Modified t cells and methods of making and using the same

Abstract

To provide modified T cells, and to provide methods of making and using the same.SOLUTION: There are disclosed herein modified primary human T cells and populations thereof comprising a genome in which the CTLA4, PD1, TCRA, TCRB, and / or B2M genes have been edited to generate universal CAR T cells readily available from allogeneic healthy donors, the modified primary human T cells and populations thereof being able to be administered to any patient while reducing or eliminating the risk of immune rejection or graft versus host disease, and not being prone to T cell inhibition; and a method for allogeneic administration of such cells to reduce the likelihood that the cells will trigger a host immune response when the cells are administered to a subject in need of such cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 139,479, filed March 27, 2015, the entire teachings of which are incorporated herein by reference.

[0002] government aid This invention was made with government support under R01DK097768 awarded by the National Institutes of Health. The government has rights in this invention. [Background technology]

[0003] Background of the Invention Chimeric antigen receptor (CAR)-based T cell therapy is a major breakthrough in cancer immunotherapy (e.g., adoptive immunotherapy) and a novel treatment modality for viral and fungal infections, among others. Currently, several obstacles, such as the risk of autoreactivity of endogenous T cell receptors (TCRs), T cell inhibition by cancerous or infected cells, and reliance on autologous T cell transplantation, prevent the safe repurposing of CAR T therapy. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for engineered T cells and methods for producing and using such T cells that overcome endogenous TCR autoreactivity, T cell inhibition, and enable the use of allogeneic T cells in CAR T therapy. The present invention is directed to further solutions that address this need, in addition to possessing other desirable characteristics. To overcome the above obstacles, the present invention utilizes genome editing, such as CRISPR and / or TALEN systems. In the CRISPR / Cas system, the Cas protein can be, for example, Cas9 or Cpf1. For example, to overcome autoreactivity, the studies detailed herein designed and tested CRISPR / Cas guide RNAs (gRNAs) targeting the TCRRa and TCRb chains, and demonstrated high on-target activity and loss of TCR surface expression in Jurkat T cells and primary human T cells. For example, the multiplexing capabilities of genome editing using CRISPR / Cas9 or CRISPR / Cpf1 systems also allow for targeting TCRa and / or TCRb in combination with other molecules (e.g., B2M, CTLA4, PD-1) to overcome T cell inhibition and enable CAR T therapy. The compositions and methods of the present invention are suitable for clinical translation and improve existing and emerging T cell therapies (e.g., adoptive immunotherapy). [Means for solving the problem]

[0005] In some embodiments, the invention provides modified primary human T cells comprising a modified genome, wherein a first genomic modification is a truncation or edited gene encoding the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2, e.g., by deleting a first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell; a second genomic modification is a truncation or edited gene encoding the programmed cell death 1 (PD1) gene on chromosome 2, e.g., by deleting a second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell; (i) a third genomic modification is a gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14, e.g., by deleting a third contiguous stretch of genomic DNA; and / or (ii) a gene encoding the TCR beta chain locus on chromosome 7, e.g., by deleting a fourth contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell. and a fourth genomic modification that has been truncated or edited to, for example, delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating B2M expression or activity and / or MHC class I molecule surface expression and / or activity in the cell, wherein each cell optionally comprises (i) at least one chimeric antigen receptor, or an exogenous nucleic acid encoding said at least one chimeric antigen receptor, that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof, and / or (ii) at least one exogenous protein, or an exogenous nucleic acid encoding said protein, that modulates a biological effect of interest in an adjacent cell, tissue, or organ.

[0006] In some embodiments, the invention provides an engineered primary human T cell comprising a modified genome, wherein the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to delete a first contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, wherein the 3' end of the genomic DNA upstream of the 5' end of the deleted first contiguous stretch of genomic DNA is covalently linked to the 5' end of the genomic DNA downstream of the 3' end of the deleted first contiguous stretch of genomic DNA, resulting in a modified CTLA4 gene on chromosome 2 lacking the first contiguous stretch of genomic DNA, thereby reducing CTLA4 receptor surface expression and / or activity in the cell. and / or a programmed cell death 1 (PD1) gene on chromosome 2 has been edited to delete a second contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, wherein the 3' end of the genomic DNA upstream of the deleted second contiguous stretch of genomic DNA is covalently linked to the 5' end of the genomic DNA downstream of the 3' end of the deleted second contiguous stretch of genomic DNA, thereby resulting in a modified PD1 gene on chromosome 2 lacking the second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell.

[0007] In some embodiments, the invention provides an engineered primary human T cell comprising an engineered genome, wherein the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA was deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. and / or a second genomic modification in which the programmed cell death 1 (PD1) gene on chromosome 2 has been edited to delete a second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell, wherein the second contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a second pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945.

[0008] In some embodiments, the first pair of ribonucleic acids comprises SEQ ID NO:128 and SEQ ID NO:72, and the second pair of ribonucleic acids comprises SEQ ID NO:462 and SEQ ID NO:421.

[0009] In certain embodiments, the modified primary human T cell further comprises (i) a third genomic modification, in which the gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14 has been edited to delete a third contiguous stretch of genomic DNA comprising at least a portion of a coding exon; and / or (ii) a fourth genomic modification, in which the gene encoding the TCR beta chain locus on chromosome 7 has been edited to delete a fourth contiguous stretch of genomic DNA comprising at least a portion of a coding exon, thereby reducing or eliminating TCR surface expression and / or activity in the cell.

[0010] In some embodiments, a third contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a third pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9545, and / or a fourth contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fourth pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In certain aspects, the third pair of ribonucleic acids comprises SEQ ID NO: 550 and SEQ ID NO: 573, and / or the fourth pair of ribonucleic acids comprises SEQ ID NO: 657 and SEQ ID NO: 662.

[0011] In certain embodiments, the modified primary human T cell further comprises a fifth genomic modification in which the beta2-microglobulin (B2M) gene on chromosome 15 has been edited to delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In certain aspects, the fifth contiguous stretch of genomic DNA is deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fifth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, the fifth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778.

[0012] In some embodiments, the modified primary human T cells further comprise a chimeric antigen receptor or an exogenous nucleic acid encoding the chimeric antigen receptor. For example, the chimeric antigen receptor can specifically bind to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof.

[0013] In certain embodiments, the modified primary human T cells further comprise at least one exogenous protein or exogenous nucleic acid encoding said protein that modulates a desired biological effect in an adjacent cell, tissue, or organ. In some aspects, the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tissue-infiltrating lymphocytes, and combinations thereof. In certain aspects, the cells are obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD).

[0014] A method for producing modified primary human T cells, comprising: (a) editing the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 in a primary human T cell to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell; (b) editing the programmed cell death 1 (PD1) gene on chromosome 2 in the cell to delete a second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell; (c)(i) editing the T cell receptor (TC) on chromosome 14 in the cell. (R) editing the gene encoding the alpha chain locus to delete a third contiguous stretch of genomic DNA, and / or (c)(ii) editing the gene encoding the TCR beta chain locus on chromosome 7 in the cell to delete a fourth contiguous stretch of genomic DNA, thereby reducing or eliminating TCR surface expression and / or activity in the cell; and (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell to delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell;and optionally (e)(i) causing the cell to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof, and / or (e)(ii) causing the cell to express at least one protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ, wherein the editing in (a)-(d) deletes a first contiguous stretch of genomic DNA from the cell and the Cas protein or nucleic acid encoding the Cas protein, and the gene in (a). Also disclosed are methods comprising contacting a gene with at least one first pair of guide RNA sequences for deleting a second contiguous stretch of genomic DNA from the gene in (b), at least one second pair of guide RNA sequences for deleting a second contiguous stretch of genomic DNA from the gene in (c)(i), at least one third pair of guide RNA sequences for deleting a third contiguous stretch of genomic DNA from the gene in (c)(ii), and / or at least one fourth pair of guide RNA sequences for deleting a fourth contiguous stretch of genomic DNA from the gene in (c)(ii), and at least one fifth pair of guide RNA sequences for deleting a fifth contiguous stretch of genomic DNA from the gene in (d);

[0015] A method for producing modified primary human T cells, comprising: (a) editing a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 in a primary human T cell to delete a first contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, and covalently linking a 3' end of the genomic DNA upstream of the 5' end of the deleted first contiguous stretch of genomic DNA to a 5' end of the genomic DNA downstream of the 3' end of the deleted first contiguous stretch of genomic DNA, thereby generating a modified CTLA4 gene on chromosome 2 lacking the first contiguous stretch of genomic DNA, thereby reducing CTLA4 receptor surface expression and / or activity in the cell. and / or (b) editing the programmed cell death 1 (PD1) gene on chromosome 2 in a primary human T cell to delete a second contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of the adjacent upstream exon and at least a portion of the adjacent downstream exon, and covalently linking the 3' end of the genomic DNA upstream of the deleted second contiguous stretch of genomic DNA to the 5' end of the genomic DNA downstream of the 3' end of the deleted second contiguous stretch of genomic DNA, thereby generating a modified PD1 gene on chromosome 2 lacking the second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell.

[0016]

[0010] Also disclosed herein are methods for producing modified primary human T cells, the method comprising: (a) contacting a primary human T cell with a Cas protein or a nucleic acid encoding the Cas protein and a first pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637, thereby editing a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 to delete a first contiguous stretch of genomic DNA and reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell; and / or (b) contacting a primary human T cell with a Cas protein or a nucleic acid encoding the Cas protein and a second pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945, thereby editing a programmed cell death 1 (PD1) gene on chromosome 2 to delete a second contiguous stretch of genomic DNA and reduce or eliminate PD1 receptor surface expression and / or activity in the cell.

[0017] In some embodiments, the method for producing an engineered primary human T cell further comprises (c)(i) editing a gene encoding a T cell receptor (TCR) alpha chain locus on chromosome 14 in the cell to delete a third contiguous stretch of genomic DNA comprising at least a portion of a coding exon, and / or (c)(ii) editing a gene encoding a TCR beta chain locus on chromosome 7 in the cell to delete a fourth contiguous stretch of genomic DNA comprising at least a portion of a coding exon, thereby reducing or eliminating TCR surface expression and / or activity in the cell. In certain embodiments, the editing in (c)(i) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a third pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750, and / or the editing in (c)(ii) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fourth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532.

[0018] In some embodiments, the method for producing modified primary human T cells further comprises (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell to delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In certain embodiments, the editing in (d) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fifth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257.

[0019] In certain embodiments of the invention disclosed herein, a first pair of ribonucleic acids comprises SEQ ID NO: 128 and SEQ ID NO: 72, and a second pair of ribonucleic acids comprises SEQ ID NO: 462 and SEQ ID NO: 421. In certain embodiments of the invention disclosed herein, a third pair of ribonucleic acids comprises SEQ ID NO: 550 and SEQ ID NO: 573, and / or a fourth pair of ribonucleic acids comprises SEQ ID NO: 657 and SEQ ID NO: 662. In certain embodiments of the invention disclosed herein, a fifth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778.

[0020] In some embodiments, the method for producing modified primary human T cells further comprises causing the cells to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof.

[0021] In some embodiments, the methods for producing modified primary human T cells further comprise causing the cells to express at least one protein that modulates a desired biological effect in an adjacent cell, tissue, or organ when the cell is in proximity to the adjacent cell, tissue, or organ.

[0022] In certain embodiments of the invention disclosed herein, the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tissue-infiltrating lymphocytes, and combinations thereof. In certain embodiments of the invention disclosed herein, the cells are obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD).

[0023] Also disclosed herein are methods of treating a patient in need thereof, comprising: (a)(i) administering to a patient in need thereof the engineered T cells of any one of claims 1-15; (a)(ii) administering to a patient in need thereof the engineered T cells produced according to the method of any one of claims 16-29; or (a)(iii) administering to a patient in need thereof the composition of claim 30. For example, the treatment can include adoptive immunotherapy.

[0024] In some embodiments, the method for treating a patient further comprises expanding the engineered T cells prior to the administering step. In some aspects, the patient is suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD).

[0025] Also disclosed herein are compositions comprising a chimeric nucleic acid, wherein the chimeric nucleic acid comprises: (a) a nucleic acid sequence encoding a Cas protein; and (b) at least one ribonucleic acid sequence selected from the group consisting of: (i) SEQ ID NOs: 1-195 and 797-3637; (ii) SEQ ID NOs: 196-531 and 4047-8945; (iii) SEQ ID NOs: 532-609 and 9102-9750; (iv) SEQ ID NOs: 610-765 and 9798-10532; (v) SEQ ID NOs: 766-780 and 10574-13257; and (vi) a combination of (i)-(v). For example, the pair of ribonucleic acid sequences in (b) is selected from the group consisting of: (i) SEQ ID NO: 128 and SEQ ID NO: 72; (ii) SEQ ID NO: 462 and SEQ ID NO: 421; (iii) SEQ ID NO: 550 and SEQ ID NO: 573; (iv) SEQ ID NO: 657 and SEQ ID NO: 662; (v) SEQ ID NO: 773 and SEQ ID NO: 778; and (vi) a combination of (i) to (v).

[0026] In some embodiments, the composition comprising the chimeric nucleic acid further comprises a nucleic acid sequence encoding a detectable marker. In some embodiments, the composition comprising the chimeric nucleic acid further comprises a promoter optimized for increased expression in human cells, operably linked to the chimeric nucleic acid, the promoter being selected from the group consisting of a cytomegalovirus (CMV) early enhancer element and a chicken beta-actin promoter, a chicken beta-actin promoter, an elongation factor-1 alpha promoter, and a ubiquitin promoter.

[0027] In some embodiments, the Cas protein comprises a Cas9 protein or a functional portion thereof. For example, the nucleic acid encoding the Cas protein comprises a messenger RNA (mRNA) encoding the Cas9 protein. In certain embodiments, the mRNA comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thiouridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. In certain embodiments of the invention, the chimeric nucleic acid comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thiouridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0028] Also disclosed herein is a method for altering a target CTLA4 polynucleotide sequence in a cell, comprising contacting the CTLA4 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize to a target motif in the target CTLA4 polynucleotide sequence, cleaving the target CTLA4 polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In certain embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637, respectively. For example, the two ribonucleic acids can comprise SEQ ID NO: 128 and SEQ ID NO: 72.

[0029] Also disclosed herein is a method for altering a target PD1 polynucleotide sequence in a cell, comprising contacting the PD1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize to a target motif in the target PD1 polynucleotide sequence, cleaving the target PD1 polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In certain embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945, respectively. For example, the two ribonucleic acids can comprise SEQ ID NOs: 462 and 421.

[0030] Also disclosed herein is a method for altering a target TCRA polynucleotide sequence in a cell, comprising contacting the TCRA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize with a target motif in the target TCRA polynucleotide sequence, cleaving the target TCRA polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In certain embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750, respectively. For example, the two ribonucleic acids can comprise SEQ ID NOs: 550 and 573.

[0031] Also disclosed herein is a method for altering a target TCRB polynucleotide sequence in a cell, comprising contacting the TCRB polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize with a target motif in the target TCRB polynucleotide sequence, cleaving the target TCRB polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In certain embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532, respectively. For example, the two ribonucleic acids may comprise SEQ ID NOs: 657 and 662.

[0032] In some embodiments, the invention is directed to modified primary human T cells, each cell comprising a modified genome comprising: (a) a first genomic modification in which the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3638-4046; and / or (b) a second genomic modification in which the programmed cell death 1 (PD1) gene on chromosome 2 has been edited to reduce or eliminate PD1 receptor surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein, and a second ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 8946-9101.

[0033] In some embodiments, the modified primary human T cells further comprise (c)(i) a third genomic modification in which a gene encoding a T cell receptor (TCR) alpha chain locus on chromosome 14 has been edited; and / or (c)(ii) a fourth genomic modification in which a gene encoding a TCR beta chain locus on chromosome 7 has been edited, thereby reducing or eliminating TCR surface expression and / or activity in the cell. In some aspects, the third contiguous stretch of genomic DNA has been edited by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a third ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 9751-9797, and / or the fourth contiguous stretch of genomic DNA has been edited by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a fourth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 10533-10573.

[0034] In some embodiments, the modified primary human T cell further comprises (d) a fifth genomic modification in which the beta2-microglobulin (B2M) gene on chromosome 15 has been edited, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In certain aspects, the fifth contiguous stretch of genomic DNA has been edited by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein, and a fifth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 13258-13719.

[0035] In some embodiments, the modified primary human T cells further comprise a chimeric antigen receptor or an exogenous nucleic acid encoding the chimeric antigen receptor. In certain embodiments, the chimeric antigen receptor specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof.

[0036] In some embodiments, the modified primary human T cells further comprise at least one exogenous protein or exogenous nucleic acid encoding said protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ.

[0037]

[0010] Also disclosed herein are methods for producing modified primary human T cells, the methods comprising: (a) contacting a primary human T cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3638-4046, thereby editing a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 to reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell; and / or (b) contacting a primary human T cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a second ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 8946-9101, thereby editing a programmed cell death 1 (PD1) gene on chromosome 2 to reduce or eliminate PD1 receptor surface expression and / or activity in the cell.

[0038] In some embodiments, the method further comprises (c)(i) editing in the cell a gene encoding a T cell receptor (TCR) alpha chain locus on chromosome 14, and / or (c)(ii) editing in the cell a gene encoding a TCR beta chain locus on chromosome 7, thereby reducing or eliminating TCR surface expression and / or activity in the cell. In certain aspects, the editing in (c)(i) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a third ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 9751-9797, and / or the editing in (c)(ii) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fourth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 10533-10573.

[0039] In some embodiments, the method further comprises (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In certain aspects, the editing in (d) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fifth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 13258-13719.

[0040] In some embodiments, the method further comprises causing the cells to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof.

[0041] In some embodiments, the methods disclosed herein further include causing the cell to express at least one protein that modulates a desired biological effect in an adjacent cell, tissue, or organ when the cell is in proximity to the adjacent cell, tissue, or organ.

[0042] In certain embodiments of the invention disclosed herein, the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tissue-infiltrating lymphocytes, and combinations thereof. In certain embodiments of the invention disclosed herein, the cells are obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD).

[0043] Also disclosed herein are compositions comprising the inventive cells disclosed herein or the cells produced according to the inventive methods disclosed herein.

[0044] Also disclosed herein is a composition comprising a chimeric nucleic acid, wherein the chimeric nucleic acid comprises: (a) a nucleic acid sequence encoding a Cas protein; (b) a ribonucleic acid sequence selected from the group consisting of (i) SEQ ID NOs: 3638-4046; (ii) SEQ ID NOs: 8946-9101; (iii) SEQ ID NOs: 9751-9797; (iv) SEQ ID NOs: 10533-10573; (v) SEQ ID NOs: 13258-13719; and (vi) a combination of (i)-(v).

[0045] In some embodiments, the chimeric nucleic acid further comprises a nucleic acid sequence encoding a detectable marker. In some aspects of the invention disclosed herein, the Cas protein comprises a Cpf1 protein or a functional portion thereof. For example, the nucleic acid encoding the Cas protein can comprise a messenger RNA (mRNA) encoding the Cpf1 protein. In certain aspects, the mRNA comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thiouridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0046] In some embodiments, the chimeric nucleic acid comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate.

[0047] Also disclosed herein is a method for altering a target CTLA4 polynucleotide sequence in a cell, comprising contacting the CTLA4 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif in the target CTLA4 polynucleotide sequence, and the target CTLA4 polynucleotide sequence is cleaved, wherein the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 3638-4046.

[0048] Also disclosed herein is a method for altering a target PD1 polynucleotide sequence in a cell, comprising contacting the PD1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif in the target PD1 polynucleotide sequence, and the target PD1 polynucleotide sequence is cleaved, wherein the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 8946-9101.

[0049] Also disclosed is a method for altering a target TCRA polynucleotide sequence in a cell, comprising contacting the TCRA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes with a target motif in the target TCRA polynucleotide sequence, and the target TCRA polynucleotide sequence is cleaved, and the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 9751-9797.

[0050] Also disclosed is a method for altering a target TCRB polynucleotide sequence in a cell, comprising contacting the TCRB polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif in the target TCRB polynucleotide sequence, and the target TCRB polynucleotide sequence is cleaved, wherein the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 10533-10573.

[0051] The above-discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention.

[0052] These and other features of the present invention will be more fully understood by reference to the following detailed description taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. In certain embodiments, for example, the following items are provided: (Item 1) 1. A modified primary human T cell comprising a modified genome, (a) a first genomic modification in which the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell; (b) a second genomic modification in which the programmed cell death 1 (PD1) gene on chromosome 2 has been edited to delete a second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell; (c)(i) a third genomic modification in which the gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14 has been edited to delete a third contiguous stretch of genomic DNA; and / or (c)(ii) a fourth genomic modification in which the gene encoding the TCR beta chain locus on chromosome 7 has been edited to delete a fourth contiguous stretch of genomic DNA, thereby reducing or eliminating TCR surface expression and / or activity in the cell; and (d) a fifth genomic modification in which the beta2-microglobulin (B2M) gene on chromosome 15 has been edited to delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. Including, Each cell may optionally (e)(i) at least one chimeric antigen receptor, or an exogenous nucleic acid encoding said at least one chimeric antigen receptor, that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof; and / or (e)(ii) A modified primary human T cell comprising at least one exogenous protein, or exogenous nucleic acid encoding the protein, that modulates a desired biological effect in an adjacent cell, tissue, or organ. (Item 2) 1. A modified primary human T cell comprising a modified genome, (a) a first genomic modification in which a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to delete a first contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, wherein the 3' end of the genomic DNA upstream of the 5' end of the deleted first contiguous stretch of genomic DNA is covalently linked to the 5' end of the genomic DNA downstream of the 3' end of the deleted first contiguous stretch of genomic DNA, resulting in a modified CTLA4 gene on chromosome 2 lacking the first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell; and / or (b) a second genomic modification in which the programmed cell death 1 (PD1) gene on chromosome 2 is edited to delete a second contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, wherein the 3' end of the genomic DNA upstream of the deleted second contiguous stretch of genomic DNA is covalently linked to the 5' end of the genomic DNA downstream of the 3' end of the deleted second contiguous stretch of genomic DNA, resulting in a modified PD1 gene on chromosome 2 lacking the second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell. 1. A modified primary human T cell comprising: (Item 3) 1. A modified primary human T cell comprising a modified genome, (a) a first genome modification in which the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell, wherein the first contiguous stretch of genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637; and / or (b) a second genome modification in which the programmed cell death 1 (PD1) gene on chromosome 2 is edited to delete a second continuous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell, wherein the second continuous stretch of genomic DNA is deleted by contacting the cell with the Cas protein or a nucleic acid encoding the Cas protein and a second pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. 1. A modified primary human T cell comprising: (Item 4) 4. The cell of item 3, wherein the first pair of ribonucleic acids comprises SEQ ID NO: 128 and SEQ ID NO: 72, and the second pair of ribonucleic acids comprises SEQ ID NO: 462 and SEQ ID NO: 421. (Item 5) (c)(i) a third genomic modification in which the gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14 has been edited to delete a third contiguous stretch of genomic DNA that includes at least a portion of a coding exon; and / or (c)(ii) a fourth genomic modification in which the gene encoding the TCR β chain locus on chromosome 7 has been edited to delete a fourth contiguous stretch of genomic DNA comprising at least a portion of a coding exon, thereby reducing or eliminating TCR surface expression and / or activity in the cell. 5. The cell according to any one of items 2 to 4, further comprising: (Item 6) a third contiguous stretch of the genomic DNA has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a third pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 532 to 609 and 9102 to 9545; and / or 6. The cell of item 5, wherein a fourth contiguous stretch of the genomic DNA is deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fourth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. (Item 7) 7. The cell of item 6, wherein the third pair of ribonucleic acids comprises SEQ ID NO: 550 and SEQ ID NO: 573, and / or the fourth pair of ribonucleic acids comprises SEQ ID NO: 657 and SEQ ID NO: 662. (Item 8) (d) the cell of any one of Items 2 to 7, further comprising a fifth genomic modification in which the beta2-microglobulin (B2M) gene on chromosome 15 has been edited to delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. (Item 9) 9. The cell of item 8, wherein a fifth contiguous stretch of the genomic DNA is deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fifth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. (Item 10) 10. The cell of item 9, wherein the fifth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778. (Item 11) 11. The cell according to any one of items 2 to 10, further comprising a chimeric antigen receptor or an exogenous nucleic acid encoding the chimeric antigen receptor. (Item 12) 12. The cell of item 11, wherein the chimeric antigen receptor specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof. (Item 13) 13. The cell of any one of items 2 to 12, further comprising at least one exogenous protein or exogenous nucleic acid encoding said protein that modulates a biological effect of interest in an adjacent cell, tissue or organ. (Item 14) 14. The cell according to any one of items 1 to 13, wherein the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a memory T cell, a regulatory T cell, a tissue-infiltrating lymphocyte, and a combination thereof. (Item 15) 15. The cell of any one of items 1 to 14, wherein the cell is obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD). (Item 16) 1. A method for producing modified primary human T cells, comprising: (a) editing the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 in primary human T cells to delete a first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in said cells; (b) editing the programmed cell death 1 (PD1) gene on chromosome 2 in said cell to delete a second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in said cell; (c)(i) editing the gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14 in the cell to delete a third contiguous stretch of genomic DNA; and / or (c)(ii) editing the gene encoding the TCR beta chain locus on chromosome 7 in the cell to delete a fourth contiguous stretch of genomic DNA, thereby reducing or eliminating TCR surface expression and / or activity in the cell; and (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell to delete a fifth continuous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. Including, in some cases, (e)(i) causing the cells to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof; and / or (e)(ii) causing the cells to express at least one protein that modulates a desired biological effect in an adjacent cell, tissue, or organ. Including, The editing in (a) to (d) comprises the cell, a Cas protein or a nucleic acid encoding the Cas protein, and at least one first pair of guide RNA sequences for deleting a first contiguous stretch of genomic DNA from the gene in (a); at least one second pair of guide RNA sequences for deleting a second contiguous stretch of the genomic DNA from the gene in (b); (c) at least one third pair of guide RNA sequences for deleting a third contiguous stretch of the genomic DNA from the gene in (i); and / or (c) at least one fourth pair of guide RNA sequences for deleting a fourth contiguous stretch of the genomic DNA from the gene in (ii); and and contacting the gene in (d) with at least one fifth pair of guide RNA sequences to delete a fifth consecutive stretch of the genomic DNA from the gene. 1. A method for producing modified primary human T cells, comprising: (a) editing a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 in a primary human T cell to delete a first contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, and covalently linking the 3' end of the genomic DNA upstream of the 5' end of the deleted first contiguous stretch of genomic DNA to the 5' end of the genomic DNA downstream of the 3' end of the deleted first contiguous stretch of genomic DNA, thereby generating a modified CTLA4 gene on chromosome 2 lacking the first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell; and / or (b) editing the programmed cell death 1 (PD1) gene on chromosome 2 in a primary human T cell to delete a second contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, and covalently linking the 3' end of the genomic DNA upstream of the deleted second contiguous stretch of genomic DNA to the 5' end of the genomic DNA downstream of the 3' end of the deleted second contiguous stretch of genomic DNA, thereby generating a modified PD1 gene on chromosome 2 lacking the second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell. A method comprising: (Item 18) 1. A method for producing modified primary human T cells, comprising: (a) contacting a primary human T cell with a Cas protein or a nucleic acid encoding the Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637, thereby editing the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 to delete a first contiguous stretch of genomic DNA and reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell; and / or (b) contacting primary human T cells with the Cas protein or a nucleic acid encoding the Cas protein and a second pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945, thereby editing the programmed cell death 1 (PD1) gene on chromosome 2 to delete a second contiguous stretch of genomic DNA and reduce or eliminate PD1 receptor surface expression and / or activity in the cells. A method comprising: (Item 19) 19. The method of claim 18, wherein the first pair of ribonucleic acids comprises SEQ ID NO: 128 and SEQ ID NO: 72, and the second pair of ribonucleic acids comprises SEQ ID NO: 462 and SEQ ID NO: 421. (Item 20) (c)(i) editing a gene encoding a T cell receptor (TCR) alpha chain locus on chromosome 14 in the cell to delete a third contiguous stretch of genomic DNA comprising at least a portion of a coding exon; and / or (c)(ii) editing the gene encoding the TCR beta chain locus on chromosome 7 in the cell to delete a fourth contiguous stretch of genomic DNA comprising at least a portion of a coding exon, thereby reducing or eliminating TCR surface expression and / or activity in the cell. 20. The method according to any one of items 17 to 19, further comprising: (Item 21) (c) the editing in (i) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a third pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750; and / or 21. The method of claim 20, wherein the editing in (c)(ii) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fourth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. (Item 22) 22. The method of claim 21, wherein the third pair of ribonucleic acids comprises SEQ ID NO: 550 and SEQ ID NO: 573, and / or the fourth pair of ribonucleic acids comprises SEQ ID NO: 657 and SEQ ID NO: 662. (Item 23) (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell to delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. (Item 24) 24. The method of claim 23, wherein the editing in (d) comprises contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fifth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. (Item 25) 25. The method of claim 24, wherein the fifth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778. (Item 26) The method of any one of Items 17 to 25, further comprising causing the cells to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof. (Item 27) 27. The method of any one of items 17 to 26, further comprising causing the cell to express at least one protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ when the cell is in proximity to the adjacent cell, tissue, or organ. (Item 28) 28. The method according to any one of items 16 to 27, wherein the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tissue-infiltrating lymphocytes, and combinations thereof. (Item 29) 29. The method of any one of items 16 to 28, wherein the cells are obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD). (Item 30) A composition comprising the cells according to any one of items 1 to 15 or the cells produced according to the method according to any one of items 16 to 29. (Item 31) 1. A method of treating a patient in need thereof, comprising: (a)(i) administering the modified T cells of any one of items 1 to 15 to a patient in need of such cells; (a)(ii) administering modified T cells produced according to the method of any one of paragraphs 16 to 29 to a patient in need of such cells; or (a)(iii) administering the composition of item 30 to a patient in need of such cells. A method comprising: (Item 32) 32. The method of claim 31, wherein the treatment comprises adoptive immunotherapy. (Item 33) 32. The method of claim 30 or 31, further comprising expanding the modified T cells prior to the administering step. (Item 34) 34. The method of any one of items 30 to 33, wherein the patient is suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD). (Item 35) A composition comprising a chimeric nucleic acid, the chimeric nucleic acid comprising: (a) a nucleic acid sequence encoding a Cas protein; (b) (i) SEQ ID NOs: 1 to 195 and 797 to 3637; (ii) SEQ ID NOs: 196-531 and 4047-8945; (iii) SEQ ID NOs: 532-609 and 9102-9750; (iv) SEQ ID NOs: 610-765 and 9798-10532; (v) SEQ ID NOs: 766-780 and 10574-13257; and (vi) Combination of (i) to (v) At least one ribonucleic acid sequence selected from the group consisting of A composition comprising: (Item 36) The pair of ribonucleic acid sequences in (b) is (i) SEQ ID NO: 128 and SEQ ID NO: 72; (ii) SEQ ID NO: 462 and SEQ ID NO: 421; (iii) SEQ ID NO: 550 and SEQ ID NO: 573; (iv) SEQ ID NO: 657 and SEQ ID NO: 662; (v) SEQ ID NO: 773 and SEQ ID NO: 778; and (vi) Combination of (i) to (v) 36. The composition according to item 35, selected from the group consisting of: (Item 37) 36. The composition of item 34 or 35, further comprising a nucleic acid sequence encoding a detectable marker. (Item 38) 38. The composition of any one of items 34 to 37, wherein the Cas protein comprises a Cas9 protein or a functional portion thereof. (Item 39) 39. The composition of any one of Items 34 to 38, further comprising a promoter optimized for increased expression in human cells operably linked to the chimeric nucleic acid, wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) early enhancer element and a chicken β-actin promoter, a chicken β-actin promoter, an elongation factor-1α promoter, and a ubiquitin promoter. (Item 40) 40. The composition of any one of Items 34 to 39, wherein the chimeric nucleic acid comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytodine, 2-thiouridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. (Item 41) 41. The composition of any one of items 34 to 40, wherein the nucleic acid encoding the Cas protein comprises messenger RNA (mRNA) encoding a Cas9 protein. (Item 42) 42. The composition of claim 41, wherein the mRNA comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thiouridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. (Item 43) 1. A method for altering a target CTLA4 polynucleotide sequence in a cell, comprising contacting the CTLA4 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize with a target motif in the target CTLA4 polynucleotide sequence, thereby cleaving the target CTLA4 polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. (Item 44) Item 44. The method according to Item 43, wherein the one or two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 1 to 195 and 797 to 3637, respectively. (Item 45) 45. The method of item 43 or 44, wherein the two ribonucleic acids comprise SEQ ID NO: 128 and SEQ ID NO: 72. (Item 46) 1. A method for altering a target PD1 polynucleotide sequence in a cell, comprising contacting the PD1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize to a target motif in the target PD1 polynucleotide sequence, thereby cleaving the target PD1 polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. (Item 47) Item 47. The method according to Item 46, wherein the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 196 to 531 and 4047 to 8945, respectively. (Item 48) 47. The method of item 45 or 46, wherein the two ribonucleic acids comprise SEQ ID NO: 462 and SEQ ID NO: 421. (Item 49) A method for altering a target TCRA polynucleotide sequence in a cell, comprising contacting the TCRA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize with a target motif in the target TCRA polynucleotide sequence, thereby cleaving the target TCRA polynucleotide sequence, and at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. (Item 50) Item 49. The method according to item 49, wherein the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 532 to 609 and 9102 to 9750, respectively. (Item 51) 51. The method of item 49 or 50, wherein the two ribonucleic acids comprise SEQ ID NO: 550 and SEQ ID NO: 573. (Item 52) 1. A method for altering a target TCRB polynucleotide sequence in a cell, comprising contacting the TCRB polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize with a target motif in the target TCRB polynucleotide sequence, thereby cleaving the target TCRB polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. (Item 53) 53. The method according to Item 52, wherein the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 610 to 765 and 9798 to 10532, respectively. (Item 54) 54. The method of item 52 or 53, wherein the two ribonucleic acids comprise SEQ ID NO: 657 and SEQ ID NO: 662. (Item 55) 1. Modified primary human T cells, each cell comprising: (a) a first genome modification in which the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein, and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3638 to 4046; and / or (b) a second genome modification in which the programmed cell death 1 (PD1) gene on chromosome 2 is edited to reduce or eliminate PD1 receptor surface expression and / or activity in the cell by contacting the cell with the Cas protein or a nucleic acid encoding the Cas protein, and a second ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 8946 to 9101. 1. A modified primary human T cell comprising a modified genome comprising: (Item 56) (c)(i) a third genomic modification in which a gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14 is edited; and / or (c)(ii) a fourth genomic modification in which a gene encoding a TCR beta chain locus on chromosome 7 is edited, thereby reducing or eliminating TCR surface expression and / or activity in the cell. 56. The cell of item 55, further comprising: (Item 57) a third contiguous stretch of genomic DNA has been edited by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a third ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 9751 to 9797; and / or 57. The cell of item 56, wherein a fourth contiguous stretch of genomic DNA has been edited by contacting the cell with the Cas protein or a nucleic acid encoding the Cas protein and a fourth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 10533 to 10573. (Item 58) (d) the cell of any one of paragraphs 55 to 57, further comprising a fifth genomic modification in which the beta2-microglobulin (B2M) gene on chromosome 15 has been edited, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in said cell. (Item 59) 59. The cell of item 58, wherein a fifth contiguous stretch of genomic DNA has been edited by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a fifth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 13258 to 13719. (Item 60) 60. The cell of any one of items 55 to 59, further comprising a chimeric antigen receptor or an exogenous nucleic acid encoding said chimeric antigen receptor. (Item 61) 61. The cell of paragraph 60, wherein the chimeric antigen receptor specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof. (Item 62) 62. The cell of any one of items 55-61, further comprising at least one exogenous protein or exogenous nucleic acid encoding said protein that modulates a biological effect of interest in an adjacent cell, tissue or organ. (Item 63) 63. The cell of any one of items 55 to 62, wherein the T cell is selected from the group consisting of a cytotoxic T cell, a helper T cell, a memory T cell, a regulatory T cell, a tissue-infiltrating lymphocyte, and a combination thereof. (Item 64) 64. The cell of any one of items 55-63, wherein the cell is obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD). (Item 65) 1. A method for producing modified primary human T cells, comprising: (a) contacting a primary human T cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3638-4046, thereby editing the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 to reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell; and / or (b) contacting a primary human T cell with the Cas protein or a nucleic acid sequence encoding the Cas protein and a second ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 8946 to 9101, thereby editing the programmed cell death 1 (PD1) gene on chromosome 2 to reduce or eliminate PD1 receptor surface expression and / or activity in the cell. A method comprising: (Item 66) (c)(i) editing a gene encoding a T cell receptor (TCR) alpha chain locus on chromosome 14 in the cell; and / or (c)(ii) editing a gene encoding a TCR beta chain locus on chromosome 7 in said cell, thereby reducing or eliminating TCR surface expression and / or activity in said cell. Item 66. The method of item 65, further comprising: (Item 67) (c) the editing in (i) comprises contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a third ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 9751 to 9797; and / or 67. The method of item 66, wherein the editing in (c)(ii) comprises contacting the cell with the Cas protein or a nucleic acid sequence encoding the Cas protein and a fourth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 10533 to 10573. (Item 68) (d) the method of any one of Items 65 to 67, further comprising editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. (Item 69) 69. The method of item 68, wherein the editing in (d) comprises contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein, and a fifth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 13258 to 13719. (Item 70) 71. The method of any one of Items 65 to 69, further comprising causing the cells to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof. 71. The method of any one of items 65 to 70, further comprising causing the cell to express at least one protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ when the cell is in proximity to the adjacent cell, tissue, or organ. (Item 72) 72. The method according to any one of items 65 to 71, wherein the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tissue-infiltrating lymphocytes, and combinations thereof. (Item 73) 73. The method of any one of paragraphs 65 to 72, wherein the cells are obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD). (Item 74) A composition comprising the cells according to any one of items 55 to 64 or the cells produced according to the method according to any one of items 65 to 73. (Item 75) A composition comprising a chimeric nucleic acid, the chimeric nucleic acid comprising: (a) a nucleic acid sequence encoding a Cas protein; (b) (i) SEQ ID NOs: 3638 to 4046; (ii) SEQ ID NOs: 8946 to 9101; (iii) SEQ ID NOs: 9751 to 9797; (iv) SEQ ID NOs: 10533 to 10573; (v) SEQ ID NOs: 13258 to 13719; and (vi) Combination of (i) to (v) a ribonucleic acid sequence selected from the group consisting of A composition comprising: (Item 76) 76. The composition of item 75, further comprising a nucleic acid sequence encoding a detectable marker. (Item 77) 77. The composition of any one of items 75 to 76, wherein the Cas protein comprises a Cpf1 protein or a functional part thereof. (Item 78) 78. The composition of any one of items 75 to 77, further comprising a promoter optimized for increased expression in human cells operably linked to the chimeric nucleic acid, wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) early enhancer element and a chicken β-actin promoter, a chicken β-actin promoter, an elongation factor-1α promoter, and a ubiquitin promoter. (Item 79) 79. The composition of any one of items 75 to 78, wherein the chimeric nucleic acid comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate and 5-azauridine-5'-triphosphate. (Item 80) 80. The composition of any one of items 75 to 79, wherein the nucleic acid encoding the Cas protein comprises messenger RNA (mRNA) encoding a Cpf1 protein. (Item 81) 81. The composition of item 80, wherein the mRNA comprises at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thiouridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate. (Item 82) 1. A method for altering a target CTLA4 polynucleotide sequence in a cell, comprising contacting the CTLA4 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif in the target CTLA4 polynucleotide sequence, and the target CTLA4 polynucleotide sequence is cleaved, wherein the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 3638-4046. (Item 83) 1. A method for altering a target PD1 polynucleotide sequence in a cell, comprising contacting the PD1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif in the target PD1 polynucleotide sequence, and the target PD1 polynucleotide sequence is cleaved, wherein the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 8946-9101. (Item 84) A method for altering a target TCRA polynucleotide sequence in a cell, comprising contacting the TCRA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes with a target motif in the target TCRA polynucleotide sequence, and the target TCRA polynucleotide sequence is cleaved, and the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 9751-9797. (Item 85) 1. A method for altering a target TCRB polynucleotide sequence in a cell, comprising contacting the TCRB polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and a ribonucleic acid, wherein the ribonucleic acid directs the Cas protein to and hybridizes to a target motif in the target TCRB polynucleotide sequence, and the target TCRB polynucleotide sequence is cleaved, wherein the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 10533-10573. (Item 86) 1. A modified primary human T cell comprising a modified genome, (a) a first genomic modification in which the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell; (b) a second genomic modification in which the programmed cell death 1 (PD1) gene on chromosome 2 has been edited to reduce or eliminate PD1 receptor surface expression and / or activity in said cell; (c)(i) a third genomic modification in which a gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14 has been edited to reduce or eliminate TCR surface expression and / or activity in the cell; and / or (c)(ii) a fourth genomic modification in which a gene encoding a TCR beta chain locus on chromosome 7 has been edited to reduce or eliminate TCR surface expression and / or activity in the cell; and (d) a fifth genomic modification, in which the beta2-microglobulin (B2M) gene on chromosome 15 is edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell. Including, Each cell may optionally (e)(i) at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof, or an exogenous nucleic acid encoding said at least one chimeric antigen receptor; and / or (e)(ii) at least one exogenous protein, or an exogenous nucleic acid encoding said protein, that modulates a biological effect of interest in an adjacent cell, tissue, or organ; 1. A modified primary human T cell comprising: [Brief explanation of the drawings]

[0053] [Figure 1] Figure 1 is a schematic diagram of adoptive immunotherapy using tumor-infiltrating lymphocytes (TILs). T cells are isolated from the tumor, expanded ex vivo, and then reinfused into the patient to target tumor cells. Because the tumor environment does not support sufficient T cell proliferation, this method allows T cells to be activated and expanded ex vivo before reinfusion to mount an immune attack against the tumor (Restifo et al., 2012).

[0054] [Figure 2] Figure 2 is a schematic diagram of three generations of CAR T cells. The tumor-associated antigen (TAA) binding domain, which determines antigen specificity, is shown in green. The intracellular domain incorporates different aspects of the TCR transduction machinery: (CD3ζ chain / ZAP70), blue (CD28 / PI3K), and yellow (4-1BB or OX40 / TRAF) (adapted from Casucci and Bondanza, 2011).

[0055] [Figure 3]Figure 3 is a schematic diagram of T cell activation and inhibition mechanisms. Dashed arrows indicate T cell activation via MHC-TCR and CD28-B7 interactions. Solid arrows represent inhibitory effects mediated through CTLA4 and PD1 on T cells. B7-1 and B7-2 are CD80 and CD86, respectively (adapted from Drake et al., CG, 2014).

[0056] [Figure 4] Figure 4 shows an exemplary amino acid sequence of a Cas protein. Yellow highlighting indicates the Ruv-C-like domain. Underlining indicates the HNH nuclease domain.

[0057] [Figure 5-1] Figure 5 shows exemplary gRNA sequences useful for targeting the CTLA4 gene using Cas9. gRNAs described in the experimental examples are identified in red text. [Figure 5-2] Same as above [Figure 5-3] Same as above [Figure 5-4] Same as above [Figure 5-5] Same as above [Figure 5-6] Same as above [Figure 5-7] Same as above [Figure 5-8] Same as above [Figure 5-9] Same as above [Figure 5-10] Same as above [Figure 5-11] Same as above [Figure 5-12] Same as above [Figure 5-13] Same as above [Figure 5-14] Same as above [Figure 5-15] Same as above [Figure 5-16] Same as above [Figure 5-17] Same as above [Figure 5-18] Same as above [Figure 5-19] Same as above [Figure 5-20] Same as above [Figure 5-21] Same as above [Figure 5-22] Same as above [Figure 5-23] Same as above [Figure 5-24] Same as above [Figure 5-25] Same as above [Figure 5-26] Same as above [Figure 5-27] Same as above [Figure 5-28] Same as above [Figure 5-29] Same as above [Figure 5-30] Same as above [Figure 5-31] Same as above [Figure 5-32] Same as above [Figure 5-33] Same as above [Figure 5-34] Same as above [Figure 5-35] Same as above [Figure 5-36] Same as above [Figure 5-37] Same as above [Figure 5-38] Same as above [Figure 5-39] Same as above [Figure 5-40] Same as above [Figure 5-41] Same as above [Figure 5-42] Same as above [Figure 5-43] Same as above [Figure 5-44] Same as above [Figure 5-45] Same as above

[0058] [Figure 6-1] Figure 6 shows exemplary gRNA sequences useful for targeting the PD1 gene using Cas9. gRNAs described in the experimental examples are identified in red text. [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 6-4] Same as above [Figure 6-5] Same as above [Figure 6-6] Same as above [Figure 6-7] Same as above [Figure 6-8] Same as above [Figure 6-9] Same as above [Figure 6-10] Same as above [Figure 6-11] Same as above [Figure 6-12] Same as above [Figure 6-13] Same as above [Figure 6-14] Same as above [Figure 6-15] Same as above [Figure 6-16] Same as above [Figure 6-17] Same as above [Figure 6-18] Same as above [Figure 6-19] Same as above [Figure 6-20] Same as above [Figure 6-21] Same as above [Figure 6-22] Same as above [Figure 6-23] Same as above [Figure 6-24] Same as above [Figure 6-25] Same as above [Figure 6-26] Same as above [Figure 6-27] Same as above [Figure 6-28] Same as above [Figure 6-29] Same as above [Figure 6-30] Same as above [Figure 6-31] Same as above [Figure 6-32] Same as above [Figure 6-33] Same as above [Figure 6-34] Same as above [Figure 6-35] Same as above [Figure 6-36] Same as above [Figure 6-37] Same as above [Figure 6-38] Same as above [Figure 6-39] Same as above [Figure 6-40] Same as above [Figure 6-41] Same as above [Figure 6-42] Same as above [Figure 6-43] Same as above [Figure 6-44] Same as above [Figure 6-45] Same as above [Figure 6-46] Same as above [Figure 6-47] Same as above [Figure 6-48] Same as above [Figure 6-49] Same as above [Figure 6-50] Same as above [Figure 6-51] Same as above [Figure 6-52] Same as above [Figure 6-53] Same as above [Figure 6-54] Same as above [Figure 6-55] Same as above [Figure 6-56] Same as above [Figure 6-57] Same as above [Figure 6-58] Same as above [Figure 6-59] Same as above [Figure 6-60] Same as above [Figure 6-61] Same as above [Figure 6-62] Same as above [Figure 6-63] Same as above [Figure 6-64] Same as above [Figure 6-65] Same as above [Figure 6-66] Same as above [Figure 6-67] Same as above [Figure 6-68] Same as above [Figure 6-69] Same as above [Figure 6-70] Same as above [Figure 6-71] Same as above [Figure 6-72] Same as above [Figure 6-73] Same as above [Figure 6-74] Same as above [Figure 6-75] Same as above [Figure 6-76] Same as above [Figure 6-77] Same as above [Figure 6-78] Same as above

[0059] [Figure 7-1] Figure 7 shows exemplary gRNA sequences useful for targeting the TCR alpha locus using Cas9. gRNAs described in the experimental examples are identified in red text. [Figure 7-2] Same as above [Figure 7-3] Same as above [Figure 7-4] Same as above [Figure 7-5] Same as above [Figure 7-6] Same as above [Figure 7-7] Same as above [Figure 7-8] Same as above [Figure 7-9] Same as above [Figure 7-10] Same as above [Figure 7-11] Same as above [Figure 7-12] Same as above [Figure 7-13] Same as above [Figure 7-14] Same as above

[0060] [Figure 8-1] Figure 8 shows exemplary gRNA sequences useful for targeting the human TCRβ locus using Cas9. gRNAs described in the experimental examples are identified in red text. [Figure 8-2] Same as above [Figure 8-3] Same as above [Figure 8-4] Same as above [Figure 8-5] Same as above [Figure 8-6] Same as above [Figure 8-7] Same as above [Figure 8-8] Same as above [Figure 8-9] Same as above [Figure 8-10] Same as above [Figure 8-11] Same as above [Figure 8-12] Same as above [Figure 8-13] Same as above [Figure 8-14] Same as above [Figure 8-15] Same as above [Figure 8-16] Same as above [Figure 8-17] Same as above [Figure 8-18] Same as above [Figure 8-19] Same as above

[0061] [Figure 9-1] Figure 9 shows exemplary gRNA sequences useful for targeting and editing the human B2M gene using Cas9. [Figure 9-2] Same as above [Figure 9-3] Same as above [Figure 9-4] Same as above [Figure 9-5] Same as above [Figure 9-6] Same as above [Figure 9-7] Same as above [Figure 9-8] Same as above [Figure 9-9] Same as above [Figure 9-10] Same as above [Figure 9-11] Same as above [Figure 9-12] Same as above [Figure 9-13] Same as above [Figure 9-14] Same as above [Figure 9-15] Same as above [Figure 9-16] Same as above [Figure 9-17] Same as above [Figure 9-18] Same as above [Figure 9-19] Same as above [Figure 9-20] Same as above [Figure 9-21] Same as above [Figure 9-22] Same as above [Figure 9-23] Same as above [Figure 9-24] Same as above [Figure 9-25] Same as above [Figure 9-26] Same as above [Figure 9-27]Same as above [Figure 9-28] Same as above [Figure 9-29] Same as above [Figure 9-30] Same as above [Figure 9-31] Same as above [Figure 9-32] Same as above [Figure 9-33] Same as above [Figure 9-34] Same as above [Figure 9-35] Same as above [Figure 9-36] Same as above [Figure 9-37] Same as above [Figure 9-38] Same as above [Figure 9-39] Same as above [Figure 9-40] Same as above [Figure 9-41] Same as above [Figure 9-42] Same as above

[0062] [Figure 10] Figure 10 demonstrates an exemplary TCR targeting strategy of the present invention. Figure 10 is a schematic diagram showing the location of CRISPR gRNAs targeting the first coding exons of the TCR alpha and beta chains, respectively.

[0063] [Figure 11] Figures 11A and 11B demonstrate T cell receptor deletion in Jurkat T cells. Figure 11A shows the results of FACS analysis using an anti-CD3 antibody, revealing successful TCR deletion in Jurkat T cells stably expressing Cas9 nuclease. Figure 11B shows the results of a SURVEYOR™ assay, confirming cleavage at the TCRa and TCRb loci.

[0064] [Figure 12]Figures 12A and 12B demonstrate TCR deletion in primary human CD3+ T cells. Figure 12A shows the results of a SURVEYOR™ assay demonstrating CRISPR cleavage at the TCRa and TCRb loci in CD3+ T cells from two independent donors. Figure 12B shows the loss of TCR surface expression demonstrated by FACS analysis.

[0065] [Figure 13] Figures 13A, 13B, and 13C demonstrate exemplary PD-1 locus targeting strategies of the present invention. Figure 13A is a schematic diagram of the PD-1 targeting strategy. Figure 13B demonstrates that the dual CRISPR strategy results in cleavage by both CRISPRs targeting the PD-1 locus in HEK293T cells. Figure 13C is a schematic diagram of sequencing that confirmed the predicted deletion at the PD-1 locus after transfection of two CRISPRs targeting the PD-1 gene (PDCD1), as shown with reference to SEQ ID NOs: 793 and 794.

[0066] [Figure 14] Figures 14 and 14B demonstrate loss of PD-1 expression in Jurkat T cells. Figure 14A shows the results of a FACS analysis demonstrating loss of PD-1 expression in activated Jurkat T cells. Figure 14B shows the results of a SURVEYOR™ assay confirming truncation at the PD-1 locus.

[0067] [Figure 15]Figure 15A, Figure 15B and Figure 15C demonstrate exemplary CTLA4 locus targeting strategies of the present invention. Figure 15A is a schematic diagram of the CTLA4 targeting strategy. Figure 15B demonstrates that the double CRISPR strategy results in cleavage by both CRISPRs targeting the CTLA4 locus in HEK293T cells. Figure 15C is a schematic diagram of sequencing that confirmed the predicted deletion in the CTLA4 locus after transfection of two CRISPRs targeting the CTLA4 gene (CTLA4), as shown with reference to SEQ ID NOs: 795 and 796.

[0068] [Figure 16] Figures 16A and 16B demonstrate cleavage at the CTLA-4 locus in Jurkat T cells. Figure 16A demonstrates that the dual CRISPR strategy results in cleavage by both CRISPRs targeting the CTLA4 locus in Jurkat T cells. Figure 16B shows the results of a SURVEYOR™ assay demonstrating successful cleavage by both CTLA4 CRISPRs in Jurkat T cells.

[0069] [Figure 17-1] Figure 17 shows exemplary gRNA sequences useful for targeting the CTLA4 gene using Cpf1. [Figure 17-2] Same as above [Figure 17-3] Same as above [Figure 17-4] Same as above [Figure 17-5] Same as above [Figure 17-6] Same as above

[0070] [Figure 18-1] Figure 18 shows exemplary gRNA sequences useful for targeting the PD1 gene using Cpf1. [Figure 18-2] Same as above [Figure 18-3] Same as above

[0071] [Figure 19] Figure 19 shows exemplary gRNA sequences useful for targeting the TCR alpha locus using Cpf1.

[0072] [Figure 20] Figure 20 shows exemplary gRNA sequences useful for targeting the human TCR β locus using Cpf1.

[0073] [Figure 21-1] Figure 21 shows exemplary gRNA sequences useful for targeting and editing the human B2M gene using Cpf1. [Figure 21-2] Same as above [Figure 21-3] Same as above [Figure 21-4] Same as above [Figure 21-5] Same as above [Figure 21-6] Same as above [Figure 21-7] Same as above

[0074] [Figure 22] Figure 22 demonstrates the B2M deletion efficiency of the selection guides in 293T cells. The arrow in the Surveyor assay indicates the nuclease cleavage junction.

[0075] [Figure 23] FIG. 23 demonstrates a comparison of B2M surface expression in 293T cells when transfected with AsCpf1 and guide crB2M.

[0076] [Figure 24] FIG. 24 demonstrates a comparison of B2M surface expression in 293T cells when transfected with LbCpf1 and guide crB2M.

[0077] [Figure 25] Figure 25 shows the design and cloning information of Cpf1 crRNA.

[0078] [Figure 26A] Figures 26A, 26B, 26C, 26D, 26E, 26F, and 26G demonstrate the generation and characterization of B2M KO JEG3 cells using TALENs. Figure 26A shows the design of the B2M TALEN and the induced mutations. Figure 26B shows the analysis of B2M at the transcript and protein levels. Figure 26C demonstrates the analysis of B2M at the surface expression level. Figure 26D demonstrates that ΔB2M clones lack MHC-I surface expression. Figure 26E demonstrates that ΔB2M clones lack HLA-G surface expression. Figure 26F demonstrates that ΔB2M clones lack HLA-C surface expression. Figure 26G demonstrates that ΔB2M clones lack HLA-E surface expression. [Figure 26B] Same as above [Figure 26C] Same as above [Figure 26D] Same as above [Figure 26E] Same as above [Figure 26F] Same as above [Figure 26G] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0079] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0080] Detailed Description of the Invention T cell therapy has emerged as a major breakthrough in cancer immunotherapy, and there are currently several clinical trials using engineered T cells in the treatment of primarily B cell malignancies. T cells can be genetically engineered to express tumor-specific chimeric antigen receptors (CARs) with specificity derived from the variable domains of monoclonal antibodies to focus the immune response on the tumor without major histocompatibility complex (MHC) restriction.

[0081] Despite these initial successes, existing CAR T therapies face several obstacles. The primary obstacle is the presence of endogenous T cell receptors (TCRs) that prevent allogeneic transplantation of CAR T cells. Even if the cells are returned to the same patient, administration to the patient at high doses can result in autoimmune attacks if the cells are cross-reactive with autoantigens. A second obstacle in T cell therapy is that tumors and viruses have evolved mechanisms to suppress T cells by exploiting critical checkpoint regulators of T cell activity. The present invention utilizes the potential of gene editing systems (e.g., CRISPR / Cas or TALEN systems) to overcome the above-mentioned obstacles that prevent the safe clinical translation of this new treatment option. In particular, the studies described herein demonstrate the feasibility of generating readily available, universal CAR T cells derived from allogeneic healthy donors that can be administered to any patient without the risk of immune rejection or graft-versus-host disease (GvHD) and that are not prone to T cell inhibition. The work described herein also demonstrates the feasibility of developing CRISPR guide sequences (gRNAs) that efficiently target endogenous TCRs and key checkpoint regulators of T cell activity. The work described herein provides gRNAs designed and tested to (1) prevent autoreactivity by targeting genes encoding the TCR α and β chains, (2) remove allobarriers by targeting TCR and B2M genes, and / or (3) overcome autoreactivity by targeting the checkpoint inhibitors PD-1 and CTLA-4.

[0082] The present invention contemplates, for example, using TALEN or CRISPR / Cas systems to alter target polynucleotide sequences by any method available to those skilled in the art. Such CRISPR / Cas systems can use various Cas proteins (Haft et al., PLoS Comput Biol. 2005;1(6)e60). In some embodiments, the CRISPR / Cas system is a type I CRISPR system. In some embodiments, the CRISPR / Cas system is a type II CRISPR system. In some embodiments, the CRISPR / Cas system is a type V CRISPR system. Although examples of methods using CRISPR / Cas (e.g., Cas9 and Cpf1) and TALEN are described in detail herein, it should be understood that the present invention is not limited to the use of these methods / systems. Other methods known to those skilled in the art for targeting polynucleotide sequences to reduce or eliminate expression in target cells can be used herein.

[0083] According to the methods of the present invention, one or more target polynucleotide sequences in a cell are altered (e.g., modified or truncated). The present invention contemplates altering a target polynucleotide sequence in a cell for any purpose, but particularly to reduce or eliminate the expression or activity of an encoded product. In some embodiments, a target polynucleotide sequence in a cell is altered to produce a mutant cell. As used herein, a "mutant cell" refers to a cell whose resulting genotype differs from its original genotype. In some cases, for example, when a normally functioning gene is altered using the CRISPR / Cas system of the present invention, a "mutant cell" exhibits a mutant phenotype. In other cases, for example, when a mutant genotype is corrected using the CRISPR / Cas system, a "mutant cell" exhibits a wild-type phenotype. In some embodiments, a target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, a target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0084] In some embodiments, the change is an indel. As used herein, "indel" refers to a mutation resulting from insertion, deletion, or a combination thereof. As those skilled in the art will recognize, an indel in the coding region of a genome sequence will result in a frameshift mutation unless the length of the indel is a multiple of three. In some embodiments, the change is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one nucleotide. The CRISPR / Cas system can be used to induce indels or point mutations of any length in target polynucleotide sequences.

[0085] In some embodiments, the alteration results in knockout of the target polynucleotide sequence or a portion thereof. For example, knockout of a target polynucleotide sequence in a cell can be performed in vitro, in vivo, or ex vivo for both therapeutic and research purposes. Knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing a disorder associated with the expression of the target polynucleotide sequence (e.g., by knocking out a mutant allele in a cell ex vivo and introducing the cell containing the knocked-out mutant allele into a subject). As used herein, "knockout" includes deleting all or a portion of a target polynucleotide sequence in a manner that disrupts the function of the target polynucleotide sequence or its expression product.

[0086] In some embodiments, the change results in a reduction in the expression of the target polynucleotide sequence. The terms "reduce," "reduced," "reduction," and "reduction" are all used herein to generally refer to a statistically significant reduction. However, for the avoidance of doubt, "reduced," "reduced," "reduction," and "reduction" include a reduction of at least 10% compared to the reference level, such as at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or a reduction of up to 100% (inclusive) (i.e., non-existent levels compared to the reference sample), or any reduction of 10-100% compared to the reference level.

[0087] The terms "increased," "increase," or "enhance" or "activate" are all used herein generally to mean an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased," "increase," or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% increase or up to and including a 100% increase compared to a reference level, or any increase between 10 and 100% compared to a reference level, or at least about a 2-fold increase, or at least about a 3-fold increase, or at least about a 4-fold increase, or at least about a 5-fold increase, or at least about a 10-fold increase or more compared to a reference level.

[0088] The term "statistically significant" or "significantly" refers to statistical significance and generally means a marker concentration that is two standard deviations (2SD) below (i.e., lower than) normal. The term refers to statistical evidence that there is a difference. It is defined as the probability of rejecting the null hypothesis when the null hypothesis is actually correct. The determination is often made using a p-value.

[0089] In some embodiments, the alteration is a homozygous alteration. In some embodiments, the alteration is a heterozygous alteration.

[0090] In some embodiments, the change results in the correction of the target polynucleotide sequence from an undesired sequence to a desired sequence. The CRISPR / Cas system can be used to correct any kind of mutation or error in the target polynucleotide sequence. For example, the CRISPR / Cas system can be used to insert a nucleotide sequence that has been deleted from the target polynucleotide sequence. The CRISPR / Cas system can also be used to delete or excise a nucleotide sequence from the target polynucleotide sequence by insertion mutation. In some cases, the CRISPR / Cas system can be used to replace an incorrect nucleotide sequence with a correct nucleotide sequence (for example, to restore the function of the target polynucleotide sequence that has been impaired by a loss-of-function mutation, i.e., SNP).

[0091] CRISPR / Cas systems can alter target polynucleotides with surprisingly high efficiency. In certain embodiments, the efficiency of alteration is at least about 5%. In certain embodiments, the efficiency of alteration is at least about 10%. In certain embodiments, the efficiency of alteration is between about 10% and about 80%. In certain embodiments, the efficiency of alteration is between about 30% and about 80%. In certain embodiments, the efficiency of alteration is between about 50% and about 80%. In some embodiments, the efficiency of alteration is greater than or equal to about 80%. In some embodiments, the efficiency of alteration is greater than or equal to about 85%. In some embodiments, the efficiency of alteration is greater than or equal to about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some embodiments, the efficiency of alteration is equal to about 100%.

[0092] The CRISPR / Cas system can be used to change any target polynucleotide sequence in a cell. Those skilled in the art will readily recognize that the desired target polynucleotide sequence to be changed in any particular cell can correspond to any genomic sequence whose expression is associated with a disorder or promotes the invasion of a pathogen into the cell. For example, the desired target polynucleotide sequence to be changed in a cell can be a polynucleotide sequence corresponding to a genomic sequence containing a disease-associated single polynucleotide polymorphism (SNP). In such an example, the CRISPR / Cas system can be used to correct the disease-associated SNP in a cell by replacing it with a wild-type allele. As another example, the polynucleotide sequence of a target gene involved in the invasion or proliferation of a pathogen into a cell can be a suitable target for deletion or insertion to disrupt the function of the target gene to prevent the pathogen from invading or proliferating in the cell.

[0093] In some embodiments, the target polynucleotide sequence is a genomic sequence. In some embodiments, the target polynucleotide sequence is a human genomic sequence. In some embodiments, the target polynucleotide sequence is a mammalian genomic sequence. In some embodiments, the target polynucleotide sequence is a vertebrate genomic sequence.

[0094] Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) In some embodiments, the target polynucleotide sequence is CTLA4 or a homolog, ortholog, or variant thereof (Gene ID: 1493, also known as CD; GSE; GRD4; ALPS5; CD152; CTLA-4; IDDM12; CELIAC3). An exemplary CTLA4 human target polynucleotide sequence is shown in Table 1 below (NG_011502.1 RefSeqGene; SEQ ID NO: 782). [Table 1-1] [Table 1-2] [Table 1-3]

[0095] The CTLA4 gene is a member of the immunoglobulin superfamily, and its polynucleotide sequence encodes a protein that transmits inhibitory signals to T cells. The protein has a V domain, a transmembrane domain, and a cytoplasmic tail. Various isoforms encoded by alternative splice variants have been characterized. The membrane-bound isoform acts as a homodimer held together by disulfide bonds, whereas the soluble isoform acts as a monomer. CTLA4 gene mutations have been reported to be associated with insulin-dependent diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, celiac disease, systemic lupus erythematosus, thyroid-associated orbitopathy, and other autoimmune diseases.

[0096] In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which the CTLA4 gene on chromosome 2 has been edited using a gene editing system (e.g., TALEN, CRISPR / Cas, etc.) to reduce or eliminate CTLA4 expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or population. In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which the CTLA4 gene on chromosome 2 has been edited to delete a contiguous stretch of genomic DNA, e.g., from SEQ ID NO: 782, thereby reducing or eliminating CTLA4 expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or population. A continuous stretch of genomic DNA can be deleted by contacting primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or a population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637 (i.e., CRISPR CTLA4 gRNA pairs, e.g., at least one gRNA pair, at least two gRNA pairs, at least three gRNA pairs, at least four gRNA pairs, at least five gRNA pairs, etc.).

[0097] As used herein, the term "contacting" (e.g., contacting a polynucleotide sequence with a clustered regularly interspaced short palindromic repeats (Cas) protein and / or ribonucleic acid) is intended to include incubating the Cas protein and / or ribonucleic acid together in cells in vitro (e.g., adding a Cas protein or a nucleic acid encoding the Cas protein to cells in culture) or contacting cells ex vivo. The step of contacting the target polynucleotide sequence with the Cas protein and / or ribonucleic acid disclosed herein can be carried out in any suitable manner. For example, cells can be treated in adherent or suspension culture. It is also understood that cells contacted with the Cas protein and / or ribonucleic acid disclosed herein can be simultaneously or sequentially contacted with another agent, such as a growth factor or other differentiation agent or environment, to stabilize or further differentiate the cells.

[0098] The present disclosure contemplates reducing or eliminating CTLA4 expression and / or activity in any cell line or primary human cell (e.g., immune cell, such as T cell, natural killer cell, etc.) population to produce cells that reduce or eliminate T cell inhibition.The primary human cell used for genome editing can be obtained from a subject suffering from, being treated for, being diagnosed with, being at risk of developing, or suspected of having a disorder selected from the group consisting of autoimmune disorder, cancer, chronic infectious disease, and graft-versus-host disease (GVHD).Cells can also be obtained from normal healthy subjects that do not suffer from, are not treated for, are not diagnosed with, are not suspected of having, or do not have a high risk of developing the disorder.

[0099] The present invention contemplates genome editing of primary human cells to ablate the CTLA4 gene sequence, and editing the genome of such cells to alter one or more additional target polynucleotide sequences (e.g., PD1, TCRA, TCRB, B2M, etc.). It should be recognized that ablation of the CTLA4 gene sequence using one or more of the gRNAs or gRNA pairs described herein may result in partial or complete deletion of the CTLA4 genomic DNA sequence (e.g., SEQ ID NO: 782).

[0100] In some aspects, the invention provides modified primary human T cells, or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a first genomic modification) that reduces or eliminates CTLA4 receptor surface expression and / or activity in the cell, wherein (a) the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to delete a first contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of the flanking upstream exons and at least a portion of the flanking downstream exons, wherein the 3' end of the genomic DNA upstream of the 5' end of the deleted first contiguous stretch of genomic DNA is covalently linked to the 5' end of the genomic DNA downstream of the 3' end of the deleted first contiguous stretch of genomic DNA, resulting in a modified CTLA4 gene on chromosome 2 lacking the first contiguous stretch of genomic DNA, thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell.

[0101] Those skilled in the art will recognize that the deletion of the first continuous stretch of CTLA4 genomic DNA can be achieved using any pair of CRISPR gRNAs targeting exons in the CTLA4 gene, where the first CTLA4 gRNA targets the first exon located upstream of the second exon downstream of the first exon.Similarly, this strategy can be used to target any pair of exons, resulting in the cleavage of the first continuous stretch of genomic DNA, so that the DNA repair mechanism of the modified cell covalently binds the 3' end of the genomic DNA upstream of the CTLA4 gRNA cleavage site in the first exon to the 5' end of the genomic DNA downstream of the CTLA4 gRNA cleavage site.An example of this strategy is shown in Figure 15A and Figure 15B, using a first pair of ribonucleic acids comprising SEQ ID NO: 128 (CR1) and SEQ ID NO: 72 (CR2).In this example, the first pair of CTLA4 gRNAs is shown to target exon 2 and exon 3 of the CTLA4 gene. Of course, any two adjacent exons in the CTLA4 gene can be targeted using this strategy (e.g., exons 1 and 2, exons 3 and 4). It should be further recognized that any portion of such a CTLA4 exon containing a CTLA4 target motif can be targeted using this strategy, so long as each CTLA4 gRNA in a first pair of CTLA4 gRNAs is directed to one CTLA4 target motif in each adjacent CTLA4 exon. In other words, to achieve this strategy, one skilled in the art need only select a first CTLA4 gRNA from among SEQ ID NOS: 1-195 and 797-3637 that targets a motif in the first exon, and then select a second CTLA4 gRNA from among SEQ ID NOS: 1-195 and 797-3637 that targets a motif in a second (e.g., adjacent) exon upstream or downstream of the first exon.In this way, the first pair of gRNAs will guide the Cas protein in the cell to the first and second exons, respectively, and cleave those exons and introns (or any other sequences between them), thereby allowing the cell's DNA repair machinery to covalently bind to the genomic DNA at the two cleavage sites to create a modified cell or population thereof having a CTLA4 gene lacking the first continuous stretch of genomic DNA. In addition to or instead of targeting adjacent exons, the first pair of gRNAs can be selected to target any two exons in the CTLA4 gene (e.g., exons 1 and 3, exons 1 and 4, exons 2 and 4, etc.) such that the genomic DNA sequence between the cleavage sites in the exons is deleted and the genomic DNA sequences flanking those cleavage sites are covalently linked, resulting in a modified cell or population thereof having a CTLA4 gene lacking the first continuous stretch of genomic DNA.

[0102] Table 2 below shows the genomic sequence of each of the four exons in an exemplary human CTLA4 gene. Using the CTLA4 targeting strategy outlined herein, one skilled in the art could easily select a pair of CTLA4 gRNAs from among SEQ ID NOS: 1-195 and 797-3637 to target the exons containing SEQ ID NOS: 783-786 shown in Table 2 below, and implement such a strategy in a variety of ways. Alternatively, using the CTLA4 targeting strategy outlined herein, one skilled in the art could easily select at least one CTLA4 gRNA from among SEQ ID NOS: 3638-4046 to target the exons containing SEQ ID NOS: 783-786 shown in Table 2 below, and implement such a strategy in a variety of ways. The size of at least a portion of the upstream exon and at least a portion of the downstream exon deleted using this strategy depends on the position at which the CTLA4 gRNA directs cleavage in each respective exon. For example, the entire part of the exon downstream of the cleavage site in upstream exon and the entire part of the exon upstream of the cleavage site in downstream exon will be deleted using this strategy.Therefore, by using this strategy, by selecting the CTLA4 gRNA that targets the motif that is closest to the 5' end of upstream exon and closest to the 3' end of adjacent downstream exon, larger parts of target exon can be deleted.On the other hand, by selecting the CTLA4 gRNA that targets the motif that is farthest from the 5' end of upstream exon and farthest from the 3' end of adjacent downstream exon, smaller parts of target exon can be deleted. [Table 2-1] [Table 2-2]

[0103] In some aspects, the invention provides modified primary human T cells, or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a first genomic modification) in which (a) the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to delete a contiguous stretch of genomic DNA (e.g., a first contiguous stretch), thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cell, wherein the contiguous stretch of genomic DNA (e.g., the first contiguous stretch) has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a pair (e.g., a first pair) of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, the first pair of ribonucleic acids comprises SEQ ID NO: 128 and SEQ ID NO: 72.

[0104] In some aspects, the invention provides methods for altering a target CTLA4 polynucleotide sequence in a cell, comprising contacting the CTLA4 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids hybridize to the CTLA4 polynucleotide sequence (e.g., directing the Cas protein to a target motif in the target CTLA4 polynucleotide sequence), cleaving the target CTLA4 polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637, respectively. In some embodiments, the two ribonucleic acids comprise SEQ ID NO: 128 and SEQ ID NO: 72.

[0105] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a first genomic modification) in which (a) the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 has been edited to reduce or eliminate CTLA4 receptor surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3638-4046.

[0106] In some embodiments, the invention provides methods for altering a target CTLA4 polynucleotide sequence in a cell, comprising contacting the CTLA4 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, wherein the at least one ribonucleic acid orients the Cas protein to and hybridizes to a target motif in the target CTLA4 polynucleotide sequence, resulting in cleavage of the target CTLA4 polynucleotide sequence, and the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 3638-4046. In certain embodiments, subsequent alterations to the target CTLA4 polynucleotide sequence in the cell result in a second cleavage of the target CTLA4 polynucleotide sequence, thereby editing the target CTLA4 polynucleotide sequence to delete the first contiguous stretch of genomic DNA.

[0107] Programmed cell death 1 (PD1) In some embodiments, the target polynucleotide sequence is PD1 or a homolog, ortholog, or variant thereof (Gene ID: 5133, also known as PD-1, CD279, SLEB2, hPD-1, hPD-I, and hSLE1). An exemplary PD1 human target polynucleotide sequence is shown in Table 3 below (NG_012110.1 ReiSeqGene; SEQ ID NO: 787). [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0108] The PD1 gene encodes an immunoglobulin superfamily cell surface membrane protein that is expressed in pro-B cells and is thought to be involved in their differentiation. Injection of mice with anti-CD3 antibodies induces expression of this gene in the thymus, resulting in massive thymocyte apoptosis. The product of this gene is also important for T cell function and is thought to play a role in preventing autoimmune diseases.

[0109] In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which the PD1 gene on chromosome 2 has been edited using a gene editing system (e.g., TALEN, CRISPR / Cas, etc.) to reduce or eliminate PD1 expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or population. In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which the PD1 gene on chromosome 2 has been edited, e.g., by deleting a contiguous stretch of genomic DNA from SEQ ID NO: 787, thereby reducing or eliminating PD1 expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or population. A continuous stretch of genomic DNA can be deleted by contacting primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or a population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one pair of ribonucleic acids (i.e., CRISPR PD1 gRNA pairs, e.g., at least one gRNA pair, at least two gRNA pairs, at least three gRNA pairs, at least four gRNA pairs, at least five gRNA pairs, etc.), at least one of which is selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945.

[0110] The present disclosure contemplates reducing or eliminating PD1 expression and / or activity in any cell line or primary human cell (e.g., immune cell, such as T cell, natural killer cell, etc.) population to produce cells that reduce or eliminate T cell inhibition. The primary human cells used for genome editing can be obtained from a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of autoimmune disorders, cancer, chronic infectious diseases, and graft-versus-host disease (GVHD). Cells can also be obtained from normal healthy subjects that do not suffer from, are not treated for, are not diagnosed with, are not suspected of, or are not at high risk of developing the disorder.

[0111] The present invention contemplates genome editing of primary human cells to ablate the PD1 gene sequence, and editing the genome of such cells to alter one or more additional target polynucleotide sequences (e.g., CTLA4, TCRA, TCRB, B2M, etc.). It should be recognized that ablation of the PD1 gene sequence using one or more gRNAs or gRNA pairs described herein may result in partial or complete deletion of the PD1 genomic DNA sequence (e.g., SEQ ID NO: 787).

[0112] In some aspects, the invention provides modified primary human T cells, or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a second genomic modification) that reduces or eliminates PD1 receptor surface expression and / or activity in the cell, wherein (b) the PD1 gene on chromosome 2 has been edited to delete a second contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of the flanking upstream exon and at least a portion of the flanking downstream exon, wherein the 3' end of the genomic DNA upstream of the 5' end of the deleted second contiguous stretch of genomic DNA is covalently linked to the 5' end of the genomic DNA downstream of the 3' end of the deleted second contiguous stretch of genomic DNA, resulting in a modified PD1 gene on chromosome 2 that lacks the second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell.

[0113] Those skilled in the art will recognize that the deletion of the second continuous stretch of PD1 genomic DNA can be achieved using any pair of CRISPR gRNAs targeting exons in the PD1 gene, where the first PD1 gRNA targets a first exon located upstream of a second exon downstream of the first exon.Similarly, this strategy can be used to target any pair of exons, resulting in the cleavage of the second continuous stretch of genomic DNA, so that the DNA repair mechanism of the modified cell covalently binds the 3' end of the genomic DNA upstream of the PD1 gRNA cleavage site in the first exon to the 5' end of the genomic DNA downstream of the PD1 gRNA cleavage site.An example of this strategy is shown in Figure 13A and Figure 13B, using a first pair of ribonucleic acids comprising SEQ ID NO: 462 (CR1) and SEQ ID NO: 421 (CR2).In this example, the first pair of PD1 gRNAs is shown to target exon 2 and exon 3 of the PD1 gene. Of course, any two adjacent exons in the PD1 gene can be targeted using this strategy (e.g., exons 1 and 2, exons 3 and 4, exons 4 and 5). It should be further recognized that any portion of such a PD1 exon containing a CRISPR gRNA PD1 target motif can be targeted using this strategy, as long as each PD1 gRNA of the first pair of gRNAs is directed to one CRISPR gRNA PD1 target motif in each adjacent PD1 exon. In other words, to achieve this strategy, one skilled in the art simply selects a first PD1 gRNA from among SEQ ID NOs: 196-531 and 4047-8945 that targets a motif in the first exon, and then selects a second PD1 gRNA from among SEQ ID NOs: 196-531 and 4047-8945 that targets a motif in a second (e.g., adjacent) exon upstream or downstream of the first exon. In this way, the first pair of gRNAs will guide the Cas protein in the cell to the first and second exons, respectively, and cleave those exons and introns (or any other sequences between them), thereby allowing the cell's DNA repair machinery to covalently bind to the genomic DNA at the two cleavage sites to create a modified cell or population thereof having a PD1 gene lacking the second continuous stretch of genomic DNA. In addition to or instead of targeting adjacent exons, the first pair of gRNAs can be selected to target any two exons in the PD1 gene (e.g., exons 1 and 3, exons 1 and 4, exons 1 and 5, exons 2 and 4, exons 2 and 5, etc.) such that the genomic DNA sequence between the cleavage sites in the exons is deleted and the genomic DNA sequences flanking those cleavage sites are covalently linked, resulting in a modified cell or population thereof having a PD1 gene lacking the second continuous stretch of genomic DNA.

[0114] Table 4 below shows the genomic sequence of each of the five exons in an exemplary human PD1 gene. Those skilled in the art can easily select a pair of PD1 gRNAs from among SEQ ID NOS: 196-531 and 4047-8945 to target the exons containing SEQ ID NOS: 788-792 shown in Table 4 below, using the PD1 targeting strategy outlined herein, and implement such a strategy in a variety of ways. Alternatively, those skilled in the art can easily select at least one PD1 gRNA from among SEQ ID NOS: 8946-9101 to target the exons containing SEQ ID NOS: 788-792 shown in Table 4 below, and implement such a strategy in a variety of ways, using the PD1 targeting strategy outlined herein. The size of at least a portion of the upstream exon and at least a portion of the downstream exon deleted using this strategy depends on the position at which the PD1 gRNA directs cleavage in each respective exon. For example, the entire part of the exon downstream of the cleavage site in the upstream exon and the entire part of the exon upstream of the cleavage site in the downstream exon will be deleted using this strategy.Therefore, by using this strategy, by selecting the PD1 gRNA that targets the motif that is closest to the 5' end of the upstream exon and closest to the 3' end of the adjacent downstream exon, larger parts of the target exon can be deleted.On the other hand, by selecting the PD1 gRNA that targets the motif that is farthest from the 5' end of the upstream exon and farthest from the 3' end of the adjacent downstream exon, smaller parts of the target exon can be deleted. [Table 4-1] [Table 4-2]

[0115] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a second genomic modification) in which (b) the PD1 gene on chromosome 2 has been edited to delete a contiguous stretch of genomic DNA (e.g., a second contiguous stretch), thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell, wherein the contiguous stretch of genomic DNA (e.g., the second contiguous stretch) has been deleted by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein and a pair (e.g., a second pair) of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, the second pair of ribonucleic acids comprises SEQ ID NO: 462 and SEQ ID NO: 421.

[0116] In some aspects, the invention provides methods for altering a target PD1 polynucleotide sequence in a cell, the method comprising contacting the PD1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize to a target motif in the target PD1 polynucleotide sequence, cleaving the target PD1 polynucleotide sequence, and at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945, respectively. In some embodiments, the two ribonucleic acids comprise SEQ ID NO: 462 and SEQ ID NO: 421.

[0117] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a second genomic modification) in which (b) the PD1 gene on chromosome 2 has been edited to reduce or eliminate PD1 receptor surface expression and / or activity in the cell by contacting the cell with a Cas protein or a nucleic acid encoding the Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 8946-9101.

[0118] In some embodiments, the invention provides methods for altering a target PD1 polynucleotide sequence in a cell, comprising contacting the PD1 polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, wherein the at least one ribonucleic acid orients the Cas protein to and hybridizes to a target motif in the target PD1 polynucleotide sequence, resulting in cleavage of the target PD1 polynucleotide sequence, and the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 8946-9101. In certain embodiments, subsequent alterations to the target PD1 polynucleotide sequence in the cell result in a second cleavage of the target PD1 polynucleotide sequence, thereby editing the target PD1 polynucleotide sequence to delete a second contiguous stretch of genomic DNA.

[0119] T cell receptor alpha chain (TCRA) and T cell receptor beta chain (TCRB) gene loci In some embodiments, the target polynucleotide sequence is the T cell receptor alpha locus (TCRA) or a homolog, ortholog, or variant thereof (Gene ID: 5133, also known as IMD7, TCRD, TRA@, TRAC, and referred to herein as TCRa, TCRA, TCRα, etc.). An exemplary TCRA, human target polynucleotide sequence is NCBI Reference Sequence: NC_000014.9. In some embodiments, the target polynucleotide is the T cell receptor alpha locus (TCRB) or a homolog, ortholog, or variant thereof (Gene ID: 6957, TCRB; also known as TRB@, and referred to herein as TCRb, TCRB, TCRβ, etc.). Antigen recognition by T-lymphocytes occurs through a mechanism similar to that using immunoglobulins made by B cells. Two major mature T cell subtypes exist (i.e., those expressing α and β chains, and those expressing γ and δ chains). In contrast to secretory Ig molecules, T cell receptor chains are membrane-bound and function through cell-cell contact. The genes encoding the T cell receptor α chain are clustered on chromosome 14.

[0120] T cell receptor α chains are formed when one of at least 70 variable (V) genes encoding N-terminal antigen recognition domains rearranges with one of 61 joining (J) gene segments to form functional V region exons, which are transcribed and spliced ​​into constant region (TRAC) gene segments encoding the C-terminal portion. T cell receptor β chains are formed when one of 52 variable (V) genes encoding N-terminal antigen recognition domains rearranges with diversity (D) and joining (J) genes to form functional V region exons, which are transcribed and spliced ​​into constant region (C) gene segments encoding the C-terminal portion. In contrast to the α chain locus, the β chain locus has two separate gene clusters after the V gene, each containing a D gene, several J genes, and a C gene. After synthesis, the α and β chains combine to generate the α-β T cell receptor heterodimer (Janeway et al., 2005).

[0121] In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which a TCR alpha chain locus on chromosome 14 has been edited using a gene editing system (e.g., TALEN, CRISPR / Cas, etc.) to reduce or eliminate TCR expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or populations thereof. In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which a TCR alpha chain locus on chromosome 14 has been edited, e.g., by deleting a contiguous stretch of genomic DNA comprising a coding exon, thereby reducing or eliminating TCR expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or populations thereof. A continuous stretch of genomic DNA can be deleted by contacting primary human cells (e.g., immune cells, such as T cells, natural killer cells, etc.) or a population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750 (i.e., CRISPR TCRA gRNA pairs, e.g., at least one gRNA pair, at least two gRNA pairs, at least three gRNA pairs, at least four gRNA pairs, at least five gRNA pairs, etc.).

[0122] In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which a TCR beta chain locus on chromosome 7 has been edited using a gene editing system (e.g., TALEN, CRISPR / Cas, etc.) to reduce or eliminate TCR expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or populations thereof. In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which a TCR beta chain locus on chromosome 7 has been edited, e.g., by deleting a contiguous stretch of genomic DNA comprising a coding exon, thereby reducing or eliminating TCR expression (e.g., cell surface expression) and / or activity (e.g., protein activity) in the cells or populations thereof. A continuous stretch of genomic DNA can be deleted by contacting primary human cells (e.g., immune cells, such as T cells, natural killer cells, etc.) or a population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532 (i.e., CRISPR TCRA gRNA pairs, e.g., at least one gRNA pair, at least two gRNA pairs, at least three gRNA pairs, at least four gRNA pairs, at least five gRNA pairs, etc.).

[0123] The present disclosure contemplates reducing or eliminating TCR expression and / or activity in any cell line or population of primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) to produce cells that reduce or eliminate autoreactivity. The present disclosure further contemplates genome editing of primary human cells to ablate TCR alpha and / or beta chain locus sequences, as well as editing the genome of such cells to alter one or more additional target polynucleotide sequences (e.g., CTLA4, PD1, and / or B2M). It should be recognized that ablation of TCR alpha and / or beta chain locus sequences using one or more gRNAs or gRNA pairs and Cas proteins described herein can result in partial or complete deletion of the target TCR alpha and / or beta chain locus DNA sequence (e.g., a coding exon, e.g., the first coding exon).

[0124] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising: (c)(i) a genomic modification (e.g., a third genomic modification) in which a TCR alpha chain locus on chromosome 14 has been edited to delete a contiguous stretch of genomic DNA (e.g., a third contiguous stretch) that comprises at least a portion of a coding exon; and / or (c)(ii) a genomic modification (e.g., a fourth genomic modification) in which a TCR beta chain locus on chromosome 7 has been edited to delete a contiguous stretch of genomic DNA (e.g., a fourth contiguous stretch) that comprises at least a portion of a coding exon; wherein the deletion of the contiguous stretch of genomic DNA from the TCR alpha chain locus on chromosome 14 and / or the deletion of the contiguous stretch of genomic DNA from the TCR beta chain locus on chromosome 7 reduces or eliminates TCR surface expression and / or TCR activity in the cell or population thereof.

[0125] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising (c)(i) a genomic modification (e.g., a third genomic modification) in which the TCR α chain locus on chromosome 14 has been edited to delete a contiguous stretch of genomic DNA (e.g., a third contiguous stretch), thereby reducing or eliminating TCR surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA (e.g., the third contiguous stretch) has been deleted by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a pair (e.g., a third pair) of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, the third pair of ribonucleic acids comprises SEQ ID NO: 550 and SEQ ID NO: 573.

[0126] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a fourth genomic modification) in which (c)(ii) the TCR β chain locus on chromosome 7 has been edited to delete a contiguous stretch of genomic DNA (e.g., a fourth contiguous stretch), thereby reducing or eliminating TCR surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA (e.g., the fourth contiguous stretch) has been deleted by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a pair (e.g., a fourth pair) of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, the fourth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778.

[0127] In some aspects, the invention provides methods for altering a target TCRA polynucleotide sequence in a cell, comprising contacting the TCRA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize to a target motif in the target TCRA polynucleotide sequence, cleaving the target TCRA polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750, respectively. In some embodiments, the two ribonucleic acids comprise SEQ ID NO: 550 and SEQ ID NO: 573.

[0128] In some aspects, the invention provides methods for altering a target TCRB polynucleotide sequence in a cell, the method comprising contacting the TCRB polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize to a target motif in the target TCRB polynucleotide sequence, cleaving the target TCRB polynucleotide sequence, and at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10321. In some embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10321, respectively. In some embodiments, the two ribonucleic acids comprise SEQ ID NOs: 657 and 662.

[0129] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising (c)(i) a genomic modification (e.g., a third genomic modification) in which the TCR alpha chain locus on chromosome 14 has been edited to reduce or eliminate TCR surface expression and / or activity in the cell. In some embodiments, the third genomic modification occurs by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 9751-9797.

[0130] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising (c)(ii) a genomic modification (e.g., a fourth genomic modification) in which the TCR β chain locus on chromosome 7 has been edited to reduce or eliminate TCR surface expression and / or activity in the cell. In some embodiments, the fourth genomic modification occurs by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein, and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 10533-10573.

[0131] In some embodiments, the invention provides methods for altering a target TRCA polynucleotide sequence in a cell, comprising contacting the TCRA polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, wherein the at least one ribonucleic acid orients the Cas protein to and hybridizes to a target motif in the target TCRA polynucleotide sequence, resulting in cleavage of the target TCRA polynucleotide sequence, and the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 9751-9797. In certain embodiments, subsequent alterations to the target TRCA polynucleotide sequence in the cell result in a second cleavage of the target TRCA polynucleotide sequence, thereby editing the target TRCA polynucleotide sequence to delete a third contiguous stretch of genomic DNA.

[0132] In some embodiments, the invention provides methods for altering a target TRCB polynucleotide sequence in a cell, comprising contacting the TCRB polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, wherein the at least one ribonucleic acid orients the Cas protein to and hybridizes to a target motif in the target TCRB polynucleotide sequence, resulting in cleavage of the target TCRB polynucleotide sequence, and the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 10533-10573. In certain embodiments, subsequent alterations to the target TRCB polynucleotide sequence in the cell result in a second cleavage of the target TRCB polynucleotide sequence, thereby editing the target TRCB polynucleotide sequence to delete a fourth contiguous stretch of genomic DNA.

[0133] β2 microglobulin (B2M) In some embodiments, the target polynucleotide sequence is β2-microglobulin (B2M; Gene ID: 567). The B2M polynucleotide sequence encodes a serum protein associated with the heavy chain of major histocompatibility complex (MHC) class I molecules, which are expressed on the surface of virtually all nucleated cells. The B2M protein contains a β-pleated sheet structure that has been found to form amyloid fibrils in certain pathological conditions. The B2M gene has four exons spanning approximately 8 kb. B2M has been observed in the serum of normal individuals and in high amounts in the urine of patients with Wilson's disease, cadmium poisoning, and various conditions leading to renal tubular dysfunction. Other pathological conditions known to be associated with B2M include, but are not limited to, homozygous mutations in the B2M gene (e.g., ala11pro), which have been reported in individuals with familial hypercatabolic hypoproteinemia, and heterozygous mutations in the B2M gene (e.g., asp76asn), which have been reported in individuals with familial visceral amyloidosis.

[0134] In some embodiments, the target polynucleotide sequence is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.

[0135] In some aspects, the disclosure provides modified primary human cells (e.g., immune cells, e.g., T cells, natural killer cells, etc.) or populations thereof comprising a genome in which the beta2-microglobulin (B2M) gene on chromosome 15 has been edited using a gene editing system (e.g., TALEN, CRISPR / Cas, etc.) to reduce or eliminate surface expression of MHC class I molecules in the cells or populations thereof. In some aspects, the disclosure provides modified primary human T cells or populations thereof comprising a genome in which the B2M gene on chromosome 15 has been edited to delete a contiguous stretch of genomic DNA of NCBI Reference Sequence: NG__012920.1, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof. A continuous stretch of genomic DNA can be deleted by contacting a cell or population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one ribonucleic acid or at least one pair of ribonucleic acids selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13719.

[0136] The present disclosure contemplates removing MHC class I molecule surface expression in any cell line or primary human cell population (e.g., immune cells, such as T cells, natural killer cells, etc.) to produce cells that reduce or eliminate the likelihood of triggering an undesirable host immune response when transplanted (e.g., allogeneic transplant). B2M is an accessory chain of MHC class I protein and is required for expression of MHC class I protein on the cell surface. It is believed that artificial cells (e.g., mutant cells) lacking surface MHC class I may reduce the likelihood of the artificial cells being detected by cytotoxic T cells when administered to a host. Thus, in some embodiments, truncation of a target polynucleotide sequence encoding BM2 in a cell or cell population reduces the likelihood of the resulting cell or cells triggering a host immune response when administered to a subject.

[0137] The present invention contemplates genome editing of primary human cells to cleave the B2M gene sequence, and editing the genome of such cells to alter one or more additional target polynucleotide sequences (e.g., CTLA4, PD1, TCRA, and / or TCRB). It should be recognized that cleavage of the B2M genomic sequence using one or more gRNAs or gRNA pairs and Cas proteins described herein may result in partial or complete deletion of the targeted B2M genomic sequence.

[0138] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome comprising a genomic modification (e.g., a fifth genomic modification) in which (d) the B2M gene on chromosome 15 has been edited to delete a contiguous stretch of genomic DNA (e.g., a fifth contiguous stretch), thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the contiguous stretch of genomic DNA (e.g., the fifth contiguous stretch) is deleted by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a pair (e.g., a fifth pair) of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, the fifth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778.

[0139] In some aspects, the invention provides methods for altering a target B2M polynucleotide sequence in a cell, comprising contacting the B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and one to two ribonucleic acids, wherein the ribonucleic acids orient the Cas protein to and hybridize to a target motif in the target B2M polynucleotide sequence, cleaving the target B2M polynucleotide sequence, and wherein at least one of the one to two ribonucleic acids is selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, the one to two ribonucleic acids are selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257, respectively. In some embodiments, the fifth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778.

[0140] In some aspects, the invention provides modified primary human T cells or populations thereof, wherein each cell comprises a modified genome including (d) a genomic modification (e.g., a fifth genomic modification) in which the B2M gene on chromosome 15 has been edited to reduce or eliminate MHC class I molecule surface expression and / or activity in the cell. In some embodiments, the fifth genomic modification occurs by contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and at least one ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 13258-13719.

[0141] In some embodiments, the invention provides methods for altering a target B2M polynucleotide sequence in a cell, the method comprising contacting the B2M polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein and at least one ribonucleic acid, wherein the at least one ribonucleic acid orients the Cas protein to and hybridizes to a target motif in the target B2M polynucleotide sequence, resulting in cleavage of the target B2M polynucleotide sequence, and the ribonucleic acid is selected from the group consisting of SEQ ID NOs: 13258-13719. In certain embodiments, subsequent alterations to the target B2M polynucleotide sequence in the cell result in a second cleavage of the target B2M polynucleotide sequence, thereby editing the target B2M polynucleotide sequence to delete a fifth contiguous stretch of genomic DNA.

[0142] Chimeric Antigen Receptor An embodiment of the present invention relates to primary human cells modified to contain chimeric antigen receptors. Chimeric antigen receptors (CARs) are molecules designed to target immune cells to specific molecular targets expressed on the cell surface. In their most basic form, they are receptors introduced into cells that couple a specificity domain expressed on the outside of the cell to a signaling pathway inside the cell, so that the cell is activated when the specificity domain interacts with its target. CARs are often made from variants of T cell receptors (TCRs) in which a specificity domain, such as an scFv or some types of receptor, is fused to the signaling domain of the TCR. These constructs are then introduced into T cells, and when the T cells are activated in the presence of cells expressing the target antigen, the activated T cells attack the target cells in an MHC-independent manner (see Chicaybam et al. (2011) Int Rev Immunol 30:294-311). Tumor-specific CARs targeting various tumor antigens are currently being tested in clinical trials for the treatment of a variety of different cancers. Examples of these cancers and their targeting antigens include follicular lymphoma (CD20 or GD2), neuroblastoma (CD171), non-Hodgkin's lymphoma (CD20), lymphoma (CD19), glioblastoma (IL13Rα2), chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL) (both CD19).Virus-specific CARs have also been developed to attack cells carrying viruses such as HIV.For example, clinical trials have begun using CARs specific to Gp100 for the treatment of HIV (Chicaybam, supra).

[0143] As used herein, a "chimeric antigen receptor" (CAR) is an artificially constructed hybrid protein or polypeptide containing a specificity or recognition (i.e., binding) domain linked to an immune receptor involved in signal transduction in lymphocytes. Typically, the binding domain is derived from a Fab antibody fragment that has been converted into a single-chain scFv via the introduction of a flexible linker between the antibody chains within the specificity domain. Other possible specificity domains include the signaling portion of a hormone or cytokine molecule, the extracellular domain of a receptor, and peptide ligands or peptides isolated by library (e.g., phage) screening (see Ramos and Dotti, (2011) Expert Opin Bio Ther 11(7):855). Because flexibility between the signaling and binding portions of a CAR can be a desirable feature for enabling more optimal interactions between the targeting and binding domains, a hinge region is often included. One example of a structure that can be used is the CH2-CH3 region from an immunoglobulin, such as an IgG molecule. A typical CAR signaling domain includes the intracellular domain of the TCR-CD3 complex, such as the ζ chain. Alternatively, the γ chain of an Fc receptor can be used. The transmembrane portion of a typical CAR can include the transmembrane portion of a protein such as CD4, CD8, or CD28 (Ramos and Dotti, supra). Some CAR features include the ability to redirect T cell specificity and reactivity to a selected target in an MHC-independent manner. Non-MHC-dependent target recognition confers on CAR-expressing T cells the ability to recognize targets independently of antigen processing, thereby circumventing a major mechanism of tumor escape.

[0144] At least three different generations of chimeric antigen receptors contemplated for use in the engineered cells, compositions, methods, and kits of the present invention are shown in Figure 2. So-called "first-generation" CARs often contain a single internal signaling domain, such as the CD3ζ chain, and are considered somewhat ineffective in clinical settings, likely due to incomplete activation. To increase the performance of T cells bearing these CARs, second-generation CARs have been created that have the ability to provide additional T cell activation signals by including additional stimulatory domains, often derived from the intracellular domains of other receptors, such as CD28, CD134 / OX40, CD137 / 4-1BB, Lck, ICOS, and DAP10. In addition, third-generation CARs have also been developed in which the CAR contains three or more stimulatory domains (Ramos and Dotti, supra). In some cases, the CAR may contain an extracellular hinge domain, a transmembrane domain, and optionally an intracellular hinge domain containing CD8, and an intracellular T cell receptor signaling domain containing CD28, 4-1BB, and CD3ζ. CD28 is a T cell marker important in T cell costimulation. CD8 is also a T cell marker. 4-1BB transmits a strong costimulatory signal to T cells, promoting differentiation and enhancing the long-term survival of T lymphocytes. CD3ζ associates with TCR to generate signals and contains an immunoreceptor tyrosine-based activation motif (ITAM). In other cases, CARs may include an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain comprising CD28 and CD3ζ. In further cases, CARs may include an extracellular hinge domain, a transmembrane domain comprising CD8, and an intracellular T cell receptor signaling domain comprising CD28 and CD3ζ.

[0145] In some embodiments, the modified primary human cells (e.g., immune cells, such as T cells, natural killer cells, etc.) further comprise a chimeric antigen receptor or an exogenous nucleic acid encoding the chimeric antigen receptor. The chimeric antigen receptor specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof. Numerous cancer antigens are known in the art and can be targeted by specific CARs. For non-limiting examples, see Table 5 for tumor-associated antigens that can be targeted by CARs (see Ramos and Dotti, supra, and Orentas et al., (2012), Front in Oncol 2:1). [Table 5-1] [Table 5-2]

[0146] In some embodiments, the CAR can have specificity for a tumor antigen, and the specificity domain of the CAR is an scFv. In other embodiments, the CAR can be specific for a tumor antigen, and the specificity domain of the CAR comprises a ligand or polypeptide. Non-limiting exemplary CARs include CD33 (see Dutour et al., (2012) Adv Hematol 2012;2012:683065), GD2 (Louis et al., (2011) Blood 118(23):650-6), CD 19 (Savoldo et al., (2011) J Clin Invest 121(5):1822 and Torikai et al., (2012) Blood 119(24):5697), IL-11Rα (Huang et al., (2012) Cancer Res 72(1):271-81), CD20 (Till et al., (2012) Blood 119(17):3940-50), NY-ESO-1 (Schuberth et al., (2012) Gene Ther doi:10.1038 / gt2012.48), ErbB2 (Zhao et al., (2009) J Immunol 183(9):5563-74), CD70 (Shaffer et al., (2011) Blood 116(16):4304-4314), CD38 (Bhattacharayya et al., (2012) Blood Canc J2(6)p.e75), CD22 (Haso et al., (2012) Canc Res 72(8)S1,doi:1158 / 1158-7445 AM2012-3504), CD74 (Stein et al., (2004) Blood 104:3705-3711), CAIX (Lamers et al., (2011) Blood 117(1):72-82), STEAP1 (for a review of targets, see Kiessling et al., (2012) Cancer Res 4:193-217), including those targeted to VEGF-R2 (U.S. Patent Application Publication No. 20120213783), folate receptor (WO 2012099973), and IL-13 Ra (U.S. Patent No. 7,514,537).

[0147] Exogenous molecules delivered via engineered cells Embodiments of the present invention relate to using the modified primary human cells or populations thereof (e.g., immune cells, e.g., T cells, natural killer cells, etc.) of the present invention to deliver exogenous molecules to cells, tissues, or organs to modulate a desired biological activity / effect in the cells, tissues, or organs. For example, the modified primary human cells or populations thereof can be used to deliver therapeutic products (e.g., immunomodulatory cytokines or antagonists thereof to mediate autoimmune activity versus anti-inflammatory Th2-type responses, see, e.g., Johnson and Tuohy, "Targeting Antigen-Specific T Cells for Gene Therapy of Autoimmune Disease," Madame Curie, 1999). As further described in the Bioscience Database, engineered T cells can be engineered to deliver regenerative products (e.g., for repairing damaged tissue, generating new or artificial tissue, or both, such as the use of engineered T cells to deliver nerve growth factor (NGF) to the central nervous system (CNS), platelet-derived growth factor-A (PDGF-A) to treat experimental autoimmune encephalomyelitis (EAE)) to sites of inflammation and tissue destruction, modulate cellular interactions (e.g., modulation of cell-cell interactions, e.g., modulation of signal transduction pathways, apoptosis induction, arrest epitope spreading, tolerance induction, tolerance reversal, and specificity programming, etc.), or correct their own genetic defects to ameliorate disease (e.g., autoimmune disease).

[0148] An "exogenous" molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical, or other methods. "Normal presence in a cell" is determined with respect to the cell's particular developmental stage and environmental conditions. Thus, for example, a molecule that is only present during embryonic development of a neuron is an exogenous molecule with respect to an adult neuron cell. An exogenous molecule can include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0149] Exogenous molecules can be, inter alia, small molecules, such as those produced by combinatorial chemical processes, or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, and can be single-stranded or double-stranded; linear, branched, or circular; and can be of any length. Nucleic acids include those that can form duplexes and triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0150] Exogenous molecules can be the same type of molecules as endogenous molecules, such as exogenous proteins or nucleic acids.In this case, exogenous molecules are introduced into cells at a higher concentration than the endogenous molecules in cells.In some cases, exogenous nucleic acids can include infectious virus genomes, plasmids or episomes introduced into cells, or chromosomes that are not normally present in cells.Methods for introducing exogenous molecules into cells are known to those skilled in the art, and include but are not limited to lipid-mediated transfer (i.e., liposomes containing neutral lipids and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer.

[0151] In some embodiments, the exogenous molecule comprises a fusion molecule (e.g., a fusion protein or a nucleic acid). A "fusion" molecule is a molecule in which two or more subunit molecules are preferably covalently linked. The subunit molecules can be molecules of the same chemical type or molecules of different chemical types. Examples of the first type of fusion molecule include, but are not limited to, a fusion protein (e.g., a fusion between a CRISPR DNA binding domain and a cleavage domain); a fusion nucleic acid (e.g., a nucleic acid encoding the fusion protein), and a fusion between a nucleic acid and a protein (e.g., a CRISPR / Cas nuclease system). Examples of the second type of fusion molecule include, but are not limited to, a fusion between a triplex-forming nucleic acid and a polypeptide, and a fusion between a minor groove binder and a nucleic acid.

[0152] Expression of a fusion molecule in a cell can occur by delivering the fusion molecule to the cell, for example, in the case of a fusion protein, by delivering the fusion protein to the cell, or by delivering a polynucleotide encoding the fusion protein to the cell, where the polynucleotide is transcribed and the transcript is translated to produce the fusion protein. Trans-splicing, polypeptide cleavage, and polypeptide ligation can also be involved in the expression of a protein in a cell. Methods for delivering polynucleotides and / or polypeptides to cells are provided elsewhere in this disclosure.

[0153] For purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product (see below) and all DNA regions that regulate the production of the gene product (whether or not such regulatory sequences flank the coding and / or transcribed sequence). Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0154] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0155] "Modulation" of gene expression refers to a change in the expression level of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Modulation can also be complete (i.e., gene expression is completely inactivated or activated to wild-type levels or above); or it can be partial, where gene expression is partially reduced or partially activated to a fraction of wild-type levels. "Eukaryotic" cells include, but are not limited to, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, and human cells (e.g., T cells).

[0156] The terms "operably linked" and "operably linked" (or "operably linked") are used interchangeably in reference to the juxtaposition of two or more components (e.g., sequence elements) such that the components are positioned to allow both components to function normally and at least one of the components to mediate a function exerted by at least one of the other components. Illustratively, a transcriptional regulatory sequence such as a promoter is operably linked to a coding sequence if it controls the level of transcription of the coding sequence depending on the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence operably linked to a coding sequence, even if the enhancer and coding sequence are not contiguous.

[0157] With respect to fusion polypeptides, the term "operably linked" can refer to the fact that each component, when linked to another component, performs the same function as if it were not so linked. For example, with respect to a fusion polypeptide in which a Cas DNA-binding domain is fused to a cleavage domain, the DNA-binding domain and the cleavage domain are operably linked if, in the fusion polypeptide, the DNA-binding domain portion is capable of binding to its target site and / or its binding site, and the cleavage domain is capable of cleaving DNA near the target site. Similarly, with respect to a fusion polypeptide in which a Cas DNA-binding domain is fused to an activation or repression domain, the DNA-binding domain and the activation or repression domain are operably linked if, in the fusion polypeptide, the DNA-binding domain portion is capable of binding to its target site and / or its binding site, and the activation domain is capable of upregulating gene expression, or the repression domain is capable of downregulating gene expression.

[0158] A "functional fragment" of a protein, polypeptide, or nucleic acid is a protein, polypeptide, or nucleic acid whose sequence is not identical to that of the full-length protein, polypeptide, or nucleic acid, but which retains the same function as the full-length protein, polypeptide, or nucleic acid. A functional fragment may have more, fewer, or the same number of residues as the corresponding native molecule, and / or may contain one or more amino acid or nucleotide substitutions. Methods for determining the function of a nucleic acid (e.g., coding function, ability to hybridize to another nucleic acid) are well known in the art. Similarly, methods for determining the function of a protein are well known. For example, the DNA binding function of a polypeptide can be determined, for example, by filter binding, electrophoretic mobility shift, or immunoprecipitation assays. DNA cleavage can be assayed by gel electrophoresis. See Ausubel et al., supra. The ability of a protein to interact with another protein can be determined, for example, by co-immunoprecipitation, two-hybrid assays, or both genetic and biochemical methods. See, e.g., Fields et al. (1989) Nature 340:245-246; U.S. Patent No. 5,585,245 and WO 98 / 44350.

[0159] A "vector" is capable of transferring a gene sequence into a target cell. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and transferring the gene sequence into a target cell. Thus, the term includes cloning and expression vehicles as well as integrating vectors.

[0160] A "reporter gene" or "reporter sequence" refers to any sequence that preferably, but not necessarily, produces a protein product that is easily measured in a routine assay. Suitable reporter genes include, but are not limited to, sequences encoding proteins that mediate antibiotic resistance (e.g., ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), colored or fluorescent or luminescent proteins (e.g., green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), and proteins that mediate enhanced cell growth and / or gene amplification (e.g., dihydrofolate reductase). Epitope tags include, for example, one or more copies of FLAG, His, myc, Tap, HA, or any detectable amino acid sequence. "Expression tags" include sequences encoding reporters that can be operably linked to a desired gene sequence to monitor the expression of the gene of interest.

[0161] In some embodiments, the modified primary human cells or population thereof further comprise at least one exogenous protein or exogenous nucleic acid encoding said protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ.

[0162] Methods for producing modified cells Aspects of the present invention relate to methods for producing modified primary human cells (e.g., immune cells, such as T cells, natural killer cells, etc.). In some embodiments, the method for producing modified primary human T cells or populations thereof comprises (a) editing the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 in primary human T cells or populations thereof to delete a contiguous stretch of genomic DNA (e.g., a first contiguous stretch), thereby reducing or eliminating CTLA4 receptor surface expression and / or activity in the cells or populations thereof.

[0163] In some embodiments, the method for producing an engineered primary human T cell or population thereof comprises (b) editing the programmed cell death 1 (PD1) gene on chromosome 2 in the cell or population thereof to delete a contiguous stretch of genomic DNA (e.g., a second contiguous stretch), thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell or population thereof.

[0164] In some embodiments, a method for producing an engineered primary human T cell or population thereof comprises (c)(i) editing a gene encoding a T cell receptor (TCR) alpha chain locus on chromosome 14 in the cell or population thereof to delete a contiguous stretch of genomic DNA (e.g., a third contiguous stretch), and / or (c)(ii) editing a gene encoding a TCR beta chain locus on chromosome 7 in the cell or population thereof to delete a contiguous stretch of genomic DNA (e.g., a fourth contiguous stretch), thereby reducing or eliminating TCR surface expression and / or activity in the cell or population thereof.

[0165] In some embodiments, the method for producing an engineered primary human T cell or population thereof comprises (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell or population thereof to delete a continuous stretch of genomic DNA (e.g., the fifth continuous stretch), thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell or population thereof.

[0166] In some embodiments, the editing step in (a) to (d) comprises combining the cell or population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein, and at least one pair (e.g., a first pair) of guide RNA sequences for deleting a contiguous stretch (e.g., a first contiguous stretch) of genomic DNA from the gene in (a), at least one pair (e.g., a second pair) of guide RNA sequences for deleting a contiguous stretch (e.g., a second contiguous stretch) of genomic DNA from the gene in (b), and at least one pair (e.g., a second pair) of guide RNA sequences for deleting a contiguous stretch (e.g., a second contiguous stretch) of genomic DNA from the gene in (c)(i). (c)(ii) with at least one pair of guide RNA sequences (e.g., a third pair) for deleting a contiguous stretch of genomic DNA (e.g., a third contiguous stretch), and / or (c)(ii) with at least one pair of guide RNA sequences (e.g., a fourth pair) for deleting a contiguous stretch of genomic DNA (e.g., a fourth contiguous stretch) from the gene in (c)(ii), and (d) with at least one pair of guide RNA sequences (e.g., a fifth pair) for deleting a contiguous stretch of genomic DNA (e.g., a fifth contiguous stretch) from the gene in (d).

[0167] In some embodiments, methods for producing modified primary human T cells or populations thereof optionally include (e)(i) causing the cells or populations thereof to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof, and / or (e)(ii) causing the cells or populations thereof to express at least one protein that modulates a biological effect of interest in an adjacent cell, tissue, or organ.

[0168] In some aspects, the invention provides a method for producing a modified primary human T cell or population thereof, comprising: (a) editing a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 in the primary human T cell or population thereof to delete a first contiguous stretch of genomic DNA comprising an intron flanked by at least a portion of an adjacent upstream exon and at least a portion of an adjacent downstream exon, and covalently linking a 3' end of the genomic DNA upstream of the 5' end of the deleted first contiguous stretch of genomic DNA to a 5' end of the genomic DNA downstream of the 3' end of the deleted first contiguous stretch of genomic DNA, thereby generating a modified CTLA4 gene on chromosome 2 lacking the first contiguous stretch of genomic DNA, thereby generating a CTLA4 receptor surface region in the cell or population thereof; and / or (b) editing the programmed cell death 1 (PD1) gene on chromosome 2 in a primary human T cell or population thereof to delete a second contiguous stretch of genomic DNA comprising an intron adjacent to at least a portion of the adjacent upstream exon and at least a portion of the adjacent downstream exon, and covalently linking the 3' end of the genomic DNA upstream of the deleted second contiguous stretch of genomic DNA to the 5' end of the genomic DNA downstream of the 3' end of the deleted second contiguous stretch of genomic DNA, thereby generating a modified PD1 gene on chromosome 2 lacking the second contiguous stretch of genomic DNA, thereby reducing or eliminating PD1 receptor surface expression and / or activity in the cell or population thereof.

[0169] In some embodiments, the present invention provides a method for producing modified primary human T cells or a population thereof, comprising: (a) contacting primary human T cells or a population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637, thereby deleting a first contiguous stretch of genomic DNA from the cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 and altering CTLA4 receptor surface expression and / or activity in the cells or population thereof; and / or (b) contacting the primary human T cell or population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a second pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945, thereby editing the programmed cell death 1 (PD1) gene on chromosome 2 to delete a second contiguous stretch of genomic DNA and reduce or eliminate PD1 receptor surface expression and / or activity in the cell or population thereof.

[0170] In some embodiments, the first pair of ribonucleic acids comprises SEQ ID NO:128 and SEQ ID NO:72, and the second pair of ribonucleic acids comprises SEQ ID NO:462 and SEQ ID NO:421.

[0171] In some embodiments, the method for producing an engineered primary human T cell or population thereof further comprises (c)(i) editing the gene encoding the T cell receptor (TCR) alpha chain locus on chromosome 14 in the cell or population thereof to delete a third contiguous stretch of genomic DNA that comprises at least a portion of a coding exon, and / or (c)(ii) editing the gene encoding the TCR beta chain locus on chromosome 7 in the cell or population thereof to delete a fourth contiguous stretch of genomic DNA that comprises at least a portion of a coding exon, thereby reducing or eliminating TCR surface expression and / or activity in the cell or population thereof. In some embodiments, the editing step in (c)(i) comprises contacting the cell or population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a third pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750, and / or the editing step in (c)(ii) comprises contacting the cell or population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a fourth pair of ribonucleic acids having a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, the third pair of ribonucleic acids comprises SEQ ID NO: 550 and SEQ ID NO: 573, and / or the fourth pair of ribonucleic acids comprises SEQ ID NO: 657 and SEQ ID NO: 662.

[0172] In some embodiments, the method for producing an engineered primary human T cell or population thereof further comprises (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cell or population thereof to delete a fifth contiguous stretch of genomic DNA, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cell or population thereof. In some embodiments, the editing step in (d) comprises contacting the cell with a Cas protein or a nucleic acid sequence encoding the Cas protein and a fifth pair of ribonucleic acids having sequences selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, the fifth pair of ribonucleic acids comprises SEQ ID NO: 773 and SEQ ID NO: 778.

[0173] In some embodiments, the invention provides methods for producing modified primary human T cells or populations thereof, the methods comprising: (a) contacting primary human T cells or populations thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a first ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3638-4046, thereby editing a cytotoxic T-lymphocyte-associated protein 4 (CTLA4) gene on chromosome 2 to reduce or eliminate CTLA4 receptor surface expression and / or activity in the cells or population; and / or (b) contacting primary human T cells or populations thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a second ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 8946-9101, thereby editing a programmed cell death 1 (PD1) gene on chromosome 2 to reduce or eliminate PD1 receptor surface expression and / or activity in the cells or populations thereof.

[0174] In certain embodiments, subsequent alterations to the target TRCB polynucleotide sequence in the cell result in a second cleavage of the target TRCB polynucleotide sequence, thereby editing the target TRCB polynucleotide sequence to delete a fourth contiguous stretch of genomic DNA.

[0175] In some embodiments, the method for producing an engineered primary human T cell or a population thereof further comprises (c)(i) editing a gene encoding a T cell receptor (TCR) alpha chain locus on chromosome 14 in the cell or a population thereof, and / or (c)(ii) editing a gene encoding a TCR beta chain locus on chromosome 7 in the cell or a population thereof, thereby reducing or eliminating TCR surface expression and / or activity in the cell or a population thereof. In some embodiments, the editing step in (c)(i) comprises contacting the cell or a population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a third ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 9751-9797, and / or the editing step in (c)(ii) comprises contacting the cell or a population thereof with a Cas protein or a nucleic acid sequence encoding the Cas protein and a fourth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 10533-10573.

[0176] In some embodiments, the method for producing modified primary human T cells or populations thereof further comprises (d) editing the beta2-microglobulin (B2M) gene on chromosome 15 in the cells or population thereof, thereby reducing or eliminating MHC class I molecule surface expression and / or activity in the cells or population thereof. In some embodiments, the editing step in (d) comprises contacting the cells with a Cas protein or a nucleic acid sequence encoding the Cas protein, and a fifth ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 13258-13719.

[0177] In some embodiments, editing of a gene described herein (e.g., CTLA4, PD1, TCRA, TCRB, and / or B2M) results in a truncation of the polynucleotide sequence of the gene. In certain aspects, subsequent application of the method to produce modified primary human T cells causes a second truncation of the polynucleotide sequence of that gene, thereby deleting a continuous stretch of genomic DNA.

[0178] In some embodiments, the method for producing an engineered primary human T cell or population thereof further comprises causing the cells or population thereof to express at least one chimeric antigen receptor that specifically binds to an antigen or epitope of interest expressed on the surface of at least one of damaged cells, dysplastic cells, infected cells, immunogenic cells, inflammatory cells, malignant cells, metaplastic cells, mutated cells, and combinations thereof.

[0179] In some embodiments, the method for producing an engineered primary human T cell or population thereof further comprises causing the cell or population thereof to express at least one protein that modulates a desired biological effect in an adjacent cell, tissue, or organ when the cell or population thereof is in proximity to the adjacent cell, tissue, or organ.

[0180] It should be appreciated that the CRISPR / Cas system of the present invention can cleave a target polynucleotide sequence in a variety of ways. In some embodiments, the target polynucleotide sequence is cleaved to create a double-stranded break. In some embodiments, the target polynucleotide sequence is cleaved to create a single-stranded break.

[0181] The method of the present invention can be used to change any target polynucleotide sequence in a cell, as long as the target polynucleotide sequence in the cell contains a suitable target motif that allows at least one ribonucleic acid of the CRISPR / Cas system to direct the Cas protein to the target motif and hybridize thereto.Those skilled in the art will recognize that the target motif that targets a particular polynucleotide depends on the CRISPR / Cas system used and the sequence of the polynucleotide to be targeted.

[0182] In some embodiments, the target motif is 17-23 bp in length. In some embodiments, the target motif is at least 20 bp in length. In some embodiments, the target motif is a 20-nucleotide DNA sequence. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence and immediately precedes an NRG motif. In some aspects, the NRG motif is NGG or NAG. In some embodiments, the target motif is a 20-nucleotide DNA sequence and immediately precedes an NGG motif recognized by a Cas protein. In some embodiments, the target motif is a 20-nucleotide DNA sequence and immediately precedes an NAG motif recognized by a Cas protein. In some embodiments, the target motif is a 20-nucleotide DNA sequence starting with G and immediately precedes an NGG motif recognized by a Cas protein. In some embodiments, the target motif is a G(N) 19 In some embodiments, the target motif is (N) 20 It's NGG.

[0183] In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNGRRT motif. In some embodiments, the target motif is a 20 nucleotide DNA sequence immediately preceding an NNGRRT motif. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNNRRT motif. In some embodiments, the target motif is a 20 nucleotide DNA sequence immediately preceding an NNNRRT motif. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNAGAAW motif. In some embodiments, the target motif is a 20 nucleotide DNA sequence immediately preceding an NNAGAAW motif. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNNNGATT motif. In some embodiments, the target motif is a 20 nucleotide DNA sequence immediately preceding an NNNNGATT motif. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding an NAAAAC motif. In some embodiments, the target motif is a 20-nucleotide DNA sequence immediately preceding the NAAAAC motif. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence with a 5' T-rich region (e.g., a TTTN motif). In some embodiments, the target motif is a 20-nucleotide DNA sequence with a 5' T-rich region (e.g., a TTTN motif).

[0184] In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding the NRG motif (e.g., NGG or NAG) recognized by the S. pyogenes Cas9 protein. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding the NNGRRT motif recognized by the S. aureus Cas9 protein. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding the NNNRRT motif recognized by the S. aureus Cas9 protein. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding the NNAGAAW motif recognized by the S. thermophilus Cas9 protein. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding the NNNNGATT motif recognized by the N. meningitides Cas9 protein. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence immediately preceding the NAAAAC motif recognized by the T. denticola Cas9 protein. In some embodiments, the target motif is a 17-23 nucleotide DNA sequence with a 5' T-rich region (e.g., a TTTN motif) recognized by the Acidaminococcus or Lachnospiraceae Cpf1 protein.

[0185] The target motif of the present invention can be selected to minimize the off-target effect of the CRISPR / Cas system of the present invention.In some embodiments, the target motif is selected to contain at least two mismatches when compared with all other genomic nucleotide sequences in cells.In some embodiments, the target motif is selected to contain at least one mismatch when compared with all other genomic nucleotide sequences in cells.Those skilled in the art will recognize that various techniques can be used to select suitable target motifs to minimize off-target effect (for example, bioinformatics analysis).

[0186] In some embodiments, the target motif is a G(N) in the CTLA gene. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in the PD1 gene. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in the TCRA gene. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in the TCRB gene. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in the B2M gene. 19 NGG or (N) 20 Contains the DNA sequence of NGG.

[0187] In some embodiments, the target motif is G(N) in SEQ ID NO: 782. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 783. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 784. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 785. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 786. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 787. 19 NGG or (N) 20In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 788. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 789. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 790. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 791. 19 NGG or (N) 20 In some embodiments, the target motif comprises the DNA sequence of G(N) in SEQ ID NO: 792. 19 NGG or (N) 20 Contains the DNA sequence of NGG.

[0188] In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-4046. In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-9101. In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9797. In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10573. In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13719.

[0189] In some embodiments, the target motif is a G(N) in the CTLA gene. 19 NGG or (N) 20In some embodiments, the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif is G(N) in the PD1 gene. 19 NGG or (N) 20 In some embodiments, the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif comprises a G(N) in the TCRA gene. 19 NGG or (N) 20 In some embodiments, the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif comprises a G(N) in the TCRB gene. 19 NGG or (N) 20 In some embodiments, the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif comprises a G(N) in the B2M gene. 19 NGG or (N) 20 It includes a DNA sequence that contains at least two nucleotide mismatches compared to the DNA sequence of NGG.

[0190] In some embodiments, the target motif or at least a portion of the target motif is a G(N) in a DNA sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-2602. 19 NGG or (N) 20 In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif or at least a portion of the target motif comprises a G(N) in a DNA sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8128. 19 NGG or (N) 20 In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif or at least a portion of the target motif comprises a G(N) in a DNA sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9545. 19 NGG or (N) 20In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif or at least a portion of the target motif comprises a G(N) in a DNA sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10321. 19 NGG or (N) 20 In some embodiments, the target motif or at least a portion of the target motif comprises a DNA sequence containing at least two nucleotide mismatches compared to the DNA sequence of NGG. In some embodiments, the target motif or at least a portion of the target motif comprises a G(N) in a DNA sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-12300. 19 NGG or (N) 20 It includes a DNA sequence that contains at least two nucleotide mismatches compared to the DNA sequence of NGG.

[0191] In some embodiments, the CRISPR / Cas system of the present invention uses homology-directed repair to correct target polynucleotide sequences. In some embodiments, homology-directed repair occurs following the cleavage of the target polynucleotide sequence. In some embodiments, homology-directed repair is carried out using an exogenously introduced DNA repair template. The exogenously introduced DNA repair template can be single-stranded or double-stranded. The exogenously introduced DNA repair template can be of any length. Those skilled in the art will recognize that the length of any specific DNA repair template depends on the target polynucleotide sequence to be corrected. The DNA repair template can be designed to repair or replace any target polynucleotide sequence, particularly target polynucleotide sequences containing disease-related polymorphisms (e.g., SNPs). For example, homology-directed repair of mutant alleles containing such SNPs can be achieved using the CRISPR / Cas system by selecting two target motifs flanking the mutant allele and designing a DNA repair template that matches the wild-type allele.

[0192] In some embodiments, the CRISPR / Cas system of the present invention comprises a Cas protein or a nucleic acid sequence encoding the Cas protein, and at least one to two ribonucleic acids (e.g., gRNAs) capable of directing and hybridizing the Cas protein to a target motif in a target polynucleotide sequence. In some embodiments, the CRISPR / Cas system of the present invention comprises at least one pair of a Cas protein or a nucleic acid sequence encoding the Cas protein, and a ribonucleic acid (e.g., gRNA) capable of directing and hybridizing the Cas protein to a target motif in a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues joined by peptide bonds (i.e., a polymer of amino acids), including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments, and other equivalents, variants, and analogs of the above.

[0193] In some embodiments, the Cas protein comprises one or more amino acid substitutions or modifications. In some embodiments, the one or more amino acid substitutions comprise conservative amino acid substitutions. In some cases, the substitutions and / or modifications may prevent or reduce proteolysis and / or extend the half-life of the polypeptide in cells. In some embodiments, the Cas protein may comprise peptide bond substitutions (e.g., urea, thiourea, carbamate, sulfonylurea, etc.). In some embodiments, the Cas protein may comprise naturally occurring amino acids. In some embodiments, the Cas protein may comprise alternative amino acids (e.g., D-amino acids, β-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, the Cas protein may comprise modifications including moieties (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0194] In some embodiments, the Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas9. In some embodiments, the Cas protein comprises an E. coli subtype Cas protein (also known as CASS2). Exemplary E. coli subtype Cas proteins include, but are not limited to, Cse1, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, the Cas protein comprises a Ypest subtype Cas protein (also known as CASS3). Exemplary Ypest subtype Cas proteins include, but are not limited to, Csy1, Csy2, Csy3, and Csy4. In some embodiments, the Cas protein comprises an Nmeni subtype Cas protein (also known as CASS4). Exemplary Nmeni subtype Cas proteins include, but are not limited to, Csn1 and Csn2. In some embodiments, the Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csd1, Csd2, and Cas5d. In some embodiments, the Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cst1, Cst2, and Cas5t. In some embodiments, the Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to, Csh1, Csh2, and Cas5h. In some embodiments, the Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5). Exemplary Cas proteins of the Apern subtype include, but are not limited to, Csa1, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, the Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6).Exemplary Cas proteins of the Mtube subtype include, but are not limited to, Csm1, Csm2, Csm3, Csm4, and Csm5. In some embodiments, the Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6.

[0195] In some embodiments, the Cas protein is a Streptococcus pyogenes Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Staphylococcus aureus Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Streptococcus thermophilic Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Neisseria meningitides Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Treponema denticola Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein is a Cas9 protein or a functional portion thereof from any bacterial species. Cas9 proteins are typically members of a type II CRISPR system that includes a trans-coding small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and a Cas protein. The Cas9 protein (also known as the CRISPR-associated endonuclease Cas9 / Csn1) is a polypeptide containing 1368 amino acids. An exemplary amino acid sequence of the Cas9 protein (SEQ ID NO:781) is shown in Figure 4. Cas9 contains two endonuclease domains, including a RuvC-like domain (residues 7-22, 759-766, and 982-989) that cleaves target DNA non-complementary to the crRNA and an HNH nuclease domain (residues 810-872) that cleaves target DNA complementary to the crRNA. In Figure 4, the RuvC-like domain is highlighted in yellow, and the HNH nuclease domain is underlined.

[0196] In some embodiments, the Cas protein is a Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is Cpf1 or a functional portion thereof from any bacterial species. In some aspects, the Cpf1 is a Francisella novicida U112 protein or a functional portion thereof. In some aspects, the Cpf1 is an Acidaminococcus sp. BV3L6 protein or a functional portion thereof. In some aspects, the Cpf1 is a Lachnospiraceae bacterium ND2006 protein or a functional portion thereof. The Cpf1 protein is a member of a type V CRISPR system. The Cpf1 protein is a polypeptide comprising approximately 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Cpf1 uses a single ribonuclease domain to cleave target DNA in a staggered pattern. The staggered DNA double-strand breaks result in 4- or 5-nt 5' overhangs.

[0197] As used herein, "functional portion" refers to a portion of a peptide that retains the ability to complex with at least one ribonucleic acid (e.g., a guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA-binding domain, at least one RNA-binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked Cpf1 protein functional domains selected from the group consisting of a DNA-binding domain, at least one RNA-binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, the functional portion of the Cas9 protein comprises a functional portion of an HNH nuclease domain. In some embodiments, the functional portion of the Cpf1 protein comprises a functional portion of a RuvC-like domain.

[0198] It should be appreciated that the present invention contemplates various methods of contacting a target polynucleotide sequence with a Cas protein (e.g., Cas9). In some embodiments, the exogenous Cas protein may be introduced into a cell in polypeptide form. In certain embodiments, the Cas protein may be conjugated or fused to a cell-penetrating polypeptide or cell-penetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refer to a polypeptide or peptide, respectively, that facilitates the uptake of a molecule into a cell. The cell-penetrating polypeptide may contain a detectable label.

[0199] In certain embodiments, the Cas protein may be conjugated or fused to a charged protein (e.g., having a positive, negative, or neutral charge). Such a linkage may be covalent. In some embodiments, to significantly increase the cell penetration ability of the Cas protein, the Cas protein may be fused to a highly positively charged GFP (Cronican et al., ACS Chem Biol. 2010;5(8):747-52). In certain embodiments, to facilitate cell entry, the Cas protein may be fused to a protein transduction domain (PTD). Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a superpositively charged GFP. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a PTD. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a tat domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to an oligoarginine domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a penetratin domain. In some embodiments, the Cpf1 protein comprises a Cpf1 polypeptide fused to a superpositively charged GFP.

[0200] In some embodiments, the Cas protein may be introduced into a cell containing a target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein (e.g., Cas9 or Cpfl). The process of introducing the nucleic acid into the cell may be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises modified DNA as described herein. In some embodiments, the nucleic acid comprises mRNA. In some embodiments, the nucleic acid comprises modified mRNA as described herein (e.g., synthetic modified mRNA).

[0201] In some embodiments, the nucleic acid encoding the Cas protein and the nucleic acid encoding at least one or two ribonucleic acids are introduced into the cell via viral transduction (e.g., lentiviral transduction).

[0202] In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic, modified mRNA) described herein.

[0203] The methods of the present invention contemplate the use of any ribonucleic acid that can direct and hybridize a Cas protein to a target motif in a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises a tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises a CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that directs and hybridizes a Cas protein to a target motif in a target polynucleotide sequence in a cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that directs and hybridizes a Cas protein to a target motif in a target polynucleotide sequence in a cell. In some embodiments, both of the one to two ribonucleic acids comprise guide RNAs that direct and hybridize a Cas protein to a target motif in a target polynucleotide sequence in a cell. As one of skill in the art will recognize, ribonucleic acids of the present invention can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR / Cas system and target polynucleotide sequence used. The one to two ribonucleic acids may also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif containing at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif containing at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein. In some embodiments, the one to two ribonucleic acids are each designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by a Cas protein (which is adjacent to a mutant allele located between the target motifs).

[0204] In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 766-780 and 10574-13257.

[0205] In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 3638-4046. In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 8946-9101. In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 9751-9797. In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 10533-10573. In some embodiments, at least one ribonucleic acid comprises a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 13258-13719.

[0206] In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, at least one of the one to two ribonucleic acids comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 766-780 and 10574-13257.

[0207] In some embodiments, at least one ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 3638-4046. In some embodiments, at least one ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 8946-9101. In some embodiments, at least one ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 9751-9797. In some embodiments, at least one ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 10533-10573. In some embodiments, at least one ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of the ribonucleic acid sequences of SEQ ID NOs: 13258-13719.

[0208] In some embodiments, the one to two ribonucleic acids each comprise a guide RNA that directs the Cas protein to and hybridizes to a target motif in a target polynucleotide sequence in a cell. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to a sequence on the same strand of the target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to a sequence on opposite strands of the target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are not complementary to and / or do not hybridize to a sequence on opposite strands of the target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to overlapping target motifs in the target polynucleotide sequence. In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to offset target motifs in a target polynucleotide sequence.

[0209] The present invention also contemplates multiplex genome editing. Those skilled in the art will recognize that the above description of genome editing of a single gene is equally applicable to the below-described embodiments of multiplex genome editing.

[0210] As used herein, the terms "administration," "introduction," and "implantation" are used interchangeably in the context of placing cells, such as the cells described herein containing a target polynucleotide sequence altered according to the methods of the present invention, into a subject by a method or route that results in at least partial localization of the introduced cells at the desired site. The cells can be directly implanted at the desired site or administered by any suitable route that results in delivery to the desired location in the subject, where at least some of the implanted cells or components of the cells remain viable. The survival period of the cells after administration to the subject can be as short as a few hours, e.g., 24 hours to several days, to as long as several years. In some cases, to maintain the implanted cells at the implanted location and prevent migration of the implanted cells, the cells can also be administered to a location other than the desired site, e.g., the liver or subcutaneously, e.g., in a capsule.

[0211] In ex vivo methods, the cells may include autologous cells, i.e., one or more cells derived from the subject requiring alteration of the target polynucleotide sequence in one or more cells (i.e., the donor and recipient are the same individual). Autologous cells have the advantage of avoiding any immune system rejection of the cells. Alternatively, the cells may be xenogeneic (e.g., harvested from the donor). The second subject may be of the same or a different species. Typically, if the cells are donor-derived, they will be from a donor that is sufficiently immunologically compatible with the recipient (i.e., will not result in transplant rejection and reduce or eliminate the need for immunosuppression). In some embodiments, the cells are harvested from a xenogeneic source (i.e., a non-human mammal genetically engineered to be sufficiently immunologically compatible with the recipient or the recipient's species). Methods for determining immunological compatibility are known in the art and include tissue typing, which assesses donor-recipient compatibility of HLA and ABO determinants. See, e.g., Transplantation Immunology, Bach and Auchincloss, Eds. (Wiley, John & Sons, Incorporated 1994).

[0212] Any suitable cell culture medium can be used in the ex vivo methods of the present invention.

[0213] The terms "subject" and "individual" are used interchangeably herein and refer to, for example, an animal, e.g., a human, from which cells can be obtained and / or treatment (including prophylactic treatment) using the cells described herein is provided. For the treatment of an infection, symptom, or disease state specific to a particular animal, such as a human subject, the term subject refers to that particular animal. As used interchangeably herein, "non-human animal" and "non-human mammal" include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. However, preferably, the subject is a mammal, e.g., a human or other mammal, such as a domestic mammal, e.g., a dog, cat, horse, or livestock mammal, e.g., a cow, sheep, pig, or the like.

[0214] In some embodiments, the alteration results in reduced expression of the target polynucleotide sequence. In some embodiments, the alteration results in knockout of the target polynucleotide sequence. In some embodiments, the alteration results in correction of the target polynucleotide sequence from an undesired sequence to a desired sequence. In some embodiments, each alteration is a homozygous alteration. In some embodiments, the efficiency of alteration at each locus is between about 5% and about 80%. In some embodiments, the efficiency of alteration at each locus is between about 10% and about 80%. In some embodiments, the efficiency of alteration at each locus is between about 30% and about 80%. In some embodiments, the efficiency of alteration at each locus is between about 50% and about 80%. In some embodiments, the efficiency of alteration at each locus is greater than or equal to about 80%. In some embodiments, the efficiency of alteration at each locus is greater than or equal to about 85%. In some embodiments, the efficiency of alteration at each locus is greater than or equal to about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the efficiency of alteration at each locus is about 100%.

[0215] In some embodiments, each target polynucleotide sequence is cleaved to create a double-stranded break. In some embodiments, each target polynucleotide sequence is cleaved to create a single-stranded break.

[0216] In some embodiments, the target polynucleotide sequence comprises multiple different portions of CTLA4. In some embodiments, the target polynucleotide sequence comprises multiple different portions of PD1. In some embodiments, the target polynucleotide sequence comprises multiple different portions of TCRA. In some embodiments, the target polynucleotide sequence comprises multiple different portions of TCRB. In some embodiments, the target polynucleotide sequence comprises multiple different portions of B2M.

[0217] In some embodiments, each target motif is a 17-23 nucleotide DNA sequence. In some embodiments, each target motif is a 20 nucleotide DNA sequence. In some embodiments, each target motif is a 20 nucleotide DNA sequence with a 5' T-rich region. In some embodiments, each target motif is a 20 nucleotide DNA sequence beginning with G and immediately preceding the NGG motif recognized by the Cas protein. In some embodiments, each target motif is a 20 nucleotide DNA sequence immediately preceding the NGG motif recognized by the Cas protein. In some embodiments, each target motif is a G(N) 19 In some embodiments, each target motif is NGG. 20 In some embodiments, each target motif is selected to contain at least two mismatches when compared with all other genomic nucleotide sequences in cell.In some embodiments, each target motif is selected to contain at least two mismatches when compared with all other genomic nucleotide sequences in cell.

[0218] In some embodiments, each target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNGRRT motif. In some embodiments, each target motif is a 20 nucleotide DNA sequence immediately preceding an NNGRRT motif. In some embodiments, each target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNNRRT motif. In some embodiments, each target motif is a 20 nucleotide DNA sequence immediately preceding an NNNRRT motif. In some embodiments, each target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNAGAAW motif. In some embodiments, each target motif is a 20 nucleotide DNA sequence immediately preceding an NNAGAAW motif. In some embodiments, each target motif is a 17-23 nucleotide DNA sequence immediately preceding an NNNNGATT motif. In some embodiments, each target motif is a 20 nucleotide DNA sequence immediately preceding an NNNNGATT motif. In some embodiments, each target motif is a 17-23 nucleotide DNA sequence immediately preceding an NAAAAC motif. In some embodiments, each target motif is a 20-nucleotide DNA sequence immediately preceding a NAAAAC motif. In some embodiments, each target motif is a 17-23 nucleotide DNA sequence with a 5' T-rich region (e.g., a TTTN motif). In some embodiments, each target motif is a 20-nucleotide DNA sequence with a 5' T-rich region (e.g., a TTTN motif).

[0219] In some embodiments, the cleavage of target polynucleotide sequence is followed by homology-directed repair.In some embodiments, homology-directed repair is carried out using exogenously introduced DNA repair template.In some embodiments, the exogenously introduced DNA repair template is single-stranded.In some embodiments, the exogenously introduced DNA repair template is double-stranded.

[0220] In some embodiments, the Cas protein (e.g., Cas9 or Cpfl) complexes with at least one ribonucleic acid. In some embodiments, the Cas protein (e.g., Cas9) complexes with multiple ribonucleic acids. In some embodiments, the multiple ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence (e.g., multiple alterations in a single target polynucleotide sequence). In some embodiments, the multiple ribonucleic acids are selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence (e.g., one or more alterations in multiple target polynucleotide sequences). In some embodiments, each of the multiple ribonucleic acids hybridizes to a target motif containing at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, each of the multiple ribonucleic acids hybridizes to a target motif containing at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, each of the multiple ribonucleic acids is designed to hybridize to a target motif immediately adjacent to the deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the multiple ribonucleic acids is designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein (which flanks a mutant allele located between the target motifs).

[0221] In some embodiments, the Cas protein (e.g., Cpfl) complexes with a single ribonucleic acid. In some embodiments, the ribonucleic acid is selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence (e.g., multiple variations in a single target polynucleotide sequence). In some embodiments, the ribonucleic acid is selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence (e.g., one or more variations in multiple target polynucleotide sequences). In some embodiments, the ribonucleic acid hybridizes to a target motif that contains at least two mismatches when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the ribonucleic acid hybridizes to a target motif that contains at least one mismatch when compared to all other genomic nucleotide sequences in the cell. In some embodiments, the ribonucleic acid is designed to hybridize to a target motif that is immediately adjacent to the deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, the ribonucleic acid is designed to hybridize to a target motif immediately adjacent to the deoxyribonucleic acid motif recognized by the Cas protein, which is adjacent to the mutant allele located between the target motifs.

[0222] It should be appreciated that any nucleic acid or ribonucleic acid encoding a Cas protein (e.g., SEQ ID NOS: 1-780 and 797-13719) can be expressed from a plasmid. In some embodiments, any Cas protein or ribonucleic acid is expressed using a promoter optimized for increased expression in stem cells (e.g., human stem and / or progenitor cells). In some embodiments, the promoter is selected from the group consisting of a cytomegalovirus (CMV) early enhancer element and a chicken β-actin promoter, a chicken β-actin promoter, an elongation factor-1α promoter, and a ubiquitin promoter.

[0223] In some embodiments, the methods of the present invention further comprise selecting cells that express the Cas protein. The present invention contemplates any suitable method for selecting cells. In some embodiments, the selection of cells comprises FACS. In some embodiments, FACS is used to select cells that co-express the Cas protein and a fluorescent protein selected from the group consisting of green fluorescent protein and red fluorescent protein.

[0224] Treatment method The present invention contemplates treating and / or preventing a variety of disorders associated with the expression of target polynucleotide sequences.

[0225] The present invention also contemplates the modified primary human cells of the invention, compositions comprising those cells, and various treatment methods using the compositions (e.g., chimeric nucleic acids) of the invention. The terms "treat," "treating," "treatment," and the like, as applied to isolated cells, include subjecting the cells to any type of process or condition, or performing any type of operation or procedure on the cells. When applied to a subject, the term refers to administering to the individual a cell or cell population in which a target polynucleotide sequence (e.g., CTLA4, PD1, TCRA, TCRB, B2M, etc.) has been altered ex vivo according to the methods described herein. Typically, the individual is sick or injured, or at increased risk of developing a disease compared to the average member of the population, and requires attention, care, or management.

[0226] As used herein, the terms "treating" and "treatment" refer to administering to a subject an effective amount of cells having a target polynucleotide sequence altered ex vivo according to the methods described herein, such that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical result. For purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a decrease in the extent of the disease, stabilization (i.e., non-worsening) of the disease state, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and remission (whether partial or complete). Treatment may refer to prolonging survival compared to expected survival in the absence of treatment. Thus, one of skill in the art will understand that treatment may improve the disease state, but may not completely cure the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" if disease progression is reduced or halted. "Treatment" also means prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already diagnosed with a disorder associated with expression of the polynucleotide sequence as well as those who may develop such a disorder due to genetic susceptibility or other factors.

[0227] "Treating," "preventing," or "ameliorating" a disease or disorder means delaying or preventing the onset, progression, severity, or worsening of such disease or disorder, or reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression or severity of symptoms associated with such disease or disorder. In one embodiment, symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0228] It should be appreciated that the methods and compositions described herein can be used to treat or prevent disorders associated with increased expression of a target polynucleotide sequence and decreased expression of a target polynucleotide sequence in cells. Increased and decreased expression of a target polynucleotide sequence include situations in which the expression level of the target polynucleotide sequence is increased or decreased, respectively, compared to normal expression and / or activity levels, and situations in which the function and / or activity level of the expression product of the target polynucleotide sequence is increased or decreased, respectively. Those skilled in the art will recognize that treatment or prevention of disorders associated with increased expression of a target polynucleotide sequence can be assessed by contacting cells with a composition described herein and then determining whether the level and / or activity of the target polynucleotide sequence (or its expression product) is decreased in the relevant cells. Those skilled in the art will also recognize that treatment or prevention of disorders associated with decreased expression of a target polynucleotide sequence can be assessed by contacting cells with a composition described herein and then determining whether the level and / or activity of the target polynucleotide sequence (or its expression product) is increased in the relevant cells.

[0229] In some embodiments, the disorder is cancer. As used herein, the term "cancer" is defined as a hyperproliferation of cells whose unique hallmark—loss of normal control—results in unregulated growth, lack of differentiation, local tissue invasion, and metastasis. For the purposes of the methods of the present invention, cancer includes acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, hodgkin lymphoma, leukemia ... The tumor may be any cancer, including Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer, lymphoma, malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureter cancer and bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of malignant cells or tissues, and does not include benign tissues, unless otherwise specified.

[0230] In some embodiments, the disorder is a genetic disorder. In some embodiments, the disorder is a monogenic disorder. In some embodiments, the disorder is a polygenic disorder. In some embodiments, the disorder is a disorder associated with one or more SNPs. Exemplary disorders associated with one or more SNPs include complex diseases as described in U.S. Pat. No. 7,627,436, Alzheimer's disease as described in WO 2009 / 112882, inflammatory diseases as described in U.S. Pat. App. Pub. No. 2011 / 0039918, polycystic ovary syndrome as described in U.S. Pat. App. Pub. No. 2012 / 0309642, cardiovascular diseases as described in U.S. Pat. App. Pub. No. 7,732,139, Huntington's disease as described in U.S. Pat. App. Pub. No. 2012 / 0136039, thromboembolic diseases as described in European Pat. App. Pub. No. 2535424, neurovascular diseases as described in WO 2012 / 001613, psychoses as described in U.S. Pat. App. Pub. No. 2010 / 0292211, and multiple sclerosis as described in U.S. Pat. App. Pub. No. 2011 / 0319288. Examples of disorders that may be treated or prevented according to the methods of the present invention include neuropathic sclerosis, schizophrenia, schizoaffective disorder, and bipolar disorder as described in WO 2006 / 023719, bipolar disorder and other diseases as described in U.S. Patent Application Publication No. 2011 / 0104674, colorectal cancer as described in WO 2006 / 104370, disorders associated with SNPs flanking the AKT1 locus as described in U.S. Patent Application Publication No. 2006 / 0204969, eating disorders as described in WO 2003 / 012143, autoimmune diseases as described in U.S. Patent Application Publication No. 2007 / 0269827, fibrostenosing disease in Crohn's disease patients as described in U.S. Patent No. 7,790,370, and Parkinson's disease as described in U.S. Patent No. 8,187,811, each of which is incorporated herein by reference in its entirety. Other disorders associated with one or more SNPs that may be treated or prevented according to the methods of the present invention will be apparent to those skilled in the art.

[0231] In some embodiments, the disorder is a chronic infectious disease. A "chronic infectious disease" is a disease caused by an infectious agent and results in a persistent infection. Such diseases may include hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV / AIDS. Non-viral examples may include chronic fungal diseases, such as aspergillosis, candidiasis, coccidioidomycosis, and diseases associated with Cryptococcus and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents may be Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immune deficiency syndrome (AIDS).

[0232] In some embodiments, the disorder is an autoimmune disorder. The term "autoimmune disease" refers to any disease or disorder in which a subject mounts a destructive immune response against its own tissues. Autoimmune disorders can affect almost any organ system in a subject (e.g., human), including, but not limited to, diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood, and blood vessels. Examples of autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

[0233] In some embodiments, the disorder is graft-versus-host disease (GVHD).

[0234] The methods of the invention can alter target polynucleotide sequences in a variety of different cells (e.g., altering immunological checkpoint regulator genes, e.g., CTL4, PD1, etc., to reduce or eliminate T cell inhibition, altering genes encoding TCR α and β chains to reduce or eliminate T cell autoreactivity, and / or altering B2M to eliminate MHC class I surface expression, and optionally altering one or more additional target polynucleotide sequences associated with a disorder in which alteration of the target polynucleotide sequence is beneficial, and / or optionally causing the cell to express a protein that modulates at least one biological process). In some embodiments, the methods of the invention are used to alter target polynucleotide sequences in cells ex vivo, for subsequent introduction into a subject.

[0235] In some embodiments, the cells or populations thereof are primary cells. In some embodiments, the cells or populations thereof are primary T cells (e.g., human). The T cells can be any T cell, including, but not limited to, cytotoxic T cells (e.g., CD8+ cells), helper T cells (e.g., CD4+ cells), memory T cells, regulatory T cells, tissue-infiltrating lymphocytes (e.g., tumor-infiltrating lymphocytes, e.g., TILs, CD3+ cells), and combinations thereof.

[0236] In some embodiments, the cells are peripheral blood cells. In some embodiments, the cells are stem cells or pluripotent cells. In some embodiments, the cells are hematopoietic stem cells. In some embodiments, the cells are CD34+ cells. In some embodiments, the cells are CD34+ mobilized peripheral blood cells. In some embodiments, the cells are CD34+ umbilical cord blood cells. In some embodiments, the cells are CD34+ bone marrow cells. In some embodiments, the cells are CD34+CD38-CD90+CD45RA- cells. In some embodiments, the cells are CD4+ cells. In some embodiments, the cells are CD4+ T cells. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are human pluripotent cells. In some embodiments, the cells are primary human cells. In some embodiments, the cells are primary CD34+ cells. In some embodiments, the cells are primary CD34+ hematopoietic progenitor cells (HPCs). In some embodiments, the cells are primary CD4+ cells. In some embodiments, the cell is a primary CD4+ T cell. In some embodiments, the cell is an autologous primary cell. In some embodiments, the cell is an autologous primary somatic cell. In some embodiments, the cell is an allogeneic primary cell. In some embodiments, the cell is an allogeneic primary somatic cell. In some embodiments, the cell is a nucleated cell. In some embodiments, the cell is a non-transformed cell. In some embodiments, the cell is a human choriocarcinoma cell. In some embodiments, the cell is a JEG-3 cell. In some embodiments, the cell is a monocyte cell. In some embodiments, the cell is a Thp-1 cell. In some embodiments, the cell is not a cancer cell. In some embodiments, the cell is not a tumor cell. In some embodiments, the cell is not a transformed cell.

[0237] The cells or populations thereof may be obtained from any subject, for example, a subject suffering from, being treated for, diagnosed with, at risk of developing, or suspected of having a disorder selected from the group consisting of an autoimmune disorder, cancer, a chronic infectious disease, and graft-versus-host disease (GVHD).

[0238] The present invention also provides compositions comprising a Cas protein of the invention or a functional portion thereof, a nucleic acid encoding the Cas protein or a functional portion thereof, and a ribonucleic acid sequence that directs and hybridizes the Cas protein to a target motif in a target polynucleotide sequence in a cell.

[0239] In some aspects, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cas9 protein and at least one ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some aspects, disclosed herein are compositions comprising a pair of a nucleic acid sequence encoding a Cas9 protein and a ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, a first ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, a first ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, a second ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, the second ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637. In some embodiments, the pair of ribonucleic acids comprises a sequence having at least one nucleotide mismatch or at least two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 1-195 and 797-3637.

[0240] In some embodiments, the pair of ribonucleic acid sequences comprises or consists of SEQ ID NO: 128 and SEQ ID NO: 72. In some embodiments, the pair of ribonucleic acid sequences comprises sequences having at least one nucleotide mismatch or at least two nucleotide mismatches relative to SEQ ID NO: 128 and SEQ ID NO: 72.

[0241] In some embodiments, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cas9 protein and at least one ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, disclosed herein are compositions comprising a pair of a nucleic acid sequence encoding a Cas9 protein and a ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, a first ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, a first ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, a second ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, the second ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945. In some embodiments, the pair of ribonucleic acids comprises a sequence having at least one nucleotide mismatch or at least two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 196-531 and 4047-8945.

[0242] In some embodiments, the pair of ribonucleic acid sequences comprises or consists of SEQ ID NO: 462 and SEQ ID NO: 421. In some embodiments, the pair of ribonucleic acid sequences comprises sequences having at least one nucleotide mismatch or at least two nucleotide mismatches relative to SEQ ID NO: 462 and SEQ ID NO: 421.

[0243] In some aspects, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cas9 protein and at least one ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some aspects, disclosed herein are compositions comprising a pair of a nucleic acid sequence encoding a Cas9 protein and a ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, a first ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, a first ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, a second ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, the second ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750. In some embodiments, the pair of ribonucleic acids comprises a sequence having at least one nucleotide mismatch or at least two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9750.

[0244] In some embodiments, the pair of ribonucleic acid sequences comprises or consists of SEQ ID NO: 550 and SEQ ID NO: 573. In some embodiments, the pair of ribonucleic acid sequences comprises sequences having at least one nucleotide mismatch or at least two nucleotide mismatches relative to SEQ ID NO: 550 and SEQ ID NO: 573.

[0245] In some embodiments, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cas9 protein and at least one ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, disclosed herein are compositions comprising a pair of a nucleic acid sequence encoding a Cas9 protein and a ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, a first ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, a first ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, a second ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, the second ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532. In some embodiments, the pair of ribonucleic acids comprises a sequence having at least one nucleotide mismatch or at least two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532.

[0246] In some embodiments, the pair of ribonucleic acid sequences comprises or consists of SEQ ID NO: 657 and SEQ ID NO: 662. In some embodiments, the pair of ribonucleic acid sequences comprises sequences having at least one nucleotide mismatch or at least two nucleotide mismatches relative to SEQ ID NO: 550 and SEQ ID NO: 573.

[0247] In some embodiments, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cas9 protein and at least one ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, disclosed herein are compositions comprising a pair of a nucleic acid sequence encoding a Cas9 protein and a ribonucleic acid sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, a first ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, a first ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, a second ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, the second ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257. In some embodiments, the pair of ribonucleic acids comprises a sequence having at least one nucleotide mismatch or at least two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 766-780 and 10574-13257.

[0248] In some embodiments, the pair of ribonucleic acid sequences comprises or consists of SEQ ID NO: 773 and SEQ ID NO: 778. In some embodiments, the pair of ribonucleic acid sequences comprises sequences having at least one nucleotide mismatch or at least two nucleotide mismatches relative to SEQ ID NO: 773 and SEQ ID NO: 778.

[0249] In some aspects, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cpf1 protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 3638-4046. In some embodiments, the ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 3638-4046. In some embodiments, the ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 3638-4046.

[0250] In some aspects, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cpf1 protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 8946-9101. In some embodiments, the ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 8946-9101. In some embodiments, the ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 8946-9101.

[0251] In some aspects, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cpf1 protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 9751-9797. In some embodiments, the ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 9751-9797. In some embodiments, the ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 9751-9797.

[0252] In some aspects, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cpf1 protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 10533-10573. In some embodiments, the ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 10533-10573. In some embodiments, the ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 10533-10573.

[0253] In some aspects, disclosed herein are compositions comprising a nucleic acid sequence encoding a Cpf1 protein and a ribonucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 13258-13719. In some embodiments, the ribonucleic acid comprises a sequence having a single nucleotide mismatch to a sequence selected from the group consisting of SEQ ID NOs: 13258-13719. In some embodiments, the ribonucleic acid comprises a sequence having two nucleotide mismatches to a sequence selected from the group consisting of SEQ ID NOs: 13258-13719.

[0254] In some embodiments, the present invention provides compositions comprising a chimeric nucleic acid, wherein the chimeric nucleic acid comprises: (a) a nucleic acid sequence encoding a Cas protein; and (b) at least one ribonucleic acid sequence selected from the group consisting of: (i) SEQ ID NOs: 1-195 and 797-3637; (ii) SEQ ID NOs: 196-531 and 4047-8945; (iii) SEQ ID NOs: 532-609 and 9102-9750; (iv) SEQ ID NOs: 610-765 and 9798-10532; and (v) SEQ ID NOs: 766-780 and 10574-13257; and combinations of (i)-(v).

[0255] In some embodiments, the at least one ribonucleic acid sequence in (b) is selected from the group consisting of: (i) SEQ ID NO: 128 and SEQ ID NO: 72; (ii) SEQ ID NO: 462 and SEQ ID NO: 421; (iii) SEQ ID NO: 550 and SEQ ID NO: 573; (iv) SEQ ID NO: 657 and SEQ ID NO: 662; and (v) SEQ ID NO: 773 and SEQ ID NO: 778; and combinations of (i)-(v).

[0256] In some embodiments, the present invention provides compositions comprising a chimeric nucleic acid, wherein the chimeric nucleic acid comprises: (a) a nucleic acid sequence encoding a Cas protein; and (b) a ribonucleic acid sequence selected from the group consisting of: (i) SEQ ID NOs: 3638-4046; (ii) SEQ ID NOs: 8946-9101; (iii) SEQ ID NOs: 9751-9797; (iv) SEQ ID NOs: 10533-10573; and (v) SEQ ID NOs: 13258-13719; and combinations of (i)-(v).

[0257] In some embodiments, the composition further comprises a nucleic acid sequence encoding a detectable marker.

[0258] In some embodiments, the composition comprises at least one additional ribonucleic acid sequence for altering the target polynucleotide sequence. In some embodiments, the composition comprises at least two additional ribonucleic acid sequences for altering the target polynucleotide sequence. In some embodiments, the composition comprises at least three additional ribonucleic acid sequences for altering the target polynucleotide sequence. In some embodiments, the composition comprises at least four additional ribonucleic acid sequences for altering the target polynucleotide sequence.

[0259] In some embodiments, at least one of the ribonucleic acids in the composition is a modified ribonucleic acid described herein (e.g., a synthetic modified ribonucleic acid containing one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate, or any other modified nucleotide or modification described herein).

[0260] In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cas protein. In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cas9 protein or a functional portion thereof. In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cpf1 protein or a functional portion thereof.

[0261] In some embodiments, at least one of the ribonucleic acids in the composition is a modified ribonucleic acid described herein (e.g., a synthetic modified ribonucleic acid containing one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate, or any other modified nucleotide or modification described herein). In some embodiments, the pair of ribonucleic acids in the composition are modified ribonucleic acids described herein (e.g., synthetic modified ribonucleic acids containing one to two modified nucleotides selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate, or any other modified nucleotides or modifications described herein).

[0262] In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cas protein. In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cas9 protein, or a functional portion thereof. In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cas protein. In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cas9 protein, or a functional portion thereof. In some embodiments, compositions of the invention comprise a nucleic acid sequence encoding a Cpf1 protein, or a functional portion thereof.

[0263] In some embodiments, the nucleic acid encoding the Cas protein (e.g., Cas9 or Cpf1) comprises a modified ribonucleic acid described herein (e.g., a synthetic, modified mRNA described herein comprising at least one modified nucleotide selected from the group consisting of pseudouridine, 5-methylcytosine, 2-thio-uridine, 5-methyluridine-5'-triphosphate, 4-thiouridine-5'-triphosphate, 5,6-dihydrouridine-5'-triphosphate, and 5-azauridine-5'-triphosphate, or any other modified nucleotide or modification described herein).

[0264] In some embodiments, the compositions of the present invention comprise a nucleic acid sequence encoding a fluorescent protein selected from the group consisting of green fluorescent protein and red fluorescent protein. In some embodiments, the compositions of the present invention comprise a promoter operably linked to the chimeric nucleic acid. In some embodiments, the promoter is optimized for increased expression in human cells. In some embodiments, the promoter is optimized for increased expression in human stem cells. In some embodiments, the promoter is optimized for increased expression in primary human cells. In some embodiments, the promoter is selected from the group consisting of a cytomegalovirus (CMV) early enhancer element and a chicken β-actin promoter, a chicken β-actin promoter, an elongation factor-1α promoter, and a ubiquitin promoter. In some embodiments, the Cas protein comprises a Cas9 protein or a functional portion thereof. In some embodiments, the Cas protein comprises a Cpf1 protein or a functional portion thereof.

[0265] The present invention also provides kits for carrying out any of the methods of the present invention, as well as kits for altering a target polynucleotide sequence in a cell or population thereof, comprising a composition of the present invention and instructions for use of the kit.

[0266] Cell administration In some aspects, the invention provides methods of administering cells to a subject in need of such cells, comprising: (a) contacting a cell or a population of cells ex vivo with a Cas protein or a nucleic acid encoding the Cas protein, and at least one ribonucleic acid that directs the Cas protein to and hybridizes to at least one target polynucleotide sequence in the cell or population thereof, which is selected from the group consisting of target polynucleotide sequences encoding CTLA4, PD1, TCRA, TCRB, B2M, and combinations thereof, to cleave the at least one target polynucleotide sequence; and (b) administering the cells obtained from (a) to a subject in need of such cells.

[0267] In some aspects, the invention provides methods of administering cells to a subject in need of such cells, comprising: (a) contacting a cell or a population of cells ex vivo with (i) a Cas protein or a nucleic acid encoding the Cas protein, and (ii) at least one pair of ribonucleic acids that direct the Cas protein to and hybridize to at least one target polynucleotide sequence in the cell or population of cells, which is selected from the group consisting of target polynucleotide sequences encoding CTLA4, PD1, TCRA, TCRB, B2M, and combinations thereof, to cleave the target polynucleotide sequence; and (b) administering one or more cells obtained from (a) to a subject in need of such cells.

[0268] In some aspects, the invention provides methods of administering cells to a subject in need of such cells, comprising: (a) contacting a cell or a population of cells ex vivo with (i) a Cas protein or a nucleic acid encoding the Cas protein, and (ii) a ribonucleic acid that directs the Cas protein to and hybridizes to a target polynucleotide sequence in the cell or population of cells, the target polynucleotide sequence being selected from the group consisting of target polynucleotide sequences encoding CTLA4, PD1, TCRA, TCRB, B2M, and combinations thereof, to cleave the target polynucleotide sequence; and (b) administering one or more cells obtained from (a) to a subject in need of such cells.

[0269] It is contemplated that the method of administering cells can be adapted for any purpose for which administration of such cells is desirable (e.g., allogeneic administration of cells to a subject in need of such cells). In some embodiments, the subject in need of cell administration suffers from a disorder. For example, the subject may suffer from a disorder in which the function or number of a particular cell is reduced, and it may be desirable to administer functional cells obtained from a healthy or normal individual in which the particular cell is functioning properly, and to administer a sufficient number of healthy cells to the individual to restore the function provided by that cell (e.g., a hormone-producing cell in reduced number or function, an immune cell in reduced number or function, etc.). In such cases, the healthy cells can be engineered to reduce the likelihood of host rejection of the healthy cells. In some embodiments, the disorder comprises a genetic disorder. In some embodiments, the disorder comprises an infectious disease. In some embodiments, the disorder comprises HIV or AIDS. In some embodiments, the disorder comprises cancer. In some embodiments, the disorder comprises an autoimmune disease.

[0270] Before administering the cells, the cell population can be sorted (e.g., using FACS) to select cells with edited genomes. The selected cells can then be expanded to the amount of cells required for transplantation for a specific disorder in which a second subject requires such cells. In some embodiments, the method can include, before the administration step, contacting the genomically modified cells with a Cas protein and one or more guide RNA sequences targeting one or more additional target polynucleotides associated with the disorder in which the second subject (i.e., recipient) requires such cells. For example, in the case of HIV, the genomically modified cells can be contacted with a Cas protein and one or more guide RNA sequences targeting the CCR5 and / or CXCR4 genes, thereby editing the genome of the genomically modified cells to eliminate or reduce the surface expression of CCR5 and / or CXCR4. Such cells would be beneficial for administration to a second subject (e.g., suffering from HIV or AIDS) because the lack of MHC class I molecule surface expression would eliminate or reduce the likelihood of an unwanted host immune response, and the lack of CCR5 and / or CXCR4 surface expression would result in less or no susceptibility to HIV infection.

[0271] As used herein, "nucleic acid" in its broadest sense includes any compound and / or substance comprising a polymer of nucleotides linked by phosphodiester bonds. Exemplary nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof. They may also include RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, tRNA, RNA that induces triple helix formation, aptamers, vectors, etc. In some embodiments, the nucleic acid encoding the Cas protein is mRNA. In some embodiments, the Cas protein is encoded by a modified nucleic acid (e.g., a synthetic modified mRNA described herein).

[0272] The present invention contemplates the use of any nucleic acid modification available to those skilled in the art. The nucleic acids of the present invention can include any number of modifications. In some embodiments, the nucleic acids are selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, and 5-taurinomethyl-2-thio-uridine. , 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-meth Deaza-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl -1-Deaza-pseudoisocytidine, Zebularine, 5-Aza-Zebularine, 5-Methyl-Zebularine, 5-Aza-2-Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytidine, 2-Methoxy-5-Methyl-Cytidine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, 2-Aminopurine, 2,6-Diaminopurine, 7-Deaza-Adenine, 7-Deaza-8-Aza-Adenine, 7-Deaza-2-Aminopurine, 7-Deaza-8-Aza-2-Aminopurine, 7-Deaza-2,6-Diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, 7-deazaguaiazen ... Inosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof.

[0273] Preparation of modified nucleosides and nucleotides used in the production or synthesis of modified RNAs of the present invention can involve the protection and deprotection of various chemical groups. Those skilled in the art can readily determine the need for protection and deprotection and the selection of appropriate protecting groups.

[0274] The chemistry of protecting groups can be found, for example, in Greene et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0275] Modified nucleosides and nucleotides can be prepared according to the synthetic methods described in Ogata et al., Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al., Nucleic Acids Research 22(1):72-78, 1994; Fukuhara et al., Biochemistry 1(4):563-568, 1962; and Xu et al., Tetrahedron 48(9):1729-1740, 1992, each of which is incorporated herein by reference in its entirety.

[0276] Modified nucleic acids (e.g., ribonucleic acids) need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and / or backbone structures can be present at various positions in the nucleic acid. Those skilled in the art will recognize that nucleotide analogs or other modifications can be placed at any position in the nucleic acid so that the function of the nucleic acid is not substantially reduced. Modifications can also be 5' or 3' terminal modifications. Nucleic acids can contain a minimum of one and a maximum of 100% modified nucleotides, or any intermediate percentage, e.g., at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.

[0277] In some embodiments, at least one ribonucleic acid is a modified ribonucleic acid. In some embodiments, at least one of the one to two ribonucleic acids is a modified ribonucleic acid. In some embodiments, each of the one to two ribonucleic acids is a modified ribonucleic acid. In some embodiments, at least one of the plurality of ribonucleic acids is a modified ribonucleic acid. In some embodiments, a majority of the plurality of ribonucleic acids are modified. In some embodiments, each of the plurality of ribonucleic acids is modified. Those of skill in the art will recognize that a modified ribonucleic acid can include one or more of the nucleic acid modifications described herein.

[0278] In some aspects, provided herein are synthetic modified RNA molecules encoding a polypeptide, the synthetic modified RNA molecules comprising one or more modifications such that introduction of the synthetic modified RNA molecule into a cell results in a reduced innate immune response compared to a cell contacted with a synthetic RNA molecule encoding a polypeptide that does not comprise the one or more modifications. In some embodiments, the Cas protein comprises a synthetic modified RNA molecule encoding a Cas protein. In some embodiments, the Cas protein comprises a synthetic modified RNA molecule encoding a Cas9 protein. In some embodiments, the Cas protein comprises a synthetic modified RNA molecule encoding a Cpf1 protein.

[0279] The synthetic modified RNAs described herein include modifications that prevent rapid degradation by endonucleases and exonucleases, and avoid or reduce the cell's innate immune or interferon response to RNA.Modifications include, but are not limited to, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, dephosphorylation, conjugation, reverse linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotide, reverse linkage, etc.), (b) base modifications, such as modified bases, stabilized bases, destabilized bases, or bases that base-pair with expanded repertoire partners, or conjugated bases, (c) sugar modifications (e.g., at the 2'- or 4'-position) or sugar replacement, and (d) internucleoside linkage modifications, including modifications or replacement of phosphodiester linkages. To the extent that such a modification prevents translation (i.e., results in a 50% or greater reduction in translation compared to the absence of the modification in, for example, an in vitro rabbit reticulocyte translation assay), the modification is not suitable for the methods and compositions described herein. Specific examples of synthetic modified RNA compositions useful in the methods described herein include, but are not limited to, RNA molecules containing modified or non-natural internucleoside linkages. Synthetic modified RNAs with modified internucleoside linkages include, among others, those that do not have a phosphorus atom in the internucleoside linkage. In other embodiments, the synthetic modified RNA has a phosphorus atom in its internucleoside linkage.

[0280] Non-limiting examples of modified internucleoside linkages include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, 3'-aminophosphoramidates and aminoalkylphosphoramidates, phosphoramidates, including thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linkage analogs, and those with reverse polarity (adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2').Various salts, mixed salts, and free acid forms are also included.

[0281] Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321 ,131; U.S. Patent No. 5,399,676; U.S. Patent No. 5,405,939; U.S. Patent No. 5,453,496; U.S. Patent No. 5,455,233; U.S. Patent No. 5,466,677; U.S. Patent No. 5,476,925; U.S. Patent No. 5,519,126; U.S. Patent No. 5,536,821; U.S. Patent No. 5,541,316; U.S. Patent No. 5,550,111; U.S. Patent No. 5,563,253; U.S. Patent No. 5,571,799; U.S. Patent No. 5,58 7,361; U.S. Patent No. 5,625,050; U.S. Patent No. 6,028,188; U.S. Patent No. 6,124,445; U.S. Patent No. 6,160,109; U.S. Patent No. 6,169,170; U.S. Patent No. 6,172,209; U.S. Patent No. 6,239,265; U.S. Patent No. 6,277,603; U.S. Patent No. 6,326,199; U.S. Patent No. 6,346,614; U.S. Patent No. 6,444,423; U.S. Patent No. 6,531,590; U.S. Patent No. 6,5 34,639; U.S. Patent No. 6,608,035; U.S. Patent No. 6,683,167; U.S. Patent No. 6,858,715; U.S. Patent No. 6,867,294; U.S. Patent No. 6,878,805; U.S. Patent No. 7,015,315; U.S. Patent No. 7,041,816; U.S. Patent No. 7,273,933; U.S. Patent No. 7,321,029; and U.S. Patent No. RE39464 (each of which is incorporated by reference herein in its entirety).

[0282] Modified internucleoside linkages that do not contain a phosphorus atom include short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or internucleoside linkages formed by one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, those with amide backbones, and others with mixed N, O, S, and CH constituent moieties.

[0283] Representative United States patents that teach the preparation of modified oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; and 5,488,681. Nos. 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439 (each of which is incorporated by reference herein in its entirety).

[0284] Some embodiments of the synthetic, modified RNAs described herein include nucleic acids with phosphorothioate internucleoside linkages, and oligonucleosides with heteroatom internucleoside linkages, and in particular the amide backbones of -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as methylene(methylimino) or MMI], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [natural phosphodiester internucleoside linkages are represented as -OPO-CH2-] of the above-cited U.S. Pat. No. 5,489,677, and the above-cited U.S. Pat. No. 5,602,240, both of which are incorporated herein by reference in their entireties. In some embodiments, the nucleic acid sequences featured herein have the morpholino backbone structures of the above-cited U.S. Patent No. 5,034,506, which is incorporated herein by reference in its entirety.

[0285] The synthetic, modified RNAs described herein can also contain one or more substituted sugar moieties. Nucleic acids featured herein can include one of the following at the 2' position: H (deoxyribose); OH (ribose); F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl (wherein alkyl, alkenyl, and alkynyl are C1-C10 alkyl or C2-C10 alkenyl and alkynyl, which may be substituted or unsubstituted). Exemplary modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments, the synthetic modified RNA comprises one of the following at the 2' position: C1-C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, reporter group, intercalator, group for improving the pharmacokinetic properties of RNA, or group for improving the pharmacodynamic properties of synthetic modified RNA, and other substituents with similar properties. In some embodiments, the modification comprises 2' methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2)2.

[0286] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the nucleic acid sequence, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked nucleotides, and the 5' position of 5' terminal nucleotide. Synthetic modified RNAs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; and 5,576 ,427; U.S. Patent No. 5,591,722; U.S. Patent No. 5,597,909; U.S. Patent No. 5,610,300; U.S. Patent No. 5,627,053; U.S. Patent No. 5,639,873; U.S. Patent No. 5,646,265; U.S. Patent No. 5,658,873; U.S. Patent No. 5,670,633; and U.S. Patent No. 5,700,920 (some of which are commonly owned with the present application and each of which is incorporated by reference herein in its entirety).

[0287] As non-limiting examples, the synthetic modified RNAs described herein can contain at least one modified nucleoside, including a 2'-O-methyl modified nucleoside, a nucleoside containing a 5' phosphorothioate group, a 2'-amino-modified nucleoside, a 2'-alkyl-modified nucleoside, a morpholino nucleoside, a phosphoramidate or unnatural base containing nucleoside, or any combination thereof.

[0288] In some embodiments of this and all other such aspects described herein, the at least one modified nucleoside is 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3,2'-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2'-fluorouridine, pseudouridine, 2'-O-methyluridine (Um), 2'-deoxyuridine (2'dU), 4-thiouridine (s4U), 5-methyluridine (5mC ... and inosine (I).

[0289] Alternatively, synthetic modified RNA can contain at least two modified nucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more nucleotides of maximum total length. At a minimum, synthetic modified RNA molecules containing at least one modified nucleoside contain a single nucleoside with the modification described herein. It is not necessary for all positions in a given synthetic modified RNA to be uniformly modified; in fact, multiple of the above modifications can be incorporated in a single synthetic modified RNA, or even in a single nucleoside within a synthetic modified RNA. However, while it is preferred, it is not absolutely necessary that every occurrence of a given nucleoside in the molecule be modified (e.g., each cytosine is a modified cytosine, e.g., 5mC). However, it is also contemplated that different occurrences of the same nucleoside can be modified in different ways in a given synthetic modified RNA molecule (e.g., some cytosines are modified as 5mC, while others are modified as 2'-O-methylcytidine or other cytosine analogs). The modification need not be the same for each of multiple modified nucleosides in a synthetic modified RNA. Furthermore, in some embodiments of the aspects described herein, the synthetic modified RNA comprises at least two different modified nucleosides. In some such preferred embodiments of the aspects described herein, the at least two different modified nucleosides are 5-methylcytidine and pseudouridine. The synthetic modified RNA can also contain a mixture of both modified and unmodified nucleosides.

[0290] As used herein, "unmodified" or "natural" nucleosides or nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). In some embodiments, synthetic modified RNAs contain at least one nucleoside ("base") modification or substitution. Modified nucleosides include other synthetic and natural nucleobases, such as inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2(amino)adenine, 2-(aminoalkyl)adenine, 2(aminopropyl)adenine, 2(methylthio)N6(isopentenyl)adenine, 6(alkyl)adenine, 6(methyl)adenine, 7(deaza)adenine, and the like. N6-(isopentyl)adenine, N6-(methyl)adenine, N6,N6-dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(isopentyl)aden ... (al)guanine, 7(methyl)guanine, 7(deaza)guanine, 8(alkyl)guanine, 8-(alkenyl)guanine, 8(alkynyl)guanine, 8-(amino)guanine, 8(halo)guanine, 8-(hydroxyl)guanine, 8(thioalkyl)guanine, 8-(thiol)guanine, N(methyl)guanine, 2-(thio)cytosine, 3(deaza)5(aza)cytosine, 3-(alkyl)cytosine, 3(methyl)cytosine, 5-(alkyl)cytosine cytosine, 5-(alkynyl)cytosine, 5(halo)cytosine, 5(methyl)cytosine, 5(propynyl)cytosine, 5(propynyl)cytosine, 5(trifluoromethyl)cytosine, 6-(azo)cytosine, N4(acetyl)cytosine, 3(3amino-3carboxypropyl)uracil, 2-(thio)uracil, 5(methyl)2(thio)uracil, 5(methylaminomethyl)-2(thio)uracil, 4-(thio)uracil, 5(methyl)4(thio)uracil,5(methylaminomethyl)-4(thio)uracil, 5(methyl)2,4(dithio)uracil, 5(methylaminomethyl)-2,4(dithio)uracil, 5(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5(aminoallyl)uracil, 5(aminoalkyl)uracil, 5(guanidinium alkyl)uracil, 5(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5(dimethicone) (methylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3(methyl)uracil, 5-uracil (i.e., pseudouracil), 2(thio)pseudouracil, 4(thio)pseudouracil, 2,4-(dithio)pseudouracil , 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4(thio)pseudouracil, 5-(methyl)-4(thio)pseudouracil, 5-(alkyl)-2,4(dithio)pseudouracil, 5-(methyl)-2,4(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil Uracil, 1(aminocarbonylethylenyl)-pseudouracil, 1(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1(aminocarbonylethylenyl)-4(thio)pseudouracil, 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1(aminoalkylaminocarbonylethylenyl)-4(thio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3- (aza)-phenthiazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidinium alkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6- (Dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl,9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, difluorotrimethylphenyl 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidine, N2-substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole, pyrrolo-pyrimidin-2-one-3-one 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-(aminoalkyl) Modified nucleosides include (but are not limited to) 2-(2-(2-methyl-2-phenyl)pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, or any O- or N-alkylated derivative thereof. Modified nucleosides also include conjugate moieties, such as natural bases containing ligands. As discussed hereinabove, RNA containing modified nucleosides must be translatable in host cells (i.e., do not prevent translation of the polypeptide encoded by the modified RNA). For example, transcripts containing s2U and m6A are poorly translated in rabbit reticulocyte lysate, while pseudouridine, m5U, and m5C are compatible with efficient translation. In addition,It is known in the art that 2'-fluoro-modified bases, useful for increasing the nuclease resistance of transcripts, result in very inefficient translation. One skilled in the art can assay translation using, for example, a rabbit reticulocyte lysate translation assay.

[0291] Further modified nucleobases include those disclosed in U.S. Pat. No. 3,687,808, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; International Patent Application No. PCT / US09 / 038,425, filed March 26, 2009; The Concise Encyclopedia Of Polymer Science and those disclosed in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0292] Representative U.S. patents that teach the preparation of some of the above and other modified nucleobases include, but are not limited to, the above-mentioned U.S. Pat. No. 3,687,808 as well as U.S. Pat. No. 4,845,205; U.S. Pat. No. 5,130,300; U.S. Pat. No. 5,134,066; U.S. Pat. No. 5,175,273; U.S. Pat. No. 5,367,066; U.S. Pat. No. 5,432,272; U.S. Pat. No. 5,457,187; U.S. Pat. No. 5,457,191; U.S. Pat. No. 5,459,255; U.S. Pat. No. 5,484,908; U.S. Pat. No. 5,502,177; U.S. Pat. No. 5,525,711; U.S. Pat. No. 5,552,540; U.S. Pat. No. 5,587,469; U.S. Pat. No. 5,594,121; U.S. Pat. No. 5,594,121; U.S. Pat. No. 5,595,122; U.S. Pat. No. 5,596,123; U.S. Pat. No. 5,597,124; U.S. Pat. No. 5,598,125; U.S. Pat. No. 5,599,121; U.S. Pat. No. 5,599,122; U.S. Pat. No. 5,599,123; U.S. Pat. No. 5,599,1 ,596,091; U.S. Patent No. 5,614,617; U.S. Patent No. 5,681,941; U.S. Patent No. 6,015,886; U.S. Patent No. 6,147,200; U.S. Patent No. 6,166,197; U.S. Patent No. 6,222,025; U.S. Patent No. 6,235,887; U.S. Patent No. 6,380,368; U.S. Patent No. 6,528,640; U.S. Patent No. 6,639,062; U.S. Patent No. 6,617,438; U.S. Patent No. 7,045,610; U.S. Patent No. 7,427,672; and U.S. Patent No. 7,495,088 (each of which is incorporated by reference herein in its entirety) and U.S. Patent No. 5,750,692 (also incorporated by reference herein in its entirety).

[0293] Another modification for use in the synthetic modified RNAs described herein involves chemically linking one or more ligands, moieties, or conjugates to the RNA that enhance the activity, cellular distribution, or cellular uptake of the RNA. The synthetic modified RNAs described herein can further comprise a 5' cap. In some embodiments of the aspects described herein, the synthetic modified RNA comprises a 5' cap comprising a modified guanine nucleotide linked to the 5' end of the RNA molecule using a 5'-5' triphosphate linkage. As used herein, the term "5' cap" also refers to, for example, a 5' diguanosine cap, a tetraphosphate cap analog having a methylene-bis(phosphonate) moiety (see, e.g., Rydzik, AM et al., (2009) Org Biomol Chem 7(22):4763-76), a dinucleotide cap analog having a phosphorothioate modification (see, e.g., Kowalska, J et al., (2008) RNA 14(6):1119-1131), a cap analog having a sulfur substitution for a non-bridging oxygen (see, e.g., Grudzien-Nogalska, E et al., (2007) RNA 13(10):1745-1755), an N7-benzylated dinucleoside tetraphosphate analog (see, e.g., Grudzien, E et al., (2004) RNA 10(9):1479-1487), or anti-reverse cap analogs (see, e.g., Jemielity, J et al., (2003) RNA 9(9):1108-1122 and Stepinski, J et al., (2001) RNA 7(10):1486-1495). In one such embodiment, the 5' cap analog is a 5' diguanosine cap. In some embodiments, the synthetic, modified RNA does not contain a 5' triphosphate.

[0294] The 5' cap is important for initiating translation, recognizing mRNA, and attaching mRNA to ribosomes. The 5' cap also protects synthetic modified RNA from 5' exonuclease-mediated degradation. It is not absolutely necessary for synthetic modified RNA to contain a 5' cap, and therefore, in other embodiments, synthetic modified RNA lacks a 5' cap. However, due to the longer half-life of synthetic modified RNA containing a 5' cap and the increased translation efficiency, synthetic modified RNA containing a 5' cap is preferred herein.

[0295] The synthetic modified RNAs described herein may further comprise 5' and / or 3' untranslated regions (UTRs). Untranslated regions are regions of RNA before the start codon (5') and after the stop codon (3') that are not translated by the translational machinery. Because untranslated regions can interfere with ribonucleases and other proteins involved in RNA degradation, modifying RNA molecules with one or more untranslated regions can improve mRNA stability. In addition, modifying RNAs with 5' and / or 3' untranslated regions can enhance translation efficiency by altering ribosome binding to mRNA through binding proteins. Modifying RNAs with 3' UTRs can be used to maintain cytoplasmic localization of the RNA, allowing translation to occur in the cytoplasm of the cell. In one embodiment, the synthetic modified RNAs described herein do not comprise a 5' or 3' UTR. In another embodiment, the synthetic modified RNAs comprise a 5' or 3' UTR. In another embodiment, the synthetic modified RNAs described herein comprise both a 5' and a 3' UTR. In one embodiment, the 5' and / or 3' UTR is selected from an mRNA known to have high stability in cells (e.g., mouse alpha-globin 3' UTR). In some embodiments, the 5' UTR, 3' UTR, or both, comprise one or more modified nucleosides.

[0296] In some embodiments, the synthetic modified RNA described herein further comprises a Kozak sequence. A "Kozak sequence" refers to a sequence on eukaryotic mRNAs that has the consensus (gcc)gccRccAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G." The Kozak consensus sequence is recognized by the ribosome, initiating polypeptide translation. Typically, initiation occurs at the first AUG codon encountered by the translation machinery proximal to the 5' end of the transcript. However, in some cases, this AUG codon can be bypassed in a process called leaky scanning. The presence of a Kozak sequence near an AUG codon reinforces that codon as the initiation site for translation, so that translation of the correct polypeptide occurs. Furthermore, the addition of a Kozak sequence to a synthetic modified RNA promotes more efficient translation, even when there is no ambiguity regarding the start codon. Thus, in some embodiments, the synthetic, modified RNAs described herein further comprise a Kozak consensus sequence for initiation of translation at a desired site to produce a polypeptide of the correct length. In some such embodiments, the Kozak sequence comprises one or more modified nucleosides.

[0297] In some embodiments, the synthetic modified RNAs described herein further comprise a "poly(A) tail," which refers to a 3' homopolymeric tail of adenine nucleotides, which can vary in length (e.g., at least five adenine nucleotides) and can be up to several hundred adenine nucleotides. The inclusion of a 3' poly(A) tail can protect the synthetic modified RNA from degradation in the cell and also promote extranuclear localization, enhancing translation efficiency. In some embodiments, the poly(A) tail comprises between 1 and 500 adenine nucleotides; in other embodiments, the poly(A) tail comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more adenine nucleotides. In one embodiment, the poly(A) tail comprises between 1 and 150 adenine nucleotides. In another embodiment, the poly(A) tail comprises between 90 and 120 adenine nucleotides. In some such embodiments, the poly(A) tail comprises one or more modified nucleosides.

[0298] It is contemplated that one or more modifications to the synthetic modified RNA described herein may allow for greater stability of the synthetic modified RNA in cells. To the extent that such modifications allow translation and do not reduce or exacerbate the cell's innate immune or interferon response to the synthetic modified RNA having the modifications, such modifications are particularly contemplated for use herein. Generally, the more stable a synthetic modified RNA is, the more protein can be produced from the synthetic modified RNA. Typically, the presence of an AU-rich region in mammalian mRNA tends to destabilize the transcript, since cellular proteins are recruited to the AU-rich region and stimulate the removal of the poly(A) tail of the transcript. Loss of the poly(A) tail of the synthetic modified RNA can result in increased RNA degradation. Therefore, in one embodiment, the synthetic modified RNA described herein does not contain an AU-rich region. In particular, it is preferred that the 3'UTR is substantially devoid of AUUUA sequence elements.

[0299] In one embodiment, the ligand changes the cellular uptake, targeting in cells, or half-life of the synthetic modified RNA that the ligand is incorporated into.In some embodiments, the ligand achieves enhanced affinity for selected targets, such as molecules, cells or cell types, intracellular compartments, such as mitochondria, cytoplasm, peroxisomes, lysosomes, for example, compared to compositions that lack such ligands.Preferred ligands do not interfere with the expression of polypeptides from synthetic modified RNA.

[0300] The ligand can be, for example, a substance, e.g., a drug, that can increase the uptake of the synthetic modified RNA or composition thereof into cells by, for example, disrupting the cytoskeleton of the cell, e.g., disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0301] In another embodiment, the ligand is a moiety, such as a vitamin, that is taken up by host cells.Exemplary vitamins include vitamins A, E, and K.Other exemplary vitamins include vitamin B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by cancer cells.Also included are HSA and low-density lipoprotein (LDL).

[0302] In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennopedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. The helical agent is preferably an α-helical agent, preferably having a lipophilic and lipophobic phase.

[0303] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or Ceropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two key amino acids (e.g., PR-39 or indolicidin). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and NLS of the SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0304] The synthetic modified RNAs described herein can be synthesized and / or modified by methods well established in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference in its entirety. Transcription methods are further described in the Examples herein. In one embodiment of the aspects described herein, templates for the synthetic modified RNAs are synthesized using "splint-mediated ligation," which allows for the rapid synthesis of DNA constructs by controlled concatemer formation of long oligos and / or dsDNA PCR products, without the need to introduce restriction sites at the junction regions. This can be used to add general untranslated regions (UTRs) to the coding sequence of a gene during T7 template generation. Splint-mediated ligation can also be used to add nuclear localization sequences to open reading frames and to create dominant-negative constructs with point mutations starting from the wild-type open reading frame. Briefly, single-stranded and / or denatured dsDNA building blocks are annealed to splint oligos, thereby connecting desired ends, and the ends are ligated by thermostable DNA ligase, and the desired construct is amplified by PCR.Then, synthetic modified RNA is synthesized from the template using RNA polymerase in vitro.After the synthesis of synthetic modified RNA is completed, the DNA template is removed from the transcription reaction before being used in the method described herein.

[0305] In some embodiments of these aspects, the synthetic, modified RNA is further treated with alkaline phosphatase.

[0306] Those skilled in the art will readily recognize that the present invention is well adapted to carry out the objects and obtain the aims and advantages mentioned, as well as those inherent therein. The details of the descriptions and examples herein are representative of particular embodiments and are exemplary and are not intended to limit the scope of the invention. Those skilled in the art will recognize modifications therein and other uses. These modifications are encompassed within the spirit of the invention. It will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0307] The articles "a" and "an," as used herein, should be understood to include plural referents unless the specification and claims clearly indicate to the contrary. A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise apparent from the context. The invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process. Furthermore, it should be understood that the invention provides for all variations, combinations, and permutations, and that one or more limits, elements, clauses, descriptive terms, etc. from one or more of the claims listed herein may be introduced into another claim in accordance with the same base claim (or any other claim, if relevant), unless otherwise indicated or unless a contradiction or inconsistency would be apparent to one skilled in the art. All embodiments described herein are contemplated as being applicable to all different aspects of the invention, where appropriate. It is also contemplated that any of the embodiments or aspects may be freely combined with one or more other such embodiments or aspects, where deemed appropriate. When elements are presented as a list, e.g., in a Markush group or similar format, it should be understood that each subgroup of elements is also disclosed, and that any element may be removed from the group. In general, when the invention, or aspects of the invention, are referred to as comprising particular elements, features, etc., it should be understood that the particular embodiment or aspect of the invention consists of, or consists essentially of, such elements, features, etc.For the sake of brevity, these embodiments have not in every instance been specifically described in so many words herein. It should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a specific exclusion is set forth in the specification. For example, any one or more active agents, additives, ingredients, optional drugs, organisms, disorders, subjects, or combinations thereof may be excluded.

[0308] Where a claim or description is directed to a subject composition, it should be understood that methods of making or using the subject composition by any of the methods disclosed herein, and methods of using the subject composition for any of the purposes disclosed herein, are aspects of the invention unless otherwise indicated or unless a contradiction or inconsistency would arise...

Claims

[Claim 1] The invention described in the present specification.

Images