Engineered immune cells and uses thereof

Abstract

Provided herein are compositions and methods that include engineered immune cells (e.g., engineered T cells) with reduced expression of endogenous T-cell receptor (TCR) chains and expression of exogenous TCR chains. The compositions and methods described herein can increase the efficiency of down-regulating the endogenous TCR and expressing the exogenous TCR.

Description

ENGINEERED IMMUNE CELLS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 658,185, filed on June 10, 2024, the entire content of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] TCR-T therapy has emerged as an important intervention for targeting diseases such as cancer or autoimmune diseases. Two approaches used for preparing therapeutic T cells involve lentiviral transduction that integrates therapeutic T-cell receptor (TCR) sequence into host genome in a non-directed manner; and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9-based insertion of therapeutic TCR sequence into a pre-defined locus based on the homology of the guide RNA with the host genome. These approaches have been met with difficulties in low yield transduction and generating potentially autoimmune T cells due to mispairing of therapeutic TCR with endogenous one. There is a need for new strategies for improving transduction and expression of therapeutic TCRs in T cells for TCR-T therapy.BRIEF SUMMARY

[0003] Recognized herein is a need for improved methods for producing engineered cells expressing therapeutic TCRs. Provided herein are compositions and methods that include engineered T cells with nucleic acid sequences to (i) down-regulate endogenous TCR using a gene editing system and (ii) express an exogenous TCR chain. The present disclosure illustrates this approach and methods for generating engineered T cells for the use in treating diseases and conditions. The combination of nucleic acid sequences can provide enhanced expression of therapeutic TCR and reduce mispairing with endogenous TCR in therapeutic T cells. The present disclosure further includes diagnostic and / or therapeutic methods using the engineered T cells, population of T cells, vectors, and pharmaceutical compositions described herein.

[0004] In an aspect, the present disclosure provides an engineered T cell comprising: (a) a modified genome comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC); and (b) a genome editing system, wherein the genome editing system (i) is capable of modifying or removing a genomic sequence encoding an endogenous TCR comprising the same hTRC as the exogenous TCR, and (ii) is notcapable of substantially modifying or removing the sequence encoding the exogenous TCR of the modified genome.

[0005] In some embodiments, the genome editing system comprises a guide RNA targeting an endogenous target sequence or a complement thereof that encodes at least 4 amino acids of an endogenous hTRC, and / or an intron of an endogenous hTRC.

[0006] In some aspects, the present disclosure provides a method of preparing an engineered T cell comprising: (a) integrating into the genome of a T cell a vector comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC); and (b) modifying or removing a genomic sequence encoding an endogenous TCR comprising the same hTRC as the exogenous TCR with a genome editing system; wherein the genome editing system is not capable of substantially modifying or removing the sequence encoding the exogenous TCR.

[0007] In some aspects, the present disclosure provides an engineered T cell comprising: (a) a modified genome comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC), wherein the exogenous sequence comprises an exogenous subsequence encoding the hTRC of the exogenous TCR or a portion thereof; and (b) a guide RNA, wherein the guide RNA comprises a nucleic acid sequence comprising a targeting sequence that hybridizes to an endogenous target sequence within the modified genome, wherein the endogenous target sequence or a complement thereof encodes at least 4 amino acids of an endogenous hTRC; wherein the amino acid sequence of the hTRC of the exogenous TCR or the portion thereof encoded by the exogenous subsequence of the exogenous sequence, and the amino acid sequence encoded by the endogenous target sequence or a complement thereof, are the same amino acid sequence; and wherein the endogenous target sequence and the complement thereof each have less than 100% sequence identity to the exogenous subsequence encoding the hTRC or the portion thereof.

[0008] In some embodiments, (i) the hTRC is a human TCR alpha constant domain (hTRAC) and the endogenous hTRC is an endogenous hTRAC, or (ii) the hTRC is a human TCR beta constant domain (hTRBC) and the endogenous hTRC is an endogenous hTRBC.

[0009] In some embodiments, the modified genome comprises (a) a first exogenous sequence encoding an exogenous TCR alpha chain comprising a human TCR alpha constant domain (hTRAC), wherein the first exogenous sequence comprises a first exogenous subsequence encoding the hTRAC or a portion thereof of the exogenous TCR alpha chain; and (b) a second exogenous sequence encoding an exogenous TCR beta chain comprising a human TCR betaconstant domain (hTRBC), wherein the second exogenous sequence comprises a second exogenous subsequence encoding the hTRBC or a portion thereof of the exogenous TCR beta chain.

[0010] In some embodiments, the guide RNA comprises a first guide RNA and a second guide RNA, wherein (a) the first guide RNA comprises nucleic acid sequence comprising a first targeting sequence that hybridizes to a first endogenous target sequence within the modified genome, wherein the first endogenous target sequence or a complement thereof encodes at least 4 amino acids of the endogenous hTRAC; and (b) the second guide RNA comprises nucleic acid sequence comprising a second targeting sequence that hybridizes to a second endogenous target sequence within the modified genome, wherein the second endogenous target sequence or a complement thereof encodes at least 4 amino acids of the endogenous hTRBC.

[0011] In some embodiments, (a) the amino acid sequence of the hTRAC of the first exogenous TCR or the portion thereof encoded by the first exogenous subsequence of the first exogenous sequence, and the amino acid sequence encoded by the first endogenous target sequence or a complement thereof, are the same amino acid sequence; and (b) the amino acid sequence of the hTRBC of the second exogenous TCR or the portion thereof encoded by the second exogenous subsequence of the second exogenous sequence, and the amino acid sequence encoded by the second endogenous target sequence or a complement thereof, are the same amino acid sequence.

[0012] In some embodiments, (a) the first endogenous target sequence and the complement thereof each have less than 100% sequence identity to the first exogenous subsequence encoding the hTRAC or the portion thereof; and (b) the second endogenous target sequence and the complement thereof each have less than 100% sequence identity to the second exogenous subsequence encoding the hTRBC or the portion thereof. In some embodiments, the engineered T cell is a engineered human T cell or an engineered cell derived from a human T cell.

[0013] In some embodiments, the engineered T cell has reduced cell-surface expression of an endogenous TCR when compared to an unmodified control cell. In some embodiments, the engineered T cell has increased cell-surface expression of the exogenous TCR chain when compared to a control cell comprising (a) a modified genome comprising the exogenous sequence encoding the exogenous TCR chain comprising the hTRC; and (b) a control guide RNA comprising a targeting sequence that hybridizes to the endogenous target sequence within the modified genome, but that has 100% complementarity to the exogenous subsequence encoding the hTRC or the portion thereof or that has 100% complementarity to the complement of the exogenous subsequence encoding the hTRC or the portion thereof.

[0014] In some embodiments, the targeting sequence of the guide RNA hybridizes to the exogenous subsequence encoding the hTRC or a portion thereof at least 2 fold less than to the endogenous target sequence of the guide RNA. In some embodiments, the targeting sequence of the first guide RNA hybridizes to the exogenous subsequence encoding the hTRAC or a portion thereof at least 2 fold less than to the endogenous target sequence of the first guide RNA. In some embodiments, the targeting sequence of the second guide RNA hybridizes to the exogenous subsequence encoding the hTRBC or a portion thereof at least 2 fold less than to the endogenous target sequence of second guide RNA.

[0015] In some embodiments, the endogenous target sequence comprises a protospacer adjacent motif (PAM) sequence or complement thereof. In some embodiments, the PAM sequence or complement thereof has less than 100% sequence identity to corresponding region of the exogenous subsequence encoding the hTRC or the portion thereof. In some embodiments, the engineered T cell lacks a sequence encoding a mouse TCR or a mouse TRC (mTRC).

[0016] In some aspects, the present disclosure provides an engineered T cell comprising: (a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and (b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein the first nucleic acid sequence: (i) does not hybridize to the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0017] In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

[0018] In some embodiments, the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence. In some embodiments, a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variationrelative to the target sequence or a complement thereof, and wherein the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0019] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO : 21, and SEQ ID NO.:22.

[0020] In some embodiments, a sequence downstream of the subsequence of the second nucleic acid sequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof. In some embodiments, a CRISPR-Cas system comprising the first nucleic acid sequence (i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof or (ii) recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

[0021] In some aspects, the present disclosure provides an engineered T cell comprising: (a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence or derivative thereof comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and (b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity; and wherein a CRISPR-Cas system comprising the first nucleic acid sequence: (i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof, or (ii) recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

[0022] In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length. In some embodiments, the TCR constantdomain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

[0023] In some embodiments, a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: has less than 100% sequence identity to the target sequence or a complement thereof, or comprises a mutation or variation relative to the target sequence or a complement thereof.

[0024] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO : 21, and SEQ ID NO.: 22.

[0025] In some embodiments, the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0026] In some aspects, the present disclosure provides an engineered T cell comprising: (a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and (b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, wherein (i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system, and / or (ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than downregulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence.

[0027] In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length. In some embodiments, the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

[0028] In some embodiments, the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

[0029] In some embodiments, a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

[0030] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO : 21, and SEQ ID NO.: 22.

[0031] In some embodiments, an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system. In some embodiments, the target sequence comprises a non-coding sequence. In some embodiments, the non-coding sequence comprises a portion of an intron. In some embodiments, the portion of the intron is at least 1 nucleotide in length. In some embodiments, the endogenous TCR chain is a human TCR chain and the exogenous TCR chain is a human TCR chain.

[0032] In some embodiments, the first nucleic acid sequence is transiently expressed in the engineered T cell. In some embodiments, the endogenous TCR chain is an endogenous TCR alpha chain, and the TCR constant domain is a TCR alpha constant domain. In some embodiments, the endogenous TCR chain is an endogenous TCR beta chain, and the TCR constant domain is a TCR beta constant domain. In some embodiments, the endogenous TCR chain comprises a endogenous TCR alpha chain and a endogenous TCR beta chain, and the TCR constant domain comprises a TCR alpha constant domain and a TCR beta constant domain. In some embodiments, theendogenous TCR chain is a endogenous TCR gamma chain, and the TCR constant domain is a TCR gamma constant domain. In some embodiments, the endogenous TCR chain is a endogenous TCR delta chain, and the TCR constant domain is a TCR delta constant domain. In some embodiments, the endogenous TCR chain comprises a endogenous TCR gamma chain and a endogenous TCR delta chain, and the TCR constant domain comprises a TCR gamma constant domain and a TCR delta constant domain.

[0033] In some embodiments, the first nucleic acid sequence comprises an RNA sequence. In some embodiments, the RNA sequence is a guide RNA (gRNA) sequence. In some embodiments, the gRNA sequence hybridizes to the target sequence or is complementary to the target sequence.

[0034] In some embodiments, the endogenous TCR chain comprises one or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acid sequence comprises one or more gRNA sequences targeting the one or more endogenous TCR chains. In some embodiments, the endogenous TCR chain comprises two or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acid sequence comprises two or more gRNA sequences targeting the two or more endogenous TCR chains. In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain and an endogenous TCR beta chain, and wherein the first nucleic acid sequence comprises a first gRNA comprising a targeting sequence that hybridizes to a first target sequence within a genomic sequence encoding a TCR alpha constant domain of the endogenous TCR alpha chain, and a second gRNA comprising a targeting sequence that hybridizes to a second target sequence within a genomic sequence encoding a TCR beta constant domain of the endogenous TCR beta chain.

[0035] In some embodiments, the second nucleic acid sequence comprises a sequence encoding an exogenous TCR alpha chain and a sequence encoding an exogenous TCR beta chain, wherein (A) the subsequence of the sequence encoding the exogenous TCR alpha chain (i) has less than 100% sequence identity to the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the first target sequence or a complement thereof, and the subsequence of the sequence encoding the exogenous TCR beta chain (i) has less than 100% sequence identity to the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the second target sequence or a complement thereof; and / or (B) a sequence downstream of the subsequence of the sequence encoding the exogenous TCR alphachain (i) has less than 100% sequence identity to a corresponding sequence downstream of the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the first target sequence or a complement thereof, and a sequence downstream of the subsequence of the sequence encoding the exogenous TCRbeta chain (i) has less than 100% sequence identity to a corresponding sequence downstream of the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the second target sequence or a complement thereof.

[0036] In some embodiments, the first nucleic acid sequence comprises a sequence with 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6 and 12.

[0037] In some embodiments, the first nucleic acid sequence is encoded by a DNA sequence.

[0038] In some embodiments, (i) the first nucleic acid sequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on the same nucleic acid molecule. In some embodiments, (i) the first nucleic acid sequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on different nucleic acid molecules.

[0039] In some embodiments, the corresponding sequence downstream of the target sequence or a complement thereof comprises a protospacer adjacent motif (PAM) site, and the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a mutation of the PAM site. In some embodiments, the PAM site comprises a sequence selected from the group consisting of NGG, NGRRT, NGRRN, NNNNGATT, NNNNRYAC, or NNAGAAW, where N is A, T, C, or G; R is A or G; and Y is T or C. In some embodiments, the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a sequence of SEQ Identifier A3.

[0040] In some embodiments, the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is comprised within a population of T cells, and wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or more of the T cells in the population of T cells have a reduced or eliminated expression of an endogenous TCR or an endogenous TCR chain. In some embodiments, the engineered T cell comprises a plurality of engineered T cells, and wherein at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold or more of the engineered T cells in the plurality have reduced or eliminated expression of an endogenous TCR compared to the number of engineered T cells that have reduced or eliminated expression of an endogenous TCR in an otherwise identical plurality of engineeredT cells comprising a single gRNA sequence targeting a single endogenous TCR chain. In some embodiments, the engineered T cells having a reduced or eliminated expression of an endogenous TCR is measured by a surface marker. In some embodiments, the surface marker is CD3.

[0041] In some embodiments, the engineered T cell is a plurality of transduced engineered T cells, wherein the plurality of transduced engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and transduced with the second nucleic acid sequence, and wherein the percentage of transduced engineered T cells derived from the population of T cells is higher compared to the percentage of transduced engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

[0042] In some embodiments, the percentage of transduced engineered T cells is at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more of the total T cells in the population of T cells from which the transduced engineered T cells are derived.

[0043] In some embodiments, the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence, and wherein the percentage of engineered T cells derived from the population of T cells that express the exogenous TCR is higher compared to the percentage of engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having(i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or(ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

[0044] In some embodiments, the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells comprises the first nucleic acid sequence and the second nucleic acid sequence, and wherein the plurality of engineered T cells expresses the exogenous TCR at a higher level compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstreamof the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

[0045] In some embodiments, the second nucleic acid sequence is encoded by a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the exogenous TCR chain recognizes an epitope of an antigen in complex with an MHC molecule.

[0046] In some embodiments, the epitope is associated with a cancer, an autoimmune disease, and / or an infectious disease. In some embodiments, the antigen is selected from the group consisting of NY-ESO-1, KRAS, TP53, NRAS, BRAF, and PIK3CA. In some embodiments, the first nucleic acid sequence is at least 15 nucleotides in length, at most 100 nucleotides in length, or from 15-100 nucleotides in length. In some embodiments, the targeting sequence is at least 15 nucleotides in length, at most 100 nucleotides in length, or from 15-100 nucleotides in length. In some embodiments, the amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence of SEQ ID NOs: 9 or 16. In some embodiments, the target sequence comprises a sequence of SEQ ID NOs: 8 or 15. In some embodiments, wherein the subsequence of the second nucleic acid sequence comprises a sequence of SEQ ID NOs: 8, 12, 15, or 19.

[0047] In some aspects, the present disclosure provides a method of generating an engineered T cell, the method comprising: delivering (a) a first nucleic acid sequence for down-regulating expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain, and (b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein the first nucleic acid sequence: (i) does not hybridize to the second nucleic acid sequence or a complement thereof or (ii) hybridizes to the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0048] In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length. In some embodiments, the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by thetarget sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence. In some embodiments, a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof, and wherein the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0049] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO : 21, and SEQ ID NO.: 22.

[0050] In some embodiments, a sequence downstream of the subsequence of the second nucleic acid sequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof. In some embodiments, a CRISPR-Cas system comprising the first nucleic acid sequence (i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof or (ii) recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

[0051] In some aspects, the present disclosure provides a method of generating an engineered T cell, the method comprising: delivering (a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and (b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity; and wherein a CRISPR-Cas system comprising the first nucleic acid sequence: (i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof, or (ii)recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

[0052] In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length. In some embodiments, the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence. In some embodiments, a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence (i) has less than 100% sequence identity to the target sequence or a complement thereof or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

[0053] In some embodiments, the first nucleic acid sequence (i) does not hybridize to a subsequence of the second nucleic acid sequence or a complement thereof or (ii) hybridizes to a subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence. In some embodiments, a sequence downstream of the subsequence of the second nucleic acid sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof, and wherein the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0054] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO : 21, and SEQ ID NO.: 22.

[0055] In some aspects, the present disclosure provides a method of generating an engineered T cell, the method comprising: delivering (a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and (b) a second nucleic acid sequence encoding an exogenousTCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, wherein (i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than downregulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system, (ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than down-regulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence, and / or (iii) an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system. In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length. In some embodiments, the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

[0056] In some embodiments, the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof. In some embodiments, a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

[0057] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO : 21, and SEQ ID NO.: 22.

[0058] In some embodiments, the first nucleic acid sequence is delivered into the T cell prior to, concurrently, or subsequent to delivering the second nucleic acid sequence. In some embodiments,the method further comprises, prior to delivering, activating the T cell. In some embodiments, the T cell has been activated. In some embodiments, a time period between delivering (i) and (ii) is at most 3 days, at most 2 days, at most 1 day, at most 20 hours, at most 12 hours, or less.

[0059] In some aspects, the present disclosure provides a method of generating an engineered T cell, wherein the engineered T cell has been delivered a first nucleic acid sequence for downregulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain, the method comprising: (a) delivering a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein (i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system, (ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than down-regulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence, and / or (iii) an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system.

[0060] In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length. In some embodiments, the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

[0061] In some embodiments, the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof. In some embodiments, a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutationor variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

[0062] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO : 21, and SEQ ID NO.: 22.

[0063] In some aspects, the present disclosure provides a method of generating an engineered T cell, the method comprising: delivering into an T cell a first nucleic acid sequence for downregulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence, (a) wherein the T cell has been delivered a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein (i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system, (ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than down-regulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence, and / or (iii) an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system.

[0064] In some embodiments, the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length. In some embodiments, the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

[0065] In some embodiments, the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequenceidentity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof. In some embodiments, a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

[0066] In some embodiments, the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22. In some embodiments, the endogenous TCR chain is a human TCR chain and the exogenous TCR chain is a human TCR chain. In some embodiments, the first nucleic acid sequence is transiently expressed in the engineered T cell.

[0067] In some embodiments, the endogenous TCR chain is an endogenous TCR alpha chain, and the TCR constant domain is a TCR alpha constant domain. In some embodiments, the endogenous TCR chain is an endogenous TCR beta chain, and the TCR constant domain is a TCR beta constant domain. In some embodiments, the endogenous TCR chain comprises a endogenous TCR alpha chain and a endogenous TCR beta chain, and the TCR constant domain comprises a TCR alpha constant domain and a TCR beta constant domain. In some embodiments, the endogenous TCR chain is a endogenous TCR gamma chain, and the TCR constant domain is a TCR gamma constant domain. In some embodiments, the endogenous TCR chain is a endogenous TCR delta chain, and the TCR constant domain is a TCR delta constant domain. In some embodiments, the endogenous TCR chain comprises a endogenous TCR gamma chain and a endogenous TCR delta chain, and the TCR constant domain comprises a TCR gamma constant domain and a TCR delta constant domain.

[0068] In some embodiments, the first nucleic acid sequence comprises an RNA sequence. In some embodiments, the RNA sequence is a guide RNA (gRNA) sequence. In some embodiments, the gRNA sequence hybridizes to the target sequence or is complementary to the target sequence.

[0069] In some embodiments, the endogenous TCR chain comprises one or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, a endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acid sequence comprises one or more gRNA sequences targeting the oneor more endogenous TCR chains. In some embodiments, the endogenous TCR chain comprises two or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, a endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acid sequence comprises two or more gRNA sequences targeting the two or more endogenous TCR chains. In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain and an endogenous TCR beta chain, and wherein the first nucleic acid sequence comprises a first gRNA comprising a targeting sequence that hybridizes to a first target sequence within a genomic sequence encoding a TCR alpha constant domain of the endogenous TCR alpha chain, and a second gRNA comprising a targeting sequence that hybridizes to a second target sequence within a genomic sequence encoding a TCR beta constant domain of the endogenous TCR beta chain.

[0070] In some embodiments, the second nucleic acid sequence comprises a sequence encoding an exogenous TCR alpha chain and a sequence encoding an exogenous TCR beta chain, wherein (A) the subsequence of the sequence encoding the exogenous TCR alpha chain (i) has less than 100% sequence identity to the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the first target sequence or a complement thereof, and the subsequence of the sequence encoding the exogenous TCR beta chain (i) has less than 100% sequence identity to the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the second target sequence or a complement thereof; and / or (B) a sequence downstream of the subsequence of the sequence encoding the exogenous TCR alpha chain (i) has less than 100% sequence identity to a corresponding sequence downstream of the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the first target sequence or a complement thereof, and a sequence downstream of the subsequence of the sequence encoding the exogenous TCR beta chain (i) has less than 100% sequence identity to a corresponding sequence downstream of the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the second target sequence or a complement thereof.

[0071] In some embodiments, the first nucleic acid sequence comprises a sequence with 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6 and 13. In some embodiments, the first nucleic acid sequence is encoded by a DNA sequence. In some embodiments, (i) the first nucleic acid sequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on the same nucleic acid molecule. In some embodiments, (i) the first nucleic acidsequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on different nucleic acid molecules.

[0072] In some embodiments, the corresponding sequence downstream of the target sequence or a complement thereof comprises a protospacer adjacent motif (PAM) site, and the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a mutation of the PAM site. In some embodiments, the PAM site comprises a sequence of NGG, NGRRT, NGRRN, NNNNGATT, NNNNRYAC, or NNAGAAW, where N is A, T, C, or G; R is A or G; and Y is T or C. In some embodiments, the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a sequence as set forth in SEQ Identifier A3.

[0073] In some embodiments, the amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence of SEQ ID NO: 9 or 16. In some embodiments, the target sequence comprises a sequence of SEQ ID NO: 8 or 15. In some embodiments, the subsequence of the second nucleic acid sequence comprises a sequence of SEQ ID NO: 8, 12, 15, or 19. In some embodiments, the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is comprised within a population of T cells, and wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or more of the T cells in the population of T cells have a reduced or eliminated expression of an endogenous TCR or an endogenous TCR chain. In some embodiments, the engineered T cell comprises a plurality of engineered T cells, and wherein at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold or more of the engineered T cells in the plurality have reduced or eliminated expression of an endogenous TCR compared to the number of engineered T cells that have reduced or eliminated expression of an endogenous TCR in an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain.

[0074] In some embodiments, the engineered T cells having a reduced or eliminated expression of an endogenous TCR is measured by a surface marker. In some embodiments, the surface marker is CD3.

[0075] In some embodiments, the engineered T cell is a plurality of transduced engineered T cells, wherein the plurality of transduced engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and transduced with the second nucleic acid sequence, and wherein the percentage of transduced engineered T cells derived from the population of T cells is higher compared to the percentage of transduced engineered T cells derived from anotherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

[0076] In some embodiments, the percentage of transduced engineered T cells is at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more of the total T cells in the population of T cells from which the transduced engineered T cells are derived. In some embodiments, the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence, and wherein the percentage of engineered T cells derived from the population of T cells that express the exogenous TCR is higher compared to the percentage of engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof. In some embodiments, the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells comprises the first nucleic acid sequence and the second nucleic acid sequence, and wherein the plurality of engineered T cells expresses the exogenous TCR at a higher level compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having(i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or(ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

[0077] In some embodiments, the method further comprises administering the engineered T cell to a subject in need thereof. In some embodiments, the T cell is obtained from the subject administered with the engineered T cell. In some embodiments, the T cell is obtained from a different subject from the subject administered with the engineered T cell.

[0078] In some aspects, the present disclosure provides a method of treating a disease or a condition in a subject in need thereof, the method comprising: administering the engineered T cell described herein to a subject in need thereof.

[0079] In some aspects, the present disclosure provides a vector comprising the first nucleic acid sequence and / or the second nucleic acid sequence described herein.

[0080] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector.

[0081] In some aspects, the present disclosure provides a population of T cells, wherein the population of T cells comprise at least two of the engineered T cell described herein.

[0082] In some aspects, the present disclosure provides a pharmaceutical composition comprising the engineered T cell described herein, the vector described herein, or the population of T cells described herein, and a pharmaceutically acceptable carrier.

[0083] In some aspects, the present disclosure provides use of the engineered T cell described herein, a T cell comprising the vector described herein, or the population of T cells described herein, in the manufacture of a medicament for treating a disease or a condition in a subject in need thereof.

[0084] In some embodiments, the disease or condition is a cancer, an autoimmune disease, and / or an infectious disease.

[0085] In some embodiments, the engineered T cell or the T cell is a CD8+ T cell, a CD4+ T cell, a CD4+ and CD8+ T cell, a gamma delta T cell, or a natural killer T cell.

[0086] In some aspects, the present disclosure provides the engineered T cell described herein, a T cell comprising the vector described herein, or the population of T cells described herein for use in the treatment of a disease or a condition.INCORPORATION BY REFERENCE

[0087] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and the disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles ofthe invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0089] FIGs. 1A-1H show surface expression of TCR / CD3 on CD8+ T cells after CRISPR-Cas9 editing. FIG. 1A shows surface expression of TCR / CD3 on T cells following no guide RNA (gRNA). FIG. IB shows surface expression of TCR / CD3 on T cells treated with a single gRNA (gl) targeting T-cell receptor alpha constant (TRAC). FIG. 1C shows surface expression of TCR / CD3 on T cells treated with a second single gRNA (g2) targeting TRAC. FIG. ID shows surface expression of TCR / CD3 on T cells following treatment with two gRNAs (gl&g2) for TRAC. FIG. IE shows surface expression of TCR / CD3 on T cells treated with a single gRNA (gl) for T-cell receptor beta constant (TRBC). FIG. IF shows surface expression of TCR / CD3 on T cells treated with a second single gRNA (g2) targeting TRBC. FIG. 1G shows surface expression of TCR / CD3 on T cells treated with two gRNAs (gl&g2) for TRBC. FIG. 1H shows surface expression of TCR / CD3 on T cells treated with two gRNAs for TRAC and two gRNAs for TRBC.

[0090] FIGs. 2A-2B show expression levels of 1G4 TCR (NYESO1 dextramer+) on CD8+ T cells. FIG. 2A shows expression of 1G4 TCR (NYESO1 dextramer+) on CD8+ T cells comprising no mutation of the PAM site downstream of the TRAC / TRBC sequence. FIG. 2B shows expression of 1G4 TCR (NYESO1 dextramer+) on CD8+ T cells comprising synonymous mutations of the PAM sites downstream of the TRAC / TRBC sequence.DETAILED DESCRIPTION

[0091] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed.Overview

[0092] The present disclosure provides compositions and methods for engineered cells and the use thereof in treating a disease or a condition in a subject in need thereof.

[0093] The process of targeted T cell therapy using engineered T cells can comprise three components: delivery of the transgene encoding the therapeutic T-cell receptor (TCR), stable expression of the transgene, and successful pairing between the alpha and beta chains or thegamma and delta chains of the therapeutic TCR. Two approaches that can be used for pairing of therapeutic TCRs can include (i) the use of engineered murine TCR constant regions instead of the human counterparts in the therapeutic TCR transgene; and (ii) the use of genetically “knocking out (KO)” endogenous TCR using CRISPR-Cas9 editing. For delivery and expression, lentiviral transduction of therapeutic TCR with the modified murine constant region can be used, and the CRISPR-Cas9 system can be utilized for insertion of therapeutic TCR into endogenous TCR alpha chain locus using homologous repair and disruption of TCR beta chain loci using non-homologous end joining. However, the murine TCR constant regions can carry a risk of being potentially recognized as foreign in human body and changing the nature of how human TCRs trigger downstream signaling upon antigen recognition. The editing of endogenous TCR and insertion of therapeutic TCR may yield low editing efficiency (e.g., 10-30% cells expressing therapeutic TCR).

[0094] To overcome the difficulties discussed above, methods can be developed to combine the advantages of CRISPR-Cas9 disruption of endogenous TCR gene expression with the high efficiency of lentiviral transduction of therapeutic TCR. Due to the nature of somatic rearrangement of TCR variable domains, the CRISPR editing can target the constant region of TCR alpha and beta chains (e.g., TRACs and / or TRBCs) to disrupt possible endogenous TCRs. As a result, directly combining the two approaches can yield low efficiency of expressing therapeutic TCRs because the resulting TCRs also contain the same constant region sequence and therefore become targets of CRISPR editing. To solve this problem, the present disclosure provides improved methods, which can lead to higher transduction efficiency and higher expression of exogenous TCRs. The methods described herein can comprise two components. Firstly, improved efficiency of CRISPR disruption of endogenous TCR expression by targeting (e.g., simultaneously targeting) one or more sites in the constant regions for TCR alpha and beta chains. This may be achieved by using a combination of guide RNAs for these target sites during CRISPR editing and increase the possibility of generating non-productive endogenous TCR gene. Secondly, avoiding the editing of therapeutic TCRs by modifying their constant region nucleotide sequences at target sites of CRISPR editing. Without modification, transduced TCRs may be subject to CRISPR disruption as the endogenous ones. Since CRISPR-Cas system of gene editing can use the recognition of the Protospacer Adjacent Motif (PAM) sequence and match between the guide RNA sequence and target site gene sequence, synonymous variants constant region sequence can be created that translate into the same protein sequence but do not contain the corresponding PAM or match the guide RNAs.

[0095] In the methods described herein, the T cells will be activated, transduced with lentivirus delivering therapeutic TCR, and then subjected to CRISPR-Cas (e.g., CRISPR-Cas9) mediated knockout of endogenous TCR alpha and beta chains. This strategy can significantly increase the efficiency of disrupting endogenous TCR and expressing therapeutic TCR.

[0096] The compositions and methods of the present disclosure can provide advantageous elements to produce the engineered T cells comprising the therapeutic TCRs. The methods provided herein can comprise activated T cells transduced with lentivirus comprising therapeutic T-cell receptors (TCR)s, providing increased expression of the therapeutic TCR and CRISPR- Cas9 mediated knockout of endogenous TCR.Terminology

[0097] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0098] The term “allogeneic”, as used herein, refers to any material derived from an individual that is transplanted into a genetically different recipient of the same species. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0099] The term “autologous,” as used herein, refers to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0100] The term “a” and “an” refers to one or to more than one (e.g., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0101] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0102] The term “consisting essentially of,” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0103] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0104] Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein.

[0105] As used in the specification and the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value encompasses variations of + / - 10%, + / - 5%, or + / - 1%. The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. As used herein, “about” and “approximately” may mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given range of values.

[0106] This disclosure includes every amino acid sequence described herein and all nucleotide sequences encoding the amino acid sequences. Every TCR sequence are included. Polynucleotide and amino acid sequences having from 80-99% similarity, inclusive, and including all numbers and ranges of numbers there between, with the sequences provided herein, are included in the invention. All of the amino acid sequences described herein can include amino acid substitutions,such as conservative substitutions, that do not adversely affect the function of the protein that comprises the amino acid sequences. In this regard, the disclosure provides alternative residues for certain positions in described sequences as described below. Any amino acid sequence described herein may have any single described residue change or a combination of described changes. Representative changes for particular TCRs are described the Tables provided herein. The changes can also include amino acid insertions. The disclosure includes each amino acid sequence that is encompassed by the description of alternative amino acids by reference to a specific sequence identifier and those described in the aforementioned Tables.

[0107] The compositions and methods of the present invention encompass polypeptides and nucleic acids having the sequences specified, or sequences substantially identical or similar thereto, e.g., sequences at least 80%, 85%, 90%, 95% identical or higher to the sequence specified. In the context of an amino acid sequence, the term “substantially identical” is used herein to refer to a first amino acid that contains a sufficient or minimum number of amino acid residues that are i) identical to, or ii) conservative substitutions of aligned amino acid residues in a second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and / or common functional activity. For example, amino acid sequences that contain a common structural domain having at least about 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein. In the context of nucleotide sequence, the term “substantially identical” is used herein to refer to a first nucleic acid sequence that contains a sufficient or minimum number of nucleotides that are identical to aligned nucleotides in a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity. For example, nucleotide sequences having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.5%, 99.9%, or 100% sequence identity to a reference sequence, e.g., a sequence provided herein.

[0108] The term “variant” can refer to a polypeptide that has a substantially identical amino acid sequence to a reference amino acid sequence, or is encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

[0109] Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In some embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions can then be compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).

[0110] The percent identity between the two sequences may be a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.[OHl] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CAB IOS, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleicacid molecule of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0112] The term “amino acid” can embrace all molecules, whether natural or synthetic, which include both an amino functionality and an acid functionality and capable of being included in a polymer of naturally-occurring amino acids. Amino acids can include naturally-occurring amino acids; analogs, derivatives and congeners thereof; amino acid analogs having variant side chains; and all stereoisomers of any of any of the foregoing. As used herein the term “amino acid” can comprise both the D- or L- optical isomers and peptidomimetics.

[0113] As used herein, the term “mutation” can refer to an alteration in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA. In some embodiments, the mutation may be a large-scale mutation, such as amplifications (or gene duplications) or repetitions of a chromosomal segment, deletions of large chromosomal regions, chromosomal rearrangements (e.g., chromosomal translocations, chromosomal inversions, non-homologous chromosomal crossover, and interstitial deletions), and loss of heterozygosity. In some embodiments, the mutation may be a small-scale mutation, such as insertions, deletions, and substitution mutations. As used herein, the term “substitution mutation” can refer to the transition that exchange a single nucleotide for another.

[0114] “Subject” generally refers to an animal, such as a mammal, for example a human. The methods described herein can be useful in both human therapeutics and veterinary applications. In some embodiments, the subject is a mammal, and in some embodiments, the subject is human. “Mammal” includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like.

[0115] “Patients” can be subjects suffering from or at risk of suffering from a disease, disorder, malignancy or condition, or otherwise in need of the compositions and / or methods provided herein. The terms “disease”, “disorder”, “malignancy”, and “condition” are used interchangeably.

[0116] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but arenot limited to, renal cell carcinoma, breast cancer, prostate cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, glioblastoma, brain cancer, colorectal cancer, stomach cancer, bladder cancer, testicular cancer, head and neck cancer, melanoma, prostate cancer, skin cancer, a hematological cancer, sarcoma, fibrosarcoma, angiosarcoma, osteosarcoma, rhabdomyosarcoma, leukemia, lymphoma, or myeloma and the like.

[0117] The term “T cell” and its grammatical equivalents as used herein can refer to a T cell from any origin. For example, a T cell can be a primary T cell, e.g., an autologous T cell, an allogeneic T cell, a T cell line, etc. The T cell can also be human or non-human. The term “T cell activation” or “T cell triggering” and its grammatical equivalents as used herein can refer to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation, cytokine production and / or detectable effector function. In some cases, “full T cell activation” can be similar to triggering T cell cytotoxicity. T cell activation can be measured using various assays known in the art. Said assays can be an ELISA to measure cytokine secretion, an ELISPOT, flow cytometry assays to measure intracellular cytokine expression, flow cytometry assays to measure proliferation, and cytotoxicity assays (51Cr release assay, impedance measurements, microscopic imaging measurements, or flow cytometry assays to enumerate live target cells) to determine target cell elimination. Said assays typically use controls (non-engineered cells) to compare to engineered cells (TCR-T) to determine relative activation of an engineered cell compared to a control. Additionally, said assays can compare engineered cells incubated or put in contact with a target cell not expressing the target antigen. For example, said comparison can be a TCR-T cell incubated with a target cell that does not express CD3.

[0118] The term “effective amount” or “therapeutically effective amount” may generally refer to a number of engineered T cells and / or an amount of a compound described herein that is sufficient to affect the intended application, including but not limited to disease treatment, as defined below. The therapeutically effective amount may vary depending upon the intended treatment application (in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells. The specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combinationwith other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0119] As used herein, “treatment” or “treating” generally refers to an approach for obtaining beneficial or desired results with respect to a disease, disorder, or medical condition including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. In certain embodiments, for prophylactic benefit, the compositions are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0120] The term “parenteral” administration of an immunogenic composition can include, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.

[0121] As used herein, “preventing” refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual. The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease, disease state, and / or condition.

[0122] The term “co-administration,” “administered in combination with,” and their grammatical equivalents, as used herein, encompass administration of two or more agents to an animal, including humans, so that both agents and / or their metabolites are present in the subject at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.

[0123] The terms “modulate” and "modulation" refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[0124] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, or greater in a recited variable.

[0125] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, 20 fold, 50 fold, 100 fold, or greater in a recited variable.

[0126] The term “expression” generally refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA (also referred to as a “transcript”) is subsequently translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectedly referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. The level of expression (or alternatively, the “expression level”) of a H0XA9 gene can be determined, for example, by determining the level of H0XA9 polynucleotides, polypeptides, and / or gene products. “Differentially expressed” or “differential expression” as applied to a nucleotide sequence (e.g., a gene) or polypeptide sequence in a subject, generally refers to the differential production of the mRNA transcribed and / or translated from the nucleotide sequence or the protein product encoded by the nucleotide sequence. A differentially expressed sequence may be overexpressed or under-expressed as compared to the expression level of a reference sample (i.e., a reference level). As used herein, elevated expression levels or overexpression refer to an increase in expression, generally at least 1.25 fold, or alternatively, at least 1.5 fold, or alternatively, at least 2 fold, or alternatively, at least 3 fold, or alternatively, at least 4 fold, or alternatively, at least 10 fold expression over that detected in a reference sample. As used herein, under-expression is a reduction in expression and generally is at least 1.25 fold, or alternatively, at least 1.5 fold, or alternatively, at least 2 fold, or alternatively, at least 3 fold, or alternatively, at least 4 fold, or alternatively, at least 10 fold expression under that detected in a reference sample. Under-expression also encompasses absence of expression of a particular sequence as evidenced by the absence of detectable expression in a test subject when compared to a reference sample.

[0127] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.

[0128] “Optional” or “optionally” means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not. For example, “optionally substituted aryl” means that the aryl group may or may not be substituted and that the description includes both substituted aryl groups and aryl groups having no substitution.

[0129] “Pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye, colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0130] The term “host cell” as used herein refers to a cell which can support the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells, such as yeast, insect cells, amphibian cells, or mammalian cells.

[0131] An “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0132] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0133] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Exemplary promoters include the immediate early cytomegalovirus (CMV), EF-la, ubiquitin C,or phosphoglycerokinase (PGK) promoters. A strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto can be used. Other constitutive promoter sequences may be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1 Ovian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, and the like. In some embodiments, the promoter is an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0134] The term “retroviral vector” can refer to a gammaretroviral vector. A retroviral vector may include, e.g., a promoter, a packaging signal, a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and polynucleotides of interest, e.g., a polynucleotide encoding a TCR. A retroviral vector may lack viral structural genes such as gag, pol, and env. Exemplary retroviral vectors include Murine Embryonic Stem Cell Virus (MESV), Murine Stem Cell Virus (MSCV), Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., in Maetzig et al., Viruses, 2011; 3(6): 677-713.

[0135] The term “lentivirus” refers to a genus of the Retroviridae family that may be used as a gene delivery vector as described herein. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell. HIV, SIV, and FIV are all examples of lentiviruses.

[0136] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX® vector system from Lentigenand the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0137] The term “in vivo” as used herein refers to inside the body of an organism. The terms “ex vivo” or “in vitro” as used herein refer to outside the body of the organism.

[0138] The term “engineered immune cell” as used herein refers to an immune cell that has been genetically modified as compared to a naturally occurring immune cell.

[0139] Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.Engineered Cells

[0140] In some aspects, the present disclosure provides an engineered immune cell (e.g., an engineered T cell). For example, an engineered T cell produced according to the methods described herein can carry a nucleic acid comprising a nucleotide sequence that does not naturally occur in a T cell from which it was derived.

[0141] In certain embodiments, the engineered immune cell is an engineered T cell, an engineered natural killer (NK) cell, or an engineered NK / T cell.

[0142] The engineered T cell can comprise a modified genome comprising an exogenous sequence. The exogenous sequence can encode an exogenous T-cell receptor (TCR) and / or TCR chain comprising a human TCR constant domain (hTRC). In some embodiments, the hTRC can be a human TCR alpha constant domain (hTRAC) or the hTRC can be a human TCR beta constant domain (hTRBC). In some embodiments, the hTRC can be a human TCR gamma constant domain or the hTRC can be a human TCR delta constant domain. The hTRC can be a hTRBC 1 or a hTRBC2. In some embodiments, the engineered immune cell (e.g., engineered T cell) comprises a genome editing system. The engineered T cell can comprise (a) a modified genome comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC); and (b) a genome editing system, wherein the genome editingsystem (i) is capable of modifying or removing a genomic sequence encoding an endogenous TCR comprising the same hTRC as the exogenous TCR, and (ii) is not capable of substantially modifying or removing the sequence encoding the exogenous TCR of the modified genome.

[0143] In some embodiments, the genome editing system may comprise a CRISPR system, a transcription activator-like effector (TALE)-nuclease system, and / or a zinc-finger nuclease system. The genome editing system may comprise a CRISPR system coupled with an endonuclease. The endonuclease can be an RNA-guided endonuclease. In some embodiments, the RNA-guided endonuclease is a CRISPR-associated protein 9 (Cas9) endonuclease. In some embodiments, the RNA-guided nuclease is a Cpfl nuclease. Other RNA-guided nucleases may be used. In some embodiments, the Cas9 endonuclease or Cpfl endonuclease is selected from A pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9, S. thermophilus CRISPRI Cas9, S. thermophilus CRISPR 3 Cas9, T. denticola Cas9, L. bacterium ND2006 Cpfl and Acidaminococcus sp. B V3L6 Cpfl . The genome editing system may be capable of modifying a genomic sequence. The genome editing system may be capable of removing a genomic sequence. The genomic sequence may encode an endogenous TCR. In some embodiments, the endogenous TCR is the same hTRC as the exogenous TCR. In some embodiments, the endogenous TCR is a different hTRC as the exogenous TCR. The engineered immune cell described herein can comprise a polynucleotide encoding an endonuclease described herein. For example, the engineered immune cell can comprise a polynucleotide (e.g., RNA or DNA) encoding a Cas9.

[0144] In some embodiments, the genome editing system is not capable of modifying the sequence (e.g., the exogenous sequence) encoding the exogenous TCR. In some embodiments, the genome editing system is not capable of removing the sequence (e.g., the exogenous sequence) encoding the exogenous TCR. In some embodiments, the exogenous sequence comprises a mutation. Without wishing to be bound by theory, the mutation of the exogenous sequence may prevent the genome editing system described herein from recognizing (e.g., modifying and / or removing) exogenous sequence. The genome editing system may comprise a guide nucleic acid (e.g., a guide RNA (gRNA)). The guide RNA can bind to an effector protein (e.g., an endonuclease) and form a complex. The complex may be referred to as a ribonucleoprotein complex. The guide RNA can guide the complex to a target nucleic acid. In some cases, the guide RNA is a nuclease-resistant guide RNA. For example, the guide RNA can comprise at least one 2'-OMe-phosphorothioate modified base, at least one 2'-O-methyl modified base, or at least one 2'-O-m ethyl 3' thioPACE modified base.

[0145] The guide RNA may target a nucleic acid sequence (e.g., an endogenous target sequence) or a complement thereof. The endogenous target sequence may encode at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, or greater than about 8 amino acids of an endogenous hTRC, an intron of an endogenous hTRC, or any combination thereof. The endogenous target sequence may encode at most about 8 amino acids, at most about 7 amino acids, at most about 6 amino acids, at most about 5 amino acids, at most about 4 amino acids, at most about 3 amino acids, at most about 2 amino acids, or less than about 8 amino acids of an endogenous hTRC, an intron of an endogenous hTRC, or any combination thereof.

[0146] The guide RNA can comprise a crispr RNA (crRNA), wherein the crRNA comprises a portion that can be complementary to the target nucleic acid sequence. In the CRISPR genome editing system, the crRNA may comprise a spacer region that hybridizes to a target sequence of a target nucleic acid. The spacer region can comprise the targeting sequence described herein. The spacer region can be the targeting sequence described herein. In some embodiments, the spacer region can be at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides in length. In some embodiments, the spacer region can be from about 10 nucleotides in length to about 20 nucleotides in length. In some embodiments, the spacer region can be from about 10 nucleotides to about 11 nucleotides, about 10 nucleotides to about 12 nucleotides, about 10 nucleotides to about 13 nucleotides, about 10 nucleotides to about 14 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 16 nucleotides, about 10 nucleotides to about 17 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 19 nucleotides, about 10 nucleotides to about 20 nucleotides, about 11 nucleotides to about 12 nucleotides, about 11 nucleotides to about 13 nucleotides, about 11 nucleotides to about 14 nucleotides, about 11 nucleotides to about 15 nucleotides, about 11 nucleotides to about 16 nucleotides, about 11 nucleotides to about 17 nucleotides, about 11 nucleotides to about 18 nucleotides, about 11 nucleotides to about 19 nucleotides, about 11 nucleotides to about 20 nucleotides, about 12 nucleotides to about 13 nucleotides, about 12 nucleotides to about 14 nucleotides, about 12 nucleotides to about 15 nucleotides, about 12 nucleotides to about 16 nucleotides, about 12 nucleotides to about 17 nucleotides, about 12 nucleotides to about 18 nucleotides, about 12 nucleotides to about 19 nucleotides, about 12 nucleotides to about 20 nucleotides, about 13 nucleotides to about 14 nucleotides, about 13 nucleotides to about 15 nucleotides, about 13 nucleotides to about 16nucleotides, about 13 nucleotides to about 17 nucleotides, about 13 nucleotides to about 18 nucleotides, about 13 nucleotides to about 19 nucleotides, about 13 nucleotides to about 20 nucleotides, about 14 nucleotides to about 15 nucleotides, about 14 nucleotides to about 16 nucleotides, about 14 nucleotides to about 17 nucleotides, about 14 nucleotides to about 18 nucleotides, about 14 nucleotides to about 19 nucleotides, about 14 nucleotides to about 20 nucleotides, about 15 nucleotides to about 16 nucleotides, about 15 nucleotides to about 17 nucleotides, about 15 nucleotides to about 18 nucleotides, about 15 nucleotides to about 19 nucleotides, about 15 nucleotides to about 20 nucleotides, about 16 nucleotides to about 17 nucleotides, about 16 nucleotides to about 18 nucleotides, about 16 nucleotides to about 19 nucleotides, about 16 nucleotides to about 20 nucleotides, about 17 nucleotides to about 18 nucleotides, about 17 nucleotides to about 19 nucleotides, about 17 nucleotides to about 20 nucleotides, about 18 nucleotides to about 19 nucleotides, about 18 nucleotides to about 20 nucleotides, or about 19 nucleotides to about 20 nucleotides in length.

[0147] In some cases, the spacer region can comprise a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% complementarity to a target nucleic acid sequence. The crRNA can further comprise a repeat region that interacts with an effector protein (e.g., an endonuclease).

[0148] In some embodiments, the guide RNA may comprise a trans-activating crispr RNA (tracrRNA). The tracrRNA may associate with an effector protein (e.g., an endonuclease). The guide RNA may be a single guide RNA (sgRNA). The sgRNA can comprise a tracrRNA region. The tracrRNA region can be a scaffold region. In some embodiments, the scaffold region may be at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The scaffold region can be from about 7 nucleotides to about 100 nucleotides in length. For example, the scaffold region can be from about 7 nucleotides to about 50 nucleotides, from about 7 nucleotides to about 40 nucleotides, from about 7 nucleotides to about 30 nucleotides, from about 7 nucleotides to about 25 nucleotides, from about 7 nucleotides to about 20 nucleotides, from about 7 nucleotides to about 15 nucleotides, from about 8 nucleotides to about 40 nucleotides, from about 8 nucleotides to about 30 nucleotides, from about 8 nucleotides to about 25 nucleotides, from about 8 nucleotides to about 20 nucleotides, from about 8 nucleotides to about 15 nucleotides, from about 15 nucleotides to about 100 nucleotides, from about 15 nucleotides to about 80 nucleotides, from about 15 nucleotides to about 50 nucleotides, from about 15 nucleotides to about 40 nucleotides, from about 15 nucleotides to about30 nucleotides or from about 15 nucleotides to about 25 nucleotides in length. In some embodiments, the scaffold region may be from about 10 nucleotides in length to about 20 nucleotides in length. In some embodiments, the scaffold region may be from about 10 nucleotides to about 11 nucleotides, about 10 nucleotides to about 12 nucleotides, about 10 nucleotides to about 13 nucleotides, about 10 nucleotides to about 14 nucleotides, about 10 nucleotides to about15 nucleotides, about 10 nucleotides to about 16 nucleotides, about 10 nucleotides to about 17 nucleotides, about 10 nucleotides to about 18 nucleotides, about 10 nucleotides to about 19 nucleotides, about 10 nucleotides to about 20 nucleotides, about 11 nucleotides to about 12 nucleotides, about 11 nucleotides to about 13 nucleotides, about 11 nucleotides to about 14 nucleotides, about 11 nucleotides to about 15 nucleotides, about 11 nucleotides to about 16 nucleotides, about 11 nucleotides to about 17 nucleotides, about 11 nucleotides to about 18 nucleotides, about 11 nucleotides to about 19 nucleotides, about 11 nucleotides to about 20 nucleotides, about 12 nucleotides to about 13 nucleotides, about 12 nucleotides to about 14 nucleotides, about 12 nucleotides to about 15 nucleotides, about 12 nucleotides to about 16 nucleotides, about 12 nucleotides to about 17 nucleotides, about 12 nucleotides to about 18 nucleotides, about 12 nucleotides to about 19 nucleotides, about 12 nucleotides to about 20 nucleotides, about 13 nucleotides to about 14 nucleotides, about 13 nucleotides to about 15 nucleotides, about 13 nucleotides to about 16 nucleotides, about 13 nucleotides to about 17 nucleotides, about 13 nucleotides to about 18 nucleotides, about 13 nucleotides to about 19 nucleotides, about 13 nucleotides to about 20 nucleotides, about 14 nucleotides to about 15 nucleotides, about 14 nucleotides to about 16 nucleotides, about 14 nucleotides to about 17 nucleotides, about 14 nucleotides to about 18 nucleotides, about 14 nucleotides to about 19 nucleotides, about 14 nucleotides to about 20 nucleotides, about 15 nucleotides to about 16 nucleotides, about 15 nucleotides to about 17 nucleotides, about 15 nucleotides to about 18 nucleotides, about 15 nucleotides to about 19 nucleotides, about 15 nucleotides to about 20 nucleotides, about 16 nucleotides to about 17 nucleotides, about 16 nucleotides to about 18 nucleotides, about 16 nucleotides to about 19 nucleotides, about 16 nucleotides to about 20 nucleotides, about 17 nucleotides to about 18 nucleotides, about 17 nucleotides to about 19 nucleotides, about 17 nucleotides to about 20 nucleotides, about 18 nucleotides to about 19 nucleotides, about 18 nucleotides to about 20 nucleotides, or about 19 nucleotides to about 20 nucleotides in length.

[0149] In some embodiments, the exogenous sequence of the modified genome comprises an exogenous subsequence. The exogenous subsequence may encode the hTRC of the exogenousTCR or portion thereof. The guide RNA can comprise a target sequence that hybridizes to an endogenous sequence (e.g., an endogenous target sequence) of the modified genome of the engineered T cell described herein. The hTRC of the exogenous TCR or protein thereof encoded by the exogenous subsequence of the exogenous sequence may comprise an amino acid sequence. The amino acid sequence encoded by the exogenous subsequence of the exogenous sequence and the amino acid sequence encoded by the endogenous sequence may be the same. The exogenous subsequence of the exogenous sequence and the endogenous target sequence may encode the same polypeptide (e.g., protein). In some embodiments, the endogenous target sequence and the complement thereof may have less than 100% sequence identity to the exogenous subsequence of the exogenous sequence encoding the hTRC or portion thereof. The endogenous target sequence may have at most about 100%, at most about 99.9%, at most about 99.5%, at most about 99%, at most 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, or less than about 95% sequence identity to the exogenous subsequence of the exogenous sequence encoding the hTRC or protein thereof.

[0150] In some embodiments, the modified genome of the engineered T cell described herein comprises one or more exogenous sequences. In some embodiments, the modified genome comprises 1, 2, 3, 4, 5, or more exogenous sequences. In some embodiments, the modified genome of the engineered T cell comprises a first exogenous sequence that can encode an exogenous TCR alpha chain. The exogenous TCR alpha chain may comprise a human TCR alpha constant domain (hTRAC). The first exogenous sequence can comprise a first exogenous subsequence. The first exogenous subsequence can encode the hTRAC or portion thereof of the exogenous TCR alpha chain. In some embodiments, a first exogenous sequence can encode an exogenous TCR beta chain. The exogenous TCR beta chain may comprise a human TCR beta constant domain (hTRBC). The first exogenous sequence can comprise a first exogenous subsequence. The first exogenous subsequence can encode the hTRBC or portion thereof of the exogenous TCR beta chain.

[0151] In some embodiments, the modified genome of the engineered T cell comprises a second exogenous sequence that can encode an exogenous TCR alpha chain. The exogenous TCR alpha chain may comprise a human TCR alpha constant domain (hTRAC). The second exogenous sequence can comprise a second exogenous subsequence. The second exogenous subsequence can encode the hTRAC or portion thereof of the exogenous TCR alpha chain. In some embodiments, a second exogenous sequence can encode an exogenous TCR beta chain. The exogenous TCR beta chain may comprise a human TCR beta constant domain (hTRBC). The second exogenoussequence can comprise a second exogenous subsequence. The second exogenous subsequence can encode the hTRBC or portion thereof of the exogenous TCR beta chain.

[0152] In some embodiments, the engineered immune cell (e.g., engineered T cell) described herein comprises one or more guide RNAs. In some embodiments, the engineered T cell comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10 guide RNAs. In some embodiments, the engineered T cell comprises a first guide RNA. The first guide RNA can comprise a nucleic acid sequence comprising a first targeting sequence. The first targeting sequence of the first guide RNA can hybridize to the first endogenous target sequence of the modified genome of the engineered T cell.

[0153] In some embodiments, an endogenous target sequence or a complement thereof can encode at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids of an endogenous hTRC. In some embodiments, the first endogenous target sequence or complement thereof may encode at least about 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, or greater than about 8 amino acids of an endogenous hTRAC or hTRBC. The first endogenous target sequence or complement thereof may encode at most about 8 amino acids, at most about 7 amino acids, at most about 6 amino acids, at most about 5 amino acids, at most about 4 amino acids, at most about 3 amino acids, at most about 2 amino acids, or less than about 8 amino acids of an endogenous hTRAC or hTRBC. In some embodiments, the first endogenous target sequence or complement thereof may encode about 2 amino acids to about 12 amino acids of an endogenous hTRAC or hTRBC. In some embodiments, the first endogenous target sequence or complement thereof may encode from about 2 amino acids to about 3 amino acids, about 2 amino acids to about 4 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 7 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 9 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 11 amino acids, about 2 amino acids to about 12 amino acids, about 3 amino acids to about 4 amino acids, about 3 amino acids to about 5 amino acids, about 3 amino acids to about 6 amino acids, about 3 amino acids to about 7 amino acids, about 3 amino acids to about 8 amino acids, about 3 amino acids to about 9 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 11 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 5 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 10amino acids, about 4 amino acids to about 11 amino acids, about 4 amino acids to about 12 amino acids, about 5 amino acids to about 6 amino acids, about 5 amino acids to about 7 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 9 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 11 amino acids, about 5 amino acids to about 12 amino acids, about 6 amino acids to about 7 amino acids, about 6 amino acids to about 8 amino acids, about 6 amino acids to about 9 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 11 amino acids, about 6 amino acids to about 12 amino acids, about 7 amino acids to about 8 amino acids, about 7 amino acids to about 9 amino acids, about 7 amino acids to about 10 amino acids, about 7 amino acids to about 11 amino acids, about 7 amino acids to about 12 amino acids, about 8 amino acids to about 9 amino acids, about 8 amino acids to about 10 amino acids, about 8 amino acids to about 11 amino acids, about 8 amino acids to about 12 amino acids, about 9 amino acids to about 10 amino acids, about 9 amino acids to about 11 amino acids, about 9 amino acids to about 12 amino acids, about 10 amino acids to about 11 amino acids, about 10 amino acids to about 12 amino acids, or about 11 amino acids to about 12 amino acids of an endogenous hTRAC or hTRBC.

[0154] In some embodiments, the engineered T cell comprises a second guide RNA. The second guide RNA can comprise a nucleic acid sequence comprising a second targeting sequence. The second targeting sequence of the guide RNA can hybridize to the second endogenous target sequence of the modified genome of the engineered T cell.

[0155] In some embodiments, the second endogenous target sequence or complement thereof may encode at least about 1 amino acid, at least about 2 amino acids, at least about 3 amino acids, at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 7 amino acids, at least about 8 amino acids, or greater than about 8 amino acids of an endogenous hTRAC or hTRBC. The second endogenous target sequence or complement thereof may encode at most about 8 amino acids, at most about 7 amino acids, at most about 6 amino acids, at most about 5 amino acids, at most about 4 amino acids, at most about 3 amino acids, at most about 2 amino acids, or less than about 8 amino acids of an endogenous hTRAC or hTRBC. In some embodiments, the second endogenous target sequence or complement thereof may encode about 2 amino acids to about 12 amino acids of an endogenous hTRAC or hTRBC. In some embodiments, the second endogenous target sequence or complement thereof may encode from about 2 amino acids to about 3 amino acids, about 2 amino acids to about 4 amino acids, about 2 amino acids to about 5 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 7 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids toabout 9 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 11 amino acids, about 2 amino acids to about 12 amino acids, about 3 amino acids to about 4 amino acids, about 3 amino acids to about 5 amino acids, about 3 amino acids to about 6 amino acids, about 3 amino acids to about 7 amino acids, about 3 amino acids to about 8 amino acids, about 3 amino acids to about 9 amino acids, about 3 amino acids to about 10 amino acids, about 3 amino acids to about 11 amino acids, about 3 amino acids to about 12 amino acids, about 4 amino acids to about 5 amino acids, about 4 amino acids to about 6 amino acids, about 4 amino acids to about 7 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 9 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 11 amino acids, about 4 amino acids to about 12 amino acids, about 5 amino acids to about 6 amino acids, about 5 amino acids to about 7 amino acids, about 5 amino acids to about 8 amino acids, about 5 amino acids to about 9 amino acids, about 5 amino acids to about 10 amino acids, about 5 amino acids to about 11 amino acids, about 5 amino acids to about 12 amino acids, about 6 amino acids to about 7 amino acids, about 6 amino acids to about 8 amino acids, about 6 amino acids to about 9 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 11 amino acids, about 6 amino acids to about 12 amino acids, about 7 amino acids to about 8 amino acids, about 7 amino acids to about 9 amino acids, about 7 amino acids to about 10 amino acids, about 7 amino acids to about 11 amino acids, about 7 amino acids to about 12 amino acids, about 8 amino acids to about 9 amino acids, about 8 amino acids to about 10 amino acids, about 8 amino acids to about 11 amino acids, about 8 amino acids to about 12 amino acids, about 9 amino acids to about 10 amino acids, about 9 amino acids to about 11 amino acids, about 9 amino acids to about 12 amino acids, about 10 amino acids to about 11 amino acids, about 10 amino acids to about 12 amino acids, or about 11 amino acids to about 12 amino acids of an endogenous hTRAC or hTRBC.

[0156] In some embodiments, the amino acid sequence encoded by the first exogenous subsequence of the first exogenous sequence is the same as the amino acid sequence encoded by the first endogenous target sequence (e.g., the amino acid sequence encoded by the first exogenous subsequence of the first exogenous sequence has 100% sequence identity to the amino acid sequence encoded by the first endogenous target sequence). In some embodiments, an amino acid sequence of the hTRAC of the first exogenous TCR encoded by the first exogenous subsequence of the first exogenous sequence is the same as an amino acid sequence encoded by the first target sequence (e.g., the amino acid sequence of the hTRAC of the first exogenous TCR encoded by the first exogenous subsequence of the first exogenous sequence has 100% sequence identity tothe amino acid sequence encoded by the first target sequence). In some embodiments, an amino acid sequence of the hTRBC of the first exogenous TCR encoded by the first exogenous subsequence of the first exogenous sequence is the same as an amino acid sequence encoded by the first target sequence (e.g., the amino acid sequence of the hTRBC of the first exogenous TCR encoded by the first exogenous subsequence of the first exogenous sequence has 100% sequence identity to the amino acid sequence encoded by the first target sequence). In some embodiments, the amino acid sequence encoded by the second exogenous subsequence of the second exogenous sequence is the same as the amino acid sequence encoded by the second endogenous target sequence (e.g., the amino acid sequence encoded by the second exogenous subsequence of the second exogenous sequence has 100% sequence identity to the amino acid sequence encoded by the second endogenous target sequence). In some embodiments, an amino acid sequence of the hTRAC of the second exogenous TCR encoded by the second exogenous subsequence of the second exogenous sequence is the same as an amino acid sequence encoded by the second target sequence (e.g., the amino acid sequence of the hTRAC of the second exogenous TCR encoded by the second exogenous subsequence of the second exogenous sequence has 100% sequence identity to the amino acid sequence encoded by the second target sequence). In some embodiments, an amino acid sequence of the hTRBC of the second exogenous TCR encoded by the second exogenous subsequence of the second exogenous sequence is the same as an amino acid sequence encoded by the second target sequence (e.g., the amino acid sequence of the hTRBC of the second exogenous TCR encoded by the second exogenous subsequence of the second exogenous sequence has 100% sequence identity to the amino acid sequence encoded by the second target sequence).

[0157] In some embodiments, the first endogenous target sequence and complement thereof may each be different from the first exogenous subsequence of the first exogenous sequence (e.g., the first endogenous target sequence and complement thereof each may have less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98%, less than about 97%, less than about 96%, or less than about 95% sequence identity to the first exogenous subsequence of the first exogenous sequence). In some embodiments, the first endogenous target sequence and complement thereof may each be different from the first exogenous subsequence of the first exogenous sequence encoding the hTRAC or portion thereof (e.g., the first endogenous target sequence and complement thereof each may have less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98%, less than about 97%, less than about 96%, or less than about 95% sequence identity to the first exogenous subsequenceof the first exogenous sequence encoding the hTRAC or portion thereof). In some embodiments, the first endogenous target sequence and complement thereof may each be different from the first exogenous subsequence of the first exogenous sequence encoding the hTRBC or portion thereof (e.g., the first endogenous target sequence and complement thereof each may have less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98%, less than about 97%, less than about 96%, or less than about 95% sequence identity to the first exogenous subsequence of the first exogenous sequence encoding the hTRBC or portion thereof.

[0158] In some embodiments, the second endogenous target sequence and complement thereof may each be different from the second exogenous subsequence of the second exogenous sequence (e.g., the second endogenous target sequence and complement thereof each may have less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98%, less than about 97%, less than about 96%, or less than about 95% sequence identity to the second exogenous subsequence of the second exogenous sequence). In some embodiments, the second endogenous target sequence and complement thereof may each be different from the second exogenous subsequence of the second exogenous sequence encoding the hTRAC or portion thereof (e.g., the second endogenous target sequence and complement thereof each may have less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98%, less than about 97%, less than about 96%, or less than about 95% sequence identity to the second exogenous subsequence of the second exogenous sequence encoding the hTRAC or portion thereof). In some embodiments, the second endogenous target sequence and complement thereof may each be different from the second exogenous subsequence of the second exogenous sequence encoding the hTRBC or portion thereof (e.g., the second endogenous target sequence and complement thereof each may have less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98%, less than about 97%, less than about 96%, or less than about 95% sequence identity to the second exogenous subsequence of the second exogenous sequence encoding the hTRBC or portion thereof.

[0159] For example, an engineered T cell may comprise: (a) a modified genome comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC), wherein the exogenous sequence comprises an exogenous subsequence encoding the hTRC of the exogenous TCR or a portion thereof; and (b) a guide RNA, wherein the guide RNA comprises a nucleic acid sequence comprising a targeting sequence that hybridizes to an endogenous target sequence within the modified genome, wherein the endogenous target sequence or a complement thereof encodes at least 4 amino acids of anendogenous hTRC; wherein the amino acid sequence of the hTRC of the exogenous TCR or the portion thereof encoded by the exogenous subsequence of the exogenous sequence, and the amino acid sequence encoded by the endogenous target sequence or a complement thereof, are the same amino acid sequence; and wherein the endogenous target sequence and the complement thereof each have less than 100% sequence identity to the exogenous subsequence encoding the hTRC or the portion thereof.

[0160] In some embodiments, the engineered T cell comprises an engineered human T cell. In some embodiments, the engineered T cell comprises an engineered cell derived from a human T cell. In some embodiments, the engineered T cell comprises an engineered CD8+ T cell. In some embodiments, the engineered T cell comprises an engineered CD4+ T cell. The engineered T cell described herein may comprise reduced cell surface expression of an endogenous TCR compared to expression of an unmodified control cell (e.g., an unmodified control T cell). A reduced surface expression may be measured by quantifying expression of an element in a CD3 / T-cell receptor complex. The element may comprise CD3s, CD36, CD3y, TCRa, TCR0, TCR5, TCRy, CD3^, or any combination thereof. A cell surface expression of an endogenous TCR in the engineered T cell may be reduced by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater than about 99% compared to an expression of the endogenous TCR in an unmodified T cell (e.g., a T cell that does not comprise the exogenous sequence). A cell surface expression of an endogenous TCR in the engineered T cell may be reduced by at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 94%, at most about 93%, at most about 92%, at most about 91%, at most about 90%, at most about 85%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, or less than about 50% compared to an expression of the endogenous TCR in an unmodified T cell (e.g., a T cell that does not comprise the exogenous sequence).

[0161] A cell surface expression of an endogenous TCR in the engineered T cell may be reduced from about 50% to about 98% compared to an expression of the endogenous TCR in an unmodified T cell (e.g., a T cell that does not comprise the exogenous sequence). A cell surface expression of an endogenous TCR in the engineered T cell may be reduced from about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 91%, about 50% to about 92%, about 50% to about 93%, about 50% to about 94%,about 50% to about 95%, about 50% to about 96%, about 50% to about 98%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 91%, about 60% to about 92%, about 60% to about 93%, about 60% to about 94%, about 60% to about 95%, about 60% to about 96%, about 60% to about 98%, about 70% to about 80%, about 70% to about 90%, about 70% to about 91%, about 70% to about 92%, about 70% to about 93%, about 70% to about 94%, about 70% to about 95%, about 70% to about 96%, about 70% to about 98%, about 80% to about 90%, about 80% to about 91%, about 80% to about 92%, about 80% to about 93%, about 80% to about 94%, about 80% to about 95%, about 80% to about 96%, about 80% to about 98%, about 90% to about 91%, about 90% to about 92%, about 90% to about 93%, about 90% to about 94%, about 90% to about 95%, about 90% to about 96%, about 90% to about 98%, about 91% to about 92%, about 91% to about 93%, about 91% to about 94%, about 91% to about 95%, about 91% to about 96%, about 91% to about 98%, about 92% to about 93%, about 92% to about 94%, about 92% to about 95%, about 92% to about 96%, about 92% to about 98%, about 93% to about 94%, about 93% to about 95%, about 93% to about 96%, about 93% to about 98%, about 94% to about 95%, about 94% to about 96%, about 94% to about 98%, about 95% to about 96%, about 95% to about 98%, or about 96% to about 98% compared to an expression of the endogenous TCR in an unmodified T cell (e.g., a T cell that does not comprise the exogenous sequence).

[0162] The engineered T cell described herein may comprise reduced cell surface expression of an endogenous TCR compared to expression of an unmodified control cell (e.g., an unmodified control T cell). A reduced surface expression may be measured by quantifying expression of a surface marker in a CD3 / T-cell receptor complex. The surface marker may comprise CD3s, CD36, CD3y, TCRa, TCR0, TCR5, TCRy, CD3^, or any combination thereof.

[0163] In some embodiments, an engineered T cell described herein may comprise increased cell surface expression of an exogenous TCR (e.g., an exogenous TCR chain) compared to expression of a control cell (e.g., an unmodified control T cell). The control cell may comprise a modified genome. The modified genome may comprise the exogenous sequence encoding an exogenous TCR chain. The exogenous TCR chain may comprise the hTRC or portion thereof (e.g., hTRAC and / or hTRBC). The control cell may comprise a guide RNA molecule (e.g., a control guide RNA) that can have a targeting sequence that hybridizes to an endogenous target sequence of the modified genome. In some embodiments, the targeting sequence can have at least 98%, at least 98.5%, at least 99%, at least 99.9%, or 100% complementarity to the exogenous sequence encoding the hTRC or portion thereof. In some embodiments, the targeting sequence can have atleast 98%, at least 98.5%, at least 99%, at least 99.9%, or 100% complementarity to a complement of the exogenous subsequence encoding the hTRC or portion thereof.

[0164] In some embodiments, the targeting sequence of the guide RNA may hybridize to the exogenous subsequence of the modified genome to a lesser degree than to the endogenous target sequence of the guide RNA. In some embodiments, the targeting sequence of the guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof at least about 1.1 fold less, at least about 1.2 fold less, at least about 1.3 fold less, at least about 1.4 fold less, at least about 1.5 fold less, at least about 2 fold less, at least about 3 fold less, at least about 4 fold less, at least about 5 fold less, at least about 6 fold less, at least about 7 fold less, at least about 8 fold less, at least about 9 fold less, at least about 10 fold less, or greater than about 10 fold less than to the endogenous target sequence of the guide RNA. In some embodiments, the targeting sequence of the guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof at most about 10 fold less, at most about 9 fold less, at most about 8 fold less, at most about 7 fold less, at most about 6 fold less, at most about 5 fold less, at most about 4 fold less, at most about 3 fold less, at most about 2 fold less, at most about 1.5 fold less, at most about 1.4 fold less, at most about 1.3 fold less, at most about 1.2 fold less, at most about 1.1 fold less, or less than about 1.1 fold less than to the endogenous target sequence of the guide RNA.

[0165] In some embodiments, the targeting sequence of the guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof from about 1.1 fold less to about 8 fold less than to the endogenous target sequence of the guide RNA. In some embodiments, the targeting sequence of the guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof from about 1.1 fold less to about 1.2 fold less, about 1.1 fold less to about 1.3 fold less, about 1.1 fold less to about 1.4 fold less, about 1.1 fold less to about 1.5 fold less, about 1.1 fold less to about 2 fold less, about 1.1 fold less to about 3 fold less, about 1.1 fold less to about 4 fold less, about 1.1 fold less to about 5 fold less, about 1.1 fold less to about 6 fold less, about 1.1 fold less to about 7 fold less, about 1.1 fold less to about 8 fold less, about 1.2 fold less to about 1.3 fold less, about 1.2 fold less to about 1.4 fold less, about 1.2 fold less to about 1.5 fold less, about 1.2 fold less to about 2 fold less, about 1.2 fold less to about 3 fold less, about 1.2 fold less to about 4 fold less, about 1.2 fold less to about 5 fold less, about 1.2 fold less to about 6 fold less, about 1.2 fold less to about 7 fold less, about 1.2 fold less to about 8 fold less, about 1.3 fold less to about 1.4 fold less, about 1.3 fold less to about 1.5 fold less, about 1.3 fold less to about 2 fold less, about 1.3 fold less to about 3 fold less, about 1.3 fold less to about 4 fold less, about 1.3 fold less to about 5 fold less, about 1.3 fold less to about 6 fold less, about 1.3 fold lessto about 7 fold less, about 1.3 fold less to about 8 fold less, about 1.4 fold less to about 1.5 fold less, about 1.4 fold less to about 2 fold less, about 1.4 fold less to about 3 fold less, about 1.4 fold less to about 4 fold less, about 1.4 fold less to about 5 fold less, about 1.4 fold less to about 6 fold less, about 1.4 fold less to about 7 fold less, about 1.4 fold less to about 8 fold less, about 1.5 fold less to about 2 fold less, about 1.5 fold less to about 3 fold less, about 1.5 fold less to about 4 fold less, about 1.5 fold less to about 5 fold less, about 1.5 fold less to about 6 fold less, about 1.5 fold less to about 7 fold less, about 1.5 fold less to about 8 fold less, about 2 fold less to about 3 fold less, about 2 fold less to about 4 fold less, about 2 fold less to about 5 fold less, about 2 fold less to about 6 fold less, about 2 fold less to about 7 fold less, about 2 fold less to about 8 fold less, about 3 fold less to about 4 fold less, about 3 fold less to about 5 fold less, about 3 fold less to about 6 fold less, about 3 fold less to about 7 fold less, about 3 fold less to about 8 fold less, about 4 fold less to about 5 fold less, about 4 fold less to about 6 fold less, about 4 fold less to about 7 fold less, about 4 fold less to about 8 fold less, about 5 fold less to about 6 fold less, about 5 fold less to about 7 fold less, about 5 fold less to about 8 fold less, about 6 fold less to about 7 fold less, about 6 fold less to about 8 fold less, or about 7 fold less to about 8 fold less than to the endogenous target sequence of the guide RNA.

[0166] A targeting sequence of a first and / or second guide RNA may have a decreased affinity to the exogenous subsequence encoding the hTRC or portion thereof (e.g., hTRAC and / or hTRBC) compared to an affinity to the endogenous target sequence of the first and / or second guide RNA. In some embodiments, the targeting sequence of the first and / or second guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof (e.g., hTRAC and / or hTRBC) at least about 1.1 fold less, at least about 1.2 fold less, at least about 1.3 fold less, at least about 1.4 fold less, at least about 1.5 fold less, at least about 2 fold less, at least about 3 fold less, at least about 4 fold less, at least about 5 fold less, at least about 6 fold less, at least about 7 fold less, at least about 8 fold less, at least about 9 fold less, at least about 10 fold less, or greater than about 10 fold less than to the endogenous target sequence of the first and / or second guide RNA. In some embodiments, the targeting sequence of the first and / or second guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof (e.g., hTRAC and / or hTRBC) at most about 10 fold less, at most about 9 fold less, at most about 8 fold less, at most about 7 fold less, at most about 6 fold less, at most about 5 fold less, at most about 4 fold less, at most about 3 fold less, at most about 2 fold less, at most about 1.5 fold less, at most about 1.4 fold less, at most about 1.3 fold less, at most about 1.2 fold less, at most about 1.1 fold less, or lessthan about 1.1 fold less than to the endogenous target sequence of the first and / or second guide RNA.

[0167] In some embodiments, the targeting sequence of the first and / or second guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof (e.g., hTRAC and / or hTRBC) from about 1.1 fold less to about 8 fold less than to the endogenous target sequence of the first and / or second guide RNA. In some embodiments, the targeting sequence of the first and / or second guide RNA can hybridize to the exogenous subsequence encoding the hTRC or portion thereof (e.g., hTRAC and / or hTRBC) from about 1.1 fold less to about 1.2 fold less, about 1.1 fold less to about 1.3 fold less, about 1.1 fold less to about 1.4 fold less, about 1.1 fold less to about 1.5 fold less, about 1.1 fold less to about 2 fold less, about 1.1 fold less to about 3 fold less, about 1.1 fold less to about 4 fold less, about 1.1 fold less to about 5 fold less, about 1.1 fold less to about 6 fold less, about 1.1 fold less to about 7 fold less, about 1.1 fold less to about 8 fold less, about 1.2 fold less to about 1.3 fold less, about 1.2 fold less to about 1.4 fold less, about 1.2 fold less to about 1.5 fold less, about 1.2 fold less to about 2 fold less, about 1.2 fold less to about 3 fold less, about 1.2 fold less to about 4 fold less, about 1.2 fold less to about 5 fold less, about 1.2 fold less to about 6 fold less, about 1.2 fold less to about 7 fold less, about 1.2 fold less to about 8 fold less, about 1.3 fold less to about 1.4 fold less, about 1.3 fold less to about 1.5 fold less, about 1.3 fold less to about 2 fold less, about 1.3 fold less to about 3 fold less, about 1.3 fold less to about 4 fold less, about 1.3 fold less to about 5 fold less, about 1.3 fold less to about 6 fold less, about 1.3 fold less to about 7 fold less, about 1.3 fold less to about 8 fold less, about 1.4 fold less to about 1.5 fold less, about 1.4 fold less to about 2 fold less, about 1.4 fold less to about 3 fold less, about 1.4 fold less to about 4 fold less, about 1.4 fold less to about 5 fold less, about 1.4 fold less to about 6 fold less, about 1.4 fold less to about 7 fold less, about 1.4 fold less to about 8 fold less, about 1.5 fold less to about 2 fold less, about 1.5 fold less to about 3 fold less, about 1.5 fold less to about 4 fold less, about 1.5 fold less to about 5 fold less, about 1.5 fold less to about 6 fold less, about 1.5 fold less to about 7 fold less, about 1.5 fold less to about 8 fold less, about 2 fold less to about 3 fold less, about 2 fold less to about 4 fold less, about 2 fold less to about 5 fold less, about 2 fold less to about 6 fold less, about 2 fold less to about 7 fold less, about 2 fold less to about 8 fold less, about 3 fold less to about 4 fold less, about 3 fold less to about 5 fold less, about 3 fold less to about 6 fold less, about 3 fold less to about 7 fold less, about 3 fold less to about 8 fold less, about 4 fold less to about 5 fold less, about 4 fold less to about 6 fold less, about 4 fold less to about 7 fold less, about 4 fold less to about 8 fold less, about 5 fold less to about 6 fold less, about 5 fold less to about 7 fold less, about 5 fold less to about 8 fold less, about 6 foldless to about 7 fold less, about 6 fold less to about 8 fold less, or about 7 fold less to about 8 fold less than to the endogenous target sequence of the first and / or second guide RNA.

[0168] In some embodiments, the endogenous target sequence of the modified genome of the engineered T cell comprises a protospacer adjacent motif (PAM) sequence or complement thereof (e.g., a PAM site). An effector protein (e.g., Cas9) of a gene editing system described herein may recognize a PAM site on a target nucleic acid molecule. In some embodiments, the PAM sequence can be downstream to the target sequence. In some embodiments, the PAM sequence can be 3’ to the target sequence. In some embodiments, the PAM sequence may comprise a nucleotide sequence of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10 base pairs in length. In some embodiments, the PAM sequence may comprise a nucleotide sequence of at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than about 2 base pairs in length. The PAM sequence may comprise a nucleotide sequence as set forth in Table 1.Table 1. Exemplary protospacer adjacent motif (PAM) sequences.

[0169] In some embodiments, the PAM site comprises a nucleotide sequence of NGG, NGRRN, NNNNGATT, NNNNRYAC, or NNAGAAW, where N is A, T, C, or G; R is A or G; and Y is T or C.

[0170] In some embodiments, the PAM sequence or complement thereof comprises a nucleotide sequence that is different than a corresponding region of the exogenous subsequence encoding the hTRC or the portion thereof. The corresponding region of the exogenous subsequence encoding the hTRC or the portion thereof may be mutated so as to not be a PAM sequence. In some embodiments, the PAM sequence or complement thereof comprises a nucleotide sequence that isdifferent than a corresponding region (e.g., PAM sequence) of the exogenous subsequence encoding the hTRC or the portion thereof. In some embodiments, the PAM sequence or complement thereof comprises a nucleotide sequence having at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater than about 95% sequence identity to a corresponding region (e.g., PAM sequence) of the exogenous subsequence encoding the hTRC or the portion thereof. In some embodiments, the PAM sequence or complement thereof comprises a nucleotide sequence having at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, or less than about 30% sequence identity to a corresponding region (e.g., PAM sequence) of the exogenous subsequence encoding the hTRC or the portion thereof. In some embodiments, the PAM sequence or complement thereof has less than 100% sequence identity to corresponding region (e.g., PAM sequence) of the exogenous subsequence encoding the hTRC or the portion thereof. In some embodiments, the PAM sequence or complement thereof comprises a nucleotide sequence having from about 30% to about 95% sequence identity to corresponding region (e.g., PAM sequence) of the exogenous subsequence encoding the hTRC or the portion thereof. In some embodiments, the PAM sequence or complement thereof comprises a nucleotide sequence having from about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95% sequence identity to corresponding region (e.g., PAM sequence) of the exogenous subsequence encoding the hTRC or the portion thereof.

[0171] In some embodiments, the engineered T cell may lack a sequence encoding a mouse TCR. In some embodiments, the engineered T cell may lack a sequence encoding a mouse TRC (e.g., mTRAC and / or mTRBC).

[0172] In some aspects, an engineered T cell described herein may comprise a first nucleic acid sequence and a second nucleic acid sequence. A first nucleic acid sequence may down-regulate expression of an endogenous TCR chain. The first nucleic acid sequence may comprise a targeting sequence that hybridizes to a target sequence within a genomic sequence. The target sequence within the genomic sequence can encode a TCR constant domain (e.g., a TCR constant domain of a TRAC or TRBC). The target sequence may encode an amino acid sequence of the TCR constant domain. In some embodiments, the amino acid sequence of the TCR constant domain encoded by the target sequence can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater than about 10 amino acids in length. In some embodiments, the amino acid sequence of the TCR constant domain encoded by the target sequence can be at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or less amino acids in length. A second nucleic acid sequence may encode an exogenous TCR chain. The exogenous TCR chain may comprise a TCR constant domain with the same amino acid sequence encoded by the target sequence. The exogenous TCR chain may comprise a TCR constant domain with a different amino acid sequence than that encoded by the target sequence. In some embodiments, a TCR constant domain of the exogenous TCR chain and a TCR constant domain of the endogenous TCR chain can be from the same species. In some embodiments, a TCR constant domain of the exogenous TCR chain and a TCR constant domain of the endogenous TCR chain can be derivatives of a TCR chain from the same species. As derivatives of a TCR chain from the same species, an amino acid sequence of the TCR constant domain of the exogenous TCR chain may have at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 79%, or at least about 100% sequence identity to an amino acid sequence of the TCR constant domain of the endogenous TCR chain.

[0173] For example, the engineered T cell may comprise (a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and (b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein the first nucleic acid sequence: (i) does not hybridizeto the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0174] In some embodiments, the TCR constant domain of the exogenous TCR chain can comprise the same amino acid sequence encoded by the target sequence. In some embodiments, the TCR constant domain of the exogenous TCR chain can comprise a sequence with at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or greater than about 99% sequence identity to the amino acid sequence encoded by the target sequence. In some embodiments, the TCR constant domain of the exogenous TCR chain can comprise a sequence with at most about 99%, at most about 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, or less than about 50% sequence identity to the amino acid sequence encoded by the target sequence.

[0175] In some embodiments, a TCR constant domain of the exogenous TCR chain may comprise an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or greater than about 99% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22. In some embodiments, a TCR constant domain of the exogenous TCR chain may comprise an amino acid sequence having at most about 99%, at most about 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, or less than about 70% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22.

[0176] In some embodiments, a TCR constant domain of the exogenous TCR chain may comprise an amino acid sequence having between about 50% to about 99% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22. In some embodiments, a TCR constant domain of the exogenous TCR chain may comprise an amino acid sequence having between about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 96%, about50% to about 97%, about 50% to about 98%, about 50% to about 99%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 96%, about 60% to about 97%, about 60% to about 98%, about 60% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 96%, about 70% to about 97%, about 70% to about 98%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 96%, about 75% to about 97%, about 75% to about 98%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 96% to about 97%, about 96% to about 98%, about 96% to about 99%, about 97% to about 98%, about 97% to about 99%, or about 98% to about 99% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22.

[0177] In some embodiments, a TCR constant domain of the endogenous TCR chain may comprise an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, or greater than about 99% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22. In some embodiments, a TCR constant domain of the endogenous TCR chain may comprise an amino acid sequence having at most about 99%, at most about 98.5%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, or less than about 70% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22.

[0178] In some embodiments, a TCR constant domain of the endogenous TCR chain may comprise an amino acid sequence having between about 50% to about 99% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ IDNO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22. In some embodiments, a TCR constant domain of the endogenous TCR chain may comprise an amino acid sequence having between about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 96%, about 50% to about 97%, about 50% to about 98%, about 50% to about 99%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 96%, about 60% to about 97%, about 60% to about 98%, about 60% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 96%, about 70% to about 97%, about 70% to about 98%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 75% to about 96%, about 75% to about 97%, about 75% to about 98%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 96% to about 97%, about 96% to about 98%, about 96% to about 99%, about 97% to about 98%, about 97% to about 99%, or about 98% to about 99% sequence identity to an amino acid sequence as set forth in a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22.

[0179] There may be a subsequence of the second nucleic acid sequence. The subsequence of the second nucleic acid sequence may encode an amino acid sequence that is the same amino acid sequence encoded by the target sequence. The subsequence of the second nucleic acid sequence may encode an amino acid sequence that is a different amino acid sequence than that encoded by the target sequence.

[0180] As another example, the engineered T cell may comprise (a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length; and (b)a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain comprising the same amino acid sequence encoded by the target sequence, wherein a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof, and wherein the first nucleic acid sequence: (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

[0181] In some embodiments, the first nucleic acid sequence may be at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, or greater than about 500 nucleotides in length. In some embodiments, the first nucleic acid sequence may be at least about 500, 1000, 1500, 2000, 2500, 5000, 10000, or greater than about 10000 nucleotides in length. In some embodiments, the first nucleic acid sequence may be at most about 500 nucleotides, at most about 400 nucleotides, at most about 300 nucleotides, at most about 250 nucleotides, at most about 200 nucleotides, at most about 150 nucleotides, at most about 100 nucleotides, at most about 90 nucleotides, at most about 80 nucleotides, at most about 70 nucleotides, at most about 60 nucleotides, at most about 50 nucleotides, at most about 45 nucleotides, at most about 40 nucleotides, at most about 35 nucleotides, at most about 30 nucleotides, at most about 25 nucleotides, at most about 20 nucleotides, at most about 15 nucleotides, at most about 10 nucleotides, or less than about 10 nucleotides in length. In some embodiments, the first nucleic acid sequence may be from about 10 nucleotides to about 300 nucleotides in length. In some embodiments, the first nucleic acid sequence may be from about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 75 nucleotides, about 10 nucleotides to about 100 nucleotides, about 10 nucleotides to about 150 nucleotides, about 10 nucleotides to about 200 nucleotides, about 10nucleotides to about 300 nucleotides, about 15 nucleotides to about 20 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 75 nucleotides, about 15 nucleotides to about 100 nucleotides, about 15 nucleotides to about 150 nucleotides, about 15 nucleotides to about 200 nucleotides, about 15 nucleotides to about 300 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 75 nucleotides, about 20 nucleotides to about 100 nucleotides, about 20 nucleotides to about 150 nucleotides, about 20 nucleotides to about 200 nucleotides, about 20 nucleotides to about 300 nucleotides, about 25 nucleotides to about 30 nucleotides, about 25 nucleotides to about 40 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 75 nucleotides, about 25 nucleotides to about 100 nucleotides, about 25 nucleotides to about 150 nucleotides, about 25 nucleotides to about 200 nucleotides, about 25 nucleotides to about 300 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 75 nucleotides, about 30 nucleotides to about 100 nucleotides, about 30 nucleotides to about 150 nucleotides, about 30 nucleotides to about 200 nucleotides, about 30 nucleotides to about 300 nucleotides, about 40 nucleotides to about 50 nucleotides, about 40 nucleotides to about 75 nucleotides, about 40 nucleotides to about 100 nucleotides, about 40 nucleotides to about 150 nucleotides, about 40 nucleotides to about 200 nucleotides, about 40 nucleotides to about 300 nucleotides, about 50 nucleotides to about 75 nucleotides, about 50 nucleotides to about 100 nucleotides, about 50 nucleotides to about 150 nucleotides, about 50 nucleotides to about 200 nucleotides, about 50 nucleotides to about 300 nucleotides, about 75 nucleotides to about 100 nucleotides, about 75 nucleotides to about 150 nucleotides, about 75 nucleotides to about 200 nucleotides, about 75 nucleotides to about 300 nucleotides, about 100 nucleotides to about 150 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 300 nucleotides, about 150 nucleotides to about 200 nucleotides, about 150 nucleotides to about 300 nucleotides, or about 200 nucleotides to about 300 nucleotides in length.

[0182] In some embodiments, the first nucleic acid sequence may be from about 500 nucleotides to about 20,000 nucleotides in length. In some embodiments, the first nucleic acid sequence may be from about 500 nucleotides to about 1,000 nucleotides, about 500 nucleotides to about 1,500 nucleotides, about 500 nucleotides to about 2,000 nucleotides, about 500 nucleotides to about 2,500 nucleotides, about 500 nucleotides to about 3,000 nucleotides, about 500 nucleotides toabout 3,500 nucleotides, about 500 nucleotides to about 4,000 nucleotides, about 500 nucleotides to about 4,500 nucleotides, about 500 nucleotides to about 5,000 nucleotides, about 500 nucleotides to about 10,000 nucleotides, about 500 nucleotides to about 20,000 nucleotides, about 1,000 nucleotides to about 1,500 nucleotides, about 1,000 nucleotides to about 2,000 nucleotides, about 1,000 nucleotides to about 2,500 nucleotides, about 1,000 nucleotides to about 3,000 nucleotides, about 1,000 nucleotides to about 3,500 nucleotides, about 1,000 nucleotides to about 4,000 nucleotides, about 1,000 nucleotides to about 4,500 nucleotides, about 1,000 nucleotides to about 5,000 nucleotides, about 1,000 nucleotides to about 10,000 nucleotides, about 1,000 nucleotides to about 20,000 nucleotides, about 1,500 nucleotides to about 2,000 nucleotides, about1.500 nucleotides to about 2,500 nucleotides, about 1,500 nucleotides to about 3,000 nucleotides, about 1,500 nucleotides to about 3,500 nucleotides, about 1,500 nucleotides to about 4,000 nucleotides, about 1,500 nucleotides to about 4,500 nucleotides, about 1,500 nucleotides to about 5,000 nucleotides, about 1,500 nucleotides to about 10,000 nucleotides, about 1,500 nucleotides to about 20,000 nucleotides, about 2,000 nucleotides to about 2,500 nucleotides, about 2,000 nucleotides to about 3,000 nucleotides, about 2,000 nucleotides to about 3,500 nucleotides, about 2,000 nucleotides to about 4,000 nucleotides, about 2,000 nucleotides to about 4,500 nucleotides, about 2,000 nucleotides to about 5,000 nucleotides, about 2,000 nucleotides to about 10,000 nucleotides, about 2,000 nucleotides to about 20,000 nucleotides, about 2,500 nucleotides to about 3,000 nucleotides, about 2,500 nucleotides to about 3,500 nucleotides, about 2,500 nucleotides to about 4,000 nucleotides, about 2,500 nucleotides to about 4,500 nucleotides, about 2,500 nucleotides to about 5,000 nucleotides, about 2,500 nucleotides to about 10,000 nucleotides, about2.500 nucleotides to about 20,000 nucleotides, about 3,000 nucleotides to about 3,500 nucleotides, about 3,000 nucleotides to about 4,000 nucleotides, about 3,000 nucleotides to about 4,500 nucleotides, about 3,000 nucleotides to about 5,000 nucleotides, about 3,000 nucleotides to about 10,000 nucleotides, about 3,000 nucleotides to about 20,000 nucleotides, about 3,500 nucleotides to about 4,000 nucleotides, about 3,500 nucleotides to about 4,500 nucleotides, about 3,500 nucleotides to about 5,000 nucleotides, about 3,500 nucleotides to about 10,000 nucleotides, about3.500 nucleotides to about 20,000 nucleotides, about 4,000 nucleotides to about 4,500 nucleotides, about 4,000 nucleotides to about 5,000 nucleotides, about 4,000 nucleotides to about 10,000 nucleotides, about 4,000 nucleotides to about 20,000 nucleotides, about 4,500 nucleotides to about 5,000 nucleotides, about 4,500 nucleotides to about 10,000 nucleotides, about 4,500 nucleotides to about 20,000 nucleotides, about 5,000 nucleotides to about 10,000 nucleotides, about 5,000nucleotides to about 20,000 nucleotides, or about 10,000 nucleotides to about 20,000 nucleotides in length.

[0183] In some embodiments, the targeting sequence (e.g., the targeting sequence that hybridizes to a target sequence within a genomic sequence) may be at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 250 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, or greater than about 500 nucleotides in length. In some embodiments, the targeting sequence may be at most about 500 nucleotides, at most about 400 nucleotides, at most about 300 nucleotides, at most about 250 nucleotides, at most about 200 nucleotides, at most about 150 nucleotides, at most about 100 nucleotides, at most about 90 nucleotides, at most about 80 nucleotides, at most about 70 nucleotides, at most about 60 nucleotides, at most about 50 nucleotides, at most about 45 nucleotides, at most about 40 nucleotides, at most about 35 nucleotides, at most about 30 nucleotides, at most about 25 nucleotides, at most about 20 nucleotides, at most about 15 nucleotides, at most about 10 nucleotides, or less than about 10 nucleotides in length. In some embodiments, the targeting sequence may be from about 10 nucleotides to about 300 nucleotides in length. In some embodiments, the targeting sequence may be from about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 75 nucleotides, about 10 nucleotides to about 100 nucleotides, about 10 nucleotides to about 150 nucleotides, about 10 nucleotides to about 200 nucleotides, about 10 nucleotides to about 300 nucleotides, about 15 nucleotides to about 20 nucleotides, about 15 nucleotides to about 25 nucleotides, about 15 nucleotides to about 30 nucleotides, about 15 nucleotides to about 40 nucleotides, about 15 nucleotides to about 50 nucleotides, about 15 nucleotides to about 75 nucleotides, about 15 nucleotides to about 100 nucleotides, about 15 nucleotides to about 150 nucleotides, about 15 nucleotides to about 200 nucleotides, about 15 nucleotides to about 300 nucleotides, about 20 nucleotides to about 25 nucleotides, about 20 nucleotides to about 30 nucleotides, about 20 nucleotides to about 40 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 75nucleotides, about 20 nucleotides to about 100 nucleotides, about 20 nucleotides to about 150 nucleotides, about 20 nucleotides to about 200 nucleotides, about 20 nucleotides to about 300 nucleotides, about 25 nucleotides to about 30 nucleotides, about 25 nucleotides to about 40 nucleotides, about 25 nucleotides to about 50 nucleotides, about 25 nucleotides to about 75 nucleotides, about 25 nucleotides to about 100 nucleotides, about 25 nucleotides to about 150 nucleotides, about 25 nucleotides to about 200 nucleotides, about 25 nucleotides to about 300 nucleotides, about 30 nucleotides to about 40 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 75 nucleotides, about 30 nucleotides to about 100 nucleotides, about 30 nucleotides to about 150 nucleotides, about 30 nucleotides to about 200 nucleotides, about 30 nucleotides to about 300 nucleotides, about 40 nucleotides to about 50 nucleotides, about 40 nucleotides to about 75 nucleotides, about 40 nucleotides to about 100 nucleotides, about 40 nucleotides to about 150 nucleotides, about 40 nucleotides to about 200 nucleotides, about 40 nucleotides to about 300 nucleotides, about 50 nucleotides to about 75 nucleotides, about 50 nucleotides to about 100 nucleotides, about 50 nucleotides to about 150 nucleotides, about 50 nucleotides to about 200 nucleotides, about 50 nucleotides to about 300 nucleotides, about 75 nucleotides to about 100 nucleotides, about 75 nucleotides to about 150 nucleotides, about 75 nucleotides to about 200 nucleotides, about 75 nucleotides to about 300 nucleotides, about 100 nucleotides to about 150 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 300 nucleotides, about 150 nucleotides to about 200 nucleotides, about 150 nucleotides to about 300 nucleotides, or about 200 nucleotides to about 300 nucleotides in length.Table 2. Exemplary gRNA sequences.

[0184] In some embodiments, the engineered T cell described herein comprises a target sequence encoding the TCR constant domain. In some embodiments, an amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than about 99% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9 or 16. In some embodiments, an amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence having at most about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 70%, 60%, or less than about 60% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9 or 16. In some embodiments, an amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence having from about 60% to about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9 or 16. In some embodiments, an amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence having from about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99.9%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99.9%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99.9%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99.9%, about 90% to about 95%, about 90% to about 99.9%, or about 95% to about 99.9% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 9 or 16. In some embodiments, an amino acid sequence of the TCR constant domain encoded by the target sequence comprises an amino acid sequence as set forth in SEQ ID NO: 9 or 16.

[0185] In some embodiments, a target sequence may comprise a sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than about 99% sequence identity to a sequence as set forth in SEQ ID NO: 8 or 15. In some embodiments, a target sequence may comprise a sequence having at most about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 70%, 60%, or less than about 60% sequence identity to a sequence as set forth in SEQ ID NO : 8 or 15. In some embodiments, a target sequence may comprise a sequence having from about 60% to about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 8 or 15. In some embodiments, a target sequence may comprise asequence having from about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99.9%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99.9%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99.9%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99.9%, about 90% to about 95%, about 90% to about 99.9%, or about 95% to about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 8 or 15. In some embodiments, a target sequence may comprise a sequence as set forth in SEQ ID NO: 8 or 15.

[0186] In some embodiments, the subsequence of the second nucleic acid sequence may comprise a sequence having at least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than about 99% sequence identity to a sequence as set forth in SEQ ID NO: 8, 12, 15, or 19. In some embodiments, the subsequence of the second nucleic acid sequence may comprise a sequence having at most about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 70%, 60%, or less than about 60% sequence identity to a sequence as set forth in SEQ ID NO: 8, 12, 15, or 19. In some embodiments, the subsequence of the second nucleic acid sequence may comprise a sequence having from about 60% to about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 8, 12, 15, or 19. In some embodiments, the subsequence of the second nucleic acid sequence may comprise a sequence having from about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 99.9%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 99.9%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 99.9%, about 85% to about 90%, about 85% to about 95%, about 85% to about 99.9%, about 90% to about 95%, about 90% to about 99.9%, or about 95% to about 99.9% sequence identity to a sequence as set forth in SEQ ID NO: 8, 12, 15, or 19. In some embodiments, the subsequence of the second nucleic acid sequence may comprise a sequence as set forth in SEQ ID NO: 8, 12, 15, or 19.

[0187] In some embodiments, the subsequence of the second nucleic acid sequence can have less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98.5%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, or less than about 60% sequence identity to a target sequence or a complement thereof. In someembodiments, the subsequence of the second nucleic acid sequence can have a mutation and / or variation. In some embodiments, the mutation and / or variation can be relative to the target sequence or complement thereof. In some embodiments, the subsequence of the second nucleic acid sequence can comprise one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations). In some embodiments, the mutation may comprise an insertion, a substitution, a deletion, or any combination thereof.Table 3. T-Cell Receptor (TCR) Constant Domain Sequences

[0188] In some embodiments, the first nucleic acid sequence does not hybridize to the subsequence of the second nucleic acid sequence or the complement thereof. In some embodiments, the first nucleic acid sequence can have a reduced affinity to hybridize to the subsequence of the second nucleic acid sequence or the complement thereof compared to an affinity to hybridize to the target sequence. In some embodiments, the first nucleic acid sequence may hybridize to the subsequence of the second nucleic acid sequence or the complement thereof with an affinity of at least 1.1 fold less, at least about 1.2 fold less, at least about 1.3 fold less, at least about 1.4 fold less, at least about 1.5 fold less, at least about 2 fold less, at least about 3 fold less, at least about 4 fold less, at least about 5 fold less, at least about 6 fold less, at least about 7 fold less, at least about 8 fold less, at least about 9 fold less, at least about 10 fold less, or greater than about 10 fold less than an affinity of the first nucleic acid sequence hybridizing to the target sequence. In some embodiments, the first nucleic acid sequence may hybridize to the subsequence of the second nucleic acid sequence or the complement thereof with an affinity of at most about 10 fold less, at most about 9 fold less, at most about 8 fold less, at most about 7 fold less, at most about 6 fold less, at most about 5 fold less, at most about 4 fold less, at most about 3 fold less, at most about 2 fold less, at most about 1.5 fold less, at most about 1.4 fold less, at most about 1.3fold less, at most about 1.2 fold less, at most about 1.1 fold less, or less than about 1.1 fold less than an affinity of the first nucleic acid sequence hybridizing to the target sequence.

[0189] In some embodiments, the first nucleic acid sequence may hybridize to the subsequence of the second nucleic acid sequence or the complement thereof with an affinity from about 1.1 fold less to about 8 fold less than an affinity of the first nucleic acid sequence hybridizing to the target sequence. In some embodiments, the first nucleic acid sequence may hybridize to the subsequence of the second nucleic acid sequence or the complement thereof with an affinity from about 1.1 fold less to about 1.2 fold less, about 1.1 fold less to about 1.3 fold less, about 1.1 fold less to about 1.4 fold less, about 1.1 fold less to about 1.5 fold less, about 1.1 fold less to about 2 fold less, about 1.1 fold less to about 3 fold less, about 1.1 fold less to about 4 fold less, about 1.1 fold less to about 5 fold less, about 1.1 fold less to about 6 fold less, about 1.1 fold less to about 7 fold less, about 1.1 fold less to about 8 fold less, about 1.2 fold less to about 1.3 fold less, about 1.2 fold less to about 1.4 fold less, about 1.2 fold less to about 1.5 fold less, about 1.2 fold less to about 2 fold less, about 1.2 fold less to about 3 fold less, about 1.2 fold less to about 4 fold less, about 1.2 fold less to about 5 fold less, about 1.2 fold less to about 6 fold less, about 1.2 fold less to about 7 fold less, about 1.2 fold less to about 8 fold less, about 1.3 fold less to about 1.4 fold less, about 1.3 fold less to about 1.5 fold less, about 1.3 fold less to about 2 fold less, about 1.3 fold less to about 3 fold less, about 1.3 fold less to about 4 fold less, about 1.3 fold less to about 5 fold less, about 1.3 fold less to about 6 fold less, about 1.3 fold less to about 7 fold less, about 1.3 fold less to about 8 fold less, about 1.4 fold less to about 1.5 fold less, about 1.4 fold less to about 2 fold less, about 1.4 fold less to about 3 fold less, about 1.4 fold less to about 4 fold less, about 1.4 fold less to about 5 fold less, about 1.4 fold less to about 6 fold less, about 1.4 fold less to about 7 fold less, about 1.4 fold less to about 8 fold less, about 1.5 fold less to about 2 fold less, about 1.5 fold less to about 3 fold less, about 1.5 fold less to about 4 fold less, about 1.5 fold less to about 5 fold less, about 1.5 fold less to about 6 fold less, about 1.5 fold less to about 7 fold less, about 1.5 fold less to about 8 fold less, about 2 fold less to about 3 fold less, about 2 fold less to about 4 fold less, about 2 fold less to about 5 fold less, about 2 fold less to about 6 fold less, about 2 fold less to about 7 fold less, about 2 fold less to about 8 fold less, about 3 fold less to about 4 fold less, about 3 fold less to about 5 fold less, about 3 fold less to about 6 fold less, about 3 fold less to about 7 fold less, about 3 fold less to about 8 fold less, about 4 fold less to about 5 fold less, about 4 fold less to about 6 fold less, about 4 fold less to about 7 fold less, about 4 fold less to about 8 fold less, about 5 fold less to about 6 fold less, about 5 fold less to about 7 fold less, about 5 fold less to about 8 fold less, about 6 fold less to about 7 fold less, about 6 fold less toabout 8 fold less, or about 7 fold less to about 8 fold than an affinity of the first nucleic acid sequence hybridizing to the target sequence.

[0190] In some embodiments, the subsequence of the second nucleic acid may comprise a downstream sequence. The downstream sequence may comprise less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98.5%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, or less than about 60% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof. In some embodiments, the downstream sequence comprises a mutation or variation. The mutation or variation may be relative to a corresponding sequence downstream of the target sequence or a complement thereof. The downstream sequence may comprise one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations). In some embodiments, the mutation may comprise an insertion, a substitution, a deletion, or any combination thereof. In some embodiments, the sequence downstream comprises a mutated PAM sequence or complement thereof. In some embodiments, the sequence downstream comprises at least about 1 mutation, at least about 2 mutations, at least about 3 mutations, at least about 4 mutations, at least about 5 mutations, at least about 6 mutations, at least about 7 mutations, at least about 8 mutations, at least about 9 mutations, at least about 10 mutations, or greater than about 10 mutations of a nucleotide sequence as set forth in any one of SEQ ID NOs: 1-5 or SEQ Identifier Al or A2. The sequence downstream of the subsequence of the second nucleic acid or a complement thereof can comprise a sequence of NHN or HNH, where N stands for any nucleotide of A / T / C / G and H stands for any nucleotide of A / T / C. For example, the sequence downstream of the subsequence of the second nucleic acid or a complement thereof can comprise a sequence of TAG, AGA, AGC, TGA, TGC, TTG, or TCG.

[0191] In some embodiments, the engineered T cell comprises a CRISPR / Cas system. The CRISPR-Cas system provides a gene-editing system using RNA-guided nucleases to cleave genetic elements. As provided herein, the term “effector protein”, “CRISPR effector”, “CRISPR enzyme”, or “Cas protein” can refer to a polypeptide or a fragment thereof with enzymatic activity and configured to hybridize to bind to a target nucleic acid molecule. In some embodiments, the enzymatic activity may be endonuclease activity, integrase activity, nickase activity, exonuclease activity, transposase activity, excision activity, or any combination thereof. In some cases, theeffector protein can be catalytically inactive. In some embodiments, the CRISPR-Cas system described herein comprises a Cas9 protein (e.g., a CRISPR-Cas9 system).

[0192] In some embodiments, the CRISPR-Cas system comprises the first nucleic acid sequence (e.g., guide RNA). In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may not recognize the second nucleic acid sequence or a complement thereof of the engineered T cell. In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may not bind to the second nucleic acid sequence or a complement thereof of the engineered T cell. In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may not cleave the second nucleic acid sequence or a complement thereof of the engineered T cell.

[0193] In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may recognize, bind to, or cleave the second nucleic acid sequence or a complement thereof less than the first nucleic acid sequence recognizing, binding to, or cleaving the target sequence. In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may recognize, bind to, or cleave the second nucleic acid sequence or a complement thereof at least 1.1 fold less, at least about 1.2 fold less, at least about 1.3 fold less, at least about 1.4 fold less, at least about 1.5 fold less, at least about 2 fold less, at least about 3 fold less, at least about 4 fold less, at least about 5 fold less, at least about 6 fold less, at least about 7 fold less, at least about 8 fold less, at least about 9 fold less, at least about 10 fold less, or greater than about 10 fold less than the first nucleic acid sequence recognizing, binding to, or cleaving the target sequence.

[0194] In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may recognize, bind to, or cleave the second nucleic acid sequence or a complement thereof at most about 10 fold less, at most about 9 fold less, at most about 8 fold less, at most about 7 fold less, at most about 6 fold less, at most about 5 fold less, at most about 4 fold less, at most about 3 fold less, at most about 2 fold less, at most about 1.5 fold less, at most about 1.4 fold less, at most about 1.3 fold less, at most about 1.2 fold less, at most about 1.1 fold less, or less than about 1.1 fold less than the first nucleic acid sequence recognizing, binding to, or cleaving the target sequence.

[0195] In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may recognize, bind to, or cleave the second nucleic acid sequence or a complement thereof from about 1.1 fold less to about 8 fold less than the first nucleic acid sequence recognizing, binding to, or cleaving the target sequence. In some embodiments, the CRISPR-Cas system comprising the first nucleic acid sequence may recognize, bind to, or cleave the second nucleic acid sequenceor a complement thereof from about 1.1 fold less to about 1.2 fold less, about 1.1 fold less to about 1.3 fold less, about 1.1 fold less to about 1.4 fold less, about 1.1 fold less to about 1.5 fold less, about 1.1 fold less to about 2 fold less, about 1.1 fold less to about 3 fold less, about 1.1 fold less to about 4 fold less, about 1.1 fold less to about 5 fold less, about 1.1 fold less to about 6 fold less, about 1.1 fold less to about 7 fold less, about 1.1 fold less to about 8 fold less, about 1.2 fold less to about 1.3 fold less, about 1.2 fold less to about 1.4 fold less, about 1.2 fold less to about 1.5 fold less, about 1.2 fold less to about 2 fold less, about 1.2 fold less to about 3 fold less, about 1.2 fold less to about 4 fold less, about 1.2 fold less to about 5 fold less, about 1.2 fold less to about 6 fold less, about 1.2 fold less to about 7 fold less, about 1.2 fold less to about 8 fold less, about 1.3 fold less to about 1.4 fold less, about 1.3 fold less to about 1.5 fold less, about 1.3 fold less to about 2 fold less, about 1.3 fold less to about 3 fold less, about 1.3 fold less to about 4 fold less, about 1.3 fold less to about 5 fold less, about 1.3 fold less to about 6 fold less, about 1.3 fold less to about 7 fold less, about 1.3 fold less to about 8 fold less, about 1.4 fold less to about 1.5 fold less, about 1.4 fold less to about 2 fold less, about 1.4 fold less to about 3 fold less, about 1.4 fold less to about 4 fold less, about 1.4 fold less to about 5 fold less, about 1.4 fold less to about 6 fold less, about 1.4 fold less to about 7 fold less, about 1.4 fold less to about 8 fold less, about 1.5 fold less to about 2 fold less, about 1.5 fold less to about 3 fold less, about 1.5 fold less to about 4 fold less, about 1.5 fold less to about 5 fold less, about 1.5 fold less to about 6 fold less, about 1.5 fold less to about 7 fold less, about 1.5 fold less to about 8 fold less, about 2 fold less to about 3 fold less, about 2 fold less to about 4 fold less, about 2 fold less to about 5 fold less, about 2 fold less to about 6 fold less, about 2 fold less to about 7 fold less, about 2 fold less to about 8 fold less, about 3 fold less to about 4 fold less, about 3 fold less to about 5 fold less, about 3 fold less to about 6 fold less, about 3 fold less to about 7 fold less, about 3 fold less to about 8 fold less, about 4 fold less to about 5 fold less, about 4 fold less to about 6 fold less, about 4 fold less to about 7 fold less, about 4 fold less to about 8 fold less, about 5 fold less to about 6 fold less, about 5 fold less to about 7 fold less, about 5 fold less to about 8 fold less, about 6 fold less to about 7 fold less, about 6 fold less to about 8 fold less, or about 7 fold less to about 8 fold than the first nucleic acid sequence recognizing, binding to, or cleaving the target sequence.

[0196] In some embodiments, the engineered T cell comprises a sequence downstream of the subsequence. The sequence downstream of the subsequence may comprise less than about 100%, less than about 99.9%, less than about 99.5%, less than about 99%, less than about 98.5%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, lessthan about 85%, less than about 80%, less than about 75%, less than about 70%, or less than about 60% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof. In some embodiments, the sequence downstream of the subsequence comprises a mutation or variation. The mutation or variation may be relative to a corresponding sequence downstream of the target sequence or a complement thereof. The sequence downstream of the subsequence may comprise one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations). In some embodiments, the mutation may comprise an insertion, a substitution, a deletion, or any combination thereof.

[0197] In some embodiments, an expression of the endogenous TCR (e.g., endogenous TCR chain) is down-regulated. In some embodiments, an expression of the endogenous TCR (e.g., endogenous TCR chain) is down-regulated to a great extent compared to a down-regulation of expression of the exogenous TCR (e.g., exogenous TCR chain). In some embodiments, an expression of the endogenous TCR (e.g., endogenous TCR chain) is down-regulated by the first nucleic acid sequence to a greater extent compared to a down-regulation of expression of the exogenous TCR (e.g., exogenous TCR chain) by the first nucleic acid sequence. In some embodiments, an expression of the endogenous TCR (e.g., endogenous TCR chain) is down- regulated by the first nucleic acid sequence with the CRISPR-Cas system (e.g., CRISPR-Cas9 system) to a greater extent compared to a down-regulation of expression of the exogenous TCR (e.g., exogenous TCR chain) by the first nucleic acid sequence with the CRISPR-Cas system (e.g., CRISPR-Cas9 system). In some embodiments, down-regulation of expression of the endogenous TCR (e.g., endogenous TCR chain) by the first nucleic acid sequence with the CRISPR-Cas system (e.g., CRISPR-Cas9 system) can be at least about 2x, at least about 3x, at least about 4x, at least about 5x, at least about 6x, at least about 7x, at least about 8x, at least about 9x, at least about lOx, at least about 12x, at least about 14x, at least about 16x, at least about 18x, at least about 20x, or at least about 50x greater than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system.

[0198] In some embodiments, an expression of the exogenous TCR (e.g., exogenous TCR chain) is down-regulated. In some embodiments, an expression of the exogenous TCR (e.g., exogenous TCR chain) is down-regulated by the first nucleic acid sequence to a less extent compared to a down-regulation of expression of the exogenous TCR (e.g., exogenous TCR chain) in combination with a nucleic acid sequence with complementarity (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 100% complementarity) to the subsequence of the second nucleic acid sequence. In some embodiments, an expression of the exogenous TCR (e.g., exogenous TCR chain) is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent compared to a down-regulation of expression of the exogenous TCR chain by the CRISPR- Cas system in combination with a nucleic acid sequence with complementarity (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 100% complementarity) to the subsequence of the second nucleic acid sequence. In some embodiments, down-regulation of the exogenous TCR (e.g., exogenous TCR chain) by the first nucleic acid sequence in combination with a CRISPR-Cas system can be at most about 50x, at most about 20x, at most about 18x, at most about 16x, at most about 14x, at most about 12x, at most about lOx, at most about 9x, at most about lOx, at most about lOx, at most about lOx, at most about lOx, at most about lOx, at most about lOx, or at most about 2x less than a down-regulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with complementarity (e.g., at least about 95%, 96%, 97%, 98%, 99%, or 100% complementarity) to the subsequence of the second nucleic acid sequence.

[0199] In some embodiments, an expression of the exogenous TCR (e.g., exogenous TCR chain) is not down-regulated. In some embodiments, an expression of the exogenous TCR (e.g., exogenous TCR chain) is not down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system. The CRISPR-Cas system (e.g., CRISPR-Cas9) system may not recognize, bind to, or cleave the second nucleic acid sequence encoding the exogenous TCR (e.g., exogenous TCR chain) which may prevent and / or inhibit down-regulation. In some embodiments, an expression of the exogenous TCR (e.g., exogenous TCR chain) is not substantially down- regulated. In some embodiments, an expression of the exogenous TCR (e.g., exogenous TCR chain) is not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system. An expression level that is not substantially downregulated may comprise an expression of the exogenous TCR (e.g., exogenous TCR chain) that is downregulated by at most about 20%, at most about 15%, at most about 10%, at most about 5%, at most about 4%, at most about 3%, at most about 2%, at most about 1%, or less than about 1% compared to an expression level of an exogenous TCR that has not been contacted by the nucleic acid sequence in combination with a CRISPR-Cas system.

[0200] In some embodiments, the target sequence comprises a non-coding sequence. The noncoding sequence may comprise an intron or a portion of an intron. The portion of the intron may be at least about 1 nucleotide in length, at least about 2 nucleotides in length, at least about 3 nucleotides in length, at least about 4 nucleotides in length, at least about 5 nucleotides in length, at least about 10 nucleotides in length, at least about 15 nucleotides in length, at least about 20nucleotides in length, at least about 30 nucleotides in length, at least about 40 nucleotides in length, at least about 50, or greater than about 50 nucleotides in length. The portion of the intron may be at most 50 nucleotides in length, at most about 40 nucleotides in length, at most about 30 nucleotides in length, at most about 20 nucleotides in length, at most about 15 nucleotides in length, at most about 10 nucleotides in length, at most about 5 nucleotides in length, at most about 4 nucleotides in length, at most about 3 nucleotides in length, at most about 2 nucleotides in length, at most about 1 nucleotides in length, or less than about 1 nucleotide in length.

[0201] The portion of the intron may be from about 1 nucleotide to about 20 nucleotides in length. The portion of the intron may be from about 1 nucleotide to about 2 nucleotides, about 1 nucleotide to about 3 nucleotides, about 1 nucleotide to about 4 nucleotides, about 1 nucleotide to about 5 nucleotides, about 1 nucleotide to about 6 nucleotides, about 1 nucleotide to about 7 nucleotides, about 1 nucleotide to about 8 nucleotides, about 1 nucleotide to about 9 nucleotides, about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 2 nucleotides to about 3 nucleotides, about 2 nucleotides to about 4 nucleotides, about 2 nucleotides to about 5 nucleotides, about 2 nucleotides to about 6 nucleotides, about 2 nucleotides to about 7 nucleotides, about 2 nucleotides to about 8 nucleotides, about 2 nucleotides to about 9 nucleotides, about 2 nucleotides to about 10 nucleotides, about 2 nucleotides to about 15 nucleotides, about 2 nucleotides to about 20 nucleotides, about 3 nucleotides to about 4 nucleotides, about 3 nucleotides to about 5 nucleotides, about 3 nucleotides to about 6 nucleotides, about 3 nucleotides to about 7 nucleotides, about 3 nucleotides to about 8 nucleotides, about 3 nucleotides to about 9 nucleotides, about 3 nucleotides to about 10 nucleotides, about 3 nucleotides to about 15 nucleotides, about 3 nucleotides to about 20 nucleotides, about 4 nucleotides to about 5 nucleotides, about 4 nucleotides to about 6 nucleotides, about 4 nucleotides to about 7 nucleotides, about 4 nucleotides to about 8 nucleotides, about 4 nucleotides to about 9 nucleotides, about 4 nucleotides to about 10 nucleotides, about 4 nucleotides to about 15 nucleotides, about 4 nucleotides to about 20 nucleotides, about 5 nucleotides to about 6 nucleotides, about 5 nucleotides to about 7 nucleotides, about 5 nucleotides to about 8 nucleotides, about 5 nucleotides to about 9 nucleotides, about 5 nucleotides to about 10 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 6 nucleotides to about 7 nucleotides, about 6 nucleotides to about 8 nucleotides, about 6 nucleotides to about 9 nucleotides, about 6 nucleotides to about 10 nucleotides, about 6 nucleotides to about 15 nucleotides, about 6 nucleotides to about 20 nucleotides, about 7 nucleotides to about 8 nucleotides, about 7 nucleotides to about 9 nucleotides, about 7 nucleotidesto about 10 nucleotides, about 7 nucleotides to about 15 nucleotides, about 7 nucleotides to about 20 nucleotides, about 8 nucleotides to about 9 nucleotides, about 8 nucleotides to about 10 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotides to about 20 nucleotides, about 9 nucleotides to about 10 nucleotides, about 9 nucleotides to about 15 nucleotides, about 9 nucleotides to about 20 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, or about 15 nucleotides to about 20 nucleotides in length.

[0202] In some embodiments, the endogenous TCR chain can be a human TCR chain. In some embodiments, the exogenous TCR chain can be a human TCR chain. In some embodiments, the endogenous TCR chain can be a human TCR chain and the exogenous TCR chain can be a human TCR chain.

[0203] In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain. The TCR constant domain may be a TCR alpha constant domain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR beta chain. The TCR constant domain may be a TCR beta constant domain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain and an endogenous TCR beta chain. The TCR constant domain may be a TCR alpha constant domain and a TCR beta constant domain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR gamma chain. The TCR constant domain may be a TCR gamma constant domain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR delta chain. The TCR constant domain may be a TCR delta constant domain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR gamma chain and an endogenous TCR delta chain. The TCR constant domain may be a TCR gamma constant domain and a TCR delta constant domain.

[0204] In some embodiments, the first nucleic acid sequence can be transiently expressed in the engineered T cell. Transient expression may comprise the temporary expression of the nucleic acid molecule (e.g., gene expressed by the nucleic acid molecule) following introduction into the engineered T cell. Transient expression may occur when the nucleic acid molecule is taken up by the cell but not incorporated into the genome of the cell. Transient expression may comprise production and / or detection of the nucleic acid molecule (e.g., targeting sequence) for a time period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more days. Transient expression may comprise production and / or detection of the nucleic acid molecule (e.g., targeting sequence) for a time period of at most about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than 1 day. In some embodiments, the first nucleic acid sequence can be stably expressed in theengineered T cell. Stable expression may comprise incorporation of the first nucleic acid sequence into the host genome. For example, stable expression can comprise long-term maintenance of the first nucleic acid sequence described herein in the transfected cell (e.g., the engineered T cell). In some embodiments, stable expression can be achieved through lentiviral vector transduction.

[0205] In some embodiments, the first nucleic acid sequence comprises an RNA sequence. In some embodiments, the first nucleic acid sequence comprises a DNA sequence. In some embodiments, the first nucleic acid sequence (e.g., RNA sequence) may be a gRNA sequence. The gRNA sequence may hybridize to the target sequence of the engineered T cell. The gRNA sequence may be complementary to the target sequence. In some embodiments, the first nucleic acid sequence comprises a gRNA sequence targeting an endogenous TCR chain.

[0206] The endogenous TCR chain may comprise an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain, an endogenous TCR delta chain, or any combination thereof. In some embodiments, the endogenous TCR chain may comprise one or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain, and an endogenous TCR delta chain.

[0207] In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain and the first nucleic acid sequence comprises a gRNA sequence targeting the endogenous TCR alpha chain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR beta chain and the first nucleic acid sequence comprises a gRNA sequence targeting the endogenous TCR beta chain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain and an endogenous TCR beta chain, and the first nucleic acid sequence comprises a gRNA sequence targeting the endogenous TCR alpha chain and the endogenous TCR beta chain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR gamma chain and the first nucleic acid sequence comprises a gRNA sequence targeting the endogenous TCR gamma chain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR delta chain and the first nucleic acid sequence comprises a gRNA sequence targeting the endogenous TCR delta chain. In some embodiments, the endogenous TCR chain comprises an endogenous TCR gamma chain and an endogenous TCR delta chain, and the first nucleic acid sequence comprises a gRNA sequence targeting the endogenous TCR gamma chain and the endogenous TCR delta chain.

[0208] In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain, an endogenous TCRdelta chain, or any combination thereof, and the first nucleic acid sequence comprises a gRNA sequence targeting an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain, an endogenous TCR delta chain, or any combination thereof.

[0209] In some embodiments, the first nucleic acid sequence comprises two or more gRNA sequences. The two or more gRNA sequences (e.g., a first gRNA sequence and second gRNA sequence) can target two or more endogenous TCR chains. A first gRNA may comprise a targeting sequence that hybridizes to a first target sequence. A second gRNA may comprise a targeting sequence that hybridizes to a second target sequence. In some embodiments, the endogenous TCR chain comprises two or more endogenous TCR chains comprising an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain, an endogenous TCR delta chain, or any combination thereof.

[0210] In some embodiments, a first target sequence can encode a TCR alpha constant domain of the endogenous TCR alpha chain. In some embodiments, a second target sequence can encode a TCR beta constant domain of the endogenous TCR beta chain. A first nucleic acid sequence may comprise a first gRNA comprising a targeting sequence that hybridizes to the first target sequence, a second gRNA comprising a targeting sequence that hybridizes to the second target sequence, or any combination thereof. For example, the first gRNA may comprise a targeting sequence that hybridizes to the target sequence encoding the TCR alpha constant domain of the endogenous TCR alpha chain and / or the second gRNA may comprise a targeting sequence that hybridizes to the target sequence encoding the TCR beta constant domain of the endogenous TCR beta chain. In some embodiments, a first target sequence can encode a TCR gamma constant domain of the endogenous TCR gamma chain. In some embodiments, a second target sequence can encode a TCR delta constant domain of the endogenous TCR delta chain. A first nucleic acid sequence may comprise a first gRNA comprising a targeting sequence that hybridizes to the first target sequence, a second gRNA comprising a targeting sequence that hybridizes to the second target sequence, or any combination thereof. For example, the first gRNA may comprise a targeting sequence that hybridizes to the target sequence encoding the TCR gamma constant domain of the endogenous TCR gamma chain and / or the second gRNA may comprise a targeting sequence that hybridizes to the target sequence encoding the TCR delta constant domain of the endogenous TCR delta chain.

[0211] In some embodiments, the endogenous TCR chain comprises an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain, an endogenous TCR delta chain, or any combination thereof, and the first nucleic acid sequence comprises a first gRNAsequence and a second gRNA sequence each targeting an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain, an endogenous TCR delta chain, or any combination thereof.

[0212] The second nucleic acid sequence may comprise a sequence encoding an exogenous TCR alpha chain, a sequence encoding an exogenous TCR beta chain, or any combination thereof. In some embodiments, a subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR alpha chain, a sequence encoding an exogenous TCR beta chain, or any combination thereof) may have less than 100% sequence identity (e.g., at most about 99.9%, 99.5%, 99%, 98.5%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than 20% sequence identity) to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR alpha chain, a sequence encoding an exogenous TCR beta chain, or any combination thereof) may have a mutation or variation relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR alpha chain, a sequence encoding an exogenous TCR beta chain, or any combination thereof) may have one or more mutations or variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more mutations or variations) relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. For example, a subsequence of the sequence encoding the exogenous TCR alpha chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR alpha constant domain of the endogenous TCR alpha chain. As another example, a subsequence of the sequence encoding the exogenous TCR alpha chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR alpha constant domain of the endogenous TCR alpha chain. For example, a subsequence of the sequence encoding the exogenous TCR beta chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR beta constant domain of the endogenous TCR beta chain. As another example, a subsequence of the sequence encoding the exogenous TCR beta chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR beta constant domain of the endogenous TCR beta chain.

[0213] In some embodiments, a sequence downstream of the subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR alpha chain, a sequence encoding an exogenous TCR beta chain, or any combination thereof) may have less than 100% sequence identity (e.g., at most about 99.9%, 99.5%, 99%, 98.5%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than 20% sequence identity) to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a sequence downstream of the subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR alpha chain, a sequence encoding an exogenous TCR beta chain, or any combination thereof) may have a mutation or variation relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a sequence downstream of the subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR alpha chain, a sequence encoding an exogenous TCR beta chain, or any combination thereof) may have one or more mutations or variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more mutations or variations) relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. For example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR alpha chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR alpha constant domain of the endogenous TCR alpha chain. As another example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR alpha chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR alpha constant domain of the endogenous TCR alpha chain. For example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR beta chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR beta constant domain of the endogenous TCR beta chain. As another example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR beta chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR beta constant domain of the endogenous TCR beta chain.

[0214] The second nucleic acid sequence may comprise a sequence encoding an exogenous TCR gamma chain, a sequence encoding an exogenous TCR delta chain, or any combination thereof. In some embodiments, a subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR gamma chain, a sequence encoding anexogenous TCR delta chain, or any combination thereof) may have less than 100% sequence identity (e.g., at most about 99.9%, 99.5%, 99%, 98.5%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than 20% sequence identity) to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR gamma chain, a sequence encoding an exogenous TCR delta chain, or any combination thereof) may have a mutation or variation relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR gamma chain, a sequence encoding an exogenous TCR delta chain, or any combination thereof) may have one or more mutations or variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more mutations or variations) relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. For example, a subsequence of the sequence encoding the exogenous TCR gamma chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR gamma constant domain of the endogenous TCR gamma chain. As another example, a subsequence of the sequence encoding the exogenous TCR gamma chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR gamma constant domain of the endogenous TCR gamma chain. For example, a subsequence of the sequence encoding the exogenous TCR delta chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR delta constant domain of the endogenous TCR delta chain. As another example, a subsequence of the sequence encoding the exogenous TCR delta chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR delta constant domain of the endogenous TCR delta chain.

[0215] In some embodiments, a sequence downstream of the subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR gamma chain, a sequence encoding an exogenous TCR delta chain, or any combination thereof) may have less than 100% sequence identity (e.g., at most about 99.9%, 99.5%, 99%, 98.5%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than 20% sequence identity) to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a sequence downstream of thesubsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR gamma chain, a sequence encoding an exogenous TCR delta chain, or any combination thereof) may have a mutation or variation relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. In some embodiments, a sequence downstream of the subsequence of the sequence of the second nucleic sequence (e.g., comprising the sequence encoding an exogenous TCR gamma chain, a sequence encoding an exogenous TCR delta chain, or any combination thereof) may have one or more mutations or variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more mutations or variations) relative to the first target sequence or complement thereof, second target sequence or complement thereof, or any combination thereof. For example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR gamma chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR gamma constant domain of the endogenous TCR gamma chain. As another example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR gamma chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR gamma constant domain of the endogenous TCR gamma chain. For example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR delta chain may have between about 10%-99% sequence identity to the first target sequence or complement thereof encoding the TCR delta constant domain of the endogenous TCR delta chain. As another example, a sequence downstream of the subsequence of the sequence encoding the exogenous TCR delta chain may have between about 1-10 mutations or variations relative to the first target sequence or complement thereof encoding the TCR delta constant domain of the endogenous TCR delta chain.

[0216] In some embodiments, the first nucleic acid sequence comprising the gRNA is at least about 5 nucleotides in length, at least about 6 nucleotides in length, at least about 7 nucleotides in length, at least about 8 nucleotides in length, at least about 9 nucleotides in length, at least about 10 nucleotides in length, at least about 15 nucleotides in length, at least about 20 nucleotides in length, or greater than about 20 nucleotides in length. In some embodiments, the first nucleic acid sequence comprising the gRNA is at most about 20 nucleotides in length, at most about 15 nucleotides in length, at most about 10 nucleotides in length, at most about 9 nucleotides in length, at most about 8 nucleotides in length, at most about 7 nucleotides in length, at most about 6 nucleotides in length, at most about 5 nucleotides in length, or less than about 5 nucleotides in length. In some embodiments, the first nucleic acid sequence comprises a nucleotide sequencewith at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or greater than about 99.9% sequence identity to a nucleotide sequence as set forth in any one of SEQ ID NOs: 6 or 13. In some embodiments, the first nucleic acid sequence comprises a nucleotide sequence with 100% sequence identity to a nucleotide sequence as set forth in a sequence selected from the group consisting of SEQ ID NOs: 6 or 13.

[0217] In some embodiments, the first nucleic acid sequence can be encoded by a DNA sequence. In some embodiments, a first nucleic acid sequence and a second nucleic acid sequence are on the same nucleic acid molecule. In some embodiments, a first nucleic acid sequence and a sequence encoding a second nucleic acid sequence are on the same nucleic acid molecule. In some embodiments, a sequence encoding a first nucleic acid sequence and a second nucleic acid sequence are on the same nucleic acid molecule. In some embodiments, a sequence encoding a first nucleic acid sequence and a sequence encoding a second nucleic acid sequence are on the same nucleic acid molecule. In some embodiments, a first nucleic acid sequence and a second nucleic acid sequence are on different nucleic acid molecules. In some embodiments, a first nucleic acid sequence and a sequence encoding a second nucleic acid sequence are on different nucleic acid molecules. In some embodiments, a sequence encoding a first nucleic acid sequence and a second nucleic acid sequence are on different nucleic acid molecules. In some embodiments, a sequence encoding a first nucleic acid sequence and a sequence encoding a second nucleic acid sequence are on different nucleic acid molecules.

[0218] In some embodiments, the sequence downstream of the target sequence comprises the PAM site and the sequence downstream of the subsequence comprises a mutation in a PAM site. In some embodiments, the PAM site comprises a sequence as set forth in NGG, wherein N is A, T, C, or G. In some embodiments, the PAM site located in the sequence downstream of the subsequence encoding the same amino acid sequence encoded by the target sequence comprises one or more mutations (e.g., 1, 2, 3, 4, 5, or more than 5 mutations). In some embodiments, the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a sequence with at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98.5%, at least about 99%, at least about 99.5%, at least about 99.9%, or at least about 100% sequence identity to a sequence as set forth in SEQ Identifier: A3. In some embodiments, the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a sequence of NHN orHNH, where N stands for any nucleotide of A / T / C / G and H stands for any nucleotide of A / T / C. For example, the sequence downstream of the subsequence of the second nucleic acid or a complement thereof can comprise a sequence of TAG, AGA, AGC, TGA, TGC, TTG, or TCG.

[0219] In some embodiments, the engineered T cell comprises a plurality of engineered T cells. The plurality of engineered T cells may be part of a population of T cells. The plurality of engineered T cells may comprise a percentage of a population of T cells. In some embodiments, a percentage of T cells (e.g., non-engineered T cells) of the population of T cells can have a reduced expression of the endogenous TCR, the endogenous TCR chain, or any combination thereof. In some embodiments, the population of T cells comprises at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or greater than about 99% of T cells having a reduced expression of the endogenous TCR, the endogenous TCR chain, or any combination thereof. In some embodiments, the population of T cells comprises at most about 99%, at most about 98%, at most about 97%, at most about 96%, at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less than about 10% of T cells having a reduced expression of the endogenous TCR, the endogenous TCR chain, or any combination thereof.

[0220] In some embodiments, the population of T cells comprises from about 10% to about 95% of T cells having a reduced expression of the endogenous TCR, the endogenous TCR chain, or any combination thereof. In some embodiments, the population of T cells comprises from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 95%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 75%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% toabout 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95% of T cells having a reduced expression of the endogenous TCR, the endogenous TCR chain, or any combination thereof.

[0221] In some embodiments, the T cells of the population of T cells can show at least about, at most about, or about a 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduced expression of an endogenous TCR, an endogenous TCR chain, or any combination thereof. In some embodiments, the T cells of the population of T cells can have an eliminated expression (e.g., 100% reduced expression) of an endogenous TCR, an endogenous TCR chain, or any combination thereof.

[0222] In some embodiments, an engineered T cell of a plurality of engineered T cells comprising two or more gRNA sequences targeting an endogenous TCR, an endogenous TCR chain, or any combination thereof, may show a greater reduced expression of the endogenous TCR and / or the endogenous TCR chain compared to an expression in an engineered T cell of an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain. In some embodiments, an engineered T cell of a plurality of engineered T cells comprising two or more gRNA sequences targeting two or more of an endogenous TCR and / or an endogenous TCR chain can have at least about 1.1 fold, at least about 1.2 fold, at least about 1.3 fold, at least about 1.4 fold, at least about 1.5 fold, at least about 2.0 fold, at least about 2.5 fold, at least about 3.0 fold, at least about 3.5 fold, at least about 4.0 fold, at least about 4.5 fold, at least about 5.0 fold, at least about 6.0 fold, at least about 7.0 fold, at least about 8.0 fold, at least about 9.0 fold, at least about 10.0 fold, or greater than 10.0 fold reduced expression of the endogenous TCR and / or an endogenous TCR chain compared to an expression in an otherwise identical engineered T cell of an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain. In some embodiments, an engineered T cell of a plurality of engineered T cells comprising two or more gRNA sequences targeting two or more of an endogenous TCR and / or an endogenous TCR chain can have at most about 10.0 fold, at most about 9.0 fold, at most about 8.0 fold, at most about 7.0 fold, at most about 6.0 fold, at most about 5.0 fold, at most about 4.5 fold, at most about 4.0 fold, at most about3.5 fold, at most about 3.0 fold, at most about 2.5 fold, at most about 2.0 fold, at most about 1.5 fold, at most about 1.4 fold, at most about 1.3 fold, at most about 1.2 fold, at most about 1.1 fold, or less than about 1.1 fold reduced expression of the endogenous TCR and / or the endogenous TCR chain compared to an expression in an otherwise identical engineered T cell of an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain.

[0223] In some embodiments, an engineered T cell of a plurality of engineered T cells comprising two or more gRNA sequences targeting two or more of an endogenous TCR and / or an endogenous TCR chain can have from about 1.1 fold to about 8 fold reduced expression of the endogenous TCR and / or the endogenous TCR chain compared to an expression in an otherwise identical engineered T cell of an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain. In some embodiments, an engineered T cell of a plurality of engineered T cells comprising two or more gRNA sequences targeting two or more of an endogenous TCR and / or an endogenous TCR chain can have from about 1.1 fold to about 1.2 fold, about 1.1 fold to about 1.3 fold, about 1.1 fold to about 1.4 fold, about 1.1 fold to about 1.5 fold, about 1.1 fold to about 2 fold, about 1.1 fold to about 3 fold, about 1.1 fold to about 4 fold, about 1.1 fold to about 5 fold, about 1.1 fold to about 6 fold, about 1.1 fold to about 7 fold, about 1.1 fold to about 8 fold, about 1.2 fold to about 1.3 fold, about 1.2 fold to about 1.4 fold, about 1.2 fold to about 1.5 fold, about 1.2 fold to about 2 fold, about 1.2 fold to about 3 fold, about 1.2 fold to about 4 fold, about 1.2 fold to about 5 fold, about 1.2 fold to about 6 fold, about 1.2 fold to about 7 fold, about 1.2 fold to about 8 fold, about 1.3 fold to about 1.4 fold, about 1.3 fold to about 1.5 fold, about 1.3 fold to about 2 fold, about 1.3 fold to about 3 fold, about 1.3 fold to about 4 fold, about 1.3 fold to about 5 fold, about 1.3 fold to about 6 fold, about 1.3 fold to about 7 fold, about 1.3 fold to about 8 fold, about 1.4 fold to about 1.5 fold, about 1.4 fold to about 2 fold, about 1.4 fold to about 3 fold, about 1.4 fold to about 4 fold, about 1.4 fold to about 5 fold, about 1.4 fold to about 6 fold, about 1.4 fold to about 7 fold, about 1.4 fold to about 8 fold, about1.5 fold to about 2 fold, about 1.5 fold to about 3 fold, about 1.5 fold to about 4 fold, about 1.5 fold to about 5 fold, about 1.5 fold to about 6 fold, about 1.5 fold to about 7 fold, about 1.5 fold to about 8 fold, about 2 fold to about 3 fold, about 2 fold to about 4 fold, about 2 fold to about 5 fold, about 2 fold to about 6 fold, about 2 fold to about 7 fold, about 2 fold to about 8 fold, about 3 fold to about 4 fold, about 3 fold to about 5 fold, about 3 fold to about 6 fold, about 3 fold to about 7 fold, about 3 fold to about 8 fold, about 4 fold to about 5 fold, about 4 fold to about 6 fold, about 4 fold to about 7 fold, about 4 fold to about 8 fold, about 5 fold to about 6 fold, about 5 foldto about 7 fold, about 5 fold to about 8 fold, about 6 fold to about 7 fold, about 6 fold to about 8 fold, or about 7 fold to about 8 fold reduced expression of the endogenous TCR and / or the endogenous TCR chain compared to an expression in an otherwise identical engineered T cell of an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain.

[0224] In some embodiments, an engineered T cell of a plurality of engineered T cells comprising two or more gRNA sequences targeting two or more of an endogenous TCR and / or an endogenous TCR chain can have eliminated expression (e.g., 100% reduced expression) of the endogenous TCR and / or the endogenous TCR chain compared to an expression in an otherwise identical engineered T cell of an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain.

[0225] The reduced and / or eliminated expression of the endogenous TCR and / or the endogenous TCR chain in an engineered T cell described herein may be measured by a surface marker. In some embodiments, the surface marker can be CD3s, CD36, CD3y, TCRa, TCR0, TCR5, TCRy, CD3<^, or any combination thereof.

[0226] In some embodiments, an engineered T cell can be a plurality of transduced engineered T cells. The plurality of transduced engineered T cells may be derived from a population of T cells comprising the first nucleic acid sequence and transduced with the second nucleic acid sequence. The second nucleic acid may encode the exogenous TCR chain and comprise the subsequence encoding the same amino acid sequence encoded by a target sequence of a first nucleic acid sequence. In some embodiments, the percentage of transduced engineered T cells derived from the population of T cells can be higher compared to a percentage of transduced engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a subsequence having 100% sequence identity to the target sequence or complement thereof of the first nucleic acid sequence. In some embodiments, the percentage of transduced engineered T cells derived from the population of T cells can be higher compared to a percentage of transduced engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof of the first nucleic acid sequence. In some embodiments, thepercentage of transduced engineered T cells derived from the population of T cells can be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or greater than about 90% higher compared to a percentage of transduced engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence, wherein the second nucleic acid sequence comprises (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof. In some embodiments, the percentage of transduced engineered T cells derived from the population of T cells can be at most about 90%, at most about 80%, at most about 70%, at most about 60%, at most about 50%, at most about 40%, at most about 30%, at most about 20%, at most about 10%, or less than about 10% higher compared to a percentage of transduced engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence, wherein the second nucleic acid sequence comprises (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof.

[0227] In some embodiments, a percentage of transduced engineered T cells (e.g., engineered T cells comprising the first nucleic acid molecule (e.g., gRNA) and transduced with the second nucleic acid molecule comprising a sequence encoding the exogenous TCR or TCR chain (e.g., sequence comprising at least one mutation)) may comprise at least about 10%, at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater than about 95% of the total T cells in the population of T cells from which the transduced engineered T cells are derived. In some embodiments, a percentage of transduced engineered T cells may comprise at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 20%, at most about 10%, or less than about 10% of the total T cells in the population of T cells from which the transduced engineered T cells are derived.

[0228] In some embodiments, a percentage of transduced engineered T cells may comprise from about 10% to about 95% of the total T cells in the population of T cells from which the transduced engineered T cells are derived. In some embodiments, a percentage of transduced engineered T cells may comprise from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 95%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 75%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 95%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 95%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 95%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, about 85% to about 95%, or about 90% to about 95% of the total T cells in the population of T cells from which the transduced engineered T cells are derived.

[0229] In some embodiments, a percentage of a plurality of engineered T cells expressing the exogenous TCR and / or exogenous TCR chain is higher than a plurality of engineered T cells not expressing the exogenous TCR and / or exogenous TCR chain. A percentage of engineered T cells derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence (e.g., expressing the exogenous TCR and / or exogenous TCR chain) can be at least about 10%, at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% higher compared to a percentage of engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequenceor a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof. A percentage of engineered T cells derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence (e.g., expressing the exogenous TCR and / or exogenous TCR chain) can be at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 20%, or at most about 10% higher compared to a percentage of engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof.

[0230] A percentage of engineered T cells derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence (e.g., expressing the exogenous TCR and / or exogenous TCR chain) can be from about 10% to about 99% higher compared to a percentage of engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof. A percentage of engineered T cells derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence (e.g., expressing the exogenous TCR and / or exogenous TCR chain) can be from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 99%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 75%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 99%, about 40% to about 50%, about 40% to about60%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 99%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 99%, about 85% to about 90%, about 85% to about 99%, or about 90% to about 99% higher compared to a percentage of engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof.

[0231] In some embodiments, a plurality of engineered T cells expressing the first nucleic acid sequence and the second nucleic acid sequence described herein may express the exogenous TCR (e.g., exogenous TCR chain) at a higher level compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having(i) a subsequence with 100% sequence identity to the target sequence or a complement thereof,(ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof. An expression of the exogenous TCR (e.g., exogenous TCR chain) of a plurality of engineered T cells expressing the first nucleic acid sequence and the second nucleic acid sequence described herein may be at least about 10%, at least about 20%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% higher compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof. An expression of the exogenous TCR (e.g., exogenous TCR chain) of aplurality of engineered T cells expressing the first nucleic acid sequence and the second nucleic acid sequence described herein may be at most about 95%, at most about 90%, at most about 85%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 20%, or at most about 10% higher compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof.

[0232] An expression of the exogenous TCR (e.g., exogenous TCR chain) of a plurality of engineered T cells expressing the first nucleic acid sequence and the second nucleic acid sequence described herein may be from about 10% to about 99% higher compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof. An expression of the exogenous TCR (e.g., exogenous TCR chain) of a plurality of engineered T cells expressing the first nucleic acid sequence and the second nucleic acid sequence described herein may be from about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 75%, about 10% to about 80%, about 10% to about 85%, about 10% to about 90%, about 10% to about 99%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 75%, about 20% to about 80%, about 20% to about 85%, about 20% to about 90%, about 20% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 75%, about 30% to about 80%, about 30% to about 85%, about 30% to about 90%, about 30% to about 99%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 75%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 75%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 99%, about 60% to about 70%, about 60% to about 75%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about99%, about 70% to about 75%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 99%, about 75% to about 80%, about 75% to about 85%, about 75% to about 90%, about 75% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 99%, about 85% to about 90%, about 85% to about 99%, or about 90% to about 99% higher compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof.

[0233] In some embodiments, the second nucleic acid sequence may be encoded by a vector. In some embodiments, the vector can be a viral vector. In some embodiments, the vector may be a retroviral vector. In some embodiments, the vector may be a lentiviral vector.

[0234] In some embodiments, the exogenous TCR encoded by the second nucleic acid molecule of the engineered T cell described herein may recognize an epitope. In some embodiments, the epitope may be of an antigen. In some embodiments, the epitope may be of an antigen in complex with an MHC molecule. The epitope may be associated with a cancer, an autoimmune disease, an infectious disease, or any combination thereof. In some embodiments, the antigen can be NY- ESO-1, KRAS, TP53, NRAS, BRAF, PIK3CA, or any combination thereof.

[0235] In some embodiments, the antigen may be associated with a cancer and the cancer may be mesothelioma, renal cell carcinoma, breast cancer, prostate cancer, pancreatic cancer, ureter cancer, kidney cancer, lung cancer, liver cancer, ovarian cancer, cervical cancer, colon cancer, esophageal cancer, glioma, gastric cancer, brain cancer, stomach cancer, bladder cancer, testicular cancer, thyroid cancer, thymic carcinoma, adrenal cancer, head and neck cancer, melanoma, skin cancer, sarcoma, fibrosarcoma, angiosarcoma, osteosarcoma, rhabdomyosarcoma, cholangiocarcinoma, leukemia, lymphoma, myeloma, a neuroendocrine tumor, endometrial cancer, or glioblastoma.

[0236] In some embodiments, the antigen may be associated with an autoimmune disease and the autoimmune disease may be amyotrophic lateral sclerosis (ALS), coeliac disease (CD), ankylosing spondylitis (AS), Covid-induced multisystem inflammatory syndrome in children (MIS-C), primary Sjogren’s syndrome (PSS), Churg-Strauss syndrome, sarcoidosis, systemic lupus erythematosus (SLE), type 1 diabetes, autoimmune hepatitis (e.g., type 1 or type 2), primary sclerosing cholangitis, primary biliary cirrhosis, multiple sclerosis, Guillain-Barre syndrome and the AMAN (axonal & neuronal neuropathy), chronic inflammatory demyelinating polyneuropathy(CIDP), transverse myelitis, Tolosa-Hunt syndrome (THS), Devic’s disease (neuromyelitis optica), paraneoplastic cerebellar degeneration (PCD), Lambert-Eaton syndrome, psoriasis, scleroderma, CREST (calcinosis, Raynaud phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia) syndrome, dermatitis herpetiformis, dermatomyositis, bullous pemphigoid, cicatricial pemphigoid / benign mucosal pemphigoid, pemphigoid gestationis, rheumatoid arthritis (RA), psoriatic arthritis, relapsing polychondritis, chronic recurrent multifocal osteomyelitis (CRMO), vasculitis, Kawasaki disease, granulomatosis with polyangiitis (GPA), Behcet’s disease (vasculitis), Takayasu’s arteritis, polyarteritis nodosa, microscopic polyangiitis (MPA), leukocytoclastic vasculitis, Cogan’s syndrome, uveitis, peripheral uveitis (Pars planitis), scleritis, autoimmune inner ear disease (AIED), Crohn’s, ulcerative colitis (UC), Dressier’s syndrome, Rheumatic fever, Evans syndrome, paroxysmal nocturnal hemoglobinuria (PNH), hemolytic anemia, thrombocytopenic purpura (TTP), polymyositis, juvenile myositis (JM), including Juvenile Dermatomyositis (JDM) and Juvenile Polymyositis (JPM), ocular cicatricial pemphigoid, or Hashimoto’s thyroiditis.

[0237] In some embodiments, the antigen may be associated with an infectious disease and the infectious disease may be Epstein-Barr virus (EBV), influenza, human immunodeficiency virus (HIV), anaplasmosis, simian immunodeficiency virus (SIV), tuberculosis, malaria, meningitis, dengue, coronavirus, bocavirus, giardiasis, gastroenteritis, diphtheria, Lyme disease, hepatitis virus, human papillomavirus, or human cytomegalovirus (HCMV).

[0238] In some embodiments, the present disclosure provides a population of T cells. The population of T cells can comprise at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) of the engineered T cells as described herein.

[0239] In some embodiments, the engineered T cells described herein may comprise enhanced functioning compared to unmodified (e.g., control) T cells. The engineered T cells comprising the first nucleic acid sequence and / or the second nucleic acid sequence as described herein may comprise improved cytotoxicity compared to that of unmodified T cells. The engineered T cells comprising the first nucleic acid sequence and / or the second nucleic acid sequence as described herein may comprise improved cytotoxicity compared to engineered T cells comprising the first nucleic acid sequence and a second nucleic acid sequence comprising (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (iii) any combination thereof. The engineered T cellsdescribed herein may show an increased expression of at least one cytokine molecule. The cytokine molecule may be IFNy, TNFa, IL-4, IL-5, IL-10, IL-2, or any combination thereof.T-Cell Receptors (TCRs)

[0240] The ability of T cells to recognize antigens is conferred by its TCR, which is made up of both an alpha (a) chain and a beta (P) chain or a gamma (y) and a delta (5) chain. The proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the diversity of the TCR. This multi-subunit immune recognition receptor can associate with the CD3 complex and bind peptides presented by the MHC class I and II proteins on the surface of antigen-presenting cells (APCs). The first signal in activation of T cells can be provided by binding of the T-cell receptor to a short peptide presented by the MHC on another cell. This ensures that only a T cell with a TCR specific to that peptide is activated. The partner cell is usually an antigen-presenting cell such as a dendritic cell, B cell, macrophage, or any combination thereof. Binding of a TCR to the antigenic peptide on the APC can be a central event in T cell activation, which occurs at an immunological synapse at the point of contact between the T cell and the APC.

[0241] Each TCR can comprise variable complementarity determining regions (CDRs), as well as framework regions (FRs) and a constant region. The TCR described herein may be in soluble form. The TCR described herein may not comprise a constant region. The TCR described herein may comprise a functional fragment of a full-length TCR. The TCR described herein may comprise the variable region of the alpha chain or beta chain only. The amino acid sequence of the third complementarity-determining region (CDR3) loops of the alpha (or a) and beta (or P) chain variable domains largely determines the sequence diversity of aP T cells arising from recombination between variable (VP), diversity (DP), and joining (JP) gene segments in the P chain locus, and between analogous Va and Ja gene segments in the a chain locus, respectively. The existence of multiple such gene segments in the TCR a and P chain loci allows for a large number of distinct CDR3 sequences to be encoded. Independent addition and deletion of nucleotides at the VP-DP, Dp-jp, and Va-Ja junctions during the process of TCR gene rearrangement further increases CDR3 sequence diversity. In this respect, immunocompetence is reflected in the diversity of TCRs. The y5 TCR is distinctive from the aP TCR in that it encodes a receptor that interacts closely with the innate immune system. TCRyS, is expressed early in development, has specialized anatomical distribution, unique small-molecule specificities, and has a broad spectrum of innate and adaptive cellular interactions. Early in ontogeny, as therestricted subsets of TCRyS cells populate various tissues prenatally, a biased pattern of TCRy V and J segment expression is established.

[0242] TCRs can bind to a peptide-MHC complex. In some cases, the TCR can bind to a peptide- MHC complex comprising an epitope from an antigen or candidate antigen described herein. The binding of the TCR and the epitope bound with the MHC molecule (e.g., MHC class I or MHC class II) can trigger signal transduction pathways that lead to regulation of immune responses against the antigenic peptide. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of 1 pM to 1 nM. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of less than 500 nM, 250 nM, 150 nM, 100 nM, 50 nM, 25 nM or 10 nM. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of less than 500 nM. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of less than 250 nM. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of less than 150 nM. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of less than 100 nM. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of less than 50 nM. In some embodiments, the TCR binds to a peptide-MHC complex with a KD or an IC50 of less than 25 nM. In some embodiments, the TCR binds to an peptide-MHC complex with a KD or an IC50 of less than 10 nM.

[0243] In some embodiments, the nucleic acid is operably linked to a promoter. In various embodiments, the nucleic acid sequence encoding a TCR is codon optimized.

[0244] In some embodiments, the TCR sequence is comprised of human sequences. In some embodiments, the TCR sequence is comprised of non-human animal sequences that have been humanized using any method of humanization known in the art. The TCRs provided herein can be engineered TCRs.Gene Editing Systems

[0245] The present disclosure provides a gene editing system that may be used to down-regulate an endogenous gene. In some embodiments, the engineered T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., U.S. Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev. Genetics (2010) vl l, 636-646), or megaTAL nucleases (a fusion protein of a meganuclease toTAL repeats) methods. In this way, a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews Immunol. 9.10: 704-716, each incorporated herein by reference. For example, the engineered T cells may be modified to have reduced expression of endogenous TCR genes. In some embodiments, one or more endogenous TCR genes encoding a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain may be disrupted.

[0246] Current gene editing technologies comprise meganucleases, zinc-finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system. These four major classes of gene-editing techniques share a common mode of action in binding a user-defined sequence of DNA and mediating a double-stranded DNA break (DSB). DSB may then be repaired by either non-homologous end joining (NHEJ) or -when donor DNA is present- homologous recombination (HR), an event that introduces the homologous sequence from a donor DNA fragment. Additionally, nickase nucleases generate single-stranded DNA breaks (SSB). DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.

[0247] Genetic modification of genomic DNA can be performed using site-specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest. Methods for producing engineered, site-specific endonucleases are known in the art. For example, zinc- finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme. The zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence -18 basepairs in length. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005) Nucleic Acids Res 33, 5978). Likewise, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23 : 93 -9). In this case, however, the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair. Compact TALENs have an alternative endonuclease architecture that avoids theneed for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762). A Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-TevI homing endonuclease. Un-like Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Com-pact TALEN is functional as a monomer.

[0248] Engineered endonucleases based on the CRISPR-Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63). The CRISPR gene-editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA / TracrRNA. A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA" or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in the genome (multiplex genomic editing).

[0249] There are two classes of CRISPR systems known in the art (Adli (2018) Nat. Commun. 9: 1911), each containing multiple CRISPR types. Class I contains type I and type III CRISPR systems that are commonly found in Archaea. Class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR-Cas system is the type II CRISPR-Cas9 system, CRISPR-Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR-Cas proteins have been remodeled within last few years (Adli (2018) Nat. Commun. 9: 1911). Among these, such as Casl2a (Cpfl) proteins from Acidaminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.

[0250] Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double-stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sei. 30(7): 503-522). Meganucleases (MN) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446). In some embodiments,meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.

[0251] In addition to mentioned four major gene editing technologies, chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446). For example, A megaTAL is a single chimeric protein, which is the combination of the easy-to- tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.

[0252] In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR- Cas9 system, a gRNA and / or a polynucleotide encoding the Cas9, can be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods can be used. In some instances, physical delivery methods can be selected from the methods including but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some cases, the polynucleotide encoding the Cas9 is an RNA (e.g., mRNA), and the RNA can be delivered into the engineered immune cells via lipid nanoparticles. In some cases, the gRNA can be delivered into the engineered immune cells via lipid nanoparticles. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.

[0253] crRNA biogenesis in a Type II CRISPR system in nature requires a trans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified by endogenous RNaselll, and then hybridizes to a crRNA repeat in the pre-crRNA array. Endogenous RNaselll can be recruited to cleave the pre-crRNA. Cleaved crRNAs can be subjected to exoribonuclease trimming to produce the mature crRNA form (e.g., 5' trimming). The tracrRNA can remain hybridized to the crRNA, and the tracrRNA and the crRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNA of the crRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleic acid to which the crRNA can hybridize. Hybridization of the crRNA to the target nucleic acid can activate Cas9 for targeted nucleic acid cleavage. The target nucleic acid in a Type II CRISPR system is referred to as a protospacer adjacent motif (PAM). In nature, the PAM is essential to facilitate binding of a site-directed polypeptide (e.g., Cas9) to the target nucleic acid. Type II systems (also referred to as Nmeni or CASS4) are further subdivided into Type II- A (CASS4) and ILB (CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed that the CRISPR-Cas9 systemis useful for RNA-programmable genome editing, and international patent application publication number WO2013 / 176772 provides numerous examples and applications of the CRISPR-Cas endonuclease system for site-specific gene editing.

[0254] Type V CRISPR systems have several differences from Type II systems. For example, Cpfl is a single RNA-guided endonuclease that, in contrast to Type II systems, lacks tracrRNA. In fact, Cpfl -associated CRISPR arrays can be processed into mature crRNAs without the requirement of an additional trans-activating tracrRNA. The Type V CRISPR array can be processed into short mature crRNAs of 42-44 nucleotides in length, with each mature crRNA beginning with 19 nucleotides of direct repeat followed by 23-25 nucleotides of spacer sequence. In contrast, mature crRNAs in Type II systems can start with 20-24 nucleotides of spacer sequence followed by about 22 nucleotides of direct repeat. Also, Cpfl can utilize a T-rich protospacer- adjacent motif such that Cpfl -crRNA complexes efficiently cleave target DNA preceded by a short T-rich PAM, which is in contrast to the G-rich PAM following the target DNA for Type II systems. Thus, Type V systems cleave at a point that is distant from the PAM, while Type II systems cleave at a point that is adjacent to the PAM. In addition, in contrast to Type II systems, Cpfl cleaves DNA via a staggered DNA double-stranded break with a 4 or 5 nucleotide 5' overhang. Type II systems cleave via a blunt double-stranded break. Similar to Type II systems, Cpfl contains a predicted RuvC-like endonuclease domain, but lacks a second HNH endonuclease domain, which is in contrast to Type II systems.Vectors

[0255] In some aspects, the present disclosure provides a vector comprising the first nucleic acid sequence, the second nucleic acid sequence, or any combination thereof, as described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector, lentiviral vector, retroviral vector, or adeno-associated viral vector.

[0256] Methods of delivering the first nucleic acid sequence and / or the second nucleic acid sequence may be modified to express the polypeptides of the engineered T cells described herein. In some embodiments, one or more expression vectors may be used and comprise viral vectors. In some embodiments, a viral expression vector can be used. For example, a viral expression vectors may be used as a naked polynucleotide, or may comprise any of viral particles, including but not limited to, defective interfering particles or other replication defective viral constructs, and virus-like particles. In some embodiments, a retroviral vector adapted from a murine Moloneyleukemia virus (MLV) or a lentiviral vector may be used, such as a lentiviral vector adapted from human immunodeficiency virus type 1 (HIV-1).

[0257] The present disclosure also provides vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve longterm gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0258] The first nucleic acid sequence and the second nucleic acid sequence may be used in multi ci stronic vectors or vectors expressing several proteins in a same transcriptional unit. Such vectors may use internal ribosomal entry sites (IRES). Since IRES are not functional in all hosts and do not allow for the stoichiometric expression of multiple protein, self-cleaving peptides may be used instead. For example, several viral peptides are cleaved during translation and allow for the expression of multiple proteins from a single transcriptional unit. Such peptides include 2A- peptides, or 2A-like sequences, from members of the Picornaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22:589-594. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence described herein may be separated by a selfcleaving peptide, such as a 2A sequence, or a T2A sequence.

[0259] The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, each of which is incorporated by reference herein in their entireties). In another embodiment, the present disclosure provides a gene therapy vector.

[0260] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0261] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least oneorganism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0262] A number of virally based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0263] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0264] An example of a promoter can be the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et al., Mol. Ther. 17(8): 1453- 1464 (2009)). Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the presentdisclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.

[0265] In order to assess expression of the exogenous TCR described herein, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0266] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0267] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0268] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well- known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A LaboratoryManual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0269] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0270] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

[0271] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.Pharmaceutical Compositions

[0272] In some aspects, the present disclosure provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition can comprise an engineered T cell as described herein, the population of engineered T cells as described herein, or T cells comprising the vector as described herein, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition can comprise one or more of the engineered T cells described herein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 500, 1,000, 2,000, 5,000, 10,000, 50,000, 100,000, or more engineered T cells). The pharmaceutical composition can comprise a therapeutically effective amount of engineered T cells (e.g., antigen specific T cells, or T cells expressing the exogenous TCRs described herein) comprising at least 104T cells. In some cases, the method can compriseproducing a therapeutically effective amount of antigen specific T cells comprising at least 103T cells, at least 105T cells, at least 106T cells, at least 107T cells, at least 108T cells, at least 109T cells, at least IO10T cells, at least IO11T cells, at least 1012T cells, at least 1013T cells, at least 1014T cells, at least IO15T cells, at least 1016T cells, at least 1017T cells, at least 1018T cells, at least 1019T cells, or at least IO20T cells.

[0273] Provided herein are compositions (e.g., pharmaceutical compositions) comprising a population of immune cells. The compositions can comprise at least one antigen specific T cells comprising a T cell receptor (TCR). The compositions can comprise at least one antigen specific T cells comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence.

[0274] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active agents into preparations which can be used pharmaceutically. Proper formulation can be dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients can be used as suitable and as understood in the art.

[0275] In some cases, a pharmaceutical composition is formulated as cell based therapeutic, e.g., a T cell therapeutic. In some embodiments, the pharmaceutical composition comprises a peptide- based therapy, a nucleic acid-based therapy, an antibody based therapy, and / or a cell based therapy. In some embodiments, a pharmaceutical composition comprises a peptide-based therapeutic, or nucleic acid based therapeutic in which the nucleic acid encodes the polypeptides. In some embodiments, a pharmaceutical composition comprises a peptide-based therapeutic, or nucleic acid based therapeutic in which the nucleic acid encodes the polypeptides; wherein the peptide-based therapeutic, or nucleic acid based therapeutic are comprised in a cell, wherein the cell is a T cell. In some embodiments, a pharmaceutical composition comprises as an antibody based therapeutic. A composition can comprise T cells specific for two or more immunogenic antigen or neoantigen peptides.

[0276] In one aspect, provided herein is a pharmaceutical composition comprising (a) a population of immune cells comprising T cells from a biological sample, wherein the T cells comprise at least one antigen specific T cell and comprises a T cell receptor (TCR) specific to at least one antigen peptide sequence; and (b) a pharmaceutically acceptable excipient. The T cell may be an APC-stimulated T cell. The T cell may be an artificially stimulated T cell by anti-CD3 antibodies, anti-CD28 antibodies, or any combination thereof.

[0277] In one aspect, provided herein is a pharmaceutical composition comprising: (a) a population of immune cells from a biological sample comprising at least one antigen specific Tcell comprising a T cell receptor (TCR) specific to at least one antigen peptide sequence, and (b) a pharmaceutically acceptable excipient. In some embodiments, the at least one antigen specific T cell comprises at least one CD4+ T cell. In some embodiments, the at least one antigen specific T cell comprises at least one CD8+ T cell. In some embodiments, the at least one antigen specific T cell comprises at least one CD4 enriched T cell. In some embodiments, the at least one antigen specific T cell comprises at least one CD8 enriched T cell. In some embodiments, the at least one antigen specific T cell comprises a memory T cell. In some embodiments, the at least one antigen specific T cell comprises a memory CD4+ T cell. In some embodiments, the at least one antigen specific T cell comprises a memory CD8+ T cell. In some embodiments, a percentage of the at least one antigen specific T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of total T cells or total immune cells. In some embodiments, a percentage of at least one antigen specific CD8+ T cell in the composition is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of total CD4+ T cells, total CD8+ T cells, total T cells or total immune cells.

[0278] Pharmaceutical compositions can include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.

[0279] Acceptable carriers, excipients, or stabilizers are those that are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; saltforming counter-ions such as sodium;metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

[0280] Acceptable carriers are physiologically acceptable to the administered patient and retain the therapeutic properties of the compounds with / in which it is administered. Acceptable carriers and their formulations are generally described in, for example, Remington’ pharmaceutical Sciences (18thed. A. Gennaro, Mack Publishing Co., Easton, PA 1990). One example of carrier is physiological saline. A pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compounds from the administration site of one organ, or portion of the body, to another organ, or portion of the body, or in an in vitro assay system. Acceptable carriers are compatible with the other ingredients of the formulation and not injurious to a subject to whom it is administered.

[0281] In one aspect, provided herein are pharmaceutically acceptable or physiologically acceptable compositions including solvents (aqueous or non-aqueous), solutions, emulsions, dispersion media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration. Pharmaceutical compositions or pharmaceutical formulations therefore refer to a composition suitable for pharmaceutical use in a subject. Compositions can be formulated to be compatible with a particular route of administration (i.e., systemic or local). Thus, compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0282] In some embodiments, a composition can further comprise an acceptable additive in order to improve the stability of immune cells in the composition. Acceptable additives may not alter the specific activity of the immune cells. Examples of acceptable additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Acceptable additives can be combined with acceptable carriers and / or excipients such as dextrose. Alternatively, examples of acceptable additives include, but are not limited to, a surfactant such as polysorbate 20 or polysorbate 80 to increase stability of the peptide and decrease gelling of the solution. The surfactant can be added to the composition in an amount of 0.01% to 5% of the solution. Addition of such acceptable additives increases the stability and half-life of the composition in storage.

[0283] The pharmaceutical composition can be administered, for example, by injection. Compositions for injection include aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water,or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phenol, ascorbic acid and thimerosal. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride can be included in the composition. The resulting solutions can be packaged for use as is, or lyophilized; the lyophilized preparation can later be combined with a sterile solution prior to administration. For intravenous, injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as needed. Sterile injectable solutions can be prepared by incorporating an active ingredient in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization. Generally, dispersions are prepared by incorporating the active ingredient into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation can be vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0284] Compositions can be conventionally administered intravenously, such as by injection of a unit dose, for example. For injection, an active ingredient can be in the form of a parenterally acceptable aqueous solution which is substantially pyrogen-free and has suitable pH, isotonicity and stability. One can prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer’s Injection, Lactated Ringer’s Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as required. Additionally, compositions can be administered via aerosolization. When the compositions are considered for use in medicaments or any of the methods provided herein, it is contemplated that the composition can be substantially free of pyrogens such that the composition will not cause an inflammatory reaction or an unsafe allergic reaction when administered to a human patient. Testing compositions for pyrogens and preparing compositions substantially free of pyrogens are wellunderstood to one or ordinary skill of the art and can be accomplished using commercially available kits.

[0285] Acceptable carriers can contain a compound that acts as a stabilizing agent, increases or delays absorption, or increases or delays clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and / or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Detergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. To protect from digestion the compound can be complexed with a composition to render it resistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately resistant carrier such as a liposome. Means of protecting compounds from digestion are known in the art (e.g., Fix (1996) Pharm Res. 13: 1760 1764; Samanen (1996) J. Pharm. Pharmacol. 48: 119 135; and U.S. Pat. No. 5,391,377).

[0286] The compositions can be administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity ...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered T cell comprising:(a) a modified genome comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC); and(b) a genome editing system, wherein the genome editing system(i) is capable of modifying or removing a genomic sequence encoding an endogenous TCR comprising the same hTRC as the exogenous TCR, and(ii) is not capable of substantially modifying or removing the sequence encoding the exogenous TCR of the modified genome.

2. The engineered T cell of claim 1, wherein the genome editing system comprises a guide RNA targeting an endogenous target sequence or a complement thereof that encodes at least 4 amino acids of an endogenous hTRC, and / or an intron of an endogenous hTRC.

3. A method of preparing an engineered T cell comprising:(a) integrating into the genome of a T cell a vector comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC); and(b) modifying or removing a genomic sequence encoding an endogenous TCR comprising the same hTRC as the exogenous TCR with a genome editing system; wherein the genome editing system is not capable of substantially modifying or removing the sequence encoding the exogenous TCR.

4. An engineered T cell comprising:(a) a modified genome comprising an exogenous sequence encoding an exogenous T-cell receptor (TCR) chain comprising a human TCR constant domain (hTRC), wherein the exogenous sequence comprises an exogenous subsequence encoding the hTRC of the exogenous TCR or a portion thereof; and(b) a guide RNA, wherein the guide RNA comprises a nucleic acid sequence comprising a targeting sequence that hybridizes to an endogenous target sequence within the modified genome, wherein the endogenous target sequence or a complement thereof encodes at least 4 amino acids of an endogenous hTRC; wherein the amino acid sequence of the hTRC of the exogenous TCR or the portion thereof encoded by the exogenous subsequence of the exogenous sequence,and the amino acid sequence encoded by the endogenous target sequence or a complement thereof, are the same amino acid sequence; and wherein the endogenous target sequence and the complement thereof each have less than 100% sequence identity to the exogenous subsequence encoding the hTRC or the portion thereof.

5. The engineered T cell of claim 4, wherein (i) the hTRC is a human TCR alpha constant domain (hTRAC) and the endogenous hTRC is an endogenous hTRAC, or (ii) the hTRC is a human TCR beta constant domain (hTRBC) and the endogenous hTRC is an endogenous hTRBC.

6. The engineered T cell of claim 4 or 5, wherein the modified genome comprises(a) a first exogenous sequence encoding an exogenous TCR alpha chain comprising a human TCR alpha constant domain (hTRAC), wherein the first exogenous sequence comprises a first exogenous subsequence encoding the hTRAC or a portion thereof of the exogenous TCR alpha chain; and(b) a second exogenous sequence encoding an exogenous TCR beta chain comprising a human TCR beta constant domain (hTRBC), wherein the second exogenous sequence comprises a second exogenous subsequence encoding the hTRBC or a portion thereof of the exogenous TCR beta chain.

7. The engineered T cell of any one of claims 4-6, wherein the guide RNA comprises a first guide RNA and a second guide RNA, wherein(a) the first guide RNA comprises nucleic acid sequence comprising a first targeting sequence that hybridizes to a first endogenous target sequence within the modified genome, wherein the first endogenous target sequence or a complement thereof encodes at least 4 amino acids of the endogenous hTRAC; and(b) the second guide RNA comprises nucleic acid sequence comprising a second targeting sequence that hybridizes to a second endogenous target sequence within the modified genome, wherein the second endogenous target sequence or a complement thereof encodes at least 4 amino acids of the endogenous hTRBC.

8. The engineered T cell of claim 7, wherein(a) the amino acid sequence of the hTRAC of the first exogenous TCR or the portion thereof encoded by the first exogenous subsequence of the first exogenous sequence, and the amino acid sequence encoded by the first endogenous target sequence or a complement thereof, are the same amino acid sequence; and(b) the amino acid sequence of the hTRBC of the second exogenous TCR or the portion thereof encoded by the second exogenous subsequence of the second exogenous sequence, and the amino acid sequence encoded by the second endogenous target sequence or a complement thereof, are the same amino acid sequence.

9. The engineered T cell of claim 8, wherein(a) the first endogenous target sequence and the complement thereof each have less than 100% sequence identity to the first exogenous subsequence encoding the hTRAC or the portion thereof; and(b) the second endogenous target sequence and the complement thereof each have less than 100% sequence identity to the second exogenous subsequence encoding the hTRBC or the portion thereof.

10. The engineered T cell of any one of claims 4-9, wherein the engineered T cell is a engineered human T cell or an engineered cell derived from a human T cell.

11. The engineered T cell of any one of claims 4-10, wherein the engineered T cell has reduced cell-surface expression of an endogenous TCR when compared to an unmodified control cell.

12. The engineered T cell of any one of claims 4-11, wherein the engineered T cell has increased cell-surface expression of the exogenous TCR chain when compared to a control cell comprising(a) a modified genome comprising the exogenous sequence encoding the exogenous TCR chain comprising the hTRC; and(b) a control guide RNA comprising a targeting sequence that hybridizes to the endogenous target sequence within the modified genome, but that has 100% complementarity to the exogenous subsequence encoding the hTRC or the portion thereof or that has 100% complementarity to the complement of the exogenous subsequence encoding the hTRC or the portion thereof.

13. The engineered T cell of any one of claims 4-12, wherein the targeting sequence of the guide RNA hybridizes to the exogenous subsequence encoding the hTRC or a portion thereof at least 2 fold less than to the endogenous target sequence of the guide RNA.

14. The engineered T cell of any one of claims 4-13, wherein the targeting sequence of the first guide RNA hybridizes to the exogenous subsequence encoding the hTRAC or a portion thereof at least 2 fold less than to the endogenous target sequence of the first guide RNA.

15. The engineered T cell of any one of claims 4-14, wherein the targeting sequence of the second guide RNA hybridizes to the exogenous subsequence encoding the hTRBC or a portion thereof at least 2 fold less than to the endogenous target sequence of second guide RNA.

16. The engineered T cell of any one of claims 4-15, wherein the endogenous target sequence comprises a protospacer adjacent motif (PAM) sequence or complement thereof.

17. The engineered T cell of claim 16, wherein the PAM sequence or complement thereof has less than 100% sequence identity to corresponding region of the exogenous subsequence encoding the hTRC or the portion thereof.

18. The engineered T cell of any one of claims 4-17, wherein the engineered T cell lacks a sequence encoding a mouse TCR or a mouse TRC (mTRC).

19. An engineered T cell comprising:(a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and(b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein the first nucleic acid sequence:(i) does not hybridize to the second nucleic acid sequence or a complement thereof, or(ii) hybridizes to the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

20. The engineered T cell of claim 19, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

21. The engineered T cell of claim 20, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

22. The engineered T cell of claim 21, wherein a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence:(i) has less than 100% sequence identity to the target sequence or a complement thereof, or(ii) comprises a mutation or variation relative to the target sequence or a complement thereof, and wherein the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

23. The engineered T cell of any one of claims 19-22, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

24. The engineered T cell of any one of claims 19-23, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.:22.

25. The engineered T cell of any one of claims 22-24, wherein a sequence downstream of the subsequence of the second nucleic acid sequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

26. The engineered T cell of any one of claims 19-25, wherein a CRISPR-Cas system comprising the first nucleic acid sequence (i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof or (ii) recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

27. An engineered T cell comprising:(a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence or derivative thereof comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and(b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity; and wherein a CRISPR-Cas system comprising the first nucleic acid sequence:(i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof, or(ii) recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

28. The engineered T cell of claim 27, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

29. The engineered T cell of claim 28, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

30. The engineered T cell of claim 29, wherein a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence:(i) has less than 100% sequence identity to the target sequence or a complement thereof, or(ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

31. The engineered T cell of any one of claims 27-30, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

32. The engineered T cell of any one of claims 27-31, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

33. The engineered T cell of any one of claims 30-32, wherein the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

34. An engineered T cell comprising:(a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and(b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, wherein(i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system, and / or(ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than downregulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence.

35. The engineered T cell of claim 34, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

36. The engineered T cell of claim 35, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

37. The engineered T cell of claim 36, wherein the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence:(i) has less than 100% sequence identity to the target sequence or a complement thereof, or(ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

38. The engineered T cell of claim 37, wherein a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

39. The engineered T cell of any one of claims 34-38, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

40. The engineered T cell of any one of claims 34-39, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

41. The engineered T cell of claim 34, wherein an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system.

42. The engineered T cell of any one of claims 19-41, wherein the target sequence comprises a non-coding sequence.

43. The engineered T cell of claim 42, wherein the non-coding sequence comprises a portion of an intron.

44. The engineered T cell of claim 43, wherein the portion of the intron is at least 1 nucleotide in length.

45. The engineered T cell of any one of claims 19-44, wherein the endogenous TCR chain is a human TCR chain and the exogenous TCR chain is a human TCR chain.

46. The engineered T cell of any one of claims 19-45, wherein the first nucleic acid sequence is transiently expressed in the engineered T cell.

47. The engineered T cell of any one of claims 19-46, wherein the endogenous TCR chain is an endogenous TCR alpha chain, and the TCR constant domain is a TCR alpha constant domain.

48. The engineered T cell of any one of claims 19-47, wherein the endogenous TCR chain is an endogenous TCR beta chain, and the TCR constant domain is a TCR beta constant domain.

49. The engineered T cell of any one of claims 19-48, wherein the endogenous TCR chain comprises a endogenous TCR alpha chain and a endogenous TCR beta chain, and the TCR constant domain comprises a TCR alpha constant domain and a TCR beta constant domain.

50. The engineered T cell of any one of claims 19-49, wherein the endogenous TCR chain is a endogenous TCR gamma chain, and the TCR constant domain is a TCR gamma constant domain.

51. The engineered T cell of any one of claims 19-50, wherein the endogenous TCR chain is a endogenous TCR delta chain, and the TCR constant domain is a TCR delta constant domain.

52. The engineered T cell of any one of claims 19-51, wherein the endogenous TCR chain comprises a endogenous TCR gamma chain and a endogenous TCR delta chain, and the TCR constant domain comprises a TCR gamma constant domain and a TCR delta constant domain.

53. The engineered T cell of any one of claims 19-52, wherein the first nucleic acid sequence comprises an RNA sequence.

54. The engineered T cell of claim 53, wherein the RNA sequence is a guide RNA (gRNA) sequence.

55. The engineered T cell of claim 54, wherein the gRNA sequence hybridizes to the target sequence or is complementary to the target sequence.

56. The engineered T cell of any one of claims 19-55, wherein the endogenous TCR chain comprises one or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acidsequence comprises one or more gRNA sequences targeting the one or more endogenous TCR chains.

57. The engineered T cell of any one of claims 19-56, wherein the endogenous TCR chain comprises two or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, an endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acid sequence comprises two or more gRNA sequences targeting the two or more endogenous TCR chains.

58. The engineered T cell of any one of claims 19-57, wherein the endogenous TCR chain comprises an endogenous TCR alpha chain and an endogenous TCR beta chain, and wherein the first nucleic acid sequence comprises a first gRNA comprising a targeting sequence that hybridizes to a first target sequence within a genomic sequence encoding a TCR alpha constant domain of the endogenous TCR alpha chain, and a second gRNA comprising a targeting sequence that hybridizes to a second target sequence within a genomic sequence encoding a TCR beta constant domain of the endogenous TCR beta chain.

59. The engineered T cell of claim 58, wherein the second nucleic acid sequence comprises a sequence encoding an exogenous TCR alpha chain and a sequence encoding an exogenous TCR beta chain, wherein(A) the subsequence of the sequence encoding the exogenous TCR alpha chain (i) has less than 100% sequence identity to the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the first target sequence or a complement thereof, and the subsequence of the sequence encoding the exogenous TCR beta chain (i) has less than 100% sequence identity to the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the second target sequence or a complement thereof; and / or(B) a sequence downstream of the subsequence of the sequence encoding the exogenous TCR alpha chain (i) has less than 100% sequence identity to a corresponding sequence downstream of the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the first target sequence or a complement thereof, and a sequence downstream of the subsequence of thesequence encoding the exogenous TCR beta chain (i) has less than 100% sequence identity to a corresponding sequence downstream of the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the second target sequence or a complement thereof.

60. The engineered T cell of any one of claims 19-59, wherein the first nucleic acid sequence comprises a sequence with 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6 and 12.

61. The engineered T cell of any one of claims 19-60, wherein the first nucleic acid sequence is encoded by a DNA sequence.

62. The engineered T cell of any one of claims 19-61, wherein (i) the first nucleic acid sequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on the same nucleic acid molecule.

63. The engineered T cell of any one of claims 19-61, wherein (i) the first nucleic acid sequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on different nucleic acid molecules.

64. The engineered T cell of any one of claims 25, 38, and 59, wherein the corresponding sequence downstream of the target sequence or a complement thereof comprises a protospacer adjacent motif (PAM) site, and the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a mutation of the PAM site.

65. The engineered T cell of claim 64, wherein the PAM site comprises a sequence selected from the group consisting of NGG, NGRRT, NGRRN, NNNNGATT, NNNNRYAC, or NNAGAAW, where N is A, T, C, or G; R is A or G; and Y is T or C.

66. The engineered T cell of claim 64 or 65, wherein the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a sequence of SEQ Identifier A3.

67. The engineered T cell of any one of claims 19-66, wherein the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is comprised within a population of T cells, and wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or more of the T cells in thepopulation of T cells have a reduced or eliminated expression of an endogenous TCR or an endogenous TCR chain.

68. The engineered T cell of any one of claims 57-67, wherein the engineered T cell comprises a plurality of engineered T cells, and wherein at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold or more of the engineered T cells in the plurality have reduced or eliminated expression of an endogenous TCR compared to the number of engineered T cells that have reduced or eliminated expression of an endogenous TCR in an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain.

69. The engineered T cell of claim 67 or 68, wherein the engineered T cells having a reduced or eliminated expression of an endogenous TCR is measured by a surface marker.

70. The engineered T cell of claim 69, wherein the surface marker is CD3.

71. The engineered T cell of any one of claims 19-70, wherein the engineered T cell is a plurality of transduced engineered T cells, wherein the plurality of transduced engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and transduced with the second nucleic acid sequence, and wherein the percentage of transduced engineered T cells derived from the population of T cells is higher compared to the percentage of transduced engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

72. The engineered T cell of claim 71, wherein the percentage of transduced engineered T cells is at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more of the total T cells in the population of T cells from which the transduced engineered T cells are derived.

73. The engineered T cell of any one of claims 19-72, wherein the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence, and wherein the percentage of engineered T cells derived fromthe population of T cells that express the exogenous TCR is higher compared to the percentage of engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

74. The engineered T cell of any one of claims 19-73, wherein the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells comprises the first nucleic acid sequence and the second nucleic acid sequence, and wherein the plurality of engineered T cells expresses the exogenous TCR at a higher level compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

75. The engineered T cell of any one of claims 19-74, wherein the second nucleic acid sequence is encoded by a vector.

76. The engineered T cell of claim 75, wherein the vector is a viral vector.

77. The engineered T cell of claim 76, wherein the viral vector is a retroviral vector.

78. The engineered T cell of claim 76, wherein the viral vector is a lentiviral vector.

79. The engineered T cell of any one of claims 19-78, wherein the exogenous TCR chain recognizes an epitope of an antigen in complex with an MHC molecule.

80. The engineered T cell of claim 79, wherein the epitope is associated with a cancer, an autoimmune disease, and / or an infectious disease.

81. The engineered T cell of claim 80, wherein the antigen is selected from the group consisting of NY-ESO-1, KRAS, TP53, NRAS, BRAF, and PIK3CA.

82. The engineered T cell of any one of claims 19-81, wherein the first nucleic acid sequence is at least 15 nucleotides in length, at most 100 nucleotides in length, or from 15-100 nucleotides in length.

83. The engineered T cell of any one of claims 19-82, wherein the targeting sequence is at least 15 nucleotides in length, at most 100 nucleotides in length, or from 15-100 nucleotides in length.

84. The engineered T cell of any one of claims 19-83, wherein the amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence of SEQ ID NOs: 9 or 16.

85. The engineered T cell of any one of claims 19-84, wherein the target sequence comprises a sequence of SEQ ID NOs: 8 or 15.

86. The engineered T cell of any one of claims 19-85, wherein the subsequence of the second nucleic acid sequence comprises a sequence of SEQ ID NOs: 8, 12, 15, or 19.

87. A method of generating an engineered T cell, the method comprising: delivering(a) a first nucleic acid sequence for down-regulating expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain, and(b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein the first nucleic acid sequence:(i) does not hybridize to the second nucleic acid sequence or a complement thereof; or(ii) hybridizes to the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

88. The method of claim 87, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

89. The method of claim 88, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

90. The method of claim 89, wherein a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence:(i) has less than 100% sequence identity to the target sequence or a complement thereof, or(ii) comprises a mutation or variation relative to the target sequence or a complement thereof, and wherein the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

91. The method of any one of claims 87-90, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

92. The method of any one of claims 87-91, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

93. The method of claim 90, wherein a sequence downstream of the subsequence of the second nucleic acid sequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

94. The method of any one of claims 87-93, wherein a CRISPR-Cas system comprising the first nucleic acid sequence (i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof or (ii) recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

95. A method of generating an engineered T cell, the method comprising: delivering(a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and(b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from thesame species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity; and wherein a CRISPR-Cas system comprising the first nucleic acid sequence:(i) does not recognize, bind to or cleave the second nucleic acid sequence or a complement thereof, or(ii) recognizes, binds to or cleaves the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence recognizes, binds to or cleaves to the target sequence.

96. The method of claim 95, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

97. The method of claim 96, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

98. The method of claim 97, wherein a subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence (i) has less than 100% sequence identity to the target sequence or a complement thereof or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

99. The method of claim 98, wherein the first nucleic acid sequence (i) does not hybridize to a subsequence of the second nucleic acid sequence or a complement thereof or (ii) hybridizes to a subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

100. The method of claim 98 or 99, wherein a sequence downstream of the subsequence of the second nucleic acid sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the target sequence or a complement thereof, and wherein the first nucleic acid sequence (i) does not hybridize to the subsequence of the second nucleic acid sequence or a complement thereof, or (ii) hybridizes to the subsequence of the second nucleic acid sequence or a complement thereof at least 2 fold less than the first nucleic acid sequence hybridizes to the target sequence.

101. The method of any one of claims 95-100, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

102. The method of any one of claims 95-101, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

103. A method of generating an engineered T cell, the method comprising: delivering(a) a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain; and(b) a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, wherein(i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system,(ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than downregulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence, and / or(iii) an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system.

104. The method of claim 103, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

105. The method of claim 104, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

106. The method of claim 105, wherein the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or(ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

107. The method of claim 106, wherein a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

108. The method of any one of claims 103-107, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

109. The method of any one of claims 103-108, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

110. The method of any one of claims 87-109, wherein the first nucleic acid sequence is delivered into the T cell prior to, concurrently, or subsequent to delivering the second nucleic acid sequence.

111. The method of any one of claims 87-110, further comprising, prior to delivering, activating the T cell.

112. The method of any one of claims 87-111, wherein the T cell has been activated.

113. The method of any one of claims 87-112, wherein a time period between delivering (i) and (ii) is at most 3 days, at most 2 days, at most 1 day, at most 20 hours, at most 12 hours, or less.

114. A method of generating an engineered T cell, wherein the engineered T cell has been delivered a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence encoding a TCR constant domain,the method comprising:(a) delivering a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein(i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system,(ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than downregulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence, and / or(iii) an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system.

115. The method of claim 114, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

116. The method of claim 115, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

117. The method of claim 116, wherein the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or(ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

118. The method of claim 117, wherein a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the targetsequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

119. The method of any one of claims 114-118, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

120. The method of any one of claims 114-119, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

121. A method of generating an engineered T cell, the method comprising: delivering into an T cell a first nucleic acid sequence for down-regulating an expression of an endogenous T-cell receptor (TCR) chain, wherein the first nucleic acid sequence comprises a targeting sequence that hybridizes to a target sequence within a genomic sequence,(a) wherein the T cell has been delivered a second nucleic acid sequence encoding an exogenous TCR chain having a TCR constant domain, wherein the TCR constant domain of the exogenous TCR chain and the TCR constant domain of the endogenous TCR chain are from the same species or are each derivatives of a TCR chain from the same species, and have at least 80% sequence identity, and wherein(i) an expression of the endogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a greater extent than down-regulation of expression of the exogenous TCR chain by the first nucleic acid sequence in combination with the CRISPR-Cas system,(ii) an expression of the exogenous TCR chain is down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system to a less extent than downregulation of expression of the exogenous TCR chain by the CRISPR-Cas system in combination with a nucleic acid sequence with 100% complementarity to a subsequence of the second nucleic acid sequence, and / or(iii) an expression of the exogenous TCR chain is not down-regulated or not substantially down-regulated by the first nucleic acid sequence in combination with a CRISPR-Cas system.

122. The method of claim 121, wherein the target sequence encodes an amino acid sequence of the TCR constant domain, and wherein the amino acid sequence of the TCR constant domain encoded by the target sequence is at least 4 amino acids in length.

123. The method of claim 122, wherein the TCR constant domain of the exogenous TCR chain comprises the same amino acid sequence encoded by the target sequence or comprises a sequence with at least 60% or at least 80% sequence identity to the amino acid sequence encoded by the target sequence.

124. The method of claim 123, wherein the subsequence of the second nucleic acid sequence that encodes the same amino acid sequence encoded by the target sequence: (i) has less than 100% sequence identity to the target sequence or a complement thereof, or(ii) comprises a mutation or variation relative to the target sequence or a complement thereof.

125. The method of claim 124, wherein a sequence downstream of the subsequence (i) has less than 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the target sequence or a complement thereof.

126. The method of any one of claims 121-125, wherein the TCR constant domain of the exogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

127. The method of any one of claims 121-126, wherein the TCR constant domain of the endogenous TCR chain has at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NO.: 20, SEQ ID NO.: 21, and SEQ ID NO.: 22.

128. The method of any one of claims 87-115, wherein the endogenous TCR chain is a human TCR chain and the exogenous TCR chain is a human TCR chain.

129. The method of any one of claims 87-122, wherein the first nucleic acid sequence is transiently expressed in the engineered T cell.

130. The method of any one of claims 87-129, wherein the endogenous TCR chain is an endogenous TCR alpha chain, and the TCR constant domain is a TCR alpha constant domain.

131. The method of any one of claims 87-130, wherein the endogenous TCR chain is an endogenous TCR beta chain, and the TCR constant domain is a TCR beta constant domain.

132. The method of any one of claims 87-131, wherein the endogenous TCR chain comprises a endogenous TCR alpha chain and a endogenous TCR beta chain, and the TCR constant domain comprises a TCR alpha constant domain and a TCR beta constant domain.

133. The method of any one of claims 87-132, wherein the endogenous TCR chain is a endogenous TCR gamma chain, and the TCR constant domain is a TCR gamma constant domain.

134. The method of any one of claims 87-133, wherein the endogenous TCR chain is a endogenous TCR delta chain, and the TCR constant domain is a TCR delta constant domain.

135. The method of any one of claims 87-134, wherein the endogenous TCR chain comprises a endogenous TCR gamma chain and a endogenous TCR delta chain, and the TCR constant domain comprises a TCR gamma constant domain and a TCR delta constant domain.

136. The method of any one of claims 87-135, wherein the first nucleic acid sequence comprises an RNA sequence.

137. The method of claims 136, wherein the RNA sequence is a guide RNA (gRNA) sequence.

138. The method of claim 137, wherein the gRNA sequence hybridizes to the target sequence or is complementary to the target sequence.

139. The method of any one of claims 87-138, wherein the endogenous TCR chain comprises one or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, a endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acid sequence comprises one or more gRNA sequences targeting the one or more endogenous TCR chains.

140. The method of any one of claims 87-139, wherein the endogenous TCR chain comprises two or more endogenous TCR chains selected from the group consisting of an endogenous TCR alpha chain, a endogenous TCR beta chain, an endogenous TCR gamma chain and an endogenous TCR delta chain, and wherein the first nucleic acid sequence comprises two or more gRNA sequences targeting the two or more endogenous TCR chains.

141. The method of any one of claims 87-139, wherein the endogenous TCR chain comprises an endogenous TCR alpha chain and an endogenous TCR beta chain, and wherein the first nucleic acid sequence comprises a first gRNA comprising a targeting sequence that hybridizes to a first target sequence within a genomic sequence encoding a TCR alpha constant domain of the endogenous TCR alpha chain, and a second gRNA comprising a targeting sequence that hybridizes to a second target sequence within a genomic sequence encoding a TCR beta constant domain of the endogenous TCR beta chain.

142. The method of claim 141, wherein the second nucleic acid sequence comprises a sequence encoding an exogenous TCR alpha chain and a sequence encoding an exogenous TCR beta chain, wherein(A) the subsequence of the sequence encoding the exogenous TCR alpha chain (i) has less than 100% sequence identity to the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the first target sequence or a complement thereof, and the subsequence of the sequence encoding the exogenous TCR beta chain (i) has less than 100% sequence identity to the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to the second target sequence or a complement thereof; and / or(B) a sequence downstream of the subsequence of the sequence encoding the exogenous TCR alpha chain (i) has less than 100% sequence identity to a corresponding sequence downstream of the first target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the first target sequence or a complement thereof, and a sequence downstream of the subsequence of the sequence encoding the exogenous TCR beta chain (i) has less than 100% sequence identity to a corresponding sequence downstream of the second target sequence or a complement thereof, or (ii) comprises a mutation or variation relative to a corresponding sequence downstream of the second target sequence or a complement thereof.

143. The method of any one of claims 87-142, wherein the first nucleic acid sequence comprises a sequence with 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 6 and 13.

144. The method of any one of claims 87-143, wherein the first nucleic acid sequence is encoded by a DNA sequence.

145. The method of any one of claims 87-144, wherein (i) the first nucleic acid sequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on the same nucleic acid molecule.

146. The method of any one of claims 87-144, wherein (i) the first nucleic acid sequence or a sequence encoding the first nucleic acid sequence and (ii) the second nucleic acid sequence or a sequence encoding the second nucleic acid sequence are on different nucleic acid molecules.

147. The method of any one of claims 87-146, wherein the corresponding sequence downstream of the target sequence or a complement thereof comprises a protospacer adjacent motif (PAM) site, and the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a mutation of the PAM site.

148. The method of claim 147, wherein the PAM site comprises a sequence of NGG, NGRRT, NGRRN, NNNNGATT, NNNNRYAC, or NNAGAAW, where N is A, T, C, or G; R is A or G; and Y is T or C.

149. The method of claim 147 or 148, wherein the sequence downstream of the subsequence that encodes the same amino acid sequence encoded by the target sequence comprises a sequence as set forth in SEQ Identifier A3.

150. The method of any one of claims 87-149, wherein the amino acid sequence of the TCR constant domain encoded by the target sequence comprises a sequence of SEQ ID NO: 9 or 16.

151. The method of any one of claims 87-150, wherein the target sequence comprises a sequence of SEQ ID NO: 8 or 15.

152. The method of any one of claims 87-151, wherein the subsequence of the second nucleic acid sequence comprises a sequence of SEQ ID NO: 8, 12, 15, or 19.

153. The method of any one of claims 87-148, wherein the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is comprised within a population of T cells, and wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or more of the T cells in the population ofT cells have a reduced or eliminated expression of an endogenous TCR or an endogenous TCR chain.

154. The method of any one of claims 140-153, wherein the engineered T cell comprises a plurality of engineered T cells, and wherein at least 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold or more of the engineered T cells in the plurality have reduced or eliminated expression of an endogenous TCR compared to the number of engineered T cells that have reduced or eliminated expression of an endogenous TCR in an otherwise identical plurality of engineered T cells comprising a single gRNA sequence targeting a single endogenous TCR chain.

155. The method of claim 154, wherein the engineered T cells having a reduced or eliminated expression of an endogenous TCR is measured by a surface marker.

156. The method of claim 155, wherein the surface marker is CD3.

157. The method of any one of claims 87-156, wherein the engineered T cell is a plurality of transduced engineered T cells, wherein the plurality of transduced engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and transduced with the second nucleic acid sequence, and wherein the percentage of transduced engineered T cells derived from the population of T cells is higher compared to the percentage of transduced engineered T cells derived from an otherwise identical population of T cells comprising the first nucleic acid sequence and transduced with a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

158. The method of claim 157, wherein the percentage of transduced engineered T cells is at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more of the total T cells in the population of T cells from which the transduced engineered T cells are derived.

159. The method of any one of claims 87-158, wherein the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells is derived from a population of T cells comprising the first nucleic acid sequence and the second nucleic acid sequence, and wherein the percentage of engineered T cells derived from the population of T cells that express the exogenous TCR is higher compared to the percentage of engineered T cells derived from an otherwise identical population of Tcells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

160. The method of any one of claims 87-159, wherein the engineered T cell is a plurality of engineered T cells, wherein the plurality of engineered T cells comprises the first nucleic acid sequence and the second nucleic acid sequence, and wherein the plurality of engineered T cells expresses the exogenous TCR at a higher level compared to that of an otherwise identical plurality of T cells comprising the first nucleic acid sequence and a second nucleic acid sequence having (i) a subsequence with 100% sequence identity to the target sequence or a complement thereof, or (ii) a sequence downstream of the subsequence with 100% sequence identity to a corresponding sequence downstream of the target sequence or a complement thereof.

161. The method of any one of claims 87-160, further comprising administering the engineered T cell to a subject in need thereof.

162. The method of claim 161, wherein the T cell is obtained from the subject administered with the engineered T cell.

163. The method of claim 161, wherein the T cell is obtained from a different subject from the subject administered with the engineered T cell.

164. A method of treating a disease or a condition in a subject in need thereof, the method comprising: administering the engineered T cell of any one of claims 1-2 and 4-86 to a subject in need thereof.

165. A vector comprising the first nucleic acid sequence and / or the second nucleic acid sequence of any one of claims 1-2 and 4-86.

166. The vector of claim 165, wherein the vector is a viral vector.

167. The vector of claim 166, wherein the viral vector is a retroviral vector.

168. The vector of claim 166, wherein the viral vector is a lentiviral vector.

169. A population of T cells, wherein the population of T cells comprise at least two of the engineered T cell of any one of claims 1-2 and 4-86.

170. A pharmaceutical composition comprising the engineered T cell of any one of claims 1- 2 and 4-86, the vector of any one of claims 165-168, or the population of T cells of claim 169, and a pharmaceutically acceptable carrier.

171. Use of the engineered T cell of any one of claims 1-2 and 4-86, a T cell comprising the vector of any one of claims 165-168, or the population of T cells of claim 169, in the manufacture of a medicament for treating a disease or a condition in a subject in need thereof.

172. The use of claim 171, wherein the disease or condition is a cancer, an autoimmune disease, and / or an infectious disease.

173. The use of any one of claims 171-172, wherein the engineered T cell or the T cell is a CD8+ T cell, a CD4+ T cell, a CD4+ and CD8+ T cell, a gamma delta T cell, or a natural killer T cell.

174. The engineered T cell of any one of claims 1-2 and 4-86, a T cell comprising the vector of any one of claims 165-168, or the population of T cells of claim 169 for use in the treatment of a disease or a condition.

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