug. Sample 17: Donor of PD1-2 / 20ug. Sample 18: Donor of PD1 (double) / 20ug. Sample 19: Donor of CTLA4-3 / 20ug. Sample 20: Donor of CTLA4 (double) / 20ug.

[0123] [Figure 71] Figures 71A and 71B show the TIDE analysis of A.gRNA 2 and B.gRNA 6 in PD1 without donor nucleic acid on day 4.

[0124] [Figure 72] Figures 72A and 72B show the TIDE analysis of A.gRNA 2 and B.gRNA 3 of CTLA4 without donor nucleic acid on day 4.

[0125] [Figure 73]Figure 73 shows the FACS analysis for the detection of TCR beta on day 7 in control cells, cells electroporated with 5 micrograms of donor DNA (minicircles), or cells electroporated with 20 micrograms of donor DNA (minicircles).

[0126] [Figure 74] Figure 74 shows an overview of T cells on day 7 after electroporation with the CRISPR system and with no polynucleotide donor (control), 5 micrograms of polynucleotide donor (minicircle), or 20 micrograms of polynucleotide donor (minicircle). An overview of FACS analysis of TCR-positive cells is shown.

[0127] [Figure 75] Figure 75 shows the integration of the TCR minicircle into the PD1 gRNA#2 cleavage site in the forward direction.

[0128] [Figure 76] Figures 76A and 76B show the percentage of viable cells on day 4 using a GUIDE-Seq administration test for human T cells transfected with CRISPR and PD-1 gRNA or CISH gRNA with 5' or 3' modifications (or both), at increased double-stranded polynucleotide donor concentrations. B shows the integration efficiency at the PD-1 or CISH locus of human T cells transfected with CRISPR and PD-1-specific gRNA or CISH-specific gRNA.

[0129] [Figure 77] Figure 77 shows the GoTaq and PhusionFlex analyses of dsDNA integration at PD-1 gene sites or CISH gene sites.

[0130] [Figure 78]Figure 78 is a diagram showing FACS analysis on day 15 for human T cells transfected with 5 micrograms or 20 micrograms of minicircle DNA encoding CRISPR and an exogenous TCR.

[0131] [Figure 79] Figure 79 is a diagram showing an overview of T cells on day 15 that were electroporated with a CRISPR system and without a polynucleotide donor (control), 5 micrograms of a polynucleotide donor (minicircle), or 20 micrograms of a polynucleotide donor (minicircle). An overview of FACS analysis for TCR-positive cells is shown.

[0132] [Figure 80] Figure 80 is a diagram depicting the copy number relative to RNaseP based on digital PCR copy number data on day 4 after transfection of CRISPR and a minicircle encoding the mTCRb chain. A plasmid donor encoding the mTCRb chain was used as a control.

[0133] [Figure 81] Figures 81A and 81B are diagrams showing: A. the viability of T cells on day 3 when increasing the dose of a minicircle encoding an exogenous TCR; B. the viability of T cells on day 7 when increasing the dose of a minicircle encoding an exogenous TCR.

[0134] [Figure 82] Figures 82A and 82B are diagrams showing: A. the optimized conditions for transfection of double-stranded DNA into T cells by Lonza nucleofection (comparing the cell number with the concentration of a plasmid encoding GFP); B. the optimized conditions for Lonza nucleofection of double-stranded DNA encoding the GFP protein into T cells (showing the percentage of gene transfer in contrast to the concentration of the GFP plasmid used for transfection).

[0135] [Figure 83] Figures 83A and 83B are diagrams depicting the pDG6-AAV helper-free packaging plasmid for A. AAV TCR delivery. B. is a diagram showing a schematic of the protocol for transient transfection of AAV into 293 cells for virus production. The virus is purified and stored for gene transfer into primary human T cells.

[0136] [Figure 84] Figure 84 shows an rAAV donor encoding an exogenous TCR flanked by 900 bp homology arms for endogenous immune checkpoints (CTLA4 and PD1 shown as exemplary examples).

[0137] [Figure 85] Figure 85 shows a schematic of genomic integration for an rAAV homologous recombinant donor encoding an exogenous TCR flanked by homology arms to the AAVS1 gene.

[0138] [Figure 86] Figures 86A, 86B, 86C, and 86D are diagrams showing possible recombination events that can occur using the AAVS1 system. A. shows homology-directed repair of a double-strand break in AAVS1 by integration of a transgene. B. shows homology-directed repair by one strand of the AAVS1 gene and a non-homologous end-joining indel of the complementary strand of AAVS1. C. shows non-homologous end-joining insertion of a transgene into the AAVS1 gene site and a non-homologous end-joining indel in AAVS1. D. shows non-homologous indels at both AAVS1 positions, with random integration of the transgene into the genomic site.

[0139] [Figure 87]Figure 87 shows a combination of CRISPR targeting and rAAV targeting, which involve introducing a trans gene encoding an exogenous TCR into an immune checkpoint gene.

[0140] [Figure 88] Figures 88A and 88B show data from day 3. A. shows a CRISPR electroporation experiment using caspase and TBK inhibitors during electroporation of a 7.5 microgram minicircle donor encoding an exogenous TCR. Survival rates are plotted against the concentrations of inhibitors used. B. shows the efficiency of electroporation. The percentage of positive TCRs is shown against the concentrations of inhibitors used.

[0141] [Figure 89] Figure 89 shows FACS data for human T cells electroporated with CRISPR and minicircle DNA (7.5 micrograms) encoding exogenous TCRs. Caspase and TBK inhibitors were added during electroporation.

[0142] [Figure 90] Figures 90A and 90B show FACS data for human T cells electroporated with CRISPR and minicircle DNA (20 micrograms) encoding exogenous TCRs. A shows the electroporation efficiency of TCR-positive cells compared to the immune checkpoint-specific guide used. B shows the FACS data for electroporation efficiency of TCR-positive cells compared to the immune checkpoint-specific guide used.

[0143] [Figure 91] Figure 91 shows the expression of CRISPR and exogenous TCRs in minicircles encoding the exogenous TCR, which vary the concentration of the minicircles, 13 days after electroporation.

[0144] [Figure 92] Figures 92A and 92B are diagrams showing a cell death inhibitor study in which human T cells were pretreated with brefeldin A and an ATM inhibitor prior to transfection with CRISPR and minicircle DNA encoding an exogenous TCR. A. is a diagram showing the survival rate of T cells on the 3rd day after electroporation. B. is a diagram showing the survival rate of T cells on the 7th day after electroporation.

[0145] [Figure 93] Figures 93A and 93B are diagrams showing a cell death inhibitor study in which human T cells were pretreated with brefeldin A and an ATM inhibitor prior to transfection with CRISPR and minicircle DNA encoding an exogenous TCR. A. is a diagram showing the expression of TCR on T cells on the 3rd day after electroporation. B. is a diagram showing the expression of TCR on T cells on the 7th day after electroporation.

[0146] [Figure 94] Figure 94 is a diagram showing a splice acceptor GFP reporter assay for rapidly detecting the integration of an exogenous transgene (e.g., TCR).

[0147] [Figure 95] Figure 95 is a diagram showing a locus-specific digital PCR assay for rapidly detecting the integration of an exogenous transgene (e.g., TCR).

[0148] [Figure 96] Figure 96 is a diagram showing a recombinant (rAAV) donor construct encoding an exogenous TCR using a PGK promoter or a splice acceptor. Each construct is flanked by 850 base pair homology arms (HAs) to the AAVS1 checkpoint gene.

[0149] [Figure 97]Figure 97 shows the rAAV AAVS1-TCR gene targeting vector. It is a schematic depiction of the rAAV targeting vector used to insert a transgenic TCR expression cassette into the AAVS1 "safe harbor" locus within the intron region of the PPP1R12C gene. Key features are shown along with their nucleotide (bp) size. ITR: Internal tandem repeat; PGK: Phosphoglycerate kinase; mTCR: Mouse T cell receptor beta; SV40 polyA: Monkey virus 40 polyadenylation signal.

[0150] [Figure 98] Figure 98 shows T cells electroporated with the GFP+ trans gene 48 hours after stimulation with modified gRNA. The gRNA was modified with pseudouridine, 5'moC, 5'meC, 5'moU, 5'hmC+5'moU, m6A, or 5'moC+5'meC.

[0151] [Figure 99] Figures 99A and 99B illustrate the viability (A) and MFI (Mass First Intake) (B) of GFP-expressing T cells electroporated with the GFP+ trans gene 48 hours after stimulation with modified gRNA. The gRNAs were modified with pseudouridine, 5'moC, 5'meC, 5'moU, 5'hmC+5'moU, m6A, or 5'moC+5'meC.

[0152] [Figure 100] Figures 100A and 100B show the TIDE results for a comparison between A. modified cap-removed Cas9 protein and B. unmodified Cas9 protein. Genomic integration was measured at the CCR5 locus of T cells electroporated with 15 micrograms of Cas9 and 10 micrograms of chemically modified gRNA, for either unmodified Cas9 or cap-removed Cas9.

[0153] [Figure 101]Figures 101A and 101B show Jurkat cells expressing reverse transcriptase (RT) reporter RNA, transfected with plasmids and primers encoding RT (see table for concentrations) using the Neon Transfection System, and assayed for cell viability and GFP expression on day 3 post-transfection. A. Viability, B. Reverse transcriptase activity. GFP-positive cells represent cells with RT activity.

[0154] [Figure 102] Figures 102A and 102B show the absolute cell count of human TILs before and after stimulation. A shows the cell count of the first donor, cultured in RPMI medium or ex vivo medium, before and after stimulation. B shows the cell count of the second donor, cultured in RPMI medium, before and after stimulation.

[0155] [Figure 103] Figures 103A and 103B show cell expansion of human tumor-infiltrating lymphocytes (TILs) or control cells electroporated with a CRISPR system targeting the PD-1 locus, with or without the addition of an autologous feeder (A).

[0156] [Figure 104] Figures 104A and 104B show human T cells electroporated with the following proteins: CRISPR system alone (control); GFP plasmid (donor) alone (control); donor and CRISPR system; donor, CRISPR, and cFLP protein; donor, CRISPR, and hAd5 E1A (E1A) protein; or donor, CRISPR, and HPV18 E7 protein. FACS analysis for GFP was measured at A. 48 hours or B. 8 days after electroporation. [Modes for carrying out the invention]

[0157] Detailed explanation of disclosure The following description and examples illustrate embodiments of the present invention in detail. It should be understood that the present invention is not limited to the specific embodiments described herein and is therefore subject to change. Those skilled in the art will recognize that numerous variations and modifications of the present invention fall within its scope.

[0158] definition As used herein, the term “about” and its grammatical equivalents with respect to base numbers and their grammatical equivalents may include values ​​within a range of plus or minus 10% from that value. For example, the quantity “about 10” includes quantities from 9 to 11. The term “about” with respect to base numbers may also include values ​​within a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.

[0159] As used herein, the term “activation” and its grammatical equivalent may refer to the process by which a cell transitions from a dormant state to an active state. This process may include a response to an antigen, migration, and / or phenotypic or genetic changes to a functionally active state. For example, the term “activation” may refer to the stepwise process of T cell activation. For example, a T cell may require at least two signals to be fully activated. The first signal may occur after the engagement of an antigen-MHC complex to the TCR, and the second signal may occur due to the engagement of a co-stimulatory molecule. In vitro, anti-CD3 may mimic the first signal, and anti-CD28 may mimic the second signal.

[0160] As used herein, the term “adjacent” and its grammatical equivalents may refer to the immediate vicinity of the reference object. For example, in the context of nucleotide sequences, the term “adjacent” means that there are no nucleotides in between. For instance, polynucleotide A adjacent to polynucleotide B may mean A and B, with no nucleotides between A and B.

[0161] As used herein, the term “antigen” and its grammatical equivalent may refer to a molecule containing one or more epitopes to which one or more receptors can bind. For example, an antigen may stimulate the host’s immune system to produce a cellular antigen-specific immune response, or a humoral antibody response, when the antigen is presented. Antigens may also have the ability to induce cellular and / or humoral responses, either by themselves or in combination with other molecules. For example, tumor cell antigens may be recognized by TCRs.

[0162] As used herein, the term “epitope” and its grammatical equivalent may refer to a portion of an antigen that can be recognized by an antibody, B cell, T cell, or manipulated cell. For example, an epitope may be a cancer epitope recognized by a TCR. Multiple epitopes within an antigen can also be recognized. Epitopes may also be mutated.

[0163] As used herein, the term “autologous” and its grammatical equivalents may refer to originating from the same entity. For example, a sample (e.g., cells) can be collected, processed, and later returned to the same entity (e.g., a patient). Autologous processes are distinguished from homogeneous processes, where the donor and recipient are different entities.

[0164] The term "barcoded to ~" refers to a relationship between molecules where the first molecule contains a barcode that can be used to identify the second molecule.

[0165] As used herein, the term "cancer" and its grammatical equivalents may refer to the excessive proliferation of cells whose inherent characteristics (loss of normal control) result in unregulated growth, lack of differentiation, localized tissue invasion, and metastasis. In connection with the methods of the present invention, cancer includes acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain tumor, breast cancer, anal cancer, anal canal cancer, rectal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, cervical cancer, gallbladder cancer, or pleural cancer, nasal cancer, nasal cavity cancer, or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharyngeal cancer. It may be any cancer, including any of the following: head cancer, kidney cancer, laryngeal cancer, leukemia, humoral neoplasm, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, reticular cancer, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and / or bladder cancer. As used herein, the term “tumor” refers to an abnormal proliferation of cells or tissue, for example, malignant or benign proliferation.

[0166] As used herein, the terms “cancer neoantigen,” “neoantigen,” or “neoepitope” and their grammatical equivalents may refer to antigens not encoded within the normal, unmutated host genome. A “neoantigen” may, in some cases, represent a tumorigenic viral protein or an abnormal protein arising as a result of somatic mutation. For example, a neoantigen may arise from the disruption of cellular mechanisms via the activity of a viral protein. Another example is exposure to a carcinogenic compound, which may, in some cases, result in somatic mutation, which may ultimately lead to tumor / cancer formation.

[0167] As used herein, the term “cytotoxic effect” refers to an unintended or unwanted alteration of a cell’s normal state. The cell’s normal state may refer to a state that is manifested or present prior to exposure of the cell to a cytotoxic composition, cytotoxic agent, and / or cytotoxic state. Generally, a cell in a normal state is a cell in homeostasis. An unintended or unwanted alteration of a cell’s normal state may manifest, for example, in the form of cell death (e.g., programmed cell death), reduction of replication potential, reduction of cellular integrity such as membrane integrity, reduction of metabolic activity, reduction of developmental potential, or any of the cytotoxic effects disclosed herein.

[0168] The phrase "reducing cytotoxicity" refers to a reduction in the degree or frequency of unintended or undesirable alterations to the normal state of cells upon exposure to a cytotoxic composition, cytotoxic agent, and / or cytotoxic state. The phrase may also refer to reducing the degree of cytotoxicity within individual cells exposed to a cytotoxic composition, cytotoxic agent, and / or cytotoxic state, or it may refer to reducing the number of cells in a cell population that exhibit cytotoxicity upon exposure to a cytotoxic composition, cytotoxic agent, and / or cytotoxic state.

[0169] As used herein, the term “manipulated” and its grammatical equivalents may refer to one or more modifications to nucleic acids, for example, nucleic acids within the genome of an organism. The term “manipulated” may refer to the modification, addition, and / or deletion of genes. Manipulated cells may also refer to cells in which genes have been added, deleted, and / or modified.

[0170] As used herein, the terms “cell” or “manipulated cell” and their grammatical equivalents may refer to cells of human origin or cells of non-human animal origin.

[0171] As used herein, the term “checkpoint gene” and its grammatical equivalent may refer to any gene involved in an inhibitory process (e.g., a feedback loop) that acts to modulate the amplitude of an immune response, such as an immunosuppressive feedback loop that mitigates the uncontrolled transmission of an adverse response. These responses may include a molecular shield that protects against incidental tissue damage that may occur during an immune response to an infection, and / or a contribution to maintaining peripheral self-tolerance. Non-limiting examples of checkpoint genes may include members of the extended CD28 family of receptors and their ligands, as well as genes involved in co-inhibitory pathways (e.g., CTLA-4 and PD-1). The term “checkpoint gene” may also refer to an immune checkpoint gene.

[0172] The terms "CRISPR," "CRISPR system," or "CRISPR nuclease system," and their grammatical equivalents, may include a non-coding RNA molecule that binds to DNA (e.g., guide RNA) and a Cas protein (e.g., Cas9) with nuclease functionality (e.g., two nuclease domains). See, for example, Sander, JD et al., "CRISPR-Cas systems for editing, regulating and targeting genomes," Nature Biotechnology, Vol. 32: pp. 347-355 (2014). Also see, for example, Hsu, PD et al., "Development and applications of CRISPR-Cas9 for genome engineering," Cell, Vol. 157 (No. 6): pp. 1262-1278 (2014).

[0173] As used herein, the term "disruption" and its grammatical equivalent may refer to the process of modifying a gene, for example, by deletion, insertion, mutation, rearrangement, or any combination thereof. For example, a gene can be disrupted by knockout. Gene disruption may result in a partial reduction or complete suppression of gene expression. Gene disruption may also lead to the activation of other genes, such as downstream genes.

[0174] As used herein, the term “function” and its grammatical equivalents may refer to the ability to perform, possess, or be suitable for an intended purpose. “Functional” can include any percentage of normal function from baseline up to 100%. For example, functional may include 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and / or 100% of normal function, or approximately these percentages. In some cases, the term functional may mean more than 100% or nearly 100% of normal function, for example, 125, 150, 175, 200, 250, 300%, and / or above normal function.

[0175] As used herein, the term “gene editing” and its grammatical equivalents may refer to a genetic operation that inserts, replaces, or removes one or more nucleotides in the genome. Gene editing involves the use of nucleases (e.g., naturally occurring nucleases). This can be carried out using an enzyme or an artificially manipulated nuclease.

[0176] As used herein, the term “mutation” and its grammatical equivalents may include substitutions, deletions, and insertions of one or more nucleotides within a polynucleotide. For example, within a polynucleotide (cDNA, gene) sequence or polypeptide sequence, up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, 12, 13, 14, 15, 20, 25, 30, 40, 50, or more nucleotides / amino acids may be substituted, deleted, and / or inserted. Mutations may affect the coding sequence or regulatory sequence of a gene. Mutations may also affect the genomic sequence structure or the structure / stability of the encoded mRNA.

[0177] As used herein, the term “non-human animal” and its grammatical equivalents include all animal species other than humans, which may include naturally occurring animals and genetically modified non-human animals, and may include animal species including non-human mammals. The terms “nucleic acid,” “polynucleotide,” “polynucleic acid,” and “oligonucleotide,” and their grammatical equivalents, may be used interchangeably and may refer to deoxyribonucleotide polymers or ribonucleotide polymers that are in linear or cyclic conformation and in single-stranded or double-stranded form. For the purposes of this disclosure, these terms should not be considered limiting in terms of length. The terms may also include analogues of naturally occurring nucleotides as well as nucleotides modified in the base moiety, sugar moiety, and / or phosphate moiety (e.g., phosphorothioate skeleton). Variations of the terms may also include demethylation, addition of CpG methylation, removal of bacterial methylation, and / or addition of mammalian methylation. In general, analogues of a particular nucleotide may have the same base-pairing specificity; that is, an analogue of A may be base-paired with T.

[0178] As used herein, the term “peripheral blood lymphocyte” (PBL) and its grammatical equivalent may refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes may refer to lymphocytes that do not localize to organs. Peripheral blood lymphocytes may include T cells, NK cells, B cells, or any combination thereof.

[0179] As used herein, the term “phenotype” and its grammatical equivalents refer to a combination of observable features or traits of an organism, which may include its shape, development, biochemical or physiological characteristics, phenology, behavior, and the consequences of its behavior. Depending on the context, the term “phenotype” may, in some cases, refer to a combination of observable features or traits of a population.

[0180] As used herein, the term “protospacer” and its grammatical equivalent may refer to a nucleic acid sequence adjacent to a PAM that is capable of hybridizing to a portion of the guide RNA, such as a spacer sequence or an engineered targeting region of the guide RNA. A protospacer may be a nucleotide sequence within a gene, genome, or chromosome that is targeted to the guide RNA. In its natural state, a protospacer is adjacent to a PAM (protospacer-adjacent motif). The cleavage site of an RNA-guided nuclease is within the protospacer sequence. For example, if the guide RNA targets a specific protospacer, the Cas protein will undergo a double-strand break within the protospacer sequence, thereby cleaving the protospacer. Following cleavage, the destruction of the protospacer may result in non-homologous end joining (NHEJ) or homology-guided repair (HDR). The destruction of the protospacer may result in the deletion of the protospacer. In addition, or alternatively, disruption of a protospacer may result in the insertion of an exogenous nucleic acid sequence into or replacing the protospacer.

[0181] As used herein, the term “recipient” and its grammatical equivalent may refer to a human or a non-human animal. A recipient may also be a recipient who requires it.

[0182] As used herein, the term “recombination” and its grammatical equivalent may refer to the process of exchanging genetic information between two polynucleic acids. For the purposes of this disclosure, “homologous recombination” or “HR” may refer to a specialized form of such gene exchange, which may occur, for example, during the repair of a double-strand break. This process may require nucleotide sequence homology, for example, by using a donor molecule as a template for repairing a target molecule (e.g., a molecule that has undergone a double-strand break), and may be known in some cases as non-crossover gene conversion or short-tract gene conversion. Such transfers may also involve synthesis-dependent strand annealing, and / or related processes, in which the donor may be used to correct a mismatch in heterodouble-strand DNA formed between the target being disrupted and the donor, and / or to resynthesize genetic information that may become part of the target. Such specialized HRs often result in alterations of the target molecule’s sequence, such that some or all of the sequence of the donor polynucleotide can be incorporated into the target polynucleotide. Where applicable, the terms “recombination arm” and “homologous arm” may be used interchangeably herein.

[0183] In this specification, the terms "target vector" and "targeting vector" are used interchangeably.

[0184] As used herein, the term “trans gene” and its grammatical equivalent may refer to a gene or genetic material that is introduced into an organism. For example, a trans gene may be a sequence or segment of DNA containing a gene that is introduced into an organism. When a trans gene is introduced into an organism, the organism is referred to as a transgenic organism. In a transgenic organism, a trans gene may retain its ability to produce RNA or polypeptides (e.g., proteins). A trans gene may consist of different nucleic acids, e.g., RNA or DNA. A trans gene may encode a modified T cell receptor, e.g., a TCR trans gene. A trans gene may contain a TCR sequence. A trans gene may contain a recombinant arm. A trans gene may contain a modification site.

[0185] As used herein, the term "T cell" and its grammatical equivalent may refer to T cells of any origin. For example, a T cell may be a primary T cell, such as an autologous T cell or a cell line. A T cell may also be a human T cell or a non-human T cell.

[0186] As used herein, the terms “TIL” or “tumor-infiltrating lymphocyte” and their grammatical equivalents may refer to cells isolated from a tumor. For example, a TIL may be a cell that has migrated to a tumor. A TIL may also be a cell that has infiltrated a tumor. A TIL may be any cell found within a tumor. For example, a TIL may be a T cell, a B cell, a monocyte, a natural killer cell, or any combination thereof. A TIL may be a mixed cell population. A TIL population may include cells with different phenotypes, different degrees of differentiation, different lineages, or any combination thereof.

[0187] A "therapeutic effect" may occur when a change is observed in the treated condition. This change may be positive or negative. For example, a "positive effect" may correspond to an increase in the number of activated T cells in the subject. In another example, a "negative effect" may correspond to a decrease in the amount or size of tumor in the subject. A "change" in the treated condition is observed when there is at least a 10% improvement, preferably at least 25%, more preferably at least 50%, even more preferably at least 75%, and most preferably 100% improvement. The changes may be based on an improvement in the severity of the condition being treated in the individual, or on a difference in the frequency of improvement in the condition between a population of individuals treated with a therapeutic composition administered in combination with the composition of the present invention and a population of individuals not treated with such a composition. Similarly, the methods of the present disclosure may include a step of administering a “therapeutically effective” amount of cells to a subject. The term “therapeutably effective” should be understood to have a definition corresponding to “having a therapeutic effect.”

[0188] As used herein, the terms “safe harbor” and “immunosafe harbor,” and their grammatical equivalents, may refer to genomic sites that can be used to incorporate exogenous nucleic acids, where the incorporation, by addition of nucleic acid alone, does not produce a significant effect on the proliferation of host cells. Non-limiting examples of safe harbors may include HPRT, AAVS sites (e.g., AAVS1, AAVS2, etc.), CCR5, or Rosa26.

[0189] As used herein, the term “sequence” and its grammatical equivalents may refer to a nucleotide sequence that may be DNA or RNA; linear, circular, or branched; single-stranded or double-stranded. Sequences can be mutated. Sequences may be of any length, for example, between 2 and 1,000,000 nucleotides, or more (or any integer between or greater than these), for example, between approximately 100 and approximately 10,000 nucleotides or between approximately 200 and approximately 500 nucleotides.

[0190] Overview This specification discloses compositions and methods useful for performing intracellular genome transplantation. Intracellular genome transplantation may involve genetically modifying cells and nucleic acids for therapeutic application. The compositions and methods described throughout may utilize nucleic acid-mediated genetic engineering steps to deliver tumor-specific TCRs in a manner that improves the physiological and immunological antitumor efficacy of the manipulated cells. Effective adoptive cell transfer-based immunotherapy (ACT) may be useful for treating cancer patients (e.g., metastatic cancer). For example, autologous peripheral blood lymphocytes (PBLs) may be modified using nonviral methods to express T cell receptors (TCRs) that recognize neoantigens, which are intrinsic mutations on cancer cells, and can be used in the compositions and methods of intracellular genome transplantation disclosed. Neoantigens may be associated with tumors with large mutational burdens (Figure 58).

[0191] Figure 1 illustrates an example of a method that may identify cancer-related target sequences, and possibly neoantigens, derived from samples obtained from cancer patients using an in vitro assay (e.g., whole exome sequencing). The method may also identify TCR transgenes derived from first T cells that recognize the target sequences. The cancer-related target sequences and TCR transgenes may be obtained from samples from the same patient or from samples from different patients. The method may effectively and efficiently deliver nucleic acids containing the TCR transgenes across the membrane of second T cells. In some cases, the first and second T cells may be obtained from the same patient. In other cases, the first and second T cells may be obtained from different patients. In other cases, the first and second T cells may be obtained from different patients. The method allows for the safe and efficient integration of the TCR transgene into the T cell genome using a nonviral integration system (e.g., CRISPR, TALEN, transposon-based ZEN, meganuclease, or Mega-TAL) to create engineered T cells, thereby enabling reliable expression of the TCR transgene within the engineered T cells. These engineered T cells can be proliferated and expanded while maintaining their immunological and antitumor efficacy, and can even be administered to patients to treat cancer.

[0192] Manipulated cells can also be proliferated and expanded under conditions that may improve their performance when administered to a patient. Manipulated cells can be selected. For example, prior to cell expansion and manipulation, cell sources can be obtained from the subject by various non-limiting methods. Cells can be obtained from numerous non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, and tumors. For example, any T cell line can be used. Alternatively, cells may be derived from healthy donors, from patients diagnosed with cancer, or from patients diagnosed with infection. In another embodiment, cells may be part of a mixed cell population exhibiting different phenotypic features. Cell lines can also be obtained from transformed T cells according to the methods already described. Cells can also be obtained from cell therapy banks. Modified cells resistant to immunosuppressive treatment can also be obtained. The desired cell population can also be selected before modification. Manipulated cell populations can also be selected after modification.

[0193] In some cases, the manipulated cells can be used in autologous transplantation. Alternatively, the manipulated cells can be used in allogeneic transplantation. In some cases, the manipulated cells can be administered to the same patient whose sample was used to identify cancer-associated target sequences and / or TCR transgenes. In other cases, the manipulated cells can be administered to a different patient than the one whose sample was used to identify cancer-associated target sequences and / or TCR transgenes. One or more homologous recombination enhancers can be introduced with the cells of the present invention. The enhancers can facilitate homology-led repair of double-strand breaks. The enhancers can facilitate the incorporation of TCRs into the cells of the present invention. The enhancers can block non-homologous end joining (NHEJ) so that homology-led repair of double-strand breaks occurs preferentially.

[0194] Modifying compounds can also be used to reduce the toxicity of the exogenous polynucleic acid of the present invention. For example, modifying compounds may act on caspase 1, TBK1, IRF3, STING, DDX41, DNA-PK, DAI, IFI16, MRE11, cGAS, 2'3'-cGAMP, TREX1, AIM2, ASC, or any combination thereof. The modifying compound may be a TBK1 modifier. The modifying compound may be a caspase 1 modifier. The modifying compound may also act on innate signaling pathways and therefore may be an innate signaling modifier. In some cases, exogenous nucleic acids may be toxic to cells. Methods that inhibit the innate immune sensing response of cells may improve the cell viability of the manipulated cell product. The modifying compound may be an inhibitor of brefeldin A and / or the ATM pathway (Figures 92A, 92B, 93A, and 93B).

[0195] The modified compound can be introduced into cells before the addition of the polynucleic acid. The modified compound can be introduced simultaneously with the polynucleic acid. The modified compound may be incorporated within the polynucleic acid. These compositions and methods are efficient and low-toxicity, thereby potentially leading to the development of cell therapies, such as cancer-specific cell therapies.

[0196] One or more cytokines can be introduced with the cells of the present invention. Cytokines can be used to boost cytotoxic T lymphocytes (including tumor-specific cytotoxic T lymphocytes to be adopted) to expand within the tumor microenvironment. Optionally, IL-2 can be used to facilitate the expansion of the cells described herein. Cytokines such as IL-15 can also be utilized. Other cytokines that are appropriate in the field of immunotherapy, such as IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof, can also be used. Optionally, IL-2, IL-7, and IL-15 can be used to culture the cells of the present invention.

[0197] Cytotoxicity generally refers to the quality of a composition, drug, and / or state (e.g., exogenous DNA) that is toxic to cells. In some embodiments, the methods of this disclosure generally relate to reducing the cytotoxic effect of exogenous DNA introduced into one or more cells during genetic modification. In some embodiments, cytotoxicity, or the effect of a substance that is cytotoxic to cells, may include DNA cleavage, cell death, autophagy, apoptosis, nuclear condensation, cell lysis, necrosis, alteration of cell motility, alteration of cell rigidity, alteration of cytoplasmic protein expression, alteration of membrane protein expression, unwanted cell differentiation, swelling, loss of membrane integrity, cessation of metabolic activity, decrease in metabolic activity, increase in metabolic activity, increase in reactive oxygen species, cytoplasmic contraction, production of pro-inflammatory cytokines (e.g., as products of DNA sensing pathways), or any combination thereof. Non-exclusive examples of pro-inflammatory cytokines include interleukin-6 (IL-6), interferon-alpha (IFNα), interferon-beta (IFNβ), CC motif ligand 4 (CCL4), CC motif ligand 5 (CCL5), CXC motif ligand 10 (CXCL10), interleukin-1 beta (IL-1β), IL-18, and IL-33. In some cases, cytotoxic effects may be affected by the introduction of polynucleic acids, such as transgenes or TCRs. Incorporation of exogenous TCRs into cells is...

[0198] Changes in cytotoxicity can be measured by any of a number of methods known in the art. In one embodiment, changes in cytotoxicity can be evaluated based on the degree and / or frequency of occurrence of cytotoxicity-related effects, such as cell death or unwanted cell differentiation. In another embodiment, reduction of cytotoxicity is evaluated by measuring the amount of cytotoxicity using assays known in the art, which include standard laboratory methods such as dye exclusion, detection of morphological features associated with cell viability, damage and / or death, and measurement of enzyme activity and / or metabolic activity associated with the cell type of interest.

[0199] Generally, the T cells of the present invention can be expanded by contacting a surface conjugated with a signal and ligand that can stimulate co-stimulatory molecules on the surface of the T cell, which are CD3 TCR complex-related signals and ligands. In particular, a T cell population can be stimulated in vitro by contacting it with an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD2 antibody immobilized on its surface, or, in some cases, with a protein kinase C activator (e.g., bryostatin) together with a calcium ionophore. To co-stimulate accessory molecules on the surface of the T cell, ligands that bind to accessory molecules can be used. For example, a population of T cells can be contacted with anti-CD3 antibodies and anti-CD28 antibodies under conditions that can stimulate T cell proliferation. In some cases, cells can be stimulated using 4-1BB. For example, cells can be stimulated with 4-1BB and IL-21 or another cytokine.

[0200] Anti-CD3 antibodies and anti-CD28 antibodies can be used to stimulate the proliferation of CD4 T cells or CD8 T cells. For example, the signaling agent may be in solution or coupled to the surface. The particle-to-cell ratio may depend on the particle size relative to the target cells. In a further embodiment, cells such as T cells can be combined with drug-coated beads, in which case the beads and cells can be separated later and optionally cultured. Each bead can be coated with an anti-CD3 antibody, an anti-CD28 antibody, or optionally a combination of the two. In an alternative embodiment, the drug-coated beads and cells are not separated before culture but cultured together. Cell surface proteins can be ligated by contacting T cells with paramagnetic beads (3 × 28 beads) that can bind anti-CD3 and anti-CD28. In one embodiment, cells and beads (e.g., DYNABEAD with a ratio of 1:1) are used. The S(registered trademark) M-450 CD3 / CD28 T paramagnetic beads are combined in a buffer solution, such as phosphate-buffered saline (PBS) (without divalent cations such as calcium and magnesium). Any cell concentration can be used. The mixture can be cultured for several hours (e.g., about 3 hours) or approximately this time to 14 days or approximately 14 days, or any integer value of time in between. In another embodiment, the mixture can also be cultured for 21 days or approximately this period, or up to 21 days or approximately this period. Appropriate conditions for culturing T cells may include a suitable culture medium (e.g., minimal essential medium or RPMI Media 1640 or X-vivo 5 (Lonza)) containing factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-g, IL-4, IL-7, GM-CSF, IL-10, IL-21, IL-15, TGF beta, and TNF alpha, or any other additives for cell proliferation. Other additives for cell proliferation include, but are not limited to, surfactants, plasmanates, and reducing agents such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may contain amino acids, sodium pyruvate, and vitamins, and may be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or sufficient amounts of cytokines to promote T cell proliferation and expansion, including RPMI 1640, A1 MV, DMEM, MEM, α-MEM, F-12, X-Vivo 1 and X-Vivo 20, and Optimizer. Antibiotics, such as penicillin and streptomycin, may be incorporated only into the experimental culture, but probably not into the culture of cells to be infused into the target. Target cells can be maintained under conditions necessary to support proliferation, such as appropriate temperature (e.g., 37°C) and atmosphere (e.g., air with 5% CO2). In some cases, T cells exposed to varying stimulation times may exhibit different characteristics.Depending on the circumstances, soluble, monospecific, tetrameric antibodies against human CD3, CD28, CD2, or any combination thereof may be used.

[0201] In some cases, cells undergoing genome transplantation can be activated and expanded by co-culturing with tissue or other cells. These cells may be antigen-presenting cells. Artificial antigen-presenting cells (aAPCs) can express ligands for T cell receptors and costimulatory molecules, and may activate and expand T cells for transplantation while improving their potency and function. aAPCs can be engineered to express any gene for T cell activation. aAPCs can be engineered to express any gene for T cell expansion. aAPCs may be beads, cells, proteins, antibodies, cytokines, or any combination thereof. aAPCs can deliver signals to cell populations that may undergo genome transplantation. For example, aAPCs may deliver signal 1, signal 2, signal 3, or any combination thereof. Signal 1 may be an antigen recognition signal. For example, signal 1 could be the ligation of a peptide-MHC complex to the TCR, or the binding of an agonist antibody against CD3 that can result in activation of the CD3 signal-transduction complex. Signal 2 could be a costimulatory signal. For example, costimulatory signals could be anti-CD28, inducible costimulator (ICOS), CD27, and 4-1BB (CD137), respectively, which bind to ICOS-L, CD70, and 4-1BBL. Signal 3 could be a cytokine signal. The cytokine could be any cytokine. The cytokine could be IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof.

[0202] In some cases, artificial antigen-presenting cells (aAPCs) can be used to activate and / or expand cell populations. In some cases, artificial antigen-presenting cells may not induce allospecificity. In some cases, aAPCs may not express HLA. aAPCs are activated and Genetic modification is possible to ensure the stable expression of genes that can be used for activation and / or stimulation. In some cases, K562 cells can be used for activation. K562 cells can also be used for expansion. K562 cells can be a human erythroleukemia cell line. K562 cells can be engineered to express the gene of interest. K562 cells cannot endogenously express HLA class I molecules, HLA class II molecules, or CD1d molecules, but they can express ICAM-1 (CD54) and LFA-3 (CD58). K562 can be engineered to deliver signal 1 to T cells. For example, K562 cells can be engineered to express HLA class I. In some cases, K562 cells can be manipulated to express additional molecules such as B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, anti-CD3, anti-CD3 mAb, anti-CD28, anti-CD28 mAb, CD1d, anti-CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, cleaved CD19, or any combination thereof. In some cases, manipulated K562 cells may express cloned OKT3, a membrane form of anti-CD3 mAb, in addition to CD80 and CD83. In some cases, manipulated K562 cells may express cloned OKT3, a membrane form of anti-CD3 mAb, and a membrane form of anti-CD28 mAb, in addition to CD80 and CD83.

[0203] aAPCs can be beads. Spherical polystyrene beads can be coated with antibodies against CD3 and CD28 and used to activate T cells. The beads can be of any size. Depending on the case, the beads can be 3 and 6 micrometers or approximately this length. The beads can be 4.5 micrometers in size or approximately this size. The beads can be used in any cell-to-bead ratio. For example, a 3:1 bead-to-cell ratio can be used with 1 million cells per millimeter. aAPCs can also be rigid spherical particles, polystyrene latex microbeads, magnetic nanoparticles or magnetic microparticles, nano-sized quantum dots, poly(lactic acid-co-glycolic acid) (PLGA) microspheres, non-spherical particles, carbon nanotube bundles, ellipsoidal PLGA microparticles, nanoworms, fluid lipid bilayer-containing systems, 2D-assisted lipid bilayers (2D-SLB), liposomes, RAFTsomes / microdomain liposomes, SLB particles, or any combination thereof.

[0204] In some cases, aAPCs can enlarge CD4 T cells. For example, aAPCs can be engineered to mimic the antigen processing and presentation pathways of HLA class II-restricted CD4 T cells. K562 can be engineered to express HLA-D, DPα, DPβ chains, II, DMα, DMβ, CD80, CD83, or any combination thereof. For example, engineered K562 cells can be pulsed with HLA-restricted peptides to enlarge HLA-restricted antigen-specific CD4 T cells.

[0205] Depending on the circumstances, the use of aAPC can be combined with cytokines exogenously introduced for T cell activation, expansion, or any combination thereof. Cells can also be expanded in vivo, for example, in the blood of a subject after administration of genome-transplanted cells to the subject.

[0206] These compositions and methods for intracellular genome transplantation may offer numerous advantages in cancer treatment. For example, these compositions and methods may result in highly efficient gene transfer, expression, increased cell viability, efficient introduction of recombination-induced double-strand breaks, and a process that prioritizes homology-led repair (HDR) through a non-homologous end joining (NHEJ) mechanism, as well as efficient recovery and expansion of homologous recombinants.

[0207] Ribonucleic acid system One exemplary method for creating manipulated cells is to use ribonucleic acid for intracellular genome transplantation. This is carried out using RNA systems, such as full RNA systems or partial RNA systems. The cells being manipulated can be genetically modified with RNA or modified RNA instead of DNA to prevent, in some cases, DNA-induced toxicity and immunogenicity (e.g., double-stranded or single-stranded DNA) observed with the use of DNA. In some cases, RNA / DNA fusion polynucleic acids can also be used for genomic manipulation.

[0208] Depending on the circumstances, all RNA polynucleotide systems can be used for gene editing of primary human T cells (see, for example, Figure 5). In outline, ribonucleic acid transcribed in vitro can be delivered into the target cell and reverse transcribed into dsDNA. The DNA template can then be used for homologous recombination (HR) reactions inside the cell.

[0209] In some cases, robust genome manipulation can be achieved by increasing the amount of polynucleic acid encoding the transgene. Since introducing DNA to increase the amount of transgene can sometimes result in cytotoxicity (Figures 2 and 3), it is sometimes preferable to introduce RNA into cells for genome manipulation.

[0210] In some cases, transgenes containing exogenous receptor sequences can be introduced into cells via RNA, such as messenger RNA (mRNA), for genomic manipulation. RNA, such as mRNA, can be converted to DNA in situ. One exemplary method involves producing mRNA polynucleic acid using in vitro transcription of the polynucleic acid. The mRNA polynucleic acid can then be transfected into cells along with reverse transcriptase (RT) (either in protein form or polynucleic acid form encoding RT). In other cases, the RT protein is introduced into the cells. RT may be chicken myeloblastosis virus reverse transcriptase (AMV RT), Moloney's mouse leukemia virus reverse transcriptase (M-MLV RT), human immunodeficiency virus (HIV) reverse transcriptase (RT), derivatives thereof, or combinations thereof, or derived from them. Once transfected, the reverse transcriptase can transcribe the manipulated mRNA polynucleic acid into double-stranded DNA (dsDNA). Reverse transcriptase (RT) may be an enzyme used to synthesize complementary DNA (cDNA) from an RNA template. In some cases, the RT enzyme can synthesize a complementary DNA strand starting from a primer that uses RNA (cDNA synthesis) or single-stranded DNA as a template. In some cases, RT may function at a temperature of 37 degrees Celsius. In other cases, RT may function at a temperature below 37 degrees Celsius. In other cases, RT may function at a temperature above 37 degrees Celsius.

[0211] RT can be any enzyme used to construct complementary DNA (cDNA) from an RNA template. RT may be derived from retroviruses, hepatitis B viruses, Hepadnaviridae viruses, or any double-stranded or single-stranded viruses. RT may possess any number of biochemical activities. For example, RT may have RNA-dependent DNA polymerase activity. RT may have ribonuclease H activity. RT may have DNA-dependent DNA polymerase activity. In some cases, RT can be used to convert single-stranded RNA into double-stranded cDNA. The cDNA can then be introduced into the cell genome. Figures 101A and 101B show in vivo reverse transcription of electroporated mRNA.

[0212] RT may be HIV-1 RT derived from human immunodeficiency virus type 1. HIV-1 RT may have subunits. For example, HIV-1 RT may have two subunits. RT can also be derived from Moloney's mouse leukemia virus (M-MLV). M-MLV viruses may or may not have subunits. In some cases, M-MLV viruses are a single monomer. RT can also be, Chicken myeloblastosis virus RT (AMV RT) is also a possibility. AMV RT may have subunits. In some cases, AMV RT has two subunits. In some cases, telomerase RT is also used.

[0213] In some cases, dsDNA can be used in a subsequent homologous recombination step. This subsequent homologous recombination step allows for the introduction of an exogenous receptor sequence into the cell genome.

[0214] A method for targeting reverse transcriptase to manipulated polynucleic acids. a) Unique sequence In some cases, it may be necessary to target the introduced RT to the polynucleic acid to be introduced. The introduced polynucleic acid may be RNA or DNA. In some cases, the introduced polynucleic acid may be a combination of RNA and DNA. Targeting of the introduced RT can be performed by incorporating sequences specific to the polynucleic acid encoding the manipulated receptor (Figure 22). These specific sequences can help target the RT to a particular polynucleic acid. In some cases, specific sequences can increase the efficiency of the reaction. Table 1 lists possible specific sequences for targeting the RT to the manipulated polynucleic acid. Specific sequences may be sequences not found in any human mRNA transcript. In some cases, specific sequences can be modified from known mRNA transcripts so that they are no longer endogenous sequences. Specific sequences can be identified using bioinformatics. In some cases, specific sequences can be identified using publicly available databases. [Table 1]

[0215] A unique sequence can be of any base-pair length. It can be 1–20 base pairs, 1–30 base pairs, 1–40, 1–50, 1–60, 1–70, 1–80, 1–90, 1–100 base pairs, or any length greater than 100 base pairs, or in between. In some cases, the unique sequence is 1–20 base pairs or in between. In some cases, the unique sequence is greater than 20 base pairs. In some cases, the unique sequence is exactly 20 base pairs long. The unique sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more base pairs. In some cases, multiple unique sequences are introduced into polynucleic acids.

[0216] In some cases, the unique sequence can be incorporated into the manipulated polynucleic acid. The unique sequence can be used to target RT to the manipulated polynucleic acid. In some cases, oligonucleotides can be pre-annealed with the manipulated polynucleic acid. The pre-annealed oligonucleotide may contain a complementary unique sequence of any length within the manipulated polynucleic acid. The pre-annealed oligonucleotide may be exactly the same length as the complementary unique sequence, or it may be less than the full length of the complementary sequence of the unique sequence within the manipulated polynucleic acid.

[0217] b) Operation structure In some cases, reverse transcriptase can be targeted to a modified polynucleic acid by manipulating the polynucleic acid to have a secondary structure. The secondary structure can be any structure. In some cases, multiple secondary structures can be used.

[0218] For example, the secondary structure can be a double helix. A double helix can be formed by regions consisting of many consecutive base pairs. In some cases, the double helix is ​​a tertiary structure. A double helix can be a spiral polymer. In some cases, a double helix can be dextrorotatory. A double helix can also be levorotatory. In some cases, a double helix can be a dextrorotatory structure containing two nucleotide chains that can base pair together. In some cases, one turn of a double helix can be 10 nucleotides or approximately this length. In other cases, one turn of a double helix can be more than 10 nucleotides, less than 10 nucleotides or approximately this length. One turn of a double helix can be 1 to 5 nucleotides, 1 to 10, 1 to 20, or more than 20 nucleotides or approximately this length.

[0219] The secondary structure can also be a stem-loop structure or a hairpin structure. An RNA hairpin can be formed when two complementary sequences within a single RNA molecule associate and join together after molecular folding or link ringing. In some cases, an RNA hairpin may consist of a double-stranded RNA (dsRNA) stem and a terminal loop. Structurally, RNA hairpins can occur at different locations within different types of RNA. RNA hairpins may occur at the 5' end of ribonucleic acid, at the 3' end, or at any position between the 5' and 3' ends. RNA hairpins can differ in stem length, loop size, number and size of bulges, and nucleotide sequence. An RNA hairpin can be a hairpin of any stem length. An RNA hairpin can have a loop of any size. For example, a hairpin loop may be between 4 and 8 bases in length, or approximately between these numbers of bases. In some cases, a hairpin loop may be longer than 8 bases, or approximately between these numbers of bases. In certain cases, hairpin loops longer than or approximately 8 base pairs may have secondary structures. RNA hairpins may have bulges of any number and size. RNA hairpins can be hairpins of any base pair length. For example, RNA hairpins may be 1–100 base pairs, 1–200 base pairs, 1–300 base pairs, or more than or approximately 300 base pairs. RNA hairpins may have secondary structures, such as bulging structures.

[0220] Functionally, RNA hairpins can regulate gene expression in either a cis or trans state. For example, an RNA hairpin within an RNA molecule may regulate only that molecule (cis) or induce effects on other RNAs or pathways (trans). Hairpins can be used as binding sites for various proteins, act as substrates for enzymatic reactions, and also exhibit endogenous enzymatic activity. In some cases, hairpins can be used to target RT to a modified polynucleic acid for transcription. Hairpin structures can be located at ribosome-binding sites. Hairpin structures can facilitate translation.

[0221] Hairpin structures may possess an internal ribosome entry site (IRES). The IRES sequence may enable targeted transcription of mRNA containing the hairpin. In some cases, the hairpin structure may guide the manipulated polynucleic acid to an intracellular location. For example, the hairpin may be, or contain, a nuclear localization signal.

[0222] Depending on the circumstances, RT may target RNA hairpins of the manipulated polynucleic acid. The manipulated polynucleic acid may contain one or more hairpin regions. The hairpins are the responsibility of the manipulated polynucleic acid. It can be formed in the position of intention.

[0223] The secondary structure can also be a pseudoknot. A pseudoknot can be a secondary structure of nucleic acid in which at least two stem-loop or hairpin structures exist, and half of one stem may contain a structure inserted between two halves of another stem. Several distinctly different folding topologies can be used for pseudoknots. In some cases, an H-type pseudoknot can be used. In H-type folding, the bases within the loop of the hairpin can form intramolecular pairs with the bases outside the stem. This can result in a pseudoknot with two stems and two loops, as a result of causing the formation of a second stem and loop. The two stems may be able to stack on top of each other to form a quasi-continuous helix with one continuous chain and one discontinuous chain.

[0224] In some cases, pseudoknots can be used to initiate transcription of manipulated polynucleic acids. Pseudoknots can induce ribosomes to slip into an alternative reading frame. Pseudoknots can, in some cases, cause frame shifting.

[0225] Targeting of the nucleus with manipulated polynucleic acids In some cases, the modified polynucleic acid may need to be localized to the nucleus within the cell. The modified polynucleic acid may encode an exogenous receptor sequence or a modified receptor sequence that may need to be introduced into the cell genome. In some cases, the introduction of the receptor sequence into the cell genome can be carried out by localizing the modified polynucleic acid to an intracellular nuclease for transcription.

[0226] Modified RNA polynucleic acids can be localized to intracellular nucleases. Localization can involve any number of techniques. In some cases, nuclear localization signals can be used to localize modified polynucleic acids encoding modified receptors to the nucleus. The nuclear localization signal may be any endogenous sequence or any modified sequence.

[0227] In some cases, nuclear localization signals or sequences may originate from proteins that are strictly nuclear proteins. Proteins that are nuclear proteins may have nuclear localization signals that are not influenced by the cell state or their genomic loci. In some cases, nuclear localization may originate from sequences or structures within the transcripts of spliced ​​mature proteins.

[0228] In some cases, the nuclear localization signal may be a BMP2-OP1 responsive gene ("BORG") sequence. In some cases, multiple BORG NLS sequences are incorporated into the polynucleic acid. In some cases, one to five BORG sequences can be incorporated. In other cases, five to ten BORG sequences are incorporated. One, two, three, four, five, six, seven, eight, nine, ten, or more BORG sequences can be incorporated as nuclear localization signals within the polynucleic acid. In some cases, as many BORG sequences as can be encoded within the polynucleic acid are used. The nuclear localization signal may be a short RNA motif consisting of AGCCC, a pentamer with two sequence constraints at positions -8 and -3 relative to the pentamer's starting point. BORG sequences can be used within the manipulated polynucleic acid to localize it to the nucleus of the cell.

[0229] In some cases, nuclear localization may be mediated by interaction with tandem repeats of short sequences similar to the consensus sequence of the intronic branch region of SF1, resulting in the localization of RNA polynucleic acid to secrete nuclear membrane subdomains. BORG sequence It may function by interacting with a large number of nuclear-localized proteins or nuclear-localized protein complexes, such as transcription complexes. In other cases, nuclear localization sequences may interact with nuclear-localized RNA or chromatin-associated RNA-protein complexes that can anchor polynucleic acids containing nuclear localization motifs within the nucleus. In other cases, nuclear localization sequences may interact with factors that can interfere with the formation of export complexes, resulting in the retention of polynucleic acids within the nucleus.

[0230] Nuclear localization signals can be sequence, structure, or any combination thereof. In some cases, nuclear localization of nucleic acids may not require transport, only anchoring to the cytoskeleton (actin or intermediate filaments). In other cases, manipulated polynucleic acids can be transported to the nucleus via microtubules. Transport may occur in the form of large ribonucleoprotein (RNP) complexes or RNP transport granules. In some cases, polynucleic acids can complex with secondary proteins that localize them to the nucleus. In some cases, transported polynucleic acids can be anchored at their final destination. Some transacting factors can shuttling back to the nucleus.

[0231] In some cases, polynucleic acids can be directly introduced into the nucleus. In other cases, polynucleic acids can be synthesized in the nucleus. Polynucleic acids can be manipulated to encode at least one BORG sequence. Polynucleic acids can be manipulated to encode multiple BORG sequences. Polynucleic acids can be manipulated to encode four BORG sequences. In some cases, cells are transfected with polynucleic acids containing BORG sequences. Polynucleic acids containing BORG sequences can be localized to the nucleus within the cell, in which case they participate in homologous recombination. In some cases, polynucleic acids localized to the nucleus can encode receptor sequences. Receptor sequences can be introduced into the cell genome by BORG-mediated nuclear localization.

[0232] Modification of polynucleic acids The polynucleic acids described herein can be modified. Modifications can be performed at any position on the polynucleic acid. A single polynucleic acid can be subjected to more than one modification. After modification, the polynucleic acids may undergo quality control. Depending on the circumstances, quality control may include PAGE, HPLC, MS, or any combination thereof.

[0233] Modifications can be substitutions, insertions, deletions, chemical modifications, physical modifications, stabilizations, purifications, or any combination thereof.

[0234] Polynucleic acids also include 5'-adenylate, 5'-guanosine triphosphate cap, and 5'N 7 - Methylguanosine triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' thiophosphate, 5' phosphate, 5' thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, debase, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bibiotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, Black Hole Quencher 1, Black Hole Quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methylribonucleoside analog, sugar-modified analog, fluctuation / universal base, fluorescent dye labeling, 2'-fluoroRNA, 2'O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester GRNA can also be modified with telRNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, or any combination thereof. Representative 2'O-methylRNA-modified gRNAs are shown in Figure 31.

[0235] In some cases, modifications can be permanent. In other cases, modifications are transient. In some cases, multiple modifications are applied to polynucleic acids. Modifications to polynucleic acids can alter the physicochemical properties of nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof.

[0236] Modifications can also be phosphorothioate substitutions. In some cases, native phosphodiester bonds are susceptible to rapid degradation by intracellular nucleases, and modifications of nucleotide linkages using phosphorothioate (PS) bond substitutions may be more stable against hydrolysis by intracellular degradation. Modifications can increase the stability of polynucleic acids. Modifications can also enhance bioactivity. In some cases, phosphorothioate-enhanced RNA polynucleic acids may inhibit RNase A, RNase T1, calf serum nucleases, or any combination thereof. These properties may enable the use of PS-RNA polynucleic acids in applications where exposure to nucleases is likely, either in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5' or 3' end of the polynucleic acid, thereby inhibiting exonuclease degradation. In some cases, phosphorothioate bonds can also be added throughout the polynucleic acid to reduce attack by endonucleases.

[0237] In some cases, modifications can be screened. Screening may include, but is not limited to, tests for immunogenicity, toxicity, transcription efficiency, translation efficiency, or any combination thereof. In some cases, a modification may not be immunogenic. In some cases, a modification may not be toxic. In some cases, candidate modifications are screened before being incorporated into polynucleic acids. In other cases, polynucleic acids with different modifications are screened to determine the level of immunogenicity, toxicity, efficacy, or any combination thereof of the added modifications. In some cases, modifications are screened for their ability to assist in the reverse transcription of polynucleic acids. In some cases, the modification is a pseudouridine-5'-triphosphate modification (see, for example, Figure 59). In other cases, the modification is a 5-methylcytidine-5'-triphosphate modification (see, for example, Figure 59). Modifications may also include changes in chirality.

[0238] Polynucleic acids can be assembled by various methods, such as automated solid-phase synthesis. Polynucleic acids can be constructed using standard solid-phase DNA / RNA synthesis. Polynucleic acids can also be constructed using synthetic procedures. Polynucleic acids can also be synthesized manually or fully automated. Depending on the case, the synthetic procedure may include: first, converting a 5'-hydroxyl oligonucleotide to the corresponding 5'-H-phosphonate monoester, then oxidizing it to an activated 5'-phosphorymidazolide in the presence of imidazole, and finally reacting it with pyrophosphate on a solid support. This procedure may include post-synthesis purification steps such as PAGE, HPLC, MS, or any combination thereof.

[0239] In some cases, polynucleic acids can be modified to reduce immunogenicity and make them more stable for transfection into cells. Modified polynucleic acids can encode any number of genes. In some cases, polynucleic acids can encode transgenes. Transgenes can encode modified receptors. Receptors can be T cell receptors (TCRs), B cell receptors (BCRs), chimeric antigen receptors (CARs), or any combination thereof (for example) (See Figure 57). In some cases, the receptor may be a TCR.

[0240] In some cases, modified polynucleic acids can be used in subsequent steps. For example, modified polynucleic acids can be used in homologous recombination reactions. Homologous recombination reactions may involve introducing a transgene encoding an exogenous receptor into the cellular genome. The introduction may involve any mechanism necessary to introduce the transgene sequence into the cellular genome. In some cases, CRISPR may be used in the step of introducing the receptor sequence into the cellular genome.

[0241] Intracellular genome transplantation Intracellular genome transplantation may be a method of genetically modifying cells and nucleic acids for therapeutic application. The compositions and methods described throughout may use nucleic acid-mediated genetic engineering steps for tumor-specific TCR expression, in a form that does not disrupt the physiological and immunological antitumor efficacy of T cells. Effective adoptive cell transfer-based immunotherapy (ACT) may be useful in treating cancer patients (e.g., metastatic cancer). For example, autologous peripheral blood lymphocytes (PBLs) may be modified using nonviral methods to express T cell receptors (TCRs) that recognize neoantigens, which are intrinsic mutations on cancer cells, and can be used in the intracellular genome transplantation compositions and methods disclosed.

[0242] One exemplary method for identifying cancer-specific TCR sequences that recognize unique immunogenic mutations in a patient's cancer is described in PCT / US14 / 58796. For example, cancer-specific TCR transgenes can be inserted into the cell genome (e.g., T cells) using random or specific insertions.

[0243] In some embodiments, the methods disclosed herein include the step of introducing one or more nucleic acids (e.g., a first nucleic acid or a second nucleic acid) into a cell. Those skilled in the art will notice that nucleic acids generally refer to substances whose molecules consist of many nucleotides linked together in a long chain. Non-limiting examples of nucleic acids include artificial nucleic acid analogs (e.g., peptide nucleic acids, morpholino oligomers, loct nucleic acids, glycol nucleic acids, or threose nucleic acids), cyclic nucleic acids, DNA, single-stranded DNA, double-stranded DNA, genomic DNA, plasmids, plasmid DNA, viral DNA, viral vectors, gamma-retroviral vectors, lentiviral vectors, adeno-associated virus vectors, RNA, short hairpin RNA, psiRNA, and / or hybrids or combinations thereof. In some embodiments, the method may include nucleic acids, and the nucleic acids are synthetic nucleic acids. In some embodiments, the sample may include nucleic acids, and the nucleic acids may be fragmented. In some cases, the nucleic acids are minicircles.

[0244] In some embodiments, the nucleic acid may include a promoter region, barcode, restriction sites, cleavage sites, endonuclease recognition sites, primer-binding sites, selection markers, unique identification sequences, resistance genes, linker sequences, or any combination thereof. In some embodiments, these sites may be useful for enzymatic digestion, amplification, sequencing, binding targeting, purification, conferring resistance characteristics (e.g., antibiotic resistance), or any combination thereof. In some embodiments, the nucleic acid may include one or more restriction sites. Restriction sites generally refer to specific peptide sequences or specific nucleotide sequences that a site-specific molecule (e.g., a protease, endonuclease, or enzyme) can cleave the nucleic acid. In one example, the nucleic acid may include one or more restriction sites, in which case cleavage of the nucleic acid at the restriction site fragments the nucleic acid. In some embodiments, the nucleic acid may include at least one endonuclease recognition site. In some embodiments, the endonuclease recognition site is a type I endonuclease recognition site, a type II endonuclease recognition site, a type III endonuclease recognition site, a type IV endonuclease recognition site, or a type V endonuclease recognition site. It may contain an endonuclease recognition site. Non-limiting examples of endonuclease recognition sites include AatII recognition site, Acc65I recognition site, AccI recognition site, AclI recognition site, AatII recognition site, Acc65I recognition site, AccI recognition site, AclI recognition site, AfeI recognition site, AflII recognition site, AgeI recognition site, ApaI recognition site, ApaLI recognition site, ApoI recognition site, AscI recognition site, AseI recognition site, AsiSI recognition site, AvrII recognition site, BamHI recognition site, BclI recognition site, BglII recognition site, Bme1580I recognition site, BmtI recognition site, BsaI recognition site, BsaHI recognition site, BsiEI recognition site, BsiWI recognition site, BspEI recognition site, BspHI recognition site, BsrGI recognition site, BssHII recognition site, BstBI recognition site, BstZ17I recognition site, BtgI recognition site, ClaI recognition site, DraI recognition site, EaeI recognition site, EagI recognition site, EcoRI recognition site, EcoRV recognition site, FseI recognition site, FspI recognition site, HaeII recognition site, HincII recognition site, HindIII recognition site, HpaI recognition site, KasI recognition site, KpnI recognition site, MfeI recognition site, MluI recognition site, MscI recognition site, MspA1I recognition site, MfeI recognition site, MluI recognition site, MscI recognition site, MspA1I recognition site, NaeI recognition site, NarI recognition site, NcoI recognition site, NdeI recognition site, NgoMIV recognition site, NheI recognition site, NotI recognition site, NruI recognition site, NsiI recognition site, NspI recognition site, PacI recognition site, PciI recognition site, PmeI recognition site, PmlI recognition site, PsiI recognition site, PspOMI recognition site, PstI recognition site, PvuI recognition site, PvuII recognition site, SacI recognition site, SacII recognition site, SalI recognition site, SbfI recognition site, ScaI recognition site, SfcI recognition site, SfoI recognition site, SgrAI recognition site, SmaI recognition site, SmlI recognition site, SnaBI recognition site, SpeI recognition site, SphI recognition site, SspI recognition site, StuI recognition site, SwaI recognition site, XbaI recognition site, XhoI recognition site, and XmaI recognition site. In a specific example, the restriction site may include a NotI endonuclease recognition site.

[0245] In some cases, nucleic acids can readily bind to other nucleic acids (for example, nucleic acids may have sticky ends or nucleotide overhangs). For example, a nucleic acid may have an overhang at its first end. Generally, sticky ends or overhangs may refer to a series of unpaired nucleotides at the end of a nucleic acid. In some cases, nucleic acids may have single-strand overhangs at one or more ends. In some cases, overhangs may occur at the 3' end of a nucleic acid. In some cases, overhangs may occur at the 5' end of a nucleic acid. Overhangs may contain any number of nucleotides. For example, an overhang may contain 1, 2, 3, 4, or 5 or more nucleotides. In some cases, nucleic acids may require modification before binding to another nucleic acid (for example, nucleic acids may require digestion by an endonuclease). In some cases, modification of a nucleic acid can create a nucleotide overhang, which may contain any number of nucleotides. For example, an overhang may contain 1, 2, 3, 4, or 5 or more nucleotides. In one example, nucleic acids can contain restriction sites, in which case digesting the nucleic acid at the restriction site with a restriction enzyme (e.g., NotI) produces a 4-nucleotide overhang. In some cases, modification involves creating blunt ends at one or more ends of the nucleic acid. Generally, a blunt end may refer to a double-stranded nucleic acid where both strands are terminated by a base pair. In one example, nucleic acids can contain restriction sites, in which case digesting the nucleic acid at the restriction site with a restriction enzyme (e.g., BsaI) produces a blunt end.

[0246] A promoter is a nucleic acid sequence that controls the binding of RNA polymerase and transcription factors, and can have a significant impact on the efficiency of gene transcription, where a gene can be expressed within a cell, and / or in which cell types a gene can be expressed. A non-limiting example of a promoter is the cytomegalovirus (CMV) promoter. This includes romortas, elongation factor 1 alpha (EF1α) promoter, SV40 (simian vacuolating) virus promoter, phosphoglycerate kinase (PGK1) promoter, ubiquitin C (Ubc) promoter, human beta-actin promoter, CAG promoter, tetracycline response element (TRE) promoter, UAS promoter, Actin 5c (Ac5) promoter, polyhedron promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKIIa) promoter, GAL1 promoter, GAL10 promoter, TEF1 promoter, glyceraldehyde 3-triphosphate (phosphage) dehydrogenase (GDS) promoter, ADH1 promoter, CaMV35S promoter, Ubi promoter, human polymerase III RNA(H1) promoter, U6 promoter, or combinations thereof.

[0247] In some cases, the nucleic acid can be a viral vector, and the viral vector may contain a sequence encoding the U3-R-U5 region of the LTR (long terminal repeat), which is found on either side of the provirus of a retrovirus. In some cases, the nucleic acid can be a viral vector, and the viral vector may contain a sequence encoding U3, which is a specific region at the 3' end of the viral genomic RNA and contains a sequence necessary for the activation of viral genomic RNA transcription. In some cases, the nucleic acid can be a viral vector, and the viral vector may contain a sequence encoding R, a repeat region found in both the 5'LTR and 3'LTR of retro / lentiviral vectors. In some cases, the nucleic acid can be a viral vector, and the viral vector may contain a sequence encoding U5, a specific region at the 5' end of the viral genomic RNA. In some cases, the nucleic acid can be a viral vector, and the viral vector may contain a sequence encoding the 5'LTR, which can act as an RNA pol II promoter. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding the 5'LTR of a hybrid with a constitutive promoter such as CMV or RSV. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding a trans-activating response (TAR) element located within the R region of the LTR and capable of acting as a binding site to Tat. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding the 3'LTR, which can be used to terminate transcription initiated by the 5'LTR by adding a poly-A tract following the R sequence. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding the central polypurine tract (cPPT), which is a recognition site for proviral DNA synthesis. The presence of cPPT may affect the efficiency of gene transfer and the expression of the transgene.In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding Psi, an RNA target site for packaging by the nucleocapsid. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding the rev response element (RRE), which is the sequence to which the Rev protein binds. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding the woodchuck hepatitis virus post-transcriptional regulatory element, which is the sequence that stimulates the expression of the trans gene through increased extrusion from the nucleus. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding GAG, a precursor structural protein of a lentiviral particle containing matrix components, capsid components, and nucleocapsid components. In some cases, the nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding Pol, a precursor protein containing reverse transcriptase and integrase components. In some cases, the nucleic acid may be a viral... The nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding a Rev that can bind to RREs in unspliced ​​and partially spliced ​​transcripts to facilitate export from the nucleus. The nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding a transactivator (Tat) that can bind to a TAR to activate transcription from the LTR promoter. The nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding vesicular stomatitis virus G glycoprotein (VSVG), a broad-affinity envelope protein that can be used for pseudotyping lentiviral vectors. The nucleic acid may be a viral vector, and the viral vector may contain a sequence encoding an inverted terminal repeat (ITR) that forms a T-shaped hairpin that can be used as a starting point for viral DNA replication. The symmetry of the ITR may affect the efficient reproduction of the AAV genome. In some cases, the nucleic acid may be a viral vector, which may contain sequences encoding Rep (e.g., Rep78, Rep68, Rep52, and Rep40), which are packaging proteins required for genome replication and integration. In some cases, the nucleic acid may be a viral vector, which may contain sequences encoding capsid structural proteins (e.g., VP1, VP2, and VP3), which can be used to release AAV particles from late endosomes and / or ensure proper virion assembly.

[0248] Depending on the context, nucleic acids may include barcodes or barcode sequences. A barcode or barcode sequence refers to a natural or synthetic nucleic acid sequence composed of polynucleotides, which enables the unambiguous identification of polynucleotides and other sequences having the barcode sequence. For example, a nucleic acid containing a barcode may enable the identification of the encoded transgene. A barcode sequence may include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 45, or 50 or more consecutive nucleotide sequences. A nucleic acid may include two or more barcode sequences or their complements. A barcode sequence may include randomly assembled nucleotide sequences. A barcode sequence may be a degenerate sequence. A barcode sequence may be a known sequence. A barcode sequence may be a predefined sequence.

[0249] Depending on the circumstances, the methods disclosed herein may include nucleic acids (e.g., a first nucleic acid and / or a second nucleic acid). Depending on the circumstances, the nucleic acid may encode a trans gene. Generally, a trans gene may refer to a linear polymer comprising a plurality of nucleotide subunits. A trans gene may contain any number of nucleotides. Depending on the circumstances, a trans gene may contain fewer than about 100 nucleotides. Depending on the circumstances, a trans gene may contain at least about 100 nucleotides. Depending on the circumstances, a trans gene may contain at least about 200 nucleotides. Depending on the circumstances, a trans gene may contain at least about 300 nucleotides. Depending on the circumstances, a trans gene may contain at least about 400 nucleotides. Depending on the circumstances, a trans gene may contain at least about 500 nucleotides. Depending on the circumstances, a trans gene may contain at least about 1000 nucleotides. Depending on the circumstances, a trans gene may contain at least about 5000 nucleotides. Depending on the circumstances, a trans gene may contain at least about 10,000 nucleotides. In some cases, the trans gene may contain at least approximately 20,000 nucleotides. In some cases, the trans gene may contain at least approximately 30,000 nucleotides. In some cases, the trans gene may contain at least approximately 40,000 nucleotides. In some cases, the trans gene may contain at least approximately 50,000 nucleotides. In some cases, the trans gene may contain between approximately 500 and 5000 nucleotides. In some cases, the trans gene may contain between approximately 5000 and 10,000 nucleotides. It may include nucleotides in between. In any of the cases disclosed herein, the trans gene may include DNA, RNA, or a hybrid of DNA and RNA. In some cases, the trans gene may be single-stranded. In some cases, the trans gene may be double-stranded.

[0250] a. Random insertion One or more transgenes, as described herein, can be randomly inserted into the cell genome. These transgenes may be functional regardless of where they are inserted within the genome. For example, a transgene may encode its own promoter, or it may be inserted into a location under the control of an endogenous promoter. Alternatively, a transgene may be inserted into a gene such as an intron, exon, promoter, or non-coding region.

[0251] Nucleic acids encoding transgene sequences, such as RNA, can be randomly inserted into the chromosomes of cells. Random insertion can be achieved by any method of introducing nucleic acids, such as RNA, into cells. For example, methods may include, but are not limited to, electroporation, ultrasonic perforation, the use of gene guns, lipotransfection, calcium phosphate transfection, the use of dendrimers, microinjection, and the use of viral vectors, including adenovirus vectors, AAV vectors, and retrovirus vectors, as well as / or ribozymes of Group II.

[0252] RNA encoding a transgene can also be designed to include a reporter gene so that the presence of the transgene or its expression product can be detected through the activation of the reporter gene. Any reporter gene can be used, such as the reporter gene disclosed above. Cells containing the transgene can be selected by selecting cells in a cell culture in which the reporter gene has been activated.

[0253] The transgene to be inserted can be excised from the polynucleic acid by flanking it with a manipulation site similar to the target double-strand break site in the genome, thus enabling insertion into the double-strand break region. In some cases, the transgene can be introduced by a virus. For example, the transgene can be used to infect cells with the transgene using the AAV virus. In some cases, the transgene can be introduced into cells using a modified AAV virus or a manipulated AAV virus (Figures 83A and 83B). Modified AAV or wild-type AAV may contain homology arms to at least one genomic location (Figures 84-86D).

[0254] RNA encoding a transgene can be introduced into cells via electroporation. RNA can also be introduced into cells via lipofection, infection, or transformation. Primary cells can be transfected using electroporation and / or lipofection. Primary hematopoietic cells can be transfected using electroporation and / or lipofection. In some cases, RNA can be reverse transcribed into DNA within the cell. The DNA substrate can then be used in a homologous recombination reaction. DNA can also be introduced into the cellular genome without the use of homologous recombination. In some cases, DNA can be sandwiched between target double-strand break regions and complementary manipulation sites within the genome. In some cases, DNA can be excised from polynucleic acids and inserted into double-strand break regions without homologous recombination.

[0255] Transgene expression can be verified by expression assays, such as qPCR, or by measuring RNA levels. Expression levels can also indicate copy number. For example, extremely high expression levels may indicate that more than one copy of the transgene has been incorporated into the genome. Alternatively, high High expression may indicate that the trans gene has been integrated into a highly expressed transcriptional region, for example, near a highly expressed promoter. Expression can also be verified by measuring protein levels, such as via Western blotting. In some cases, a splice acceptor assay can be used in conjunction with a reporter system to measure the integration of the trans gene (Figure 94).

[0256] b. Site-specific insertion Insertion of one or more transgenes in any of the methods disclosed herein may be site-specific. For example, one or more transgenes may be inserted adjacent to or near a promoter. In another example, one or more transgenes may be inserted adjacent to, near, or within an exon of a gene (e.g., the PD-1 gene). Such insertions can be used to knock in a transgene (e.g., a cancer-specific TCR transgene) while simultaneously disrupting another gene (e.g., the PD-1 gene). In yet another example, one or more transgenes may be inserted adjacent to, near, or within an intron of a gene. The transgenes can be introduced by an AAV viral vector and integrated into a target genomic site (Figure 87).

[0257] Modification of targeted gene loci in cells can be achieved by introducing DNA homologous to the target locus into the cell. The DNA may contain marker genes that enable cell selection, including the integration of constructs. Complementary DNA within the target vector can recombinate chromosomal DNA at the target locus. Marker genes can be flanked by complementary DNA sequences, a 3' recombination arm, and a 5' recombination arm. Multiple loci within a cell can be targeted. For example, a trans gene with recombination arms specific to one or more target loci can be introduced simultaneously, so that modifications of multiple genomes occur in a single step.

[0258] Various enzymes can catalyze the insertion of foreign DNA into the host genome. For example, site-directed recombinases can be clustered into two protein families with distinctly different biochemical properties: tyrosine recombinases (in which case DNA is covalently joined to tyrosine residues) and serine recombinases (in which case covalent joining occurs at serine residues). Depending on the case, recombinases may include Cre, fC31 integrase (a serine recombinase derived from the Streptomyces phage fC31), or site-directed recombinases derived from bacteriophages (including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase, and phage TP901-1 integrase).

[0259] Expression regulatory sequences can also be used within the construct. For example, expression regulatory sequences may include constitutive promoters that are expressed in a wide variety of cell types. Tissue-specific promoters can also be used to direct expression to specific cell lines.

[0260] Site-directed gene editing can be achieved using nonviral gene editing methods such as CRISPR, TALEN (see U.S. Patent No. 14 / 193,037), transposon-based ZEN, meganucleases, or Mega-TAL, or transposon-based systems. For example, the PiggyBac transposon system (Moriarty, BS et al., "Modular assembly of transposon") “Integratable multigene vectors using RecWay assembly”, Nucleic Acids Research, (8 You can use the Sleeping Beauty transposon system (see page 92 (2013)) or the Sleeping Beauty transposon system (see Aronovich, EL et al., "The Sleeping Beauty transposon system: a non-viral vector for gene therapy", Hum. Mol. Genet., Vol. 20 (R1): pages R14-R20 (2011)).

[0261] Site-directed gene editing can also be achieved without homologous recombination. Exogenous polynucleic acids can be introduced into the cellular genome without the use of homologous recombination. In some cases, the trans gene can be flanked by a complementary manipulation site to the target double-strand break region in the genome. Since the trans gene can be excised from the polynucleic acid, it can be inserted into the double-strand break region without homologous recombination.

[0262] c. Transgene Transgenes can be useful for expressing endogenous genes at higher levels than in cells without transgenes, for example, for overexpression. In addition, transgenes can be used to express exogenous genes at higher levels than in the background, i.e., in cells that have not been transfected. Transgenes can also encompass other types of genes, such as dominant-negative genes.

[0263] Transgenes can be introduced into organisms, cells, tissues, or organs to produce transgene products. Polynucleic acids may contain transgenes. Polynucleic acids may encode exogenous receptors (Figures 57A, 57B, and 57C). For example, disclosed herein are polynucleic acids comprising at least one exogenous T cell receptor (TCR) sequence flanked by at least two recombinant arms having a sequence complementary to a polynucleotide in the genomic sequence, such as adenosine A2a receptor, CD276, V-set domain-containing T cell activation inhibitor 1, B lymphocyte and T lymphocyte-related, cytotoxic T lymphocyte-related protein 4, indoleamine 2,3-dioxygenase 1, killer cell immunoglobulin-like receptor, 3-domain, long cytoplasmic tail, 1, lymphocyte activation gene 3, programmed cell death 1, hepatitis A virus cell receptor 2, V-domain immunoglobulin suppressor for T cell activation, or natural killer cell receptor 2B4. One or more transgenes can be combined with one or more disruptions.

[0264] T cell receptor (TCR) T cells may contain one or more transgenes. One or more transgenes may express TCR alpha-chain proteins, TCR beta-chain proteins, TCR gamma-chain proteins, and / or TCR delta-chain proteins that recognize and bind to at least one epitope on an antigen (e.g., a cancer epitope) or bind to a mutant epitope on an antigen. TCRs may bind to cancer neoantigens. TCRs may be functional TCRs, as shown in Figures 22 and 26. TCRs may contain only one of the alpha-chain sequences or beta-chain sequences defined herein (e.g., combined with further alpha-chains or beta-chains, respectively), or both chains. TCRs may contain only one of the gamma-chain sequences or delta-chain sequences defined herein (e.g., combined with further gamma-chains or delta-chains, respectively), or both chains. Functional TCRs maintain at least substantial biological activity within the fusion protein. In the case of the alpha and / or beta chains of the TCR, this means that both chains are still capable of forming a T cell receptor (together with the unmodified alpha and / or beta chains, or with the alpha and / or beta chains of another fusion protein), which performs its biological function, in particular, binding to the TCR's specific peptide-MHC complex and / or functional signaling in peptide activation. In the case of the gamma and / or delta chains of a TCR, this means that both chains can still form a T cell receptor (together with the unmodified gamma and / or delta chain, or with the gamma and / or delta chain of another fusion protein) that performs its biological function, in particular, binding to the TCR's specific peptide-MHC complex and / or functional signaling in peptide activation. A T cell may also contain one or more TCRs. A T cell may also contain a single TCR that is specific to more than one target.

[0265] TCRs can be identified using a variety of methods. In some cases, TCRs can be identified using whole exome sequencing. For example, TCRs can target the ERBB2IP (ErbB2 interacting protein) antigen containing the E805G mutation identified by whole exome sequencing. Alternatively, TCRs can be identified from the autologous, allogeneic, or heterologous repertoire. Autologous and allogeneic identification may involve multi-step processes. In both autologous and allogeneic identification, dendritic cells (DCs) can be generated from CD14-selected monocytes, matured, and then pulsed or transfected with specific peptides. Peptide-pulsed DCs can be used to stimulate autologous or allogeneic T cells. Single-cell peptide-specific T cell clones can be isolated from these peptide-pulsed T cell lines using limiting dilution. The TCR of interest can then be identified and isolated. The α and β chains of the target TCR can be cloned, codon-optimized, and encoded into a vector or transgene. Parts of the TCR can be replaced. For example, the constant region of a human TCR can be replaced with the corresponding mouse region. Replacing the human constant region with the corresponding mouse region can increase the stability of the TCR. The TCR can also be identified ex vivo with high avidity or hyperphysiological avidity.

[0266] To successfully generate tumor-specific TCRs, appropriate target sequences must be identified. These sequences can be found by isolating rare tumor-reactive T cells, or, if this is not possible, alternative techniques can be used to generate highly active anti-tumor T cell antigens. One approach may involve immunizing transgenic mice expressing the human leukocyte antigen (HLA) system with human tumor proteins to generate T cells expressing TCRs against human antigens (see, for example, Stanislawski et al., "Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer," Nature Immunology, Vol. 2, pp. 962-970 (2001)). An alternative approach is allogeneic TCR gene transfer, in which tumor-specific T cells are isolated from patients undergoing tumor remission, and their reactive TCR sequences are transferred to T cells from another patient who shares the disease but may be unresponsive (de Witte, MA et al., "Targeting self-antigens through allogeneic TCR gene transfer," Blood, Vol. 108, pp. 870-877 (2006)). Finally, in vitro techniques can be used to modify the TCR sequence, increasing the avidity of weakly reactive tumor-specific TCRs to their target antigens and thereby enhancing their tumor-killing activity (Schmid, DA et al., "Evidence for a TCR affinity threshold delimiting maximal CD8 T cell function," J. Immunol., Vol. 184, pp. 4936-4946 (2010)). Alternatively, TCRs can be identified using whole-exome sequencing.

[0267] This functional TCR fusion protein targets epitopes presented by MHC. This is possible. The MHC may be a class I molecule, such as HLA-A. The MHC may be a class II molecule. This functional TCR fusion protein may also have peptide-based or peptide-guided functionality to target antigens. This functional TCR can be ligated; for example, it can be ligated with a 2A sequence. This functional TCR can also be ligated with furin-V5-SGSGF2A, as shown in Figure 26. This functional TCR may also contain mammalian components. For example, this functional TCR may contain a mouse constant region. This functional TCR may also, in some cases, contain a human constant region. Peptide-guided functionality can, in principle, be achieved by introducing peptide sequences into the TCR and using these peptide sequences to target tumors. These peptides may be derived from phage display libraries or synthetic peptide libraries (e.g., Arap, W. et al., "Cancer Treatment by Targeted Drug Delivery to Tumor Vasculature in a Mouse Model," Science, Vol. 279, pp. 377-380 (1998); Scott, CP et al., "Structural requirements for the biosynthesis of backbone cyclic peptide See "libraries", Vol. 8: pp. 801-815 (2001). In particular, peptides specific to breast cancer, prostate cancer, and colon cancer, as well as peptides specific to angiogenesis, have already been successfully isolated and can be used in the present invention (Samoylova, TI et al., "Peptide Phage Display: Opportunities for Development of Personalized "Anti-Cancer Strategies," Anti-Cancer Agents in Medicinal Chemistry, Vol. 6 (No. 1): pp. 9-17 (September 2006). This functional TCR fusion protein can target mutated cancer epitopes or mutated cancer antigens.

[0268] Transgenes that can be used and are specifically envisioned may include genes that exhibit a certain degree of identity and / or homology to genes disclosed herein, such as the TCR gene. Thus, genes are envisioned to be usable as transgenes if they exhibit at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology, or at least approximately these proportions of homology (at the nucleic acid level or protein level). Furthermore, genes exhibiting at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or at least approximately these proportions of identity (at the nucleic acid or protein level), may be considered usable as trans genes. In some cases, trans genes may be functional.

[0269] Transgenes can be incorporated into cells. For example, transgenes can be incorporated into the germline of an organism. Once inserted into a cell, a transgene may be a complementary DNA (cDNA) segment, which is a copy of messenger RNA (mRNA), or it may be the gene itself, located within its original region of genomic DNA (with or without introns). An X protein transgene may refer to a transgene containing the nucleotide sequence that codes for the X protein. In some cases, as used herein, an X protein-coding transgene may be a transgene that codes for 100% or approximately 100% of the amino acid sequence of the X protein. In other cases, an X protein-coding transgene may code for at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, or 92% of the amino acid sequence of the X protein. A transgene may encode an amino acid sequence in proportions of 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%, or at least approximately these proportions. Expression of a transgene may ultimately result in a functional protein, e.g., a partially, completely, or excessively functional protein. As discussed above, when a partial sequence is expressed, the final result may be a non-functional or dominant-negative protein. Non-functional or dominant-negative proteins may also compete with functional (endogenous or exogenous) proteins. A transgene may also encode RNA (e.g., mRNA, shRNA, siRNA, or microRNA). In some cases, if a transgene encodes mRNA, it can be translated into a polypeptide (e.g., a protein). Therefore, it is conceivable that a transgene may encode a protein. Depending on the case, a trans gene may encode a protein, or a portion of a protein. In addition, the protein may have one or more mutations (e.g., deletions, insertions, amino acid substitutions, or rearrangements) compared to the wild-type polypeptide. The protein may be a natural polypeptide or an artificial polypeptide (e.g., a recombinant polypeptide). A trans gene may encode a fusion protein formed by two or more polypeptides. A T cell may contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteenth, twenty, twenty, or more trans genes, or approximately these numbers. For example, a T cell may contain one or more trans genes, including the TCR gene.

[0270] Transgenes (e.g., TCR genes) can be inserted into safe harbor loci. Safe harbors may include genomic locations where transgenes can be integrated and function without disrupting endogenous activity. For example, one or more transgenes can be inserted into any one of the following: HPRT, AAVS sites (e.g., AAVS1, AAVS2, etc.), CCR5, hROSA26, and / or any combination thereof. Transgenes (e.g., TCR genes) can also be inserted into endogenous immune checkpoint genes. Endogenous immune checkpoint genes may be stimulative or inhibitory checkpoint genes. Transgenes (e.g., TCR genes) can also be inserted into stimulative checkpoint genes such as CD27, CD40, CD122, OX40, GITR, CD137, CD28, or ICOS. The locations of immune checkpoint genes are presented using the Genome Reference Consortium Human Build 38 patch release 2 (GRCh38.p2) assembly. Transgenes (e.g., TCR genes) can also be inserted into endogenous inhibitory checkpoint genes such as A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, or CISH. For example, one or more transgenes can be inserted into any one of the following and / or any combination thereof: CD27, CD40, CD122, OX40, GITR, CD137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, AAVS sites (e.g., AAVS1, AAVS2, etc.), PHD1, PHD2, PHD3, CCR5, CISH, PPP1R12C. Transgenes can also be inserted into endogenous TCR genes. Transgenes can be inserted into coding genome regions. Transgenes can also be inserted into non-coding genome regions. Transgenes can be inserted into the genome without homologous recombination.Transgene insertion may involve the step of intracellular genome transplantation. Transgenes can be inserted into the PD-1 gene (Figures 46A and 46B). In some cases, more than one guide may target immune checkpoints (Figure 47). In other cases, transgenes are used. The gene can be integrated into the CTLA-4 gene (Figures 48 and 50). In other cases, the trans gene can be integrated into the CTLA-4 gene and the PD-1 gene (Figure 49). The trans gene can also be integrated into safe harbors such as AAVS1 (Figures 96 and 97). The trans gene can be inserted into AAV integration sites. In some cases, AAV integration sites can be safe harbors. Alternative AAV integration sites may also exist, such as AAVS2 on chromosome 5 or AAVS3 on chromosome 3. Further AAV integration sites, such as AAVS2, AAVS3, AAVS4, AAVS5, AAVS6, AAVS7, and AAVS8, are also considered possible integration sites for exogenous receptors such as TCRs. As used herein, AAVS may refer to AAVS1 and related adeno-associated virus (AAVS) integration sites.

[0271] Chimeric antigen receptors may consist of an extracellular antigen-recognition domain, a transmembrane domain, and a signaling domain that regulates T cell activation. The extracellular antigen-recognition domain may be derived from a mouse monoclonal antibody, a humanized monoclonal antibody, or a fully human monoclonal antibody. Specifically, the extracellular antigen-recognition domain consists of the variable regions of the heavy and light chains of a monoclonal antibody, cloned in the form of a single-stranded variable fragment (scFv), and bound to intracellular signaling molecules and at least one costimulatory molecule of the T cell receptor (TCR) complex via a hinge domain and a transmembrane domain. In some cases, the costimulatory domain is not used.

[0272] The CARs of this disclosure can reside within the cell membrane of eukaryotic cells, for example, mammalian cells, in which case suitable mammalian cells include, but are not limited to, cytotoxic cells, T lymphocytes, stem cells, progeny cells of stem cells, progenitor cells, progeny cells of progenitor cells, and NK cells. When present within the cell membrane of eukaryotic cells, the CAR may be active in the presence of a target to which it binds. The target may be expressed on the membrane. The target may also be soluble (e.g., not bound to a cell). The target may reside on the surface of a cell, such as a target cell. The target may be presented on a solid surface, such as a lipid bilayer. The target may be soluble, such as a soluble antigen. The target may be an antigen. The antigen may reside on the surface of a cell, such as a target cell. The antigen may be presented on a solid surface, such as a lipid bilayer. In some cases, the target may be an epitope of an antigen. In some cases, the target may be a cancer neoantigen. Recent advances have focused on identifying tumor-specific mutations that, in some cases, induce antitumor T cell responses. For example, these endogenous mutations can be identified using whole-exome sequencing (Tran E et al., "Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer," Science, vol. 344: pp. 641-644 (2014)). Thus, CARs may consist of scFvs targeting tumor-specific neoantigens.

[0273] The method may identify cancer-associated target sequences derived from samples obtained from cancer patients using an in vitro assay (e.g., whole exome sequencing). The method may further identify TCR transgenes derived from first T cells that recognize the target sequences. The cancer-associated target sequences and TCR transgenes may be obtained from samples from the same patient or from samples from different patients. The cancer-associated target sequences may be encoded on the CAR transgene to make the CAR specific to the target sequence. The method may efficiently deliver nucleic acids containing the CAR transgene across the T cell membrane. In some cases, the first and second T cells may be obtained from the same patient. In other cases, the first and second T cells may be obtained from different patients. In other cases, the first and second T cells may be obtained from different patients. The method may produce engineered T cells. Therefore, it is possible to safely and efficiently integrate CAR trans genes into the T cell genome using either a non-viral or viral integration system, and thus, to reliably express CAR trans genes within manipulated T cells.

[0274] T cells may contain disruption of one or more genes and one or more trans genes. For example, the one or more genes whose expression is disrupted may include any one of the following: CD27, CD40, CD122, OX40, GITR, CD137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, PHD1, PHD2, PHD3, VISTA, CISH, PPP1R12C, and / or any combination thereof. For example, just to illustrate the variety of combinations, the one or more genes whose expression is disrupted may include PD-1, and the one or more trans genes may include TCR. In another example, the one or more genes whose expression is disrupted may also include CTLA-4, and the one or more trans genes may include TCR.

[0275] T cells may contain the repression of one or more genes and one or more trans genes. For example, the one or more genes whose expression is repressed may include any one of the following: CD27, CD40, CD122, OX40, GITR, CD137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, PHD1, PHD2, PHD3, VISTA, CISH, PPP1R12C, and / or any combination thereof. For example, just to illustrate the variety of combinations, the one or more genes whose expression is repressed may include PD-1, and the one or more trans genes may include TCR. In another example, the one or more genes whose expression is repressed may also include CTLA-4, and the one or more trans genes may include TCR.

[0276] T cells may also contain one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteenth, thirteenth, fifteenth, fifteenth, sixteenth, seventeenth, nineteenth, twentyth, or more dominant-negative transgenes, or nearly these numbers of dominant-negative transgenes. The expression of a dominant-negative transgene may suppress the expression and / or function of its wild-type counterpart. Therefore, for example, a T cell containing the dominant-negative transgene X will have a similar phenotype to a different T cell containing the X gene whose expression is suppressed. One or more dominant-negative transgenes include dominant-negative CD27, dominant-negative CD40, dominant-negative CD122, dominant-negative OX40, dominant-negative GITR, dominant-negative CD137, dominant-negative CD28, dominant-negative ICOS, dominant-negative A2AR, dominant-negative B7-H3, dominant-negative B7-H4, dominant-negative BTLA, dominant-negative CTLA-4, and dominant-negative ID. This could be O, dominant-negative KIR, dominant-negative LAG3, dominant-negative PD-1, dominant-negative TIM-3, dominant-negative VISTA, dominant-negative PHD1, dominant-negative PHD2, dominant-negative PHD3, dominant-negative CISH, dominant-negative CCR5, dominant-negative HPRT, dominant-negative AAVS sites (e.g., AAVS1, AAVS2, etc.), dominant-negative PPP1R12C, or any combination of these.

[0277] Furthermore, T cells containing one or more transgenes encoding one or more nucleic acids that can suppress gene expression, for example, knock down genes, are also presented. RNAs that suppress gene expression include shRNA, siRNA, RNAi, and microRNA. This includes, but is not limited to, siRNA, RNAi, and / or microRNAs that can be delivered to T cells to suppress gene expression. Furthermore, T cells may contain one or more transgenes encoding shRNA. The shRNA may be specific to a particular gene. For example, the shRNA may be specific to any gene described in this application, including, but not limited to, CD27, CD40, CD122, OX40, GITR, CD137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, AAVS sites (e.g., AAVS1, AAVS2, etc.), PHD1, PHD2, PHD3, CCR5, CISH, PPP1R12C, and / or any combination thereof.

[0278] One or more transgenes may originate from different species. For example, one or more transgenes may include human genes, mouse genes, rat genes, pig genes, cattle genes, dog genes, cat genes, monkey genes, chimpanzee genes, or any combination thereof. For example, a transgene may originate from a human with a human-like genetic sequence. One or more transgenes may include human genes. In some cases, one or more transgenes are not adenovirus genes.

[0279] As described above, transgenes can be inserted randomly or site-specifically into the T cell genome. For example, a transgene can be inserted into a random locus within the T cell genome. These transgenes can be functional, meaning they can be fully functional regardless of their insertion location within the genome. For instance, a transgene may encode its own promoter, or it may be inserted into a location under the control of an endogenous promoter. Alternatively, a transgene can be inserted into a gene's intron, exon, promoter, or non-coding region. A transgene can be inserted in such a way that the insertion disrupts a gene, such as an endogenous checkpoint. Transgene insertions may include endogenous checkpoint regions. Transgene insertions can be guided by recombination arms that can flank the transgene.

[0280] In some cases, more than one copy of a trans gene can be inserted into more than one random locus in the genome. For example, multiple copies can be inserted into random loci in the genome. This can result in increased overall expression compared to inserting the trans gene randomly once. Alternatively, one copy of the trans gene can be inserted into one gene, and another copy of the trans gene can be inserted into a different gene. Trans genes can be targeted so that they can be inserted into specific loci in the genome of T cells.

[0281] The expression of a trans gene can be controlled by one or more promoters. Promoters can be ubiquitous, constitutive (non-regulatory promoters that allow continuous transcription of related genes), tissue-specific, or inductive promoters. The expression of a trans gene inserted adjacent to or near a promoter can be regulated. For example, a trans gene can be inserted near or next to a ubiquitous promoter. Some ubiquitous promoters may be the CAGGS promoter, hCMV promoter, PGK promoter, SV40 promoter, or ROSA26 promoter.

[0282] The promoter may be an endogenous or exogenous promoter. For example, one or more transgenes can be inserted adjacent to or near an endogenous or exogenous ROSA26 promoter. These may be T cell-specific. For example, one or more transgenes can be inserted adjacent to or near the porcine ROSA26 promoter.

[0283] The location of expression can be controlled using tissue-specific or cell-specific promoters. For example, one or more transgenes can be inserted adjacent to or near a tissue-specific promoter. Tissue-specific promoters may include the FABP promoter, Lck promoter, CamKII promoter, CD19 promoter, keratin promoter, albumin promoter, aP2 promoter, insulin promoter, MCK promoter, MyHC promoter, WAP promoter, or Col2A promoter.

[0284] The location of expression can be controlled using tissue-specific or cell-specific promoters. For example, one or more transgenes can be inserted adjacent to or near a tissue-specific promoter. Tissue-specific promoters may include the FABP promoter, Lck promoter, CamKII promoter, CD19 promoter, keratin promoter, albumin promoter, aP2 promoter, insulin promoter, MCK promoter, MyHC promoter, WAP promoter, or Col2A promoter.

[0285] Inductive promoters can also be used in a similar manner. These inductive promoters can be turned on or off by adding or removing an inducer, if desired. Inductive promoters may be, but are not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and / or Trex.

[0286] Cells can be manipulated to knock out endogenous genes. These endogenous genes may include immune checkpoint genes. These immune checkpoint genes can be either stimulative or inhibitory. The locations of immune checkpoint genes can be shown using the Genome Reference Consortium Human Build 38 patch release 2 (GRCh38.p2) assembly.

[0287] The genes to be knocked out can be selected using a database. In some cases, certain endogenous genes are more suitable for genomic manipulation. The database may contain target sites that are permissible by epigenetics. The database may be ENCODE (Encyclopedia of DNA Elements)(http: / / www.genome.gov / 10005107). The database may identify regions with open chromatin that may be more permissible for genomic manipulation.

[0288] T cell disruption may involve the disruption of one or more genes. For example, one or more genes whose expression is disrupted include adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-associated (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), and CISH (cytokine-inducible SH2-containing (Protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD160), TIGIT (T-cell immunoreceptor with Ig and ITIM domains), CD96 molecule (CD96), CRTAM (cytotoxic and regulatory T-cell (molecule), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), FADD (Fas associated via death domain), FAS (Fas cell surface death , Transforming Growth Factor Beta Receptor II (TGFBRII), Transforming Growth Factor Beta Receptor I (TGFBR1), SMAD Family Member 2 (SMAD2), SMAD Family Member 3 (SMAD3), SMAD Family Member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto-oncogene (SKIL), TGIF1 (TGFB-induced factor homeobox 1), Interleukin-10 receptor subunit alpha (IL10RA), Interleukin-10 receptor subunit beta (IL10RB), HMOX2 (heme oxygenase 2), Interleukin-6 receptor (IL6R), IL6ST (interleukin 6 signal transducer), c-src tyrosine kinase (CSK), PAG1 (phosphoprotein membrane anchor with glycosphingolipid microdomains 1), SIT1 (signaling thresholdThis may include any one or any combination of the following: regulating transmembrane adaptor 1), FOXP3 (forkhead box P3), PR domain 1 (PRDM1), BATF (basic leucine zipper transcription factor, ATF-like), soluble guanylate cyclase 1 alpha-2 (GUCY1A2), soluble guanylate cyclase 1 alpha-3 (GUCY1A3), soluble guanylate cyclase 1 beta-2 (GUCY1B2), soluble guanylate cyclase 1 beta-3 (GUCY1B3), CISH (cytokine-inducible SH2-containing protein), and the prolyl hydroxylase domain family of proteins (PHD1, PHD2, PHD3). In some cases, endogenous TCRs may also be knocked out. For example, to illustrate the variety of combinations, one or more genes whose expression is disrupted may include PD-1, CTLA-4, and CISH.

[0289] T cell repression may involve the suppression of one or more genes. For example, one or more genes whose expression is suppressed include adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-associated (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), and programmed gene Hepatitis A virus cell receptor 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), V-domain immunoglobulin suppressor for T cell activation (VISTA), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS1), or chemokine (CC motif) receptor 5 (gene / pseudogene) (CCR5), CD160 molecule (CD160), TIGIT ( T-cell immunoreceptor with Ig and ITIM domains), CD96 molecule (CD96), CRTAM (cytotoxic and Regulatory T-cell molecule), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), sialic acid-binding Ig-like lectin 7 (SIGLEC7), sialic acid-binding Ig-like lectin 9 (SIGLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), FADD (Fas associated via death domain), FAS (Fas cell surface death , Transforming Growth Factor Beta Receptor II (TGFBRII), Transforming Growth Factor Beta Receptor I (TGFBR1), SMAD Family Member 2 (SMAD2), SMAD Family Member 3 (SMAD3), SMAD Family Member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto-oncogene (SKIL), TGIF1 (TGFB-induced factor homeobox 1), Interleukin-10 receptor subunit alpha (IL10RA), Interleukin-10 receptor subunit beta (IL10RB), HMOX2 (heme oxygenase 2), Interleukin-6 receptor (IL6R), IL6ST (interleukin 6 signal transducer), c-src tyrosine kinase (CSK), PAG1 (phosphoprotein membrane anchor with glycosphingolipid microdomains 1), SIT1 (signaling threshold regulating transmembrane adaptor 1), FOXP3 (forkhead box P3), PR domain 1 (PRDM1), BATF (basic leucine zipper transcription factor,It may include any one of the following, or any combination thereof: ATF-like, soluble guanylate cyclase 1 alpha-2 (GUCY1A2), soluble guanylate cyclase 1 alpha-3 (GUCY1A3), soluble guanylate cyclase 1 beta-2 (GUCY1B2), soluble guanylate cyclase 1 beta-3 (GUCY1B3), the prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, or CISH (cytokine-inducible SH2-containing protein). For example, to illustrate the diverse combinations, one or more genes whose expression is repressed may include PD-1, CTLA-4, and CISH.

[0290] d. Cancer targets Manipulated cells can target antigens. Manipulated cells can also target epitopes. Antigens can be tumor cell antigens. Epitopes can be tumor cell epitopes. Such tumor cell epitopes can originate from a wide variety of tumor antigens, including tumor-derived antigens arising from mutations (neoantigens or neoepitopes), common tumor-specific antigens, differentiation antigens, and antigens overexpressed within tumors. These antigens include, to name a few, alpha-actinin-4, ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDKN2A, COA-1, dek-can fusion protein, EFTUD2, elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FN1, GPNMB, LDLR-fucosyltransferase fusion protein, HLA-A2d, HLA-Al ld, hsp70-2, KIAAO205, MART2, ME1, MUM-1f, MUM-2, MUM-3, neo-PAP, myosin class I, NFYC, OGT, OS-9, p53, pml-RAR alpha fusion protein, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 fusion protein or SYT-SSX2 fusion protein, TGF-beta-RII, triosethrin isomerase, BAGE-1, GAGE-1, 2, 8, Gage 3, 4, 5 , 6, 7, GnTVf, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-Al2, MAGE-C2, mucink, NA-88, NY-ESO-1 / LAGE-2, S AGE, Sp17, SSX-2, SSX-4, TAG-1, TAG-2, TRAG-3, TRP2-INT2g, XAGE-1b, CEA, gp100 / Pmel17, kallikrein 4, mammaglobin A, Melan-A / MART-1, NY-BR-1, OA1, PSA, RAB38 / NY-MEL-1, TRP-1 / gp75, It may be derived from TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX(L), BCMA, BING-4, CPSF, cyclin D1, DKK1, ENAH(hMena), EP-CAM, EphA3, EZH2, FGF5, G250 / MN / CAIX, HER-2 / neu, IL13R alpha-2, intestinal carboxylesterase, alpha-fetoprotein, M-CSFT, MCSP, mdm-2, MMP-2, MUC1, p53, PBF, PRAME, PSMA, RAGE-1, RGS5, RNF43, RU2AS, cesernin 1, SOX10, STEAP1, sulbibin, telomerase, VEGF, and / or WT1. Tumor-associated antigens may be antigens that the host does not normally express; they may be abnormal manifestations of molecules that the host does not normally express, such as being mutated, cleaved, misfolded, or otherwise; they may be identical to molecules that are normally expressed but expressed at abnormally high levels; or they may be expressed in abnormal associations or environments. Tumor-associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, other biological molecules, or any combination thereof.

[0291] In some cases, the target is a neoantigen or neoepitope. For example, a neoantigen may be an E805G mutation within ERBB2IP. In some cases, neoantigens and neoepitopes can be identified by whole-exome sequencing. In some cases, neoantigen targets and neoepitope targets may be expressed in gastrointestinal cancer cells. Neoantigens and neoepitopes may be expressed in epithelial cancers.

[0292] e. Other targets Epitopes may be stromal epitopes. Such epitopes may be present on the stroma of the tumor microenvironment. Antigens may be stromal antigens. Such antigens may be present on the stroma of the tumor microenvironment. These antigens and these epitopes may be present in, to name a few, tumor endothelial cells, tumor vascular system, tumor fibroblasts, tumor pericytes, tumor stroma, and / or tumor mesenchymal cells. These antigens may include, for example, CD34, MCSP, FAP, CD31, PCNA, CD117, CD40, MMP4, and / or tenascin.

[0293] f. Gene disruption Transgene insertion can be performed with or without gene disruption. Transgenes can be inserted adjacent to, near, or within genes such as CD27, CD40, CD122, OX40, GITR, CD137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, AAVS sites (e.g., AAVS1, AAVS2, etc.), CCR5, PPP1R12C, or CISH to reduce or eliminate gene activity or expression. For example, a cancer-specific TCR transgene can be inserted adjacent to, near, or within a gene (e.g., PD-1) to reduce or eliminate gene activity or expression. Transgene insertion can be performed in endogenous TCR genes.

[0294] Gene disruption may be the disruption of any specific gene. Genetic phase of genes in this application It is envisioned that the target will be an identical organism (e.g., any mammalian form of a gene). For example, the disrupted gene may exhibit certain identity and / or homology to the genes disclosed herein, such as CD27, CD40, CD122, OX40, GITR, CD137, CD28, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, HPRT, CCR5, AAVS sites (e.g., AAVS1, AAVS2, etc.), PPP1R12C, or CISH. Therefore, it is assumed that genes exhibiting homology of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or homology of approximately these ratios (at the nucleic acid level or protein level), can be disrupted. Furthermore, it is conceivable that genes exhibiting 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, or identity in approximately these proportions (at the nucleic acid or protein level), may be disrupted. In this art, some gene homologs are known, but in some cases, homologs are not. However, homologous genes among mammals can be found by comparing nucleic acid (DNA or RNA) sequences or protein sequences using publicly available databases such as NCBI's BLAST.

[0295] A gene that can be disrupted may be a member of a gene family. For example, a gene that can be disrupted may improve the therapeutic potential of cancer immunotherapy. In some cases, the gene may be CISH. The CISH gene may be a member of the cytokine-inducible STAT inhibitor (CIS) protein family, also known as the SOCS (suppressor of cytokine signaling) protein family or the STAT-inducible STAT inhibitor (SSI) protein family (see, for example, Palmer et al., Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance., The Journal of Experimental Medicine, Vol. 202 (No. 12), pp. 2095-2113 (2015)). The gene may be part of the SOCS protein family, which may form part of a classic negative feedback system that can regulate cytokine signaling. The gene that can be disrupted may be CISH. CISH may be involved in the negative regulation of cytokines that signal via the JAK-STAT5 pathway, such as erythropoietin, prolactin, or interleukin-3 (IL-3) receptor. The gene may inhibit STAT5 transactivation by repressing its tyrosine phosphorylation. CISH family members are known to be cytokine-inducible negative regulators of cytokine signaling. Gene expression can be induced by IL2, IL3, GM-CSF, or EPO in hematopoietic cells. Proteasome-mediated degradation of the gene's protein may be involved in erythropoietin receptor inactivation. In some cases, the targeted gene may be expressed in tumor-specific T cells. When the targeted gene is disrupted, it can increase the invasion of manipulated cells into antigen-related tumors. In some cases, the targeted gene may be CISH.

[0296] Genes that can be disrupted may be involved in TCR signaling, functional avidity, or attenuation of immunity against cancer. In some cases, disrupted genes are upregulated when the TCR is stimulated. Genes may be involved in inhibiting cell growth, functional avidity, or cytokine multifunctionality. Genes may be involved in negatively regulating cytokine production by cells. For example, genes may be involved in inhibiting the production of effector cytokines, IFN-gamma, and / or TNF. Genes may also stimulate the TCR. Following the initial reaction, these genes may also be involved in inhibiting the expression of accessory cytokines such as IL-2. Such genes may be involved in CISH (Chronic Inflammatory Syndrome).

[0297] Gene repression can also be carried out in numerous ways. For example, gene expression can be repressed by knockout, by altering the gene promoter, and / or by administering interfering RNA. This can be done at the biological level, as well as at the tissue, organ, and / or cellular level. When knocking down one or more genes within an cell, tissue, and / or organ, one or more genes can be repressed by administering RNA interfering reagents, such as siRNA, shRNA, or microRNA. For example, nucleic acids capable of expressing shRNA can be stably transfected into cells to knock down their expression. Furthermore, nucleic acids capable of expressing shRNA can be inserted into the genome of T cells to knock down genes within T cells.

[0298] Disruption methods may also include the step of overexpressing a dominant-negative protein. This method can result in an overall reduction of the function of a functional wild-type gene. In addition, the step of expressing a dominant-negative gene may result in a phenotype similar to that of a knockout and / or knockdown.

[0299] In some cases, stop codons can be inserted or created (e.g., by nucleotide substitution) within one or more genes, which may result in a non-functional transcript or non-functional protein (sometimes referred to as a knockout). For example, if a stop codon is created in the middle of one or more genes, the resulting transcript and / or protein may be cleaved and non-functional. However, in some cases, cleavage may result in an active (partially active or overactive) protein. If the protein is overactive, this may result in a dominant-negative protein.

[0300] This dominant-negative protein can be expressed within nucleic acids under the control of any promoter. For example, the promoter can be a ubiquitous promoter. The promoter can also be an inducible promoter, a tissue-specific promoter, a cell-specific promoter, and / or a development-specific promoter.

[0301] Next, nucleic acids encoding dominant-negative proteins can be inserted into cells. Any method can be used; for example, stable transfection can be used. In addition, nucleic acids encoding dominant-negative proteins can also be inserted into the genome of T cells.

[0302] One or more genes within a T cell can be knocked out or disrupted using any method. For example, knocking out one or more genes may include deleting one or more genes from the T cell genome. Knockout may also include removing all or part of a gene sequence from the T cell. It is also conceivable that knockout may include replacing all or part of a gene in the T cell genome with one or more nucleotides. Knockout of one or more genes may also include inserting a sequence into one or more genes, thereby disrupting the expression of one or more genes. For example, a sequence insertion may create a stop codon in the middle of one or more genes. A sequence insertion may also shift the open reading frame of one or more genes.

[0303] Knockout can be performed within any cell, organ, and / or tissue, for example, within T cells, hematopoietic stem cells, bone marrow, and / or the thymus. For example, knockout can be systemic, where, for instance, the expression of one or more genes is suppressed in all human cells. Knockout can also be specific to one or more human cells, tissues, and / or organs. This can be achieved by conditional knockout, which selectively suppresses the expression of one or more genes in one or more organs, tissues, or cell types. Conditional knockout can be performed using the Cre-lox system, which expresses Cre under the control of cell-specific promoters, tissue-specific promoters, and / or organ-specific promoters. For example, one or more genes may be knocked out (or their expression may be suppressed) within one or more tissues or organs, in which case one or more tissues or organs may include the brain, lungs, liver, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, smooth muscle, skin, bone, adipose tissue, hair, thyroid gland, trachea, gallbladder, kidney, ureter, bladder, aorta, vein, esophagus, diaphragm, stomach, rectum, adrenal gland, bronchi, ear, eye, retina, genitals, hypothalamus, larynx, nose, tongue, spinal cord, or ureter, uterus, ovary, testis, and / or any combination thereof.Furthermore, one or more genes within a single cell type may also be knocked out (or have their expression suppressed), in which case the one or more cell types are hair follicles, keratinocytes, gonadotropin-producing cells, corticotropin-producing cells, thyroid-stimulating hormone-producing cells, somatotropin-producing cells, prolactin-producing cells, chromaffin cells, parafollicular cells, glomus cells, melanocytes, nevus cells, Merkel cells, odontoblasts, cementoblasts, corneal stromal cells, retinal Müller cells, retinal pigment epithelial cells, neurons, glia (e.g., oligodendrocytes, astrocytes), ependymal cells, pineal cells, lung cells (e.g., type I and type II lung cells), Clara cells, goblet cells, G cells, D cells, and enterochromaffin cells. Cells, gastric chief cells, parietal cells, gastric pit cells, K cells, D cells, I cells, goblet cells, Paneth cells, intestinal cells, M cells, hepatocytes, hepatic stellate cells (e.g., Kupffer cells derived from the mesoderm), gallbladder cells, acinar central cells, pancreatic stellate cells, pancreatic α cells, pancreatic β cells, pancreatic δ cells, pancreatic F cells, pancreatic ε cells, thyroid (e.g., follicular cells), parathyroid (e.g., parathyroid chief cells), eosinophilic cells, urothelial cells, osteoblasts, osteocytes, chondrocytes, chondrocytes, fibroblasts, fibrocytes, myoblasts, myocytes, muscle satellite cells, tendinocytes, cardiomyocytes, lipoblasts, adipocytes, interstitial cells of Cajal, angioblasts, endothelial cells, mesangial cells (e.g., intraglomerular mesangial cells and extraglomerular mesangial cells), juxtaglomerular cells, macula densa cells, stromal cells This includes cells, interstitial cells, telocytes, simple epithelial cells, podocytes, renal proximal tubular brush border cells, Sertoli cells, Leydig cells, granulosa cells, non-ciliary epithelial cells, germ cells, sperm, oocytes, lymphocytes, myeloid cells, endothelial progenitor cells, endothelial stem cells, angioblasts, mesodermal angioblasts, pericytes, parietal cells, and / or any combination thereof.

[0304] In some embodiments, the methods of the present disclosure may include the step of obtaining one or more cells from a subject. Cells may generally refer to any biological structure, including cytoplasm, proteins, nucleic acids, and / or organelles, encapsulated within a membrane. In some embodiments, cells may be mammalian cells. In some embodiments, cells may refer to immune cells. Non-limiting examples of cells may include B cells, basophils, dendritic cells, eosinophils, gamma delta T cells, granulocytes, helper T cells, Langerhans cells, lymphoid cells, innate lymphoid cells (ILCs), macrophages, mast cells, megakaryocytes, memory T cells, monocytes, myeloid cells, natural killer T cells, neutrophils, precursor cells, plasma cells, progenitor cells, regulatory T cells, T cells, thymus, any differentiated or dedifferentiated cells of these, or mixtures or combinations of any of these cells.

[0305] In some embodiments, the cells can be ILCs, which are Group 1 ILCs, Group 2 ILCs, or Group 3 ILCs. Group 1 ILCs are generally regulated by the T-bet transcription factor and respond to intracellular pathogens with IFN-gamma and TNF-ALF Group 1 ILCs can be described as cells that secrete type 1 cytokines, such as α. Group 2 ILCs can generally be described as cells that rely on GATA-3 transcription factor and ROR-alpha transcription factor and produce type 2 cytokines in response to extracellular parasitic infections. Group 3 ILCs can generally be described as cells that are regulated by ROR-gamma transcription factor and produce IL-17 and / or IL-22.

[0306] In some embodiments, cells may be positive for a given factor, or negative for a given factor. In some embodiments, cells may be CD3+ cells, CD3- cells, CD5+ cells, CD5- cells, CD7+ cells, CD7- cells, CD14+ cells, CD14- cells, CD8+ cells, CD8- cells, CD103+ cells, CD103- cells, CD11b+ cells, CD11b- cells, BDCA1+ cells, BDCA1- cells, L-selectin+ cells, L-selectin- cells, CD25+ cells, CD25- cells, CD27+ cells, CD27- cells, CD28+ cells, CD28- cells, CD44+ cells, CD4 These may be 4-cells, CD56+ cells, CD56-cells, CD57+ cells, CD57-cells, CD62L+ cells, CD62L-cells, CD69+ cells, CD69-cells, CD45RO+ cells, CD45RO-cells, CD127+ cells, CD127-cells, CD132+ cells, CD132-cells, IL-7+ cells, IL-7-cells, IL-15+ cells, IL-15-cells, lectin-like receptor G1-positive cells, lectin-like receptor G1-negative cells, or differentiated or dedifferentiated cells thereof. Examples of factors expressed by cells are not intended to be limiting, and those skilled in the art will notice that cells may be positive or negative for any factor known in the art. In some embodiments, cells may be positive for two or more factors. For example, cells may be CD4+ and CD8+. In some embodiments, cells may be negative for two or more factors. For example, cells may be CD25-, CD44-, and CD69-. In some embodiments, cells may be positive for one or more factors and negative for one or more factors. For example, cells may be CD4+ and CD8-. The selected cells can then be infused into a subject. In some embodiments, cells can be selected for having or not having one or more given factors (for example, cells can be separated based on the presence or absence of one or more factors). Separation efficiency may affect cell viability and the efficiency with which transgenes can be incorporated into and / or expressed in the cell genome.In some embodiments, the selected cells may also be magnified in vitro. The selected cells may be magnified in vitro before infusion. It should be understood that the cells used in any of the methods disclosed herein may be a mixture of any of the cells disclosed herein (e.g., two or more different cells). For example, the method of this disclosure may include cells that are a mixture of CD4+ cells and CD8+ cells. In another example, the method of this disclosure may include cells that are a mixture of CD4+ cells and naive cells.

[0307] Naive cells possess several characteristics that may be particularly useful in the methods disclosed herein. For example, naive cells are readily expandable in vitro and express T cell receptor transgenes, exhibit a small number of terminal differentiation markers (qualities that may be associated with greater efficacy after cell infusion), and possess long telomeres, suggesting greater potential for proliferation (Hinrichs, CS et al., "Human effector CD8+ T cells derived from naive rather than memory subsets possess superior traits for adoptive immunotherapy," Blood, 117(3):808-814 (2011)). The methods disclosed herein may include the selection or negative selection of markers specific to naive cells. In some embodiments, the cells may be naive cells. Naive cells are generally exposed to antigens. The term may refer to any cell that is not present. Any cell in this disclosure may be a naive cell. For example, the cell may be a naive T cell. Naive T cells can generally be described as cells that differentiate in the bone marrow, successfully undergo positive and negative processes of central selection in the thymus, and / or are characterized by the expression or absence of specific markers (e.g., surface expression of L-selectin, absence of the activation markers CD25, CD44, or CD69, absence of the memory CD45RO isoform).

[0308] In some embodiments, the cells may include cell lines (e.g., immortalized cell lines). Non-limiting examples of cell lines include human BC-1 cells, human BJAB cells, human IM-9 cells, human Jiyoye cells, human K-562 cells, human LCL cells, mouse MPC-11 cells, human Raji cells, human Ramos cells, mouse Ramos cells, human RPMI8226 cells, human RS4-11 cells, human SKW6.4 cells, human dendritic cells, mouse P815 cells, mouse RBL-2H3 cells, human HL-60 cells, human NAMALWA cells, human macrophage cells, mouse RAW 264.7 cells, human KG-1 cells, mouse M1 cells, human PBMC cells, mouse BW5147(T200-A)5.2 cells, human CCRF-CEM cells, mouse EL4 cells, human Jurkat cells, human SCID.adh cells, human U-937 cells, or any combination of these cells.

[0309] Stem cells can give rise to a variety of somatic cells and, therefore, in principle, have the potential to be used as an unlimited source of virtually any type of therapeutic cell. The reprogramming ability of stem cells also allows for further manipulation to enhance the therapeutic value of the reprogrammed cells. In any of the methods of this disclosure, one or more cells may be derived from stem cells. Non-limiting examples of stem cells include embryonic stem cells, adult stem cells, tissue-specific stem cells, neural stem cells, allogeneic stem cells, totipotent stem cells, plutopitous stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, epidermal stem cells, umbilical cord stem cells, epithelial stem cells, or adipose-derived stem cells. In one example, the cells may be lymphoid progenitor cells derived from hematopoietic stem cells. In another example, the cells may be T cells derived from embryonic stem cells. In yet another example, the cells may be T cells derived from induced pluripotent stem cells (iPSCs).

[0310] Conditional knockout can be inducible knockout, for example, by using a tetracycline-inducible promoter or a development-specific promoter. This can allow for the loss or suppression of gene / protein expression at any point in time or at a specific point in time. For example, in the case of a tetracycline-inducible promoter, tetracycline can be delivered to T cells at any point in the postnatal period. The cre / lox system can also be controlled under the control of a development-specific promoter. For example, some promoters are turned on shortly after birth or after the onset of puberty. These promoters can be used to control cre expression and therefore can be used in development-specific knockout.

[0311] Furthermore, it is conceivable that any combination of knockout techniques could be implemented. For example, tissue-specific or cell-specific knockouts could be combined with induction techniques to create tissue-specific or cell-specific inducible knockouts. In addition, other systems, such as development-specific promoters, could be used in combination with tissue-specific promoters and / or inducible knockouts.

[0312] Knockout technology may also include gene editing. For example, gene editing can be carried out using nucleases, including CRISPR-related proteins (Cas proteins, e.g., Cas9), zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and meganucleases. These may be naturally occurring nucleases, genetically modified nucleases, and / or recombinant nucleases. Gene editing can also be performed using transposon-based systems (e.g., PiggyBac, Sleeping Beauty). For example, gene editing can be performed using transposases.

[0313] CRISPR system The methods described herein may utilize CRISPR systems. At least five types of CRISPR systems exist, all of which involve RNA proteins and Cas proteins. Types I, III, and IV assemble multi-Cas protein complexes capable of cleaving nucleic acids complementary to crRNA. Both types I and III require pre-crRNA processing before assembling the processed crRNA into a multi-Cas protein complex. Types II and V CRISPR systems contain a single Cas protein complexed with at least one guide RNA.

[0314] For general mechanisms and recent advances in the CRISPR system, see: Cong, L. et al., "Multiplex genome engineering using CRISPR systems," Science, Vol. 339 (No. 6121): pp. 819-823 (2013); Fu, Y. et al., "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells," Nature Biotechnology, Vol. 31, pp. 822-826 (2013); Chu, VT et al., "Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells," Nature Biotechnology, Vol. 33, pp. 543-548 (2015); Shmakov, S. et al., "Discovery and functional characterization of diverse Class 2 CRISPR-Cas systems," Molecular Cell, Vol. 60, pp. 1-13 (2015); Makarova, KS et al., "An updated evolutionary classification" This is discussed in "of CRISPR-Cas systems," Nature Reviews Microbiology, Vol. 13, pp. 1-15 (2015). Site-specific cleavage of target DNA occurs at a location determined by both 1) base pair complementarity between guide RNA and target DNA (also called protospacer), and 2) a short motif within the target DNA called a protospacer adjacent motif (PAM). For example, the manipulated cells can be prepared using a CRISPR system, such as a type II CRISPR system. The Cas enzyme used in the methods disclosed herein may be Cas9, which catalyzes DNA cleavage.Enzymatic action by Cas9 derived from Streptococcus pyogenes, or any closely related Cas9, can hybridize to a 20-nucleotide guide sequence, and induce double-strand breaks at a target site sequence having a protospacer-adjacent motif (PAM) following the 20-nucleotide target sequence.

[0315] a. Cas protein The vector can be operably ligated to an enzyme-coding sequence that encodes a CRISPR enzyme, such as a Cas protein (CRISPR-related protein). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm 6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, their homologs, or modified forms thereof. Unmodified CRISPR enzymes, such as Cas9, may have DNA cleavage activity. CRISPR enzymes can lead to cleavage of one or both strands in a target sequence, such as within the target sequence and / or in the complement of the target sequence. For example, a CRISPR enzyme can lead to the cleavage of one or both strands within approximately one, two, three, four, five, six, seven, eight, nine, ten, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence, or within approximately these number of base pairs. A vector encoding a CRISPR enzyme mutated relative to the corresponding wild-type enzyme can be used so that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. The Cas protein can be a high-fidelity Cas protein, such as Cas9HiFi.

[0316] Vectors encoding CRISPR enzymes containing one or more nuclear localization sequences (NLSs) can be used, such as one, two, three, four, five, six, seven, eight, nine, more than ten NLSs, or nearly these numbers of NLSs. For example, a CRISPR enzyme may contain one, two, three, four, five, six, seven, eight, nine, more than ten NLSs, or nearly these numbers of NLSs at or near the amino terminus, or one, two, three, four, five, six, seven, eight, nine, more than ten NLSs, or nearly these numbers of NLSs at or near the carboxyl terminus, or any combination thereof (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxyl terminus). If more than one NLS exists, each NLS can be independently selected from other NLSs so that a single NLS may exist in combination with one or more other NLSs that exist in more than one copy and / or one or more copies.

[0317] Cas9 may refer to a polypeptide with at least 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or similarity, or at least approximately these ratios, to an exemplary wild-type Cas9 polypeptide (e.g., Cas9 derived from S. pyogenes). Cas9 may refer to a polypeptide with up to 50%, 60%, 70%, 80%, 90%, or 100% sequence identity and / or similarity, or at least approximately these ratios, to an exemplary wild-type Cas9 polypeptide (e.g., derived from S. pyogenes). Cas9 may refer to the wild-type form of the Cas9 protein, or it may refer to a modified form of the Cas9 protein, which may include amino acid changes such as deletions, insertions, substitutions, mutants, mutations, fusions, chimeras, or any combination thereof.

[0318] Polynucleotides encoding endonucleases (e.g., Cas proteins such as Cas9) can have their codons optimized for expression in specific cells, such as eukaryotic cells. This type of optimization may involve exogenous (e.g., recombinant) DNA mutations that mimic the codon preference of the intended host organism or host cell, while encoding the same protein.

[0319] The CRISPR enzyme used in the method may include an NLS. The NLS may be located at any position in the polypeptide chain, for example, near the N-terminus or C-terminus. For example, the NLS may be within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 amino acids from the N-terminus or C-terminus along the polypeptide chain, or approximately this number of amino acids. It may be within the amino acid range. In some cases, the NLS may be within 50 amino acids or more from the N-terminus or C-terminus, for example, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 amino acids, or approximately within these numbers.

[0320] The endonuclease may contain an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% amino acid sequence identity, or at least approximately these ratios, to the nuclease domain of an exemplary wild-type site-directed polypeptide (e.g., Cas9 derived from S. pyogenes).

[0321] S. pyogenes Cas9 (SpCas9) (Table 11) is generally used as a CRISPR endonuclease for genome manipulation, but it may not be the best endonuclease for every target excision site. For example, while the PAM sequence for SpCas9 (5'NGG3') is abundant throughout the human genome, the NGG sequence may not be positioned to properly target the gene desired for modification. In some cases, a different endonuclease can be used to target a specific genomic target. In some cases, mutants derived from synthetic SpCas9 with PAM sequences other than NGG can be used. In addition, other Cas9 orthologues from diverse species have been identified, and these "non-SpCas9" bind to various PAM sequences and may also be useful in the present invention. For example, the relatively large size of SpCas9 (approximately 4kb of coding sequence) means that plasmids containing SpCas9 cDNA may not be efficiently expressed in cells. Conversely, Staphylococcus aureus The coding sequence of Cas9 (SaCas9) is approximately 1 kilobase shorter than that of SpCas9, which likely allows for more efficient expression within cells. Similar to SpCas9, the SaCas9 endonuclease can modify target genes in vitro in mammalian cells and in vivo in mice.

[0322] Alternatives to S. pyogenes Cas9 may include RNA-guided endonucleases derived from the Cpf1 family that exhibit cleavage activity within mammalian cells. Unlike Cas9 nucleases, DNA cleavage mediated by Cpf1 results in double-strand breaks with short 3' overhangs. The adherent-end cleavage pattern of Cpf1 is a directional gene transfer similar to conventional restriction enzyme cloning, opening up the possibility of directional gene transfer that increases the efficiency of gene editing. Similar to the Cas9 variants and orthologs described above, Cpf1 can also expand the number of sites that can be targeted by CRISPR to AT-rich regions or AT-rich genomes that lack the NGG PAM sites preferred by SpCas9.

[0323] Any functional concentration of Cas protein can be introduced into cells. For example, 15 micrograms of Cas mRNA can be introduced into cells. In other cases, 0.5 to 100 micrograms of Cas mRNA can be introduced. 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms of Cas It is possible to introduce antigens.

[0324] b. Guide RNA As used herein, the term “guide RNA (gRNA)” and its grammatical equivalent may refer to RNA that can be specific to target DNA and can form a complex with the Cas protein. Guide RNA is a guide sequence or spacer that designates a target site and guides the RNA / Cas complex to the designated target DNA for cleavage. —may include sequences. For example, Figure 15 shows that the guide RNA can target the CRISPR complex to three genes and perform targeted double-strand breaks. Site-specific breaks of the target DNA occur at a location determined by both 1) base pairing complementarity between the guide RNA and the target DNA (also called the protospacer), and 2) a short motif within the target DNA called a protospacer adjacent motif (PAM).

[0325] The methods disclosed herein may also include the step of introducing at least one guide RNA, or a nucleic acid encoding at least one guide RNA, such as DNA, into a cell or embryo. The guide RNA interacts with an RNA-guided endonuclease to guide the endonuclease to a specific target site where, at that site, the 5' end of the guide RNA base-pairs with a specific protospacer sequence within the chromosomal sequence.

[0326] Guide RNA may consist of two RNAs, for example, CRISPR RNA (crRNA) and trans-activated crRNA (tracrRNA). In some cases, guide RNA may consist of a single guide RNA (sgRNA) formed by the fusion of portions (e.g., functional portions) of crRNA and tracrRNA. Guide RNA may also be a biRNA containing both crRNA and tracrRNA. Guide RNA may contain crRNA but lack tracrRNA. Furthermore, crRNA may hybridize with target DNA or a protospacer sequence.

[0327] As discussed above, guide RNA can be an expression product. For example, the DNA encoding guide RNA can be a vector containing the sequence encoding the guide RNA. Guide RNA can be introduced into a cell or organism by transfecting it with isolated guide RNA or isolated plasmid DNA containing the sequence encoding the guide RNA and a promoter. Guide RNA can also be introduced into a cell or organism in other ways, such as using virus-mediated gene delivery.

[0328] Guide RNA can be isolated guide RNA. For example, guide RNA can be transfected into cells or organisms in the form of isolated RNA. Guide RNA can be prepared by in vitro transcription using any in vitro transcription system. Guide RNA can be transferred into cells in the form of isolated RNA, rather than in the form of a plasmid containing the coding sequence of the guide RNA.

[0329] Guide RNA may include a DNA targeting segment and a protein-binding segment. The DNA targeting segment (or DNA targeting sequence, or spacer sequence) contains a nucleotide sequence that may be complementary to a specific sequence in the target DNA (e.g., a protospacer). The protein-binding segment (or protein-binding sequence) may interact with site-directed modifying polypeptides, such as the Cas protein, which are RNA-guided endonucleases. "Segment" means a segment / compartment / region of a molecule, e.g., a continuous sequence of nucleotides in RNA. A segment also means a region / compartment of a complex, such that the segment may contain regions of more than one molecule. For example, in some cases, the protein-binding segment of DNA targeting RNA is one RNA molecule and the protein-binding segment, and therefore contains a region of this RNA molecule. In other cases, the protein-binding segment of DNA targeting RNA contains two separate molecules hybridized along a complementary region.

[0330] Guide RNA may contain two separate RNA molecules or a single RNA molecule. An example of a single-molecule guide RNA is a DNA targeting segment and It contains both protein-binding segments.

[0331] Exemplary two-molecule DNA targeting RNAs may include a crRNA-like molecule ("CRISPR RNA," "targeter RNA," "crRNA," or "crRNA repeat") and a corresponding tracrRNA-like molecule ("trans-activated CRISPR RNA," "activator RNA," or "tracrRNA"). The first RNA molecule may be a crRNA-like molecule (targeter RNA) that may contain a DNA targeting segment (e.g., a spacer) and a sequence of nucleotides that can form half of a double helix of double-stranded RNA (dsRNA) containing the protein-binding segment of the guide RNA. The second RNA molecule may be a corresponding tracrRNA-like molecule (activator RNA) that may contain a sequence of nucleotides that can form the other half of the dsRNA double helix of the protein-binding segment of the guide RNA. In other words, the sequence of nucleotides of the crRNA-like molecule can be complementary to the sequence of nucleotides of the tracrRNA-like molecule and can hybridize with it to form the dsRNA double helix of the protein-binding domain of the guide RNA. Therefore, it can be said that each crRNA-like molecule has a corresponding tracrRNA-like molecule. In addition, the crRNA-like molecule may also have a single-stranded DNA targeting segment or spacer sequence. Thus, the crRNA-like molecule and the tracrRNA-like molecule (as a corresponding pair) can hybridize to form a guide RNA. The two-molecule guide RNA of the subject may contain any corresponding crRNA-tracrRNA pair.

[0332] The DNA targeting segment or spacer sequence of the guide RNA may be complementary to the sequence at the target site in the chromosome sequence, e.g., the protospacer sequence, so that the DNA targeting segment of the guide RNA can base-pair with the target site or protospacer. In some cases, the DNA targeting segment of the guide RNA may contain 10 nucleotides or approximately this number of nucleotides to 25 or approximately this number of nucleotides or more. For example, the base-pairing region of the first region of the guide RNA with the target site in the chromosome sequence may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length, or approximately this length. In some cases, the first region of the guide RNA may be 19, 20, or 21 nucleotides in length, or approximately this length.

[0333] Guide RNA can target nucleic acid sequences of 20 nucleotides or approximately this length. Target nucleic acids may be less than 20 nucleotides or approximately this length. Target nucleic acids may be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides, or at least approximately this length. Target nucleic acids may be up to 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides, or at most approximately this length. The target nucleic acid sequence may be 20 bases immediately 5' from the first nucleotide of the PAM, or approximately this length. Guide RNA can target nucleic acid sequences.

[0334] A guide nucleic acid, such as a guide RNA, may refer to a nucleic acid that can hybridize to another nucleic acid, such as a target nucleic acid or protospacer within the cellular genome. A guide nucleic acid can be RNA. A guide nucleic acid can be DNA. A guide nucleic acid can be programmed or designed to specifically bind to the sequence of a nucleic acid site. A guide nucleic acid can contain a polynucleotide chain and may be called a single guide nucleic acid. A guide nucleic acid can contain two polynucleotide chains and may be called a dual guide nucleic acid.

[0335] Guide nucleic acids may include one or more modifications that give the nucleic acid novel or enhanced characteristics. Guide nucleic acids may include nucleic acid affinity tags. Guide nucleic acids may include synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0336] The guide nucleic acid may contain, for example, a nucleotide sequence (e.g., a spacer) at or near its 5' or 3' end that can hybridize to a sequence (e.g., a protospacer) in the target nucleic acid. The spacer of the guide nucleic acid may interact with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). The spacer sequence may hybridize to the target nucleic acid located on the 5' or 3' side of the protospacer adjacent motif (PAM). The length of the spacer sequence may be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides, or at least approximately this length. The length of the spacer sequence may be at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more nucleotides, or at most approximately this length.

[0337] The guide RNA may also include a dsRNA double-stranded region that forms a secondary structure. For example, the secondary structure formed by the guide RNA may include a stem (or hairpin) and a loop. The lengths of the loop and stem may vary. For example, the loop may be in the range of about 3 to about 10 nucleotides in length, and the stem may be in the range of about 6 to about 20 base pairs in length. The stem may include one or more bulges of 1 to about 10 nucleotides. The total length of the second region may be in the range of about 16 to about 60 nucleotides in length. For example, the loop may be 4 nucleotides in length, or approximately this length, and the stem may be 12 base pairs, or approximately this length. The dsRNA double-stranded region may include a protein-binding segment that can form a complex with RNA-binding proteins, such as RNA-guided endonucleases, e.g., Cas proteins.

[0338] Guide RNA may also include a tail region at its 5' or 3' end, which may be essentially single-stranded. For example, the tail region may not be complementary to any chromosomal sequence in the target cell, and may not be complementary to the rest of the guide RNA. Furthermore, the length of the tail region can vary. The tail region may be longer than or approximately longer than 4 nucleotides. For example, the length of the tail region may range from 5 or about 5 to 60 or about 60 nucleotides.

[0339] Guide RNA can be introduced into cells or embryos as an RNA molecule. For example, RNA molecules can be transcribed in vitro and / or chemically synthesized. The guide RNA can then be introduced into cells or embryos as an RNA molecule. Guide RNA can also be introduced into cells or embryos in the form of nucleic acid molecules other than RNA, such as DNA molecules. For example, the DNA encoding the guide RNA can be operably ligated to a promoter control sequence for the expression of the guide RNA in the target cell or embryo. The RNA coding sequence can be operably ligated to a promoter sequence recognized by RNA polymerase III (PolIII).

[0340] The DNA molecule encoding the guide RNA can also be linear. The DNA molecule encoding the guide RNA can also be circular.

[0341] The DNA sequence encoding the guide RNA can also be part of the vector. Examples of vector components include plasmid vectors, phagemids, cosmids, artificial / minichromosomes, and tragus. Plasmid vectors may include sposons and viral vectors. For example, DNA encoding an RNA-guided endonuclease is present within a plasmid vector. Other non-limiting examples of suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and their variants. Furthermore, vectors may include additional expression regulatory sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selection marker sequences (e.g., antibiotic resistance genes), origins of replication, etc.

[0342] When both RNA-guided endonucleases and guide RNAs are introduced into cells as DNA molecules, each may be part of separate molecules (e.g., one vector containing the coding sequence for the fusion protein and a second vector containing the coding sequence for the guide RNA) or part of the same molecule (e.g., one vector containing the coding (and regulatory) sequences for both the fusion protein and the guide RNA).

[0343] Cas proteins, such as the Cas9 protein or any derivative thereof, can be pre-complexed with guide RNA to form a ribonucleoprotein (RNP) complex. The RNP complex can be introduced into primary immune cells. The introduction of the RNP complex may be time-sensitive. Cells can synchronize with other cells during the G1, S, and / or M phases of the cell cycle. The RNP complex can be delivered during cell phases that enhance HDR. The RNP complex may facilitate homology-guided repair.

[0344] Guide RNA can also be modified. Modifications may include chemical changes, synthetic modifications, nucleotide additions, and / or nucleotide deletions. Modifications may also enhance the manipulation of the CRISPR genome. Modifications may alter the chirality of the gRNA. In some cases, the chirality may be uniform or sterically pure after modification. Guide RNA can be synthetic guide RNA. Synthetic guide RNA can enhance the manipulation of the CRISPR genome. Guide RNA can also be cleaved. Cleavages can be used to reduce unwanted off-target mutagenesis. Cleavages may contain any number of nucleotide deletions. For example, a cleavage may contain 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more nucleotides. Guide RNA may contain a target complementarity region of any length. For example, the target complementarity region may be less than 20 nucleotides long. The target complementarity region may be more than 20 nucleotides long.

[0345] In some cases, double nicase can be used to introduce double-strand breaks. The Cas protein can be mutated at a known amino acid within either nuclease domain, thereby creating a nicase Cas protein capable of deactivating the activity of one nuclease domain and generating a single-strand break. A nicase with two distinctly different guide RNA targeting reverse strands can be used to generate DSBs within a target site (often referred to as a "double nic" CRISPR system or "double nicase" CRISPR system). This technique can dramatically increase target specificity because the likelihood of two off-target nicks being generated close enough to induce a DSB is small.

[0346] In some cases, GUIDE-Seq analysis can be performed to determine the specificity of the guide RNA. For general mechanisms and protocols of GUIDE-Seq profiling for off-target cleavage by CRISPR nucleases, see Tsai, S. et al., "GUIDE-Seq enables genome-wide This is discussed in "Profiling of off-target cleavage by CRISPR system nucleases," Nature, Vol. 33: pp. 187-197 (2015).

[0347] gRNA can be introduced at any functional concentration. For example, gRNA can be introduced into cells at a dose of 10 micrograms. In other cases, gRNA can be introduced at doses ranging from 0.5 to 100 micrograms. gRNA can be introduced at doses of 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 micrograms.

[0348] Depending on the case, the method is Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cm The method may include endonucleases selected from the group consisting of r5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, their homologs, or modified forms thereof. The Cas protein may be Cas9. The method may further include at least one guide RNA (gRNA). The gRNA may include at least one modification. The exogenous TCR may bind to the cancer neoantigen.

[0349] This specification discloses a method for producing engineered cells, comprising the steps of: introducing at least one polynucleic acid encoding at least one exogenous T cell receptor (TCR) sequence; introducing at least one guide RNA (gRNA) having at least one modification; and introducing at least one endonuclease; wherein the gRNA comprises at least one sequence complementary to at least one endogenous genome. The modification may be a modification at the 5' end, a modification at the 3' end, a modification between the 5' and 3' ends, a single-base modification, a 2'-ribose modification, or any combination thereof. The modification can be selected from the group consisting of base substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, and any combination thereof.

[0350] In some cases, the modification is a chemical modification. Modifications include: 5'-adenylic acid, 5'-guanosine triphosphate cap, 5'N7-methylguanosine triphosphate cap, 5'-triphosphate cap, 3'-phosphate, 3'-thiophosphate, 5'-phosphate, 5'-thiophosphate, Cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, debasement, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bibiotin, PC biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, Black Hole Quencher 1, Black Hole Quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methylribonucleoside analog, sugar-modified analog, fluctuation / universal base, fluorescent dye labeling, 2'-fluoroRNA, 2'O-methylRNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 2-O-methyl-3-phosphorothioate, or any combination thereof can be selected. The modification may be a pseudouridine modification, as shown in Figure 98. In some cases, the modifications do not need to affect the survival rate (Figures 99A and 99B).

[0351] In some cases, the modification is a 2'-O-methyl3'-phosphorothioate addition. 2'-O-methyl3'-phosphorothioate addition can be performed over 1 to 150 bases. 2'-O-methyl3'-phosphorothioate addition can be performed over 1 to 4 bases. 2'-O-methyl3'-phosphorothioate addition can be performed over 2 bases. 2'-O-methyl3'-phosphorothioate addition can be performed over 4 bases. The modification can also be a cleavage. A cleavage can be a cleavage of 5 bases.

[0352] In some cases, truncation of five bases can prevent cleavage (cut) by Cas proteins. Endonucleases can be selected from the group consisting of CRISPR systems, TALENs, zinc fingers, transposon-based ZENs, meganucleases, Mega-TAL, and any combination. Endonucleases can be Cas endonucleases. Cas endonucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, C The group can be selected from mr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, their homologs, or modified forms thereof. Modified forms of Cas may be cap-removed Cas, as shown in Figures 100A and 100B. The Cas protein may be Cas9. Cas9 can create double-strand breaks within the at least one endogenous genome. The endogenous genome may include at least one gene. The gene may be CISH, PD-1, TRA, TRB, or a combination thereof. Depending on the circumstances, double-strand breaks can be repaired using homology-mediated repair (HR), non-homologous end joining (NHEJ), microhomologous end joining (MMEJ), or any combination or derivative thereof. TCRs can be incorporated into double-strand breaks.

[0353] c. Transgene Transgene insertions (e.g., exogenous sequences) can be used, for example, to express polypeptides, to correct mutant genes, or to increase the expression of wild-type genes. Transgenes are typically not identical to the genomic sequence in which they reside. Donor transgenes may contain a non-homologous sequence flanked by two homologous regions at the target location to enable efficient HDR. In addition, transgene sequences may contain a vector molecule containing a sequence of intracellular chromatin that is not homologous to the target region. Transgenes may contain several discontinuous regions homologous to intracellular chromatin. For example, for targeted insertion of a sequence not normally present in the target region, the sequence can reside within the donor nucleic acid molecule and be flanked by homologous regions to the sequence in the target region.

[0354] The transgene polynucleic acid can be DNA or RNA, single-stranded or double-stranded, and can be introduced into cells in linear or circular form. The transgene sequence can be contained within a DNA minicircle, which can be introduced into cells in circular or linear form. When introduced in linear form, the ends of the transgene sequence can be protected in any way (e.g., from exonuclease degradation). For example, one or more dideoxynucleotide residues can be added to the 3' end of the linear molecule, and / or a self-complementary oligonucleotide can be ligated to one or both ends. Further methods for protecting exogenous polynucleotides from degradation include, but are not limited to, the addition of terminal amino groups, as well as the use of modified nucleotide linkages, such as phosphorothioate residues, phosphoramidite residues, and O-methylribose or deoxyribose residues.

[0355] Trans genes can be flanked by recombination arms. In some cases, recombination arms may include complementary regions that target the trans gene to a desired integration site. Trans genes can also be integrated into genomic regions such that the insertion disrupts an endogenous gene. Trans genes can be integrated by any method, e.g., non-recombination end joining and / or recombination-directed repair. Trans genes can also be integrated during recombination events that repair double-strand breaks. Trans genes can also be integrated using homologous recombination enhancers. For example, an enhancer may block non-homologous end joining to perform homology-guided repair to repair double-strand breaks.

[0356] A trans gene can be flanked by a recombinant arm, in which case the degree of homology between the arm and its complementary sequence is sufficient to enable homologous recombination between the two. For example, the degree of homology between the arm and its complementary sequence can be 50% or more. The two homologous non-identical sequences can be of any length, and their degree of non-homonymy can be as low as a single nucleotide (e.g., correcting a point mutation in the genome by targeted homologous recombination) or as high as 10 kilobases or more (e.g., inserting a gene into a predetermined ectopic site in a chromosome). The two polynucleotides containing homologous non-identical sequences do not need to be the same length. For example, a representative trans gene with a recombinant arm for CCR5 is shown in Figure 16. Recombinant arms can be constructed using any other gene, e.g., the genes described herein.

[0357] A trans gene can be flanked by a manipulation site complementary to the target double-strand break region in the genome. In some cases, the manipulation site is not a recombination arm. The manipulation site may have homology to the double-strand break region. The manipulation site may have homology to the gene. The manipulation site may have homology to the genome coding region. The manipulation site may have homology to the genome non-coding region. In some cases, a trans gene can be excised from a polynucleic acid and inserted into a double-strand break region without homologous recombination. A trans gene can be incorporated into a double-strand break without homologous recombination.

[0358] Polynucleotides can be introduced into cells as part of a vector molecule containing further sequences, such as genes encoding origins of replication, promoters, and antibiotic resistance. Furthermore, transgene polynucleotides can be introduced as naked nucleic acids, as nucleic acids complexed with drugs such as liposomes or poloxamers, and can be delivered by viruses (e.g., adenoviruses, AAVs, herpesviruses, retroviruses, lentiviruses, and integrase-deficient lentiviruses (IDLVs)). AAV viruses are one example of viruses capable of delivering transgenes.

[0359] Transgenes are generally inserted so that their expression is driven by an endogenous promoter at the integration site, i.e., a promoter that drives the expression of the endogenous gene into which the transgene is inserted (e.g., AAVS site (e.g., AAVS1, AAVS2, etc.), CCR5, HPRT). Transgenes may include promoters and / or enhancers, such as constitutive promoters, inductive promoters, or tissue / cell-specific promoters. Minicircle vectors may encode transgenes.

[0360] Targeting of insertions of non-coding nucleic acid sequences can also be achieved. Sequences encoding antisense RNA, RNAi, shRNA, and microRNA (miRNA) can also be used for insertion targeting.

[0361] A trans gene can be inserted into an endogenous gene to express all or part of the endogenous gene, or it can be inserted into an endogenous gene to prevent the expression of the endogenous gene. For example, a trans gene described herein can be inserted into an endogenous locus to express, for example, a portion of the endogenous sequence (the N-terminal and / or C-terminal portion relative to the trans gene) as a fusion with the trans gene, or it can be inserted into an endogenous locus to prevent the expression of the endogenous sequence as a fusion with the trans gene. In other cases, a trans gene (with or without further coding sequences, such as an endogenous gene) is incorporated into any endogenous locus, such as a safe harbor locus. For example, a TCR trans gene can be inserted into an endogenous TCR gene. For example, Figure 17 shows that a trans gene can be inserted into an endogenous CCR5 gene. A trans gene can be inserted into any gene, such as the genes described herein.

[0362] When an endogenous sequence is expressed together with a trans gene (either the endogenous sequence or a portion of the trans gene), the endogenous sequence may be a full-length sequence (wild-type or mutant) or a partial sequence. The endogenous sequence may be functional. Non-limiting examples of the functions of these full-length or partial sequences include extending the serum half-life of a polypeptide expressed by the trans gene (e.g., a therapeutic gene) and / or acting as a carrier.

[0363] Furthermore, although not required for expression, exogenous sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, 2A peptides, and / or sequences encoding polyadenylation signals.

[0364] In some cases, the exogenous sequence (e.g., trans gene) includes a fusion of the target protein and, as its fusion partner, the extracellular domain of a membrane protein that positions the fusion protein on the cell surface. In some cases, the trans gene encodes a TCR, in which case the TCR coding sequence is inserted into the safe harbor to express the TCR. In some cases, the TCR coding sequence is inserted into the PD1 locus and / or the CTLA-4 locus. In other cases, the TCR is delivered to the cell by lentivirus for random insertion, supplied with PD1-specific nuclease or CTLA-4-specific nuclease as mRNA. In some cases, the TCR is delivered via a viral vector system such as a retrovirus, AAV, or adenovirus, along with mRNA encoding a nuclease specific to the safe harbor (e.g., AAVS site (e.g., AAVS1, AAVS2, etc.), CCR5, albumin, or HPRT). Cells can also be treated with mRNA encoding PD1-specific nucleases and / or CTLA-4-specific nucleases. Optionally, polynucleotides encoding the TCR are supplied via a viral delivery system in conjunction with mRNA encoding HPRT-specific nucleases and PD1-specific or CTLA-4-specific nucleases. Cells containing the TCR-encoding nucleotide, incorporated into the HPRT locus, may be selected using 6-thioguanine, a guanine analog, along with the intact HPRT gene, which may result in cell arrest and / or induce apoptosis within the cell. TCRs that can be used with the methods and compositions of the present invention include all types of these chimeric proteins, including types with first-generation, second-generation, and third-generation designs. The present invention also envisions specificity domains derived from receptors, ligands, and manipulated polypeptides, but also includes specificity domains derived from antibodies. The CR may be particularly useful. The intercellular signaling domain may originate from the TCR chain, such as the zeta chain, as well as other members of the CD3 complex, such as the γ and ε chains. In some cases, the TCR may include further co-stimulatory domains, such as intercellular domains derived from CD28, CD137 (also known as 4-1BB), or CD134. In further cases, two types of co-stimulatory domains may be used simultaneously (e.g., CD3 zeta used with CD28 + CD137).

[0365] In some cases, manipulated cells are composed of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, exhibiting stem memory T SCM Stem memory cells can be cells, and they can also express CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting numerous functional attributes specific to stem memory cells. Manipulated cells also contain L-selectin and CCR7, which are central memory cells. CM It is also possible for these cells to be central memory cells, in which case they may secrete, for example, IL-2 but not IFNγ or IL-4. Manipulated cells are also effector memory cells containing L-selectin or CCR7. EM These cells can also be cells that produce effector cytokines, such as IFNγ and IL-4. In some cases, a population of cells can be introduced into the target. For example, the population may be a combination of T cells and NK cells. In other cases, the population may be a combination of naive cells and effector cells.

[0366] Delivery of homologous recombinant HR enhancers In some cases, homologous recombination HR enhancers can be used to suppress non-homologous end joining (NHEJ). Non-homologous end joining can result in the loss of nucleotides at the ends of double-strand breaks, and non-homologous end joining can also result in frameshifts. Therefore, homology-driven repair may be a more attractive mechanism to use when knocking in genes. HR enhancers can be delivered to suppress non-homologous end joining. In some cases, more than one HR enhancer can be delivered. HR enhancers may inhibit proteins involved in non-homologous end joining, such as KU70, KU80, and / or DNA ligase IV. In some cases, ligase IV inhibitors such as Scr7 can be delivered. In some cases, the HR enhancer may be L755507. In some cases, different ligase IV inhibitors can be used. In some cases, the HR enhancer may be a protein from adenovirus type 4, e.g., E1B55K and / or E4orf6. In some cases, a chemical inhibitor may be used.

[0367] Non-homologous end binding molecules such as KU70, KU80, and / or DNA ligase IV can be repressed by various methods. For example, non-homologous end binding molecules such as KU70, KU80, and / or DNA ligase IV can be repressed by gene silencing. For example, non-homologous end binding molecules such as KU70, KU80, and / or DNA ligase IV can be repressed by gene silencing during the transcription or translation of factors. Non-homologous end binding molecules such as KU70, KU80, and / or DNA ligase IV can also be repressed by factor degradation. Non-homologous end binding molecules such as KU70, KU80, and / or DNA ligase IV can also be inhibited. Inhibitors of KU70, KU80, and / or DNA ligase IV may include E1B55K and / or E4orf6. Non-homologous end-binding molecules, such as KU70, KU80, and / or DNA ligase IV, can also be inhibited by blockade. Gene expression can also be suppressed by knockout, by altering the gene promoter, and / or by administering interfering RNA to the factor.

[0368] HR enhancers that suppress non-homologous end joining can be delivered by plasmid DNA. In some cases, the plasmid may be a double-stranded DNA molecule. Plasmid molecules can also be single-stranded DNA. Plasmids can also contain at least one gene. Plasmids can also contain more than one gene. At least one plasmid can also be used. More than one plasmid can also be used. HR enhancers that suppress non-homologous end joining can be delivered by plasmid DNA together with CRISPR-Cas, primers, and / or modifying compounds. Modifying compounds can reduce the cytotoxicity of plasmid DNA and improve cell viability. HR enhancers and modifying compounds can be introduced into cells before genomic manipulation. HR enhancers can be small molecules. In some cases, HR enhancers can be delivered into T cell suspensions. HR enhancers can improve the viability of cells transfected with double-stranded DNA. In some cases, the introduction of double-stranded DNA can be toxic (Figures 81A and 81B).

[0369] HR enhancers that suppress non-homologous end joining can be delivered together with the HR substrate to be incorporated. The substrate may be a polynucleic acid. The polynucleic acid may contain the TCR transgene. The polynucleic acid can be delivered as mRNA (see Figures 10 and 14). The polynucleic acid may contain a recombination arm to an endogenous region of the genome for the integration of the TCR transgene. The polynucleic acid may be a vector. The vector can be inserted into another vector (e.g., a viral vector) in sense or antisense orientation. For in vitro transcription of the viral cassette, a T7, T3, or other transcription start sequence can be placed upstream of the 5'LTR region of the viral genome (see Figure 3). This vector cassette can then be used as a template for mRNA transcription in vitro. For example, when delivering this mRNA to any cell along with its cognitive reverse transcriptase, or when delivering it as mRNA or protein, a single-stranded mRNA cassette can be used as a template to create hundreds to thousands of copies in the form of double-stranded DNA (dsDNA), which can be used as HR substrates for homologous recombination events desired to integrate the transgene cassette into a desired target site in the genome. This method can avoid the need for delivery of toxic plasmid DNA for CRISPR-mediated homologous recombination. In addition, since each mRNA template becomes hundreds or thousands of copies of dsDNA, the amount of homologous recombination templates available in the cell can be extremely high. A high amount of homologous recombination templates can drive the desired homologous recombination event. Furthermore, mRNA can also be used to create single-stranded DNA. Single-stranded DNA can also be used as a template for homologous recombination, for example, in conjunction with gene targeting by recombinant AAV (rAAV). mRNA can be reverse transcribed in situ into HR enhancers for DNA homologous recombination. This strategy avoids the delivery of toxic plasmid DNA. In addition, mRNA can be used to amplify homologous recombination substrates to higher levels than plasmid DNA and / or improve the efficiency of homologous recombination.

[0370] HR enhancers that inhibit non-homologous end joining can be delivered as chemical inhibitors. For example, HR enhancers may act by interfering with ligase IV-DNA binding. HR enhancers may also activate the endogenous apoptotic pathway. HR enhancers may also be peptide mimetic agents of ligase IV inhibitors. HR enhancers can also be co-expressed with Cas9 systems. HR enhancers can also be co-expressed with viral proteins such as E1B55K and / or E4orf6. HR enhancers may also be SCR7, L755507, or any derivative thereof. HR enhancers can be delivered with compounds that reduce the toxicity of exogenous DNA insertions.

[0371] If, by any chance, only robust reverse transcription of single-stranded DNA occurs within the cell, mRNA encoding both the sense and antisense strands of the viral vector can be introduced (see Figure 3). In this case, both mRNA strands can be reverse transcribed within the cell to produce dsDNA, or / or annealed spontaneously to produce dsDNA.

[0372] HR enhancers can be delivered to primary cells. Homologous recombinant HR enhancers can be delivered by any suitable means. Homologous recombinant HR enhancers can also be delivered as mRNA. Homologous recombinant HR enhancers can also be delivered as plasmid DNA. Homologous recombinant HR enhancers can also be delivered to immune cells together with CRISPR-Cas. Homologous recombinant HR enhancers can also be delivered to immune cells together with CRISPR-Cas, polynucleic acids containing TCR sequences, and / or compounds that reduce the toxicity of exogenous DNA insertions.

[0373] Homologous recombinant HR enhancers can be delivered to any cell, such as immune cells. For example, homologous recombinant HR enhancers can be delivered to primary immune cells. Homologous recombinant HR enhancers can also be delivered to T cells and primary T cells, including but not limited to T cell lines. Homologous recombinant HR enhancers can also be delivered to CD4+ cells, CD8+ cells, and / or tumor-infiltrating cells (TILs). Homologous recombinant HR enhancers can also be delivered to immune cells together with CRISPR-Cas.

[0374] In some cases, homologous recombination HR enhancers can be used to suppress non-homologous end joining. In some cases, homologous recombination HR enhancers can be used to promote homology-driven repair. In some cases, homologous recombination HR enhancers can be used to promote homology-driven repair after CRISPR-Cas double-strand breaks. In some cases, homologous recombination HR enhancers can be used to promote homology-driven repair, as well as knock-in and knock-out, of one or more genes after CRISPR-Cas double-strand breaks. The gene to be knocked in may be a TCR. The gene to be knocked out may also be any number of endogenous checkpoint genes. For example, endogenous checkpoint genes can be selected from the group consisting of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM-3, VISTA, AAVS sites (e.g., AAVS1, AAVS2, etc.), CCR5, HPRT, PPP1R12C, or CISH. In some cases, the gene may be PD-1. In some cases, the gene may be endogenous TCT. In some cases, the gene may contain a coding region. In some cases, the gene may contain a non-coding region.

[0375] The increase in HR efficiency due to HR enhancers can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or approximately one of these percentages.

[0376] The reduction in NHEJ due to HR enhancers may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, or approximately one of these percentages.

[0377] Low toxicity cell manipulation It is possible to mitigate cytotoxicity to exogenous polynucleic acids and improve the manipulation of cells, including T cells. For example, cytotoxicity can be reduced by altering the cellular response to polynucleic acids. can.

[0378] Polynucleotides can be brought into contact with cells. Then, the polynucleotides can be introduced into the cells. In some cases, polynucleotides are used to modify the cell genome. After polynucleotide insertion, cells may die. For example, polynucleotide insertion can induce apoptosis in cells, as shown in Figure 18. The toxicity ...

Claims

1. a. Lymphocytes derived from human subjects; b. A polynucleotide targeting polynucleotide that has been engineered to hybridize to a specific region of a target gene within the genome of the lymphocyte; c. A nuclease capable of associating with a polynucleic acid that targets the polynucleic acid to form a nuclear protein complex, wherein the nuclear protein complex is a nuclease capable of causing targeted double-strand breaks of the target gene in the genome of the lymphocyte; d. A target polynucleic acid which is genomic DNA containing a double-strand break within the target gene, wherein the double-strand break within the target gene results in the disruption of the target gene function, and when the nuclear protein complex is brought into contact with a population of primary lymphocytes, the disruption of the target gene function occurs with an efficiency of at least 60%, Expanding on this, it is possible to create a clonal population of lymphocytes in which the function of the target gene has been altered, and the clonal population of lymphocytes is a genetically modified immune cell suitable for administration to humans who need it.

2. The endogenous CISH (cytokine-inducible SH2-containing) gene is disrupted in its sequence, and at least one further disruption occurs within the endogenous gene, wherein the endogenous gene is related to adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related protein (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), and KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains). Modified primary cells selected from the group consisting of long cytoplasmic tails, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS1), and chemokine (C-C motif) receptor 5 (gene / pseudogene) (CCR5).

3. a) with at least one exogenous T cell receptor (TCR); b) with at least one genomic disruption of programmed death ligand 1 (PD-1); c) comprising at least one genomic disruption of the TCR alpha (TCRA) chain gene and the TCR beta (TCRB) chain gene, Manipulated cells in which the TCR has been introduced using a lentiviral vector and the genome disruption has been performed using the CRISPR endonuclease system.

4. a. At least one exogenous T cell receptor (TCR) sequence; b. At least one nucleic acid that targets a nucleic acid nuclease complex, i. Manipulated nucleic acids that target nucleic acids, comprising at least one sequence substantially complementary to a target genome sequence; and ii. Cells containing nucleic acids, including exogenous endonucleases.

5. With at least one exogenous T cell receptor (TCR) sequence; At least one complex, a. At least one modified polynucleic acid having a sequence complementary to at least one genome sequence; and b. Manipulated cells comprising at least one complex containing at least one exogenous endonuclease.

6. a. At least one exogenous T cell receptor (TCR); b. With at least one genomic disruption of programmed death ligand 1 (PD-1); c. comprising at least one genomic disruption of at least one endogenous gene, Manipulated cells in which the TCR has been introduced using a lentiviral vector and the genome disruption has been performed using the CRISPR system.

7. A modified cell comprising the disruption of at least one gene and at least one non-viral T cell receptor (TCR) sequence, wherein the gene is disrupted by the non-viral TCR sequence.

8. Cells according to any of the preceding claims, which are manipulated using a CRISPR nuclease.

9. Cells according to any of the preceding claims, which are manipulated using an AAV vector.

10. The manipulated cell according to any one of the preceding claims, further comprising an exogenous receptor.

11. The manipulated cell according to any one of the preceding claims, wherein the exogenous receptor is selected from the group comprising a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a B cell receptor (BCR).

12. A pharmacological composition comprising the cells described in any of the preceding claims.

13. A cell according to any of the preceding claims, which can expand more than 40 times in 12 days.

14. A cell according to any of the prior claims, which is a TIL.

15. A cell, which is an immune cell, as described in any of the preceding claims.

16. A human cell, as described in any of the preceding claims.

17. The manipulated cell according to any one of the preceding claims, wherein the exogenous receptor is selected from the group comprising a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a B cell receptor (BCR).

18. A method for treating a patient in need thereof, comprising the step of administering cells as described in any of the preceding claims.

19. At least one guide RNA that binds to the endogenous CISH (cytokine-inducible SH2-containing) gene, and adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activation inhibitor 1 (VTCN1), B lymphocyte and T lymphocyte-related protein (BTLA), cytotoxic T lymphocyte-related protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3) A combination comprising a secondary guide RNA that binds to an endogenous gene selected from programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (C-C motif) receptor 5 (gene / pseudogene) (CCR5), and a combination including a secondary guide RNA that binds to an endogenous gene selected from programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration sites (AAVS sites (e.g., AAVS1, AAVS2, etc.)), or chemokine (C-C motif) receptor 5 (gene / pseudogene) (CCR5). Finished product.

20. The manipulated cell according to any one of the preceding claims, wherein the exogenous receptor is selected from the group comprising a T cell receptor (TCR), a chimeric antigen receptor (CAR), or a B cell receptor (BCR).

21. A method for efficient disruption of checkpoint inhibitors within T cells, a. A step of contacting T cells with Cas9 nuclease and guide RNA, wherein the guide RNA contains a region of 17 to 22 nucleotides that is substantially complementary to a region in the target gene; b. A step of cleaving the target gene, wherein the target gene is PD-1, and when a population of primary T cells is brought into contact with Cas9 nuclease and guide RNA, a knockout event occurs in at least 30% of the primary T cells; c. The step of destroying the checkpoint inhibitor in the T cell. A method that includes this.

22. A method for treating an object that requires it, a. The step of collecting lymphocytes from humans; b. A step of genetically modifying lymphocytes in ex vivo by contacting them with a ribonuclease capable of knocking out the function of the PD-1 protein by inducing double-strand breaks in a specific target region of genomic DNA within the lymphocytes, wherein the target region of the genomic DNA within the lymphocytes is located within the PD-1 gene, and the double-strand break occurs in a target region of genomic DNA that is 3' to the region of the target DNA capable of hybridizing to at least 15 nucleotides of the ribonuclease, and 5' to the region of the target DNA containing a protospacer adjacent motif; c. The step of expanding a population of genetically modified lymphocytes having PD-1 protein knockout to create a population of PD-1 knockout T cells; d. A step of administering the population of PD-1 knockout T cells to the subject, wherein the PD-1 knockout T cells are suitable for administration to the patient. A method that includes this.

23. The method according to claim 21, wherein the population of genetically modified lymphocytes is expanded for at least 12 days, and the population of genetically modified lymphocytes increases by at least 40 times.

24. The method according to claim 22, wherein the population of genetically modified lymphocytes increases by at least 100 times.

25. A method for producing manipulated cells, a. The step of introducing one or more polynucleic acids, each containing at least one exogenous T cell receptor (TCR) sequence, into cells using a non-viral method, with the polynucleic acid being sandwiched between recombinant arms; b. The step of bringing at least one exogenous TCR sequence into contact with a double-strand break region containing a gene. A method that includes this.

26. The method according to claim 24, wherein the recombinant arm is complementary to the portion of the gene.

27. The aforementioned gene is associated with adenosine A2a receptor (ADORA), CD276, V-set domain-containing T cell activating inhibitor 1 (VTCN1), B lymphocytes, and T lymphocyte-related ( The method according to claim 24, wherein the receptor is BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-dioxygenase 1 (IDO1), KIR3DL1 (killer cell immunoglobulin-like receptor, 3 domains, long cytoplasmic tail, 1), lymphocyte activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cell receptor 2 (HAVCR2), VISTA (V-domain immunoglobulin suppressor for T cell activation), natural killer cell receptor 2B4 (CD244), CISH (cytokine-inducible SH2-containing protein), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated virus integration site (AAVS site (e.g., AAVS1, AAVS2, etc.)), or chemokine (C-C motif) receptor 5 (gene / pseudogene) (CCR5).

28. The method according to claim 24, wherein the double-strand break region is repaired by insertion of the at least one exogenous TCR sequence.

29. The method according to claim 24, wherein the insertion of the at least one exogenous TCR sequence comprises the disruption of the at least one gene.

30. The method according to claim 28, wherein the insertion of the at least one exogenous TCR sequence is supported by a homologous recombination (HR) enhancer.

31. The method according to claim 24, wherein the insertion includes a repair guided by homology.

32. The method according to any one of the preceding claims, wherein the double-strand break is induced by a nuclease.

33. The method according to claim 31, wherein the nuclease is selected from the group consisting of Cas9, Argonaut, Cpf1, CRISPR, TALEN, transposase, ZEN, meganuclease, or Mega-TAL.

34. The method according to any one of claims 36 to 52, wherein the double-strand break region is created by CRISPR.

35. The method according to claim 32, wherein the nuclease is multiplexed.

36. The method according to claim 34, wherein the multiplexing is carried out by adding at least two guide polynucleic acids.

37. The method according to any of the preceding claims, wherein the nonviral introduction includes electroporation or nucleofection.

38. The method according to any one of the preceding claims, wherein the polynucleic acid is co-delivered with at least one modifier that alters the cellular response to the polynucleic acid.

39. A method for facilitating homology-driven restoration (HDR), a. The step of introducing mRNA, reverse transcriptase (RT), enhancer, and primer into cells using a non-viral method; b. The step of reverse transcribing the mRNA into one or more copies of a polynucleic acid; c. A step to facilitate HDR between the genome of the cell and the polynucleic acid. A method that includes this.

40. A method for reducing cytotoxicity of cells to one or more exogenously modified polynucleic acids, comprising the step of altering one or more cellular responses to the polynucleic acids by contacting the cells with the one or more exogenously modified polynucleic acids, wherein the one or more cellular responses include a cytoplasmic DNA sensing pathway.

41. The step of modifying one or more cellular responses includes DAI (DNA-dependent activator of IFN regulators), IFN-inducible protein 16 (IFI16), DEAD-box polypeptide 41 (DDX41), and AIM2 (absent in melanoma). 2) The method according to claim 39, comprising modifying DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), STING (IFN gene stimulator), TANK-binding kinase (TBK1), interleukin-1β (IL-1β), MRE11 (meiotic recombination 11), Trex1, caspase-1 (aspartate-specific cysteine ​​protease), 3' repair exonuclease, DAI (DNA-dependent activator of IRF), IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), MRE11 (meiotic recombination 11) homolog A, and / or IFN regulator (IRF) 3 or 7.

42. a. The step of bringing cells into contact with one or more signal transduction modifying compounds; b. A method for genome manipulation, comprising the step of contacting the cells with a polynucleic acid comprising at least one antigen receptor sequence flanked by at least two recombinant arms complementary to at least one genomic region.

43. The method according to claim 39, wherein the one or more signal transduction modifying compounds alter the cytoplasmic DNA sensing pathway.

44. The one or more signal transduction modifying compounds include DAI (DNA-dependent activator of IFN regulators), IFN-inducible protein 16 (IFI16), DEAD-box polypeptide 41 (DDX41), AIM2 (absent in melanoma 2), DNA-dependent protein kinase, cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), STING (IFN gene stimulator), TANK-binding kinase (TBK1), interleukin 1β (IL-1β), MRE11 (meiotic recombination 11), Trex1, caspase 1 (aspartate-specific cysteine ​​protease), 3' repair exonuclease, and DAI (DNA-dependent activator of IFN regulators). The method according to claim 39, wherein the IRF), IFI16, DDX41, DNA-dependent protein kinase (DNA-PK), MRE11 (meiotic recombination 11) homolog A, and / or IFN regulator (IRF) 3 or 7 are modified.

45. A method for creating manipulated cells, a. To the cells, i. A guide polynucleotide comprising a spacer region complementary to the target nucleic acid within the genomic region of the cell; ii. Nucleases guided by the aforementioned guide polynucleotide; and iii. Polynucleotides encoding exogenous T cell receptors Steps to implement: b. The step of site-specifically cleaving the target nucleic acid within the cell with the nuclease guided by the guide polynucleotide; c. The step of inserting the polynucleotide encoding the exogenous T cell receptor into the genomic region of the cell at the cleavage site. A method that includes this.

46. The method according to claim 44, wherein the gene is PD-1.

47. The method according to claim 44, wherein the insertion of the exogenous TCR sequence at the cleavage site results in the disruption of the gene.

48. The method according to claim 44, further comprising the step of expressing the exogenous T cell receptor within the cell.

49. The method according to any one of the preceding claims, further comprising the step of introducing the manipulated cells into a living organism.

50. The method according to any one of the preceding claims, further comprising the step of expanding the manipulated cells in ex vivo.

51. A method for producing manipulated cells, a. The step of introducing at least one exogenous T cell receptor (TCR) into at least one genome of the cell by a virus; b. A step of disrupting the genome of at least one endogenous gene; c. A step of genomically disrupting at least one immune checkpoint gene. Includes, The method by which the genome disruption is adjacent to a protospacer-adjacent motif (PAM) sequence of the cell.

52. A method for producing manipulated cells, a. The step of introducing at least one polynucleic acid encoding at least one exogenous T cell receptor (TCR) sequence by a virus; b. The step of genomically disrupting at least one gene with at least one endonuclease or a functional portion thereof. A method that includes this.

53. A method for producing manipulated cells, a) the step of introducing at least one polynucleic acid encoding at least one exogenous T cell receptor (TCR) sequence; b) The step of introducing at least one guide RNA (gRNA) which includes at least one modification; c) The step of introducing at least one endonuclease and Including; The gRNA comprises at least one sequence that is complementary to at least one endogenous genome. method.

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