JPWO2019195491A5 - - Google Patents

Description

Genetically engineered T cells containing an altered T cell receptor alpha constant (TRAC) locus are provided herein. In some aspects, the modified TRAC locus comprises a nucleic acid encoding a recombinant TCR or portion thereof, wherein the recombinant TCR or portion thereof comprises: (i) a variable alpha (Vα) domain and a constant alpha (Cα) domain; ) a TCR alpha (TCRα) chain comprising a domain, and (ii) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant beta (Cβ) domain, the nucleic acid sequence comprising: (a) a TCRβ and Vα domain; (b) an open reading frame of the endogenous TRAC locus, or a subsequence thereof, encoding at least a portion of the Ca domain of the recombinant TCR. In some of any such embodiments, a portion of Cα is encoded by an open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by a transgene sequence; The further portion of Cα is shorter than the full length of native Cα; and the further portion of Cα and/or the Cβ region encoded by the nucleic acid sequence of (a) is compared to the native Cα region and/or the native Cβ region. wherein the one or more modifications include one or more cysteine residues capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. groups and/or the Cα and/or Cβ of the recombinant TCR contain one or more non-natural cysteines.

In some embodiments, the transgene sequence integrates or integrates downstream of the 5'-most nucleotide of exon 1 and upstream of the 3'-most nucleotide of exon 1 of the open reading frame of the endogenous TRAC locus. It is. In some embodiments, at least a portion of the Cα is in at least exons 2-4 of the open reading frame of the endogenous TRAC locus, or at least a portion of exon 1 and exons 2-4 of the open reading frame of the endogenous TRAC locus. Therefore it is coded. In some embodiments, at least a portion of Cα is encoded by less than the full length of exon 1 of the open reading frame of the endogenous TRAC locus.

In some embodiments, the additional portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) has one or more modifications compared to the native Cα and/or Cβ region, optionally, Contains one or more amino acid substitutions, deletions, or insertions, optionally said one or more modifications forming one or more non-natural disulfide bridges between the alpha and beta chains. One or more cysteine residues can be introduced.

In some embodiments, the additional portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) has one or more modifications compared to the native Cα and/or Cβ region, optionally, Contains one or more amino acid substitutions, deletions, or insertions, optionally said one or more modifications forming one or more non-natural disulfide bridges between the alpha and beta chains. One or more cysteine residues can be introduced.

Also provided herein are genetically engineered T cells containing modified T cell receptor beta constant (TRBC) loci. In some of any such embodiments, the modified TRBC locus comprises a nucleic acid encoding a recombinant TCR or a portion thereof, wherein the recombinant TCR or portion thereof comprises: (i) variable beta ( a TCR beta (TCRβ) chain comprising a Vβ) domain and a constant beta (Cβ) domain; and (ii) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant alpha (Cα) domain; comprises (a) a transgene sequence encoding the TCRα and Vβ domains, and (b) an open reading frame of the endogenous TRBC locus, or a subsequence thereof, encoding at least a portion of the Cβ domain of the recombinant TCR. In some of any such embodiments, a portion of Cβ is encoded by an open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by a transgene sequence; The further portion of Cβ is shorter than the full length of native Cβ; and the further portion of Cβ and/or the Cα region encoded by the nucleic acid sequence of (a) is compared to the native Cβ region and/or the native Cα region. including one or more modifications, in some cases one or more amino acid substitutions, deletions, or insertions; in some cases, the one or more modifications include one or more modifications in the alpha chain. Introducing one or more cysteine residues that can form one or more non-natural disulfide bridges between the beta chain and

In some embodiments, at least a portion of Cβ is encoded by at least exons 2-4 of the open reading frame of the endogenous TRBC locus. In some embodiments, at least a portion of Cβ is encoded by at least a portion of exon 1 and exons 2-4 of the open reading frame of the endogenous TRBC locus. In some embodiments, at least a portion of Cβ is encoded by less than the full length of exon 1 of the open reading frame of the endogenous TRBC locus.

In some embodiments, the additional portion of Cβ and/or Cα region encoded by the nucleic acid sequence of (a) has one or more modifications compared to the native Cβ region and/or native Cα region, optionally, Contains one or more amino acid substitutions, deletions, or insertions, optionally said one or more modifications forming one or more non-natural disulfide bridges between the alpha and beta chains. One or more cysteine residues can be introduced.

Also, a composition comprising a plurality of genetically engineered T cells comprising an altered T cell receptor alpha constant (TRAC) locus, wherein the altered TRAC locus is a recombinant TCR or a The recombinant TCR or portion thereof comprises a nucleic acid encoding a portion thereof, wherein the recombinant TCR or portion thereof comprises (i) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant alpha (Cα) domain, and (ii) a variable beta (Vβ ) domain and a TCR beta (TCRβ) chain comprising a constant beta (Cβ) domain, the nucleic acid sequence comprising (a) a transgene sequence encoding the TCRβ and Vα domains, and (b) a Cα domain of the recombinant TCR. Also provided herein is a composition comprising an open reading frame encoding at least a portion of the endogenous TRAC locus, or a subsequence thereof. In some of any such embodiments, a portion of Cα is encoded by an open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by a transgene sequence; The further portion of Cα is shorter than the full length of native Cα. In some of any such embodiments, the additional portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) is one compared to the native Cα region and/or the native Cβ region. or a plurality of modifications, the one or more modifications introducing one or more cysteine residues capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. do.

Also, a composition comprising a plurality of genetically engineered T cells comprising a modified T cell receptor beta constant (TRBC) locus, the modified TRBC locus comprising a recombinant TCR or a The recombinant TCR or portion thereof comprises a nucleic acid encoding a portion thereof, wherein the recombinant TCR or portion thereof comprises (i) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant beta (Cβ) domain, and (ii) a variable alpha (Vα ) domain and a TCR alpha (TCRα) chain comprising a constant alpha (Cα) domain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCRα and Vβ domains; and (b) a Cβ domain of the recombinant TCR. Also provided herein is a composition comprising an open reading frame encoding at least a portion of the endogenous TRBC locus, or a subsequence thereof. In some of any such embodiments, a portion of Cβ is encoded by an open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by a transgene sequence; The further portion of Cβ is shorter than the full length of native Cβ. In some of any such embodiments, the additional portion of Cβ and/or Cα region encoded by the nucleic acid sequence of (a) is one compared to the native Cβ region and/or the native Cα region. or multiple modifications, optionally comprising one or more amino acid substitutions, deletions, or insertions. In some of any such embodiments, the one or more modifications are capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. A cysteine residue is introduced.

(a) (i) the T cell receptor beta (TCRβ) chain, which includes a variable beta (Vβ) domain and a constant beta (Cβ) domain, and (ii) the full length of the native T cell receptor alpha (TCRα) chain. (b) a sequence homologous to one or more regions of the open reading frame of the TRAC locus; one or more homology arms linked to the nucleic acid sequence comprising; when the TCR or antigen-binding fragment thereof is expressed from a cell into which the polynucleotide has been introduced: Cα a portion of the Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of the Cα is encoded by the transgene sequence, the further portion of the Cα being shorter than the full length of the native Cα. ; and the further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to the native Cα region and/or the native Cβ region, said one or Said polynucleotide is herein defined as having multiple modifications introducing one or more cysteine residues capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. provided. In some aspects and/or the Cα and/or Cβ of the recombinant TCR comprises one or more non-natural cysteines.

In some embodiments, the additional portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) has one or more modifications compared to the native Cα region and/or native Cβ region, optionally, Contains one or more amino acid substitutions, deletions, or insertions, optionally said one or more modifications forming one or more non-natural disulfide bridges between the alpha and beta chains. One or more cysteine residues can be introduced.

In some embodiments, the additional portion of Cβ and/or Cα region encoded by the nucleic acid sequence of (a) has one or more modifications compared to the native Cβ region and/or native Cα region, optionally, Contains one or more amino acid substitutions, deletions, or insertions, optionally said one or more modifications forming one or more non-natural disulfide bridges between the alpha and beta chains. One or more cysteine residues can be introduced.

Also provided herein are methods, such as methods of producing genetically engineered T cells. In some embodiments, (a) one or more agents capable of inducing gene disruption in the T cell at a target site within the endogenous T cell receptor alpha constant (TRAC) locus of the T cell. and (b) a transgene sequence encoding a T cell receptor beta (TCRβ) and a portion of a TCRα chain that is shorter than the full-length native T cell receptor alpha (TCRα) chain; 1. A method of producing a genetically engineered T cell comprising a modified TRAC locus, the method comprising: introducing a polynucleotide comprising an altered TRAC locus; Provided are methods of generating genetically engineered cells that are targeted for integration within the TRAC locus and thereby include a modified TRAC locus. In some of any such embodiments, upon targeted integration of the transgene: a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is The further portion of Cα encoded by the transgene sequence is shorter than the full length of native Cα; and the further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) is shorter than the native Cα region and or one or more modifications compared to the native Cβ region, the one or more modifications being capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. Introduce one or more cysteine residues that can be used. In some embodiments, the Cα and/or Cβ of the recombinant TCR comprises one or more non-natural cysteines.

Additionally, a portion of the T cell receptor beta (TCRβ) chain and the T cell receptor alpha (TCRα) chain are found in T cells with gene disruption within the endogenous T cell receptor alpha constant (TRAC) locus. 1. A method of producing a genetically engineered T cell comprising a modified TRAC locus, the method comprising: introducing a polynucleotide comprising a transgene sequence encoding; HDR)-mediated integration within the endogenous TRAC locus, thereby creating genetically engineered cells containing a modified TRAC locus, upon targeted integration of the transgene: a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is shorter than the full length of native Cα; and , the further portion of Cα and/or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to the native Cα region and/or the native Cβ region, the one or more The modification introduces one or more cysteine residues that can form one or more non-natural disulfide bridges between the alpha and beta chains and/or the Cα and/or Also provided are the aforementioned methods, wherein the Cβ comprises one or more non-natural cysteines.

から選択される配列を含む、ターゲティングドメイン
を有する、態様198～202のいずれかの方法。
204. gRNAが、配列GAGAAUCAAAAUCGGUGAAU（SEQ ID NO:31）を含むターゲティングドメインを有する、態様203の方法。
205. gRNAが、
TRBC1遺伝子およびTRBC2遺伝子の一方または両方における標的部位に相補的であり、かつ

から選択される配列を含む、ターゲティングドメイン
を有する、態様198～202のいずれかの方法。
206. gRNAが、配列

を含むターゲティングドメインを有する、態様205の方法。
207. 前記T細胞が、CD8+ T細胞またはそのサブタイプである、態様161～206のいずれかの態様の方法。
208. 前記T細胞が、CD4+ T細胞またはそのサブタイプである、態様207の方法。
209. 前記T細胞が、任意でiPSCである多分化能細胞または多能性細胞に由来している、態様161～206のいずれかの方法。
210. 前記T細胞が、対象にとって自己由来であるT細胞を含む、態様161～209のいずれかの方法。
211. 前記T細胞が、対象にとって同種異系であるT細胞を含む、態様161～209のいずれかの方法。
212. gRNAおよび/またはCas9タンパク質をコードするポリヌクレオチドおよび/または1つもしくは複数のポリヌクレオチドが、任意でウイルスベクターである、1つまたは複数のベクター中に含まれる、態様161～211のいずれかの方法。
213. 前記ベクターがAAVベクターである、態様212の方法。
214. 前記AAVベクターが、AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、またはAAV8ベクターの中から選択される、態様213の方法。
215. 前記AAVベクターが、AAV2またはAAV6ベクターである、態様214の方法。
216. 前記ウイルスベクターがレトロウイルスベクターである、態様212の方法。
217. 前記ウイルスベクターがレンチウイルスベクターである、態様216の方法。
218. 前記ポリヌクレオチドが直鎖ポリヌクレオチドである、態様161～211のいずれかの方法。
219. 前記直鎖ポリヌクレオチドが二本鎖ポリヌクレオチドである、態様218の方法。
220. 前記直鎖ポリヌクレオチドが一本鎖ポリヌクレオチドである、態様219の方法。
221. 遺伝子破壊を誘導することができる1つまたは複数の作用物質の導入、および鋳型ポリヌクレオチドの導入が、同時にまたは逐次的に、任意の順序で行われる、態様161～220のいずれかの方法。
222. 鋳型ポリヌクレオチドの導入が、遺伝子破壊を誘導することができる1つまたは複数の作用物質の導入後に行われる、態様161～221のいずれかの方法。
223. 鋳型ポリヌクレオチドが、遺伝子破壊を誘導することができる1つまたは複数の作用物質23の導入の直後に、またはその後約30秒、1分、2分、3分、4分、5分、6分、6分、8分、9分、10分、15分、20分、30分、40分、50分、60分、90分、2時間、3時間、もしくは4時間以内に導入される、態様222の方法。
224. 態様161～2のいずれかの方法を用いて生成された、操作されたT細胞または複数の操作されたT細胞。
225. 態様224の操作されたT細胞または複数の操作された細胞を含む、組成物。
226. CD4+ T細胞および/またはCD8+ T細胞を含む、態様225の組成物。
227. CD4+ T細胞およびCD8+ T細胞を含み、CD4+ T細胞対CD8+ T細胞の比が、1:3～3:1または約1:3～3:1、任意で1:1である、態様225または態様226の組成物。
228. 組成物における操作された細胞の少なくとも70％、75％、80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、もしくは99％が、内在性T細胞受容体アルファ定常領域（TRAC）遺伝子および/もしくはT細胞受容体ベータ定常領域（TRBC）遺伝子におけるもしくはその遺伝子破壊を含み；かつ/または
組成物における操作された細胞の少なくとも80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、もしくは99％が、内在性TRACもしくはTRBC遺伝子の遺伝子産物を発現しないか、もしくは検出可能なレベルで発現しない、
態様225～227のいずれかの組成物。
229. 組成物における細胞の少なくとも35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、もしくは90％が、または35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、もしくは90％よりも多くが、組換えTCRを発現し、かつ/または抗原結合を示す、態様225～228のいずれかの組成物。
230. 態様224～229のいずれかの操作された細胞、複数の操作された細胞、または組成物を対象に投与する工程を含む、処置の方法。
231. ある疾患または障害の処置のための、態様224～229のいずれかの操作された細胞、複数の操作された細胞、または組成物の使用。
232. ある疾患または障害を処置するための薬剤の製造における、態様224～229のいずれかの操作された細胞、複数の操作された細胞、または組成物の使用。
233. ある疾患または障害の処置における使用のための、態様224～229のいずれかの操作された細胞、複数の操作された細胞、または組成物。
234. 態様79～102または116～149のいずれかのポリヌクレオチドと、
TRAC遺伝子座内の標的部位で遺伝子破壊を誘導することができる1つまたは複数の作用物質と
を含む、製造物品。
235. (a) T細胞受容体ベータ（TCRβ）鎖と完全長天然T細胞受容体アルファ（TCRα）鎖よりも短いTCRα鎖の一部分とをコードする核酸配列、および
(b) TCRα鎖をコードするTRAC遺伝子座のオープンリーディングフレームの1つまたは複数の領域に相同な配列を含む、該核酸配列に連結された1つまたは複数の相同性アーム
を含むポリヌクレオチドと；
TRAC遺伝子座内の標的部位で遺伝子破壊を誘導することができる1つまたは複数の作用物質と
を含む、製造物品。
236. 前記ポリヌクレオチドが、態様79～102または125～160のいずれか由来である、態様201の製造物品。
237. 態様79～160のいずれかのポリヌクレオチドと、
TRBC遺伝子座内の標的部位で遺伝子破壊を誘導することができる1つまたは複数の作用物質と
を含む、製造物品。
238. (a) T細胞受容体アルファ（TCRα）鎖と完全長天然T細胞受容体ベータ（TCRβ）鎖よりも短いTCRβ鎖の一部分とをコードする核酸配列、および
(b) TCRβ鎖をコードするTRBC遺伝子座のオープンリーディングフレームの1つまたは複数の領域に相同な配列を含む、該核酸配列に連結された1つまたは複数の相同性アーム
を含むポリヌクレオチドと；
TRAC遺伝子座内の標的部位で遺伝子破壊を誘導することができる1つまたは複数の作用物質と
を含む、製造物品。
239. TRBC遺伝子座が、TRBC1および/またはTRBC2である、態様237または238の製造物品。
240. 前記ポリヌクレオチドが、態様103～160のいずれか由来である、態様237または238に記載の製造物品。
241. 遺伝子破壊を誘導することができる1つまたは複数の作用物質が、標的部位に特異的に結合するかまたはハイブリダイズする、DNA結合タンパク質またはDNA結合核酸を含む、態様234～241のいずれかの製造物品。
242. 遺伝子破壊を誘導することができる1つまたは複数の作用物質が、(a) DNAターゲティングタンパク質およびヌクレアーゼを含む融合タンパク質、または(b) RNA誘導型ヌクレアーゼを含む、態様241の製造物品。
243. 前記DNAターゲティングタンパク質またはRNA誘導型ヌクレアーゼが、標的部位に特異的な、ジンクフィンガータンパク質（ZFP）、TALタンパク質、またはクラスター化して規則的な配置の短い回文核酸（CRISPR）関連ヌクレアーゼ（Cas）を含む、態様242の製造物品。
244. 前記1つまたは複数の作用物質が、標的部位に特異的に結合するか、認識するか、またはハイブリダイズする、ジンクフィンガーヌクレアーゼ（ZFN）、TALエフェクターヌクレアーゼ（TALEN）、または、およびCRISPR-Cas9の組み合わせを含む、態様241～243のいずれかの製造物品。
245. 前記1つまたは複数の作用物質の各々が、少なくとも1つの標的部位に相補的であるターゲティングドメインを有するガイドRNA（gRNA）を含む、態様241～244のいずれかの製造物品。
246. 前記1つまたは複数の作用物質が、gRNAおよびCas9タンパク質を含むリボヌクレオタンパク質（RNP）複合体として導入される、態様241～245の製造物品。
247. RNPが、エレクトロポレーション、パーティクルガン、リン酸カルシウムトランスフェクション、細胞圧迫またはスクイージングを介して導入される、態様246の製造物品。
248. RNPが、エレクトロポレーションを介して導入される、態様246または態様247の製造物品。
249. 前記1つまたは複数の作用物質が、gRNAおよび/またはCas9タンパク質をコードする1つまたは複数のポリヌクレオチドとして導入される、態様245～248のいずれかの製造物品。
250. gRNAが、
TRAC遺伝子座における標的部位に相補的であり、かつ

から選択される配列を含む、ターゲティングドメイン
を有する、態様245～249のいずれかの製造物品。
251. gRNAが、配列

を含むターゲティングドメインを有する、態様250の製造物品。
252. gRNAが、
TRBC1遺伝子およびTRBC2遺伝子の一方または両方における標的部位に相補的であり、かつ

から選択される配列を含む、ターゲティングドメイン
を有する、態様245～251のいずれかの製造物品。
253. gRNAが、配列

を含むターゲティングドメインを有する、態様252の製造物品。
254. 態様234～253のいずれかの製造物品と、使用説明書とを含む、キット。
255. 前記説明書が、1つまたは複数の作用物質およびポリヌクレオチドが、細胞中に導入されることを明記する、態様254のキット。
256. 前記説明書が、1つまたは複数の作用物質およびポリヌクレオチドが、同時にまたは逐次的に、任意の順序で導入されることを明記する、態様254または255のキット。
257. 前記説明書が、ポリヌクレオチドの導入が、1つまたは複数の作用物質の導入後に行われることを明記する、態様254～257のいずれかのキット。
258. 前記説明書が、ポリヌクレオチドが、遺伝子破壊を誘導することができる1つまたは複数の作用物質の導入の直後に、またはその後約30秒、1分、2分、3分、4分、5分、6分、6分、8分、9分、10分、15分、20分、30分、40分、50分、60分、90分、2時間、3時間、もしくは4時間以内に導入されることを明記する、態様257のキット。
IX. Exemplary Embodiments Among the embodiments provided are:
1. A genetically engineered T cell comprising an altered T cell receptor alpha constant (TRAC) locus, wherein the altered TRAC locus comprises a nucleic acid encoding a recombinant TCR or a portion thereof. , the recombinant TCR or portion thereof comprises (i) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant domain; and (ii) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant domain. ) a chain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCR β and Vα domains; and (b) an open reading frame of the endogenous TRAC locus encoding at least a portion of the Cα domain of the recombinant TCR; or a partial sequence thereof.
2. The genetically engineered T cell of embodiment 1, wherein said transgene sequence is integrated via homologous recombination repair (HDR).
3. The gene of embodiment 1 or embodiment 2, wherein the modified TRAC locus comprises an in-frame fusion of (i) a transgene sequence and (ii) an open reading frame of an endogenous TRAC locus or a subsequence thereof. Engineered T cells.
4. The genetically engineered T cell of any of embodiments 1 to 3, wherein the transgene sequence does not include sequences encoding 3' untranslated regions (3' UTRs) or introns.
5. The genetically engineered T cell of any of embodiments 1 to 4, wherein said open reading frame or subsequence thereof comprises the 3' UTR of the endogenous TRAC locus.
6. A portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is encoded by the full-length native Cα. The genetically engineered T cell of any of embodiments 1 to 5, which is shorter than .
7. The genetically engineered T cell of any of embodiments 1 to 6, wherein said open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous TRAC locus.
8. The genetically engineered T cell of any of embodiments 1 to 7, wherein said transgene sequence is in frame with one or more exons of the open reading frame of the endogenous TRAC locus or a subsequence thereof.
9. Any of embodiments 1 to 8, wherein the transgene sequence is integrated downstream of the 5'-most nucleotide of exon 1 and upstream of the 3'-most nucleotide of exon 1 of the open reading frame of the endogenous TRAC locus. Genetically engineered T cells.
10. The genetically engineered T cell of any of embodiments 1 to 9, wherein at least a portion of Cα is encoded by at least exons 2 to 4 of the open reading frame of the endogenous TRAC locus.
11. The genetically engineered T cell of any of embodiments 1 to 10, wherein at least a portion of Cα is encoded by at least a portion of exon 1 and exons 2 to 4 of the open reading frame of the endogenous TRAC locus.
12. The genetically engineered T cell of any of embodiments 1 to 11, wherein at least a portion of Cα is encoded by less than the full length of exon 1 of the open reading frame of the endogenous TRAC locus.
13. The genetically engineered T cell of any of embodiments 1-12, wherein the encoded TCRα chain is capable of dimerizing with the TCRβ chain.
14. The Cα encoded by a sequence selected from any one of SEQ ID NO: 14, 15, 19, or 24, or by any one of SEQ ID NO: 14, 15, 19, or 24 at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. 14. The genetically engineered T cell of any of embodiments 1 to 13, comprising a sequence, or a subsequence thereof, exhibiting higher sequence identity than .
15. A further part of Cα is
The sequence of nucleotides starting from residue 3 to residue 3155 of the sequence set forth in SEQ ID NO:1, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:1. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 3155 or one or more exons thereof; The genetically engineered T cell of any of embodiments 6 to 14, encoded by a sequence, or subsequence thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity. .
16. The genetically engineered T cell of embodiments 6-15, wherein the further portion of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length. .
17. A further portion of Cα contains less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRAC locus. The genetically engineered T cell of embodiment 14 or embodiment 15 encoded by a sequence of nucleotides.
18. Any of embodiments 6 to 17, wherein the further portion of Cα is encoded by a portion of exon 1 of the TRAC locus, said portion of exon 1 being shorter than the full length of exon 1 of the open reading frame of the TRAC locus. Genetically engineered T cells.
19. The genetically engineered T cell of any of embodiments 6 to 18, wherein the further portion of Cα is encoded by a portion of exon 1 of the TRAC locus, the portion of exon 1 comprising the 5' portion of exon 1. .
20. A further portion of Cα is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 relative to the sequence set forth in SEQ ID NO:142, or to SEQ ID NO:142. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity, or a subsequence thereof. Genetically engineered T cells.
21. The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) has one or more modifications, optionally one or more, compared to the native Cα region and/or the native Cβ region. 1, wherein the one or more modifications can form one or more non-natural disulfide bridges between the alpha and beta chains. 21. The genetically engineered T cell of any of embodiments 6 to 20, which introduces one or more cysteine residues.
22. The genetically engineered T cell of any of embodiments 1-21 further comprising a gene disruption at the TRBC locus.
23. The genetically engineered T cell of any of embodiments 1 to 22 further comprising gene disruption at the TRBC1 and/or TRBC2 loci.
24. The further portion of Cα and/or the Cβ region encoded by the nucleic acid sequence of (a) has one or more modifications, optionally one or more, compared to the native Cα region and/or the native Cβ region. 1, wherein the one or more modifications can form one or more non-natural disulfide bridges between the alpha and beta chains. 24. The genetically engineered T cell of any of embodiments 1 to 23, which introduces one or more cysteine residues.
25. A genetically engineered T cell comprising a modified T cell receptor beta constant (TRBC) locus, wherein the modified TRBC locus comprises a nucleic acid encoding a recombinant TCR or a portion thereof. , the recombinant TCR or portion thereof comprises (i) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant domain; and (ii) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant domain. ) a chain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCRα and Vβ domains; and (b) an open reading frame of the endogenous TRBC locus encoding at least a portion of the Cβ domain of the recombinant TCR; or a partial sequence thereof.
26. The genetically engineered T cell of embodiment 25, wherein said transgene sequence is integrated via homologous recombination repair (HDR).
27. The genetically engineered T cell of embodiment 25 or embodiment 26, wherein the TRBC locus is the TRBC1 locus and/or the TRBC2 locus.
28. The gene of embodiment 25 or embodiment 26, wherein the modified TRBC locus comprises an in-frame fusion of (i) a transgene sequence and (ii) an open reading frame of an endogenous TRBC locus or a subsequence thereof. Engineered T cells.
29. The genetically engineered T cell of any of embodiments 25 to 28, wherein the transgene sequence does not include sequences encoding 3' untranslated regions (3' UTRs) or introns.
30. The genetically engineered T cell of any of embodiments 25 to 29, wherein said open reading frame or subsequence thereof comprises the 3' UTR of the endogenous TRBC locus.
31. A portion of Cβ is encoded by an open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by a transgene sequence, and the further portion of Cβ is encoded by the full-length native Cβ. The genetically engineered T cell of any of embodiments 25-30, which is shorter than.
32. The genetically engineered T cell of any of embodiments 25 to 31, wherein said open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus.
33. The genetically engineered T cell of any of embodiments 25 to 32, wherein said transgene sequence is in frame with one or more exons or subsequences thereof of the open reading frame of the endogenous TRBC locus.
34. Any of embodiments 25 to 33, wherein the transgene sequence is integrated downstream of the 5'-most nucleotide of exon 1 and upstream of the 3'-most nucleotide of exon 1 of the open reading frame of the endogenous TRBC locus. Genetically engineered T cells.
35. The genetically engineered T cell of any of embodiments 25-34, wherein at least a portion of Cβ is encoded by at least exons 2 to 4 of the open reading frame of the endogenous TRBC locus.
36. The genetically engineered T cell of any of embodiments 25-35, wherein at least a portion of Cβ is encoded by at least a portion of exon 1 and exons 2-4 of the open reading frame of the endogenous TRBC locus.
37. The genetically engineered T cell of any of embodiments 25-36, wherein at least a portion of Cβ is encoded by less than the full length of exon 1 of the open reading frame of the endogenous TRBC locus.
38. The genetically engineered T cell of any of embodiments 25-37, wherein the encoded TCRβ chain is capable of dimerizing with the TCRα chain.
39. A sequence in which the encoded Cβ is selected from any one of SEQ ID NO: 16, 17, 21, or 25, or at least 85% relative to SEQ ID NO: 16, 17, 21, or 25. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity The genetically engineered T cell according to any of embodiments 25 to 38, comprising a sex-indicating sequence, or a subsequence thereof.
40. A further part of Cα is
The sequence of nucleotides starting from residue 3 to residue 1445 of the sequence set forth in SEQ ID NO:2, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:2. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1445 or one or more exons thereof; Sequences, or subsequences thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity; or
The sequence of nucleotides starting from residue 3 to residue 1486 of the sequence set forth in SEQ ID NO:3, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:3. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1486 or one or more exons thereof; The genetically engineered T cell of any of embodiments 31 to 39, encoded by a sequence, or subsequence thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity. .
41. The genetically engineered T cell of embodiments 31-40, wherein the further portion of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length. .
42. A further portion of Cα contains less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRBC locus. A genetically engineered T cell according to embodiment 35 or embodiment 36, encoded by a sequence of nucleotides.
43. Any of embodiments 31 to 42, wherein the further portion of Cα is encoded by a portion of exon 1 of the TRBC locus, said portion of exon 1 being shorter than the full length of exon 1 of the open reading frame of the TRBC locus. Genetically engineered T cells.
44. The genetically engineered T cell of any of embodiments 31-43, wherein the further portion of Cα is encoded by a portion of exon 1 of the TRBC locus, the portion of exon 1 comprising the 5' portion of exon 1. .
45. The further portion of Cβ and/or the Cα region encoded by the nucleic acid sequence of (a) has one or more modifications, optionally one or more, compared to the native Cβ region and/or the native Cα region. 1, wherein the one or more modifications can form one or more non-natural disulfide bridges between the alpha and beta chains. 45. The genetically engineered T cell of any of embodiments 25-44, which introduces one or more cysteine residues.
46. The genetically engineered T cell of any of embodiments 25-45, further comprising a gene disruption at the TRAC locus.
47. The genetically engineered T cell of any of embodiments 1 to 46, wherein said transgene sequence comprises one or more multicistronic elements.
48. The genetically engineered T cell of embodiment 47, wherein said multicistronic element is located between a nucleic acid sequence encoding TCRα or a portion thereof and a nucleic acid sequence encoding TCRβ or a portion thereof.
49. The genetically engineered T cell of embodiment 47 or embodiment 48, wherein said one or more multicistronic elements are upstream of a nucleic acid sequence encoding a TCR or a portion of a TCR, or a nucleic acid molecule encoding a TCR.
50. The genetically engineered T cell of any of embodiments 47 to 49, wherein said multicistronic element is or comprises a ribosome skipping sequence, optionally T2A, P2A, E2A, or F2A.
51. The genetically engineered T cell of any of embodiments 1-50, further comprising one or more heterologous control or regulatory elements.
52. The transgene sequence comprises one or more heterologous or regulatory regulatory elements operably linked to modulate expression of the TCR upon expression from the introduced cell in the genetically engineered T cell. 52. The genetically engineered T cell of any of embodiments 1-51, comprising:
53. According to embodiment 51 or embodiment 52, the one or more heterologous control or regulatory elements comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, and/or a splice donor sequence. Genetically engineered T cells.
54. The genetically engineered T cell of any of embodiments 51-53, wherein said heterologous control or regulatory element comprises a heterologous promoter.
55. The genetically engineered T cell of embodiment 54, wherein said heterologous promoter is selected among constitutive promoters, inducible promoters, repressible promoters, and/or tissue-specific promoters.
56. The genetically engineered T cell of embodiment 54 or embodiment 55, wherein said heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof.
57. The genetically engineered T cell of any of embodiments 1 to 56, wherein Cα and/or Cβ of said recombinant TCR comprises one or more non-natural cysteines.
58. said recombinant TCR binds to an antigen associated with, specific for, and/or expressed on cells or tissues associated with a disease, disorder, or condition; 58. The genetically engineered T cell according to any one of aspects 1 to 57, which is capable of.
59. The genetically engineered T cell of any of embodiments 1 to 58, wherein said disease, disorder or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer.
60. The genetically engineered T cell of any of embodiments 1 to 59, wherein said antigen is a tumor antigen or a pathogenic antigen.
61. The genetically engineered T cell of embodiment 60, wherein said pathogenic antigen is a bacterial or viral antigen.
62. The antigen is a viral antigen, optionally hepatitis A, hepatitis B, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis virus infection, Epstein-Barr virus (EBV), human herpesvirus. 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV). Genetically engineered T cells.
63. The genetically engineered virus of embodiment 61 or embodiment 62, wherein the viral antigen is an HPV-derived antigen selected from HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35. T cells.
64. The genetically engineered T cell of embodiment 61 or embodiment 62, wherein said viral antigen is an HPV-16 antigen, which is an HPV-16 E6 or HPV-16 E7 antigen.
65. The viral antigen is Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), latent membrane protein LMP- 63. The genetically engineered T cell of embodiment 61 or embodiment 62, which is an EBV antigen selected from among EBV-1, LMP-2A, and LMP-2B, EBV-EA, EBV-MA, and EBV-VCA.
66. The genetically engineered T cell of embodiment 61 or embodiment 62, wherein said viral antigen is an HTLV antigen that is TAX.
67. The genetically engineered T cell of embodiment 61 or embodiment 62, wherein the viral antigen is an HBV antigen, which is hepatitis B core antigen or hepatitis B envelope antigen.
68. The genetically engineered T cell of embodiment 61 or embodiment 62, wherein said antigen is a tumor antigen.
69. The antigen is glioma-associated antigen, β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2. (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 (e.g., MUC1-8), p53, Ras, Cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE- A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, Tyrosinase (e.g. tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2 , telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate-specific membrane antigen (PSM), and prostatic acid phosphatase (PAP), neutrophil elastase, ephrinB2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, caspase 8, FRa, CD24 , CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, The genetically engineered T cell of embodiment 59 or 60 selected among glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.
70. The genetically engineered T cell of any of embodiments 1 to 69, wherein said T cell is a primary T cell derived from a subject, optionally said subject being human.
71. The genetically engineered T cell of any of embodiments 1 to 70, wherein said T cell is a CD8+ T cell or a subtype thereof.
72. The genetically engineered cell of any of embodiments 1 to 71, wherein said T cell is a CD4+ T cell or a subtype thereof.
73. The genetically engineered cell of any of embodiments 1 to 72, wherein said T cell is derived from a multipotent or pluripotent cell, optionally an iPSC.
74. A composition comprising a plurality of genetically engineered T cells of any of embodiments 1-73.
75. The composition of embodiment 74, comprising CD4+ T cells and/or CD8+ T cells.
76. Embodiments comprising CD4+ T cells and CD8+ T cells, and wherein the ratio of CD4+ T cells to CD8+ T cells is from 1:3 to 3:1 or about 1:3 to 3:1, optionally 1:1. 74 or the composition of embodiment 75.
77. At least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition. comprises a genetic disruption in or to the endogenous T cell receptor alpha constant region (TRAC) gene and/or the T cell receptor beta constant region (TRBC) gene; and/or at least 80 of the engineered cells in the composition %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% do not express or detect the gene product of the endogenous TRAC or TRBC gene not expressed at possible levels,
The composition of any of embodiments 74-76.
78. At least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition, or 35%, 40%, Embodiments 74 to 74, wherein greater than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% expresses the recombinant TCR and/or exhibits antigen binding. Any composition of 77.
79. (a) From (i) the T-cell receptor beta (TCRβ) chain, which includes a variable beta (Vβ) domain and a constant beta (Cβ) domain, and (ii) the full-length native T-cell receptor alpha (TCRα) chain. a nucleic acid sequence encoding a portion of a recombinant T-cell receptor (TCR), which also encodes a portion of a short TCRα chain;
(b) A polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRAC locus.
80. The TCRα chain contains a constant alpha region (Cα), and when the TCR or antigen-binding fragment thereof is expressed from a cell into which the polynucleotide has been introduced, at least a portion of the Cα is located at the endogenous TRAC locus. 80. The polynucleotide of embodiment 79, which is encoded by an open reading frame or a subsequence thereof.
81. The nucleic acid sequence of (a) and one of the one or more homology arms together comprise a sequence of nucleotides encoding a Cα that is shorter than the full length of native Cα, and the TCR or antigen-binding fragment thereof is 81. The polynucleotide of embodiment 79 or 80, wherein at least a portion of Cα is encoded by an open reading frame of the endogenous TRAC locus or a subsequence thereof when expressed from a cell into which the polynucleotide has been introduced.
82. The polynucleotide of any of embodiments 79-81, comprised in a viral vector.
83. The polynucleotide of any of embodiments 79-82, wherein the nucleic acid sequence encoding the TCR β chain is upstream of the nucleic acid sequence encoding a portion of the TCR α chain.
84. The polynucleotide of any of embodiments 79-83, wherein the nucleic acid sequence of (a) does not contain an intron.
85. The polynucleotide of any of embodiments 79-84, wherein the nucleic acid sequence of (a) is a sequence that is foreign or heterologous to the open reading frame of the endogenous genomic TRAC gene of a T cell, optionally a human T cell.
86. Embodiments 79-85, wherein the nucleic acid sequence of (a) is in frame with one or more exons or subsequences thereof of the open reading frame of the TRAC locus comprised in one or more homology arms. Any polynucleotide.
87. The polynucleotide of any of embodiments 79 to 86, wherein one or more exons of said open reading frame or a subsequence thereof comprises a sequence within exon 1 of the open reading frame of the TRAC locus.
88. The polynucleotide of any of embodiments 79-87, wherein the TCRα chain is capable of dimerizing with the TCRβ chain upon production from a cell into which the polynucleotide has been introduced.
89. The polynucleotide of any of embodiments 79-88, wherein a portion of the TCRα chain comprises a variable alpha (Vα) domain.
90. A portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the nucleic acid sequence of (a), and the further portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof; 89. The polynucleotide of any of embodiments 79-89, which is shorter than the full length of.
91. A further part of Cα is
The sequence of nucleotides starting from residue 3 to residue 3155 of the sequence set forth in SEQ ID NO:1, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:1. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 3155 or one or more exons thereof; 91. The polynucleotide of embodiment 90, encoded by a sequence, or subsequence thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity.
92. The polynucleotide of embodiment 91, wherein the further portion of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length.
93. A further portion of Cα contains less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRAC locus. 93. The polynucleotide of embodiment 91 or 92, encoded by a sequence of nucleotides.
94. Any of embodiments 91 to 93, wherein the further portion of Cα is encoded by a portion of exon 1 of the TRAC locus, said portion of exon 1 being shorter than the full length of exon 1 of the open reading frame of the TRAC locus. That polynucleotide.
95. The polynucleotide of any of embodiments 90-94, wherein the further portion of Cα is encoded by a portion of exon 1 of the TRAC locus, said portion of exon 1 comprising the 5' portion of exon 1.
96. A further portion of Cα is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or a subsequence thereof. polynucleotide.
97. The further portion of Cα and/or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one or more, compared to the native Cα region and/or the native Cβ region. 1, wherein the one or more modifications can form one or more non-natural disulfide bridges between the alpha and beta chains. 97. A polynucleotide according to any of embodiments 79 to 96, which introduces one or more cysteine residues.
98. The polynucleotide of any of embodiments 79-97, wherein the one or more homology arms comprises a 5' homology arm and/or a 3' homology arm.
99. The polynucleotide of embodiment 98, wherein the 5' homology arm and the 3' homology arm include nucleic acid sequences homologous to nucleic acid sequences surrounding the target site, and the target site is within the TRAC locus.
100. The polynucleotide of embodiment 99, wherein said target site is within exon 1 of the TRAC locus.
101. The 5' homology arm is
a) At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% to the sequence shown in SEQ ID NO:124 , 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides for sequences exhibiting 97%, 98%, 99%, or higher sequence identity, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides; A sequence containing consecutive nucleotides;
b) 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence set forth in SEQ ID NO:124, or at least 150, 200, 250, 300, 350; , 400, 450, 500, 550, or 600 contiguous nucleotides, or a sequence comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides;
c) The polynucleotide of any of embodiments 98-100, comprising the sequence shown in SEQ ID NO:124.
102. The 3' homology arm is
a) At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% to the sequence shown in SEQ ID NO:125 , 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides for sequences exhibiting 97%, 98%, 99%, or higher sequence identity, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides; A sequence containing consecutive nucleotides;
b) 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides, or at least 150, 200, 250, 300, 350 of the sequence set forth in SEQ ID NO:125; , 400, 450, 500, 550, or 600 contiguous nucleotides, or a sequence comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides;
c) The polynucleotide of any of embodiments 98-101, comprising the sequence shown in SEQ ID NO:125.
103. (a) From (i) the T cell receptor alpha (TCRα) chain, which includes a variable alpha (Vα) domain and a constant alpha (Cα) domain, and (ii) the full-length native T cell receptor beta (TCRβ) chain. a nucleic acid sequence encoding a portion of a recombinant T cell receptor (TCR), which also encodes a portion of a short TCR β chain;
(b) A polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRBC locus.
104. When the TCR β chain contains constant beta (Cβ) and the TCR or antigen-binding fragment thereof is expressed from a cell into which the polynucleotide has been introduced, at least a portion of the Cβ is present at the open endogenous TRAC locus. 104. The polynucleotide of embodiment 103, encoded by a reading frame or a subsequence thereof.
105. The nucleic acid sequence of (a) and one of the one or more homology arms together include a sequence of nucleotides encoding Cβ that is shorter than the full length of native Cβ, and the TCR or antigen-binding fragment thereof is 105. The polynucleotide of embodiment 103 or 104, wherein at least a portion of Cβ is encoded by an open reading frame of the endogenous TRBC locus or a subsequence thereof when expressed from a cell into which the polynucleotide has been introduced.
106. The polynucleotide of any of embodiments 103-105, comprised in a viral vector.
107. The polynucleotide of any of embodiments 103-106, wherein the TRBC locus is one or more of TRBC1 or TRBC2.
108. The polynucleotide of any of embodiments 103 to 107, wherein the nucleic acid sequence encoding the TCRα chain is upstream of the nucleic acid sequence encoding a portion of the TCRβ chain.
109. The polynucleotide of any of embodiments 103-108, wherein the nucleic acid sequence of (a) does not contain an intron.
110. The polynucleotide of any of embodiments 103 to 109, wherein the nucleic acid sequence of (a) is a sequence that is foreign or heterologous to the open reading frame of the endogenous genomic TRBC locus of a T cell, optionally a human T cell. .
111. Embodiments 103-110, wherein the nucleic acid sequence of (a) is in frame with one or more exons or subsequences thereof of the open reading frame of the TRAC locus comprised in one or more homology arms. Any polynucleotide.
112. The polypeptide of any of embodiments 103 to 111, wherein one or more exons of said open reading frame or a subsequence thereof is or comprises a sequence within exon 1 of the open reading frame of the TRBC locus. nucleotide.
113. The polynucleotide of any of embodiments 103-112, wherein the TCRβ chain is capable of dimerizing with the TCRα chain upon production from a cell into which the polynucleotide has been introduced.
114. The polynucleotide of any of embodiments 103-113, wherein a portion of the TCR β chain comprises a variable beta (Vβ) domain.
115. A portion of Cβ is encoded by the open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by the nucleic acid sequence of (a), and the further portion of Cβ is encoded by the open reading frame of the endogenous TRBC locus or a subsequence thereof; The polynucleotide of any of embodiments 103-114, which is shorter than the full length of.
116. A further part of Cβ is
The sequence of nucleotides starting from residue 3 to residue 1445 of the sequence set forth in SEQ ID NO:2, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:2. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1445 or one or more exons thereof; Sequences, or subsequences thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity; or
The sequence of nucleotides starting from residue 3 to residue 1486 of the sequence set forth in SEQ ID NO:3, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:3. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1486 or one or more exons thereof; The polynucleotide of embodiment 115 is encoded by a sequence, or subsequence thereof, that exhibits 95%, 96%, 97%, 98%, 99% or higher sequence identity.
117. The polynucleotide of embodiment 115 or 116, wherein the further portion of Cβ is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length.
118. An additional portion of Cβ encodes less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRBC locus 118. The polynucleotide of any of embodiments 115-117, encoded by a sequence of nucleotides.
119. Any of embodiments 115-118, wherein the further portion of Cβ is encoded by a portion of exon 1 of the TRBC locus, and the portion of exon 1 is shorter than the full length of exon 1 of the open reading frame of the TRBC locus. That polynucleotide.
120. The polynucleotide of embodiments 115-119, wherein the further portion of the TCRα constant domain is encoded by a portion of exon 1 of the TRAC locus, said portion of exon 1 comprising the 5' portion of exon 1.
121. The further portion of Cβ and/or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one or more, compared to the native Cβ region and/or the native Cα region. 1, wherein the one or more modifications can form one or more non-natural disulfide bridges between the alpha and beta chains. 120. The polynucleotide of any of embodiments 115-120, which introduces one or more cysteine residues.
122. The polynucleotide of any of embodiments 115-121, wherein the one or more homology arms comprises a 5' homology arm and/or a 3' homology arm.
123. The polypeptide of embodiment 122, wherein the 5' homology arm and the 3' homology arm contain a nucleic acid sequence homologous to a surrounding nucleic acid sequence, and the target site is within an open reading frame of the TRBC locus. nucleotide.
124. The polynucleotide of embodiment 123, wherein said target site is within exon 1 of the open reading frame of the TRBC locus.
125. The 5' homology arm and the 3' homology arm are independently at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or 10, 20, 30 , 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or less than or about 2000 nucleotides , 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.
126. The 5' homology arm and the 3' homology arm independently contain 100-1000 or about 100-1000 nucleotides, 100-750 or about 100-750 nucleotides, 100-600 or about 100-600 nucleotides, 100 ~400 or about 100-400 nucleotides, 100-300 or about 100-300 nucleotides, 100-200 or about 100-200 nucleotides, 200-1000 or about 200-1000 nucleotides, 200-750 or about 200-750 nucleotides, 200 ~600 or about 200-600 nucleotides, 200-400 or about 200-400 nucleotides, 200-300 or about 200-300 nucleotides, 300-1000 or about 300-1000 nucleotides, 300-750 or about 300-750 nucleotides, 300 ~600 or about 300-600 nucleotides, 300-400 or about 300-400 nucleotides, 400-1000 or about 400-1000 nucleotides, 400-750 or about 400-750 nucleotides, 400-600 or about 400-600 nucleotides, 600 The polynucleotide of embodiment 125, which is ˜1000 or about 600-1000 nucleotides, 600-750 or about 600-750 nucleotides, or 750-1000 or about 750-1000 nucleotides in length.
127. Embodiments wherein the 5' homology arm and the 3' homology arm are independently 600 nucleotides long, about 600 nucleotides long, or less than about 600 nucleotides long. 125 or the polynucleotide of embodiment 126.
128. The polynucleotide of any of embodiments 79-127, wherein the nucleic acid sequence of (a) comprises one or more multicistronic elements.
129. The polynucleotide of embodiment 128, wherein said multicistronic element is located between a nucleic acid sequence encoding TCRα or a portion thereof and a nucleic acid sequence encoding TCRβ or a portion thereof.
130. The polynucleotide of embodiment 128 or embodiment 129, wherein said one or more multicistronic elements are upstream of a nucleic acid sequence encoding a TCR or a portion of a TCR, or a nucleic acid molecule encoding a TCR.
131. The polynucleotide of any of embodiments 129 or 130, wherein said multicistronic element is or comprises a ribosome skipping sequence, optionally T2A, P2A, E2A, or F2A.
132. The polynucleotide of any of embodiments 79-131, further comprising one or more heterologous control or regulatory elements.
133. The nucleic acid sequence of (a) comprises one or more heterologous or regulatory regulatory elements operably linked to modulate expression of the TCR upon expression from a cell into which the polynucleotide has been introduced. , the polynucleotide of any one of embodiments 79-132.
134. The polypeptide of embodiment 132 or 133, wherein said one or more heterologous control or regulatory elements comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence, and/or a splice donor sequence. nucleotide.
135. The polynucleotide of any of embodiments 132-134, wherein said heterologous control or regulatory element comprises a heterologous promoter.
136. The polynucleotide of embodiment 135, wherein said heterologous promoter is selected among constitutive promoters, inducible promoters, repressible promoters, and/or tissue-specific promoters.
137. The polynucleotide of embodiment 135 or embodiment 136, wherein said heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof.
138. The polynucleotide of any of embodiments 82-102 or 106-137, wherein said viral vector is an AAV vector.
139. The polynucleotide of embodiment 138, wherein said AAV vector is selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vectors.
140. The polynucleotide of embodiment 139, wherein said AAV vector is an AAV2 or AAV6 vector.
141. The polynucleotide of any of embodiments 82-102 or 106-137, wherein said viral vector is a retroviral vector.
142. The polynucleotide of any of embodiments 82-102 or 106-137, wherein said viral vector is a lentiviral vector.
143. The polynucleotide of any of embodiments 79-81, 84-105, or 107-137, which is a linear polynucleotide.
144. The polynucleotide of embodiment 143, wherein the linear polynucleotide is a double-stranded polynucleotide.
145. The polynucleotide of embodiment 144, wherein said linear polynucleotide is a single-stranded polynucleotide.
146. The polynucleotide of any of embodiments 79-145, comprising structure: [5' homology arm]-[nucleic acid sequence of (a)]-[3' homology arm].
147. The polynucleotide of any of embodiments 128 to 146, comprising the structure: [5' homology arm]-[multicistronic element]-[nucleic acid sequence of (a)]-[3' homology arm].
148. The polynucleotide of any of embodiments 134 to 147, comprising structure: [5' homology arm]-[promoter]-[nucleic acid sequence of (a)]-[3' homology arm].
149. said recombinant TCR binds to an antigen associated with, specific for, and/or expressed on cells or tissues associated with a disease, disorder, or condition; The polynucleotide according to any one of embodiments 79 to 148, which is capable of
150. The polynucleotide of embodiment 149, wherein said disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer.
151. The polynucleotide of embodiment 149, wherein said antigen is a tumor antigen or a pathogenic antigen.
152. The polynucleotide of embodiment 151, wherein said pathogenic antigen is a bacterial or viral antigen.
153. The antigen is a viral antigen, optionally hepatitis A, hepatitis B, hepatitis C virus (HCV), human papillomavirus (HPV), hepatitis virus infection, Epstein-Barr virus (EBV), human herpesvirus. 8 (HHV-8), human T-cell leukemia virus-1 (HTLV-1), human T-cell leukemia virus-2 (HTLV-2), or cytomegalovirus (CMV). polynucleotide.
154. The polynucleotide of embodiment 152 or 153, wherein said antigen is an HPV-derived antigen selected from HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35.
155. The polynucleotide of embodiment 152 or 153, wherein said antigen is an HPV-16 antigen, which is an HPV-16 E6 or HPV-16 E7 antigen.
156. The viral antigen is Epstein-Barr nuclear antigen (EBNA)-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP), latent membrane protein LMP- 154. The polynucleotide of embodiment 152 or 153, which is an EBV antigen selected from among EBV-1, LMP-2A, and LMP-2B, EBV-EA, EBV-MA, and EBV-VCA.
157. The polynucleotide of embodiment 152 or 153, wherein said viral antigen is an HTLV antigen that is TAX.
158. The polynucleotide of embodiment 152 or 153, wherein the viral antigen is an HBV antigen, which is hepatitis B core antigen or hepatitis B envelope antigen.
159. The polynucleotide of embodiment 151, wherein said antigen is a tumor antigen.
160. The antigen is glioma-associated antigen, β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2. (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, melanin-A/MART-1, WT-1, S-100, MBP, CD63, MUC1 (e.g., MUC1-8), p53, Ras, Cyclin B1, HER-2/neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE- A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, pl5, Tyrosinase (e.g. tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2 , telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, melanotransferrin (p97), uroplakin II, prostate-specific antigen (PSA), human kallikrein (huK2), prostate-specific membrane antigen (PSM), and prostatic acid phosphatase (PAP), neutrophil elastase, ephrinB2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, caspase 8, FRa, CD24 , CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33/IL3Ra, c-Met, PSMA, The polynucleotide of embodiment 159, selected among glycolipid F77, GD-2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.
161. A genetically engineered T cell comprising a modified TRAC locus, comprising introducing the polynucleotide of any of embodiments 79-102 or 125-160 into a T cell comprising a gene disruption at the TRAC locus. How to make.
162. (a) introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the endogenous T cell receptor alpha constant (TRAC) locus of the T cell; ;and
(b) introducing into the T cell a polynucleotide comprising a transgene sequence encoding a T cell receptor beta (TCRβ) chain and a portion of a TCRα chain that is shorter than the full-length native T cell receptor alpha (TCRα) chain; A method of producing a genetically engineered T cell containing a modified TRAC locus, the method comprising the step of introducing a transgene into an endogenous TRAC locus via homologous recombination repair (HDR) Said method of producing genetically engineered cells that are targeted for integration and thereby contain a modified TRAC locus.
163. The T cell receptor beta (TCRβ) chain and a portion of the T cell receptor alpha (TCRα) chain are present in T cells with gene disruptions within the endogenous T cell receptor alpha constant (TRAC) locus. A method of producing a genetically engineered T cell comprising a modified TRAC locus, the method comprising the step of introducing a polynucleotide comprising a transgene sequence encoding a homologous recombination repair ( said method, wherein said method generates a genetically engineered cell that is targeted for integration within the endogenous TRAC locus via HDR), thereby comprising a modified TRAC locus.
164. The method of embodiment 163, wherein said gene disruption is induced by one or more agents capable of inducing gene disruption of one or more target sites within the endogenous TRAC locus.
165. The method of any of embodiments 161-164, wherein said polynucleotide is derived from any of embodiments 79-102 or 125-160.
166. The modified TRAC locus comprises a nucleic acid sequence encoding a recombinant TCR or a portion thereof, and the nucleic acid sequence combines (i) the transgene sequence, and (ii) the open reading frame of the endogenous TRAC locus or 166. The method of any of embodiments 161-165, comprising an in-frame fusion with a subsequence thereof.
167. The method of any of embodiments 161-166, wherein the transgene sequence does not include sequences encoding 3' untranslated regions (3' UTRs) or introns.
168. The method of any of embodiments 161-167, wherein said open reading frame or subsequence thereof comprises the 3' UTR of the endogenous TRAC locus.
169. A portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is encoded by the full-length native Cα The method of any of embodiments 161 to 168, wherein the method is shorter than .
170. The method of any of embodiments 161-169, wherein said open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous TRAC locus.
171. The method of any of embodiments 161-170, wherein the transgene sequence is in frame with one or more exons of the open reading frame of the endogenous TRAC locus or a subsequence thereof.
172. Any of embodiments 161-171, wherein the transgene sequence is integrated downstream of the 5'-most nucleotide of exon 1 and upstream of the 3'-most nucleotide of exon 1 of the open reading frame of the endogenous TRAC locus. That method.
173. The method of any of embodiments 161-172, wherein at least a portion of Cα is encoded by at least exons 2-4 of the open reading frame of the endogenous TRAC locus.
174. Cα encoded by a sequence selected from any one of SEQ ID NO: 14, 15, 19, or 24; at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. 174. The method of any of embodiments 161 to 173, comprising a sequence, or a subsequence thereof, exhibiting higher sequence identity than.
175. A further portion of Cα is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 relative to the sequence set forth in SEQ ID NO:142, or to SEQ ID NO:142. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or a subsequence thereof. Method.
176. The method of any of embodiments 161-75, further comprising the engineered T cell inducing gene disruption at the TRBC locus.
177. The method of any of embodiments 161-174, wherein the engineered T cell comprises a gene disruption at the TRBC1 and/or TRBC2 loci.
178. A method of producing a genetically engineered T cell comprising a modified TRBC locus, the method comprising introducing the polynucleotide of any of embodiments 103 to 160 into a T cell comprising a gene disruption at the TRBC locus. .
179. (a) introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the endogenous T cell receptor beta constant (TRBC) locus of the T cell; :and
(b) introducing into the T cell a polynucleotide comprising a transgene encoding a T cell receptor alpha (TCRα) chain and a portion of a TCRβ chain that is shorter than the full-length native T cell receptor beta (TCRβ) chain; A method of producing a genetically engineered T cell comprising an altered TRBC locus, the method comprising the step of: said method of producing a genetically engineered cell targeted for, thereby comprising an altered TRBC locus.
180. The T cell receptor alpha (TCRα) chain and a portion of the T cell receptor beta (TCRβ) chain are present in T cells with gene disruptions within the endogenous T cell receptor beta constant (TRBC) locus. A method of producing a genetically engineered T cell comprising a modified TRBC locus, the method comprising the step of introducing a polynucleotide comprising a transgene encoding ), thereby producing a genetically engineered cell containing a modified TRBC locus.
181. The method of embodiment 180, wherein said gene disruption is induced by one or more agents capable of inducing gene disruption of one or more target sites within the endogenous TRBC locus.
182. The method of any of embodiments 178-181, wherein the TRBC locus is the TRBC1 locus and/or the TRBC2 locus.
183. The method of any of embodiments 178-182, wherein said polynucleotide is derived from any of embodiments 95-149.
184. A modified TRBC locus comprises a nucleic acid sequence encoding a recombinant TCR or a portion thereof, and the nucleic acid sequence combines (i) the transgene sequence and (ii) the open reading frame of the endogenous TRBC locus or 184. The method of any of embodiments 178-183, comprising an in-frame fusion with a subsequence thereof.
185. The method of any of embodiments 178-184, wherein the transgene sequence does not include sequences encoding 3' untranslated regions (3' UTRs) or introns.
186. The method of any of embodiments 178-185, wherein said open reading frame or subsequence thereof comprises the 3' UTR of the endogenous TRBC locus.
187. A portion of Cβ is encoded by the open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by the transgene sequence, and the further portion of Cβ is encoded by the full-length native Cβ. The method of any of aspects 178 to 186, wherein the method is shorter than .
188. The method of any of embodiments 178-187, wherein said open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus.
189. The method of any of embodiments 178-188, wherein the transgene sequence is in frame with one or more exons or subsequences thereof of the open reading frame of the endogenous TRBC locus.
190. Any of embodiments 178-189, wherein the transgene sequence is integrated downstream of the 5'-most nucleotide of exon 1 and upstream of the 3'-most nucleotide of exon 1 of the open reading frame of the endogenous TRBC locus. That method.
191. The method of any of embodiments 178-190, wherein at least a portion of Cβ is encoded by at least exons 2-4 of the open reading frame of the endogenous TRBC locus.
192. A sequence in which the encoded Cβ is selected from any one of SEQ ID NO: 16, 17, 21, or 25, or at least 85% relative to SEQ ID NO: 16, 17, 21, or 25. , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity 192. The method of any one of embodiments 178 to 191, comprising a sequence indicative of sex, or a subsequence thereof.
193. The method of any of embodiments 177-192, further comprising the engineered T cell inducing gene disruption at the TRAC locus.
194. The method of any of the embodiments, wherein the one or more agents capable of inducing gene disruption comprise a DNA binding protein or DNA binding nucleic acid that specifically binds or hybridizes to a target site.
195. The method of embodiment 194, wherein the one or more agents capable of inducing gene disruption comprises (a) a fusion protein comprising a DNA targeting protein and a nuclease, or (b) an RNA-guided nuclease.
196. The DNA targeting protein or RNA-guided nuclease may be a zinc finger protein (ZFP), a TAL protein, or a clustered regularly arranged short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) specific for the target site. ).
197. A zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or, and a CRISPR-, wherein the one or more agents specifically binds to, recognizes, or hybridizes to a target site. 197. The method of any of embodiments 194-196, comprising a combination of Cas9.
198. The method of embodiments 162, 164-177, 179, or 181-197, wherein each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. Either way.
199. The method of embodiment 198, wherein the one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein.
200. The method of embodiment 199, wherein the RNP is introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing.
201. The method of embodiment 199 or embodiment 200, wherein the RNP is introduced via electroporation.
202. Any of embodiments 162, 164-177, 179, or 181-197, wherein said one or more agents are introduced as one or more polynucleotides encoding a gRNA and/or a Cas9 protein. Method.
203. gRNA is
is complementary to the target site at the TRAC locus, and

203. The method of any of embodiments 198-202, wherein the targeting domain comprises a sequence selected from .
204. The method of embodiment 203, wherein the gRNA has a targeting domain comprising the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO:31).
205. gRNA is
is complementary to a target site in one or both of the TRBC1 gene and TRBC2 gene, and

203. The method of any of embodiments 198-202, wherein the targeting domain comprises a sequence selected from .
206. gRNA is sequenced

The method of embodiment 205, having a targeting domain comprising:
207. The method of any of embodiments 161-206, wherein said T cell is a CD8+ T cell or a subtype thereof.
208. The method of embodiment 207, wherein the T cell is a CD4+ T cell or a subtype thereof.
209. The method of any of embodiments 161-206, wherein said T cells are derived from multipotent or pluripotent cells, optionally iPSCs.
210. The method of any of embodiments 161-209, wherein the T cells comprise T cells that are autologous to the subject.
211. The method of any of embodiments 161-209, wherein the T cell comprises a T cell that is allogeneic to the subject.
212. Any of embodiments 161-211, wherein the polynucleotide and/or one or more polynucleotides encoding gRNA and/or Cas9 protein are comprised in one or more vectors, optionally being viral vectors. the method of.
213. The method of embodiment 212, wherein said vector is an AAV vector.
214. The method of embodiment 213, wherein said AAV vector is selected among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vectors.
215. The method of embodiment 214, wherein the AAV vector is an AAV2 or AAV6 vector.
216. The method of embodiment 212, wherein said viral vector is a retroviral vector.
217. The method of embodiment 216, wherein said viral vector is a lentiviral vector.
218. The method of any of embodiments 161-211, wherein said polynucleotide is a linear polynucleotide.
219. The method of embodiment 218, wherein the linear polynucleotide is a double-stranded polynucleotide.
220. The method of embodiment 219, wherein the linear polynucleotide is a single-stranded polynucleotide.
221. The method of any of embodiments 161-220, wherein the introduction of one or more agents capable of inducing gene disruption and the introduction of the template polynucleotide are performed in any order, simultaneously or sequentially. .
222. The method of any of embodiments 161-221, wherein the introduction of the template polynucleotide is performed after the introduction of one or more agents capable of inducing gene disruption.
223. The template polynucleotide is immediately after, or about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes after the introduction of one or more agents 23 capable of inducing gene disruption. Deployed within 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours , the method of aspect 222.
224. An engineered T cell or a plurality of engineered T cells produced using the method of any of embodiments 161-2.
225. A composition comprising the engineered T cell or engineered cells of embodiment 224.
226. The composition of embodiment 225, comprising CD4+ T cells and/or CD8+ T cells.
227. Embodiment 225 comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CD4+ T cells to CD8+ T cells is from 1:3 to 3:1 or about 1:3 to 3:1, optionally 1:1. or the composition of embodiment 226.
228. At least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition. comprises a genetic disruption in or to the endogenous T cell receptor alpha constant region (TRAC) gene and/or the T cell receptor beta constant region (TRBC) gene; and/or at least 80 of the engineered cells in the composition %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% do not express or detect the gene product of the endogenous TRAC or TRBC gene not expressed at possible levels,
The composition of any of embodiments 225-227.
229. At least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition, or 35%, 40%, Embodiments 225 to 225, wherein greater than 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% express the recombinant TCR and/or exhibit antigen binding. Any composition of 228.
230. A method of treatment comprising administering to a subject the engineered cell, a plurality of engineered cells, or the composition of any of embodiments 224-229.
231. Use of the engineered cell, engineered cells, or composition of any of embodiments 224-229 for the treatment of a disease or disorder.
232. Use of an engineered cell, a plurality of engineered cells, or a composition of any of embodiments 224-229 in the manufacture of a medicament for treating a disease or disorder.
233. The engineered cell, engineered cells, or composition of any of embodiments 224-229 for use in the treatment of a disease or disorder.
234. The polynucleotide of any of embodiments 79-102 or 116-149;
one or more agents capable of inducing gene disruption at a target site within the TRAC locus.
235. (a) a nucleic acid sequence encoding a T cell receptor beta (TCRβ) chain and a portion of a TCRα chain that is shorter than the full-length native T cell receptor alpha (TCRα) chain, and
(b) a polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRAC locus encoding the TCR alpha chain;
one or more agents capable of inducing gene disruption at a target site within the TRAC locus.
236. The article of manufacture of embodiment 201, wherein said polynucleotide is derived from any of embodiments 79-102 or 125-160.
237. The polynucleotide of any of embodiments 79 to 160;
one or more agents capable of inducing gene disruption at a target site within the TRBC locus.
238. (a) a nucleic acid sequence encoding a T-cell receptor alpha (TCRα) chain and a portion of a TCRβ chain that is shorter than the full-length native T-cell receptor beta (TCRβ) chain, and
(b) a polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRBC locus encoding the TCR β chain;
one or more agents capable of inducing gene disruption at a target site within the TRAC locus.
239. The article of manufacture of embodiment 237 or 238, wherein the TRBC locus is TRBC1 and/or TRBC2.
240. The article of manufacture according to embodiment 237 or 238, wherein said polynucleotide is derived from any of embodiments 103-160.
241. Any of embodiments 234-241, wherein the one or more agents capable of inducing gene disruption comprise a DNA binding protein or DNA binding nucleic acid that specifically binds or hybridizes to a target site. manufactured goods.
242. The article of manufacture of embodiment 241, wherein the one or more agents capable of inducing gene disruption comprises (a) a fusion protein comprising a DNA targeting protein and a nuclease, or (b) an RNA-guided nuclease.
243. The DNA targeting protein or RNA-guided nuclease may be a zinc finger protein (ZFP), a TAL protein, or a clustered regularly arranged short palindromic nucleic acid (CRISPR)-associated nuclease (Cas) that is specific for the target site. ).
244. A zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or, and CRISPR-, wherein the one or more agents specifically binds to, recognizes, or hybridizes to a target site. The article of manufacture of any of embodiments 241-243, comprising a combination of Cas9.
245. The article of manufacture of any of embodiments 241-244, wherein each of the one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site.
246. The article of manufacture of embodiments 241-245, wherein the one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein.
247. The article of manufacture of embodiment 246, wherein the RNP is introduced via electroporation, particle gun, calcium phosphate transfection, cell compression or squeezing.
248. The article of manufacture of embodiment 246 or embodiment 247, wherein the RNP is introduced via electroporation.
249. The article of manufacture of any of embodiments 245-248, wherein said one or more agents are introduced as one or more polynucleotides encoding a gRNA and/or a Cas9 protein.
250. gRNA is
is complementary to the target site at the TRAC locus, and

The article of manufacture of any of embodiments 245-249, having a targeting domain comprising a sequence selected from .
251. gRNA is sequenced

The article of manufacture of embodiment 250, having a targeting domain comprising:
252. gRNA is
is complementary to a target site in one or both of the TRBC1 gene and TRBC2 gene, and

252. The article of manufacture of any of embodiments 245-251, having a targeting domain comprising a sequence selected from .
253. gRNA is sequenced

253. The article of manufacture of embodiment 252, having a targeting domain comprising:
254. A kit comprising the article of manufacture of any of embodiments 234-253 and instructions for use.
255. The kit of embodiment 254, wherein the instructions specify that the one or more agents and polynucleotides are introduced into the cell.
256. The kit of embodiment 254 or 255, wherein the instructions specify that the one or more agents and polynucleotides are introduced in any order, simultaneously or sequentially.
257. The kit of any of embodiments 254-257, wherein the instructions specify that the introduction of the polynucleotide occurs after the introduction of one or more agents.
258. The instructions provide that the polynucleotide is administered for about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, immediately after, or after the introduction of one or more agents capable of inducing gene disruption. Within 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours The kit of embodiment 257, specifying that the kit is introduced.

Claims (210)

A genetically engineered T cell comprising a modified T cell receptor alpha constant (TRAC) locus, the modified TRAC locus comprising a nucleic acid encoding a recombinant TCR or a portion thereof; The recombinant TCR or portion thereof comprises: (i) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant alpha (Cα) domain; and (ii) a variable beta (Vβ) domain and a constant beta (Cβ) domain. a TCR beta (TCRβ) chain comprising a TCR beta (TCRβ) chain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCRβ and Vα domains; and (b) an endogenous TRAC gene encoding at least a portion of the Cα domain of the recombinant TCR. Said genetically engineered T cell comprising an open reading frame of the locus, or a partial sequence thereof. A genetically engineered T cell comprising a modified T cell receptor alpha constant (TRAC) locus, the modified TRAC locus comprising a nucleic acid encoding a recombinant TCR or a portion thereof; The recombinant TCR or portion thereof comprises: (i) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant alpha (Cα) domain; and (ii) a variable beta (Vβ) domain and a constant beta (Cβ) domain. a TCR beta (TCRβ) chain comprising a TCR beta (TCRβ) chain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCRβ and Vα domains; and (b) an endogenous TRAC gene encoding at least a portion of the Cα domain of the recombinant TCR. comprising the open reading frame of the locus or a partial sequence thereof;
a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is greater than the full length of native Cα. short; and
The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to the native Cα region and/or the native Cβ region, the one or more modifications introduces one or more cysteine residues that can form one or more non-natural disulfide bridges between the alpha and beta chains, and/or Cα and/or Cβ contains one or more non-natural cysteines,
The genetically engineered T cell. A genetically engineered T cell comprising a modified T cell receptor alpha constant (TRAC) locus, the modified TRAC locus comprising a nucleic acid encoding a recombinant TCR or a portion thereof; The recombinant TCR or portion thereof comprises: (i) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant alpha (Cα) domain; and (ii) a variable beta (Vβ) domain and a constant beta (Cβ) domain. a TCR beta (TCRβ) chain comprising a TCR beta (TCRβ) chain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCRβ and Vα domains; and (b) an endogenous TRAC gene encoding at least a portion of the Cα domain of the recombinant TCR. a heterologous gene containing an open reading frame of the locus, or a subsequence thereof, to which the transgene sequence is operably linked such that upon expression from the introduced cell in a genetically engineered T cell, the expression of the TCR is modulated. Said genetically engineered T cell comprises one or more heterologous or regulated regulatory elements including a promoter. Genetically engineered T cell according to any one of claims 1 to 3, wherein the transgene sequence has been integrated via homologous recombination repair (HDR). The modified TRAC locus comprises an in-frame fusion of (i) a transgene sequence and (ii) an open reading frame of an endogenous TRAC locus or a subsequence thereof, optionally the transgene sequence comprising: A genetically engineered T cell according to any one of claims 1 to 4, which is in frame with one or more exons or subsequences thereof of the open reading frame of the endogenous TRAC locus. Genetically engineered T cells according to any one of claims 1 to 5, wherein the transgene sequence does not contain sequences encoding 3' untranslated regions (3' UTRs) or introns. a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is greater than the full length of native Cα. A genetically engineered T cell according to any one of claims 1 and 3 to 6, which is short. Genetically engineered T cell according to any one of claims 1 to 7, wherein the open reading frame or a subsequence thereof comprises at least one intron and at least one exon of the endogenous TRAC locus. A further portion of Cα contains nucleotides containing less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRAC locus. 9. A genetically engineered T cell according to any one of claims 2, 7 and 8 encoded by the sequence. According to any one of claims 2 and 7 to 9, the further portion of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length. Genetically engineered T cells as described. A further portion of Cα is encoded by a portion of exon 1 of the TRAC locus, said portion of exon 1 being shorter than the full length of exon 1 of the open reading frame of the TRAC locus. Genetically engineered T cells according to any one of the preceding paragraphs. 12. The transgene sequence of claims 1-11, wherein the transgene sequence is integrated downstream of the 5'-most nucleotide of exon 1 and upstream of the 3'-most nucleotide of exon 1 of the open reading frame of the endogenous TRAC locus. Genetically engineered T cells according to any one of the preceding paragraphs. The genetically engineered T according to any one of claims 1 to 2, wherein at least a part of Cα is encoded by at least a part of exon 1 and exons 2 to 4 of the open reading frame of the endogenous TRAC locus. cell. Genetically engineered T cell according to any one of claims 1 to 13, wherein the encoded TCRα chain is capable of dimerizing with the TCRβ chain. A sequence in which the encoded Cα is selected from any one of SEQ ID NO: 14, 15, 19, and 24, or for any one of SEQ ID NO: 14, 15, 19, and 24 At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Genetically engineered T cells according to any one of claims 1 to 14, comprising sequences showing high sequence identity, or subsequences thereof. A sequence in which the encoded Cα is selected from any one of SEQ ID NO: 19, 24, and 243-252, or for any one of SEQ ID NO: 19, 24, and 243-252 At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Genetically engineered T cells according to any one of claims 1 to 14, comprising sequences showing high sequence identity, or subsequences thereof. At least a portion of Cα is
The sequence of nucleotides starting from residue 3 to residue 3155 of the sequence set forth in SEQ ID NO:1, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:1. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 3155 or one or more exons thereof; Claims 2 and 7 to 16, encoded by sequences, or subsequences thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity. genetically engineered T cells. a further portion of Cα is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% relative to the sequence shown in SEQ ID NO: 142, or SEQ ID NO: 142; Any of claims 2 and 7 to 17, comprising sequences showing sequence identity of 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher, or subsequences thereof. The genetically engineered T cell according to item 1. The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one or more amino acids, compared to the native Cα region and/or the native Cβ region. one or more substitutions, deletions, or insertions in which the one or more modifications are capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. Genetic manipulation according to any one of claims 7 to 18, wherein a plurality of cysteine residues are introduced and/or Cα and/or Cβ of the recombinant TCR comprises one or more non-natural cysteines. T cells. 20. The genetically engineered T cell of claim 2 or claim 19, wherein the introduction of one or more cysteine residues comprises replacement of a non-cysteine residue by a cysteine residue. The encoded Cα region contains a cysteine at position corresponding to position 48 with numbering as shown in SEQ ID NO:24; and/or the encoded Cβ region contains a cysteine at position 48 as shown in SEQ ID NO:20; 21. The genetically engineered T cell of any one of claims 2, 19, and 20, comprising a cysteine at a position corresponding to position 57, numbered as follows. The genetically engineered protein according to any one of claims 2 and 19-21, wherein the encoded Cα comprises a sequence selected from any one of SEQ ID NO: 248-252, or a partial sequence thereof. T cells. Genetically engineered T cell according to any one of claims 1 to 22, further comprising gene disruption at the TRBC locus. A genetically engineered T cell comprising a modified T cell receptor beta constant (TRBC) locus, wherein the modified TRBC locus comprises a nucleic acid encoding a recombinant TCR or a portion thereof; The recombinant TCR or portion thereof comprises: (i) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant beta (Cβ) domain; and (ii) a variable alpha (Vα) domain and a constant alpha (Cα) domain. a TCR alpha (TCRα) chain comprising a TCR alpha (TCRα) chain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCRα and Vβ domains; and (b) an endogenous TRBC gene encoding at least a portion of the Cβ domain of the recombinant TCR. Said genetically engineered T cell comprising an open reading frame of the locus, or a partial sequence thereof. A genetically engineered T cell comprising a modified T cell receptor beta constant (TRBC) locus, wherein the modified TRBC locus comprises a nucleic acid encoding a recombinant TCR or a portion thereof; The recombinant TCR or portion thereof comprises: (i) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant beta (Cβ) domain; and (ii) a variable alpha (Vα) domain and a constant alpha (Cα) domain. a TCR alpha (TCRα) chain comprising a TCR alpha (TCRα) chain, the nucleic acid sequence comprising: (a) a transgene sequence encoding the TCRα and Vβ domains; and (b) an endogenous TRBC gene encoding at least a portion of the Cβ domain of the recombinant TCR. comprising the open reading frame of the locus or a partial sequence thereof;
a portion of Cβ is encoded by an open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by a transgene sequence, wherein the further portion of Cβ is greater than the full length of native Cβ. short; and
The further portion of Cβ and/or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one or more amino acids, compared to the native Cβ region and/or the native Cα region. one or more substitutions, deletions, or insertions in which the one or more modifications are capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. Introducing multiple cysteine residues,
The genetically engineered T cell. 26. The genetically engineered T cell of claim 24 or claim 25, wherein the transgene sequence has been integrated via homologous recombination repair (HDR). Genetically engineered T cell according to any one of claims 24 to 26, wherein the TRBC locus is the TRBC1 locus and/or the TRBC2 locus. The modified TRBC locus comprises an in-frame fusion of (i) the transgene and (ii) the open reading frame of the endogenous TRBC locus or a subsequence thereof, optionally the transgene sequence 28. A genetically engineered T cell according to any one of claims 24 to 27, which is in frame with one or more exons or subsequences thereof of the open reading frame of the sex TRBC locus. A genetically engineered T cell according to any one of claims 24 to 28, wherein the transgene sequence does not include sequences encoding 3' untranslated regions (3' UTRs) or introns. a portion of Cβ is encoded by an open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by a transgene sequence, the further portion of Cβ being greater than the full length of native Cβ. A genetically engineered T cell according to any one of claims 24 and 26-29, which is short. Genetically engineered T cell according to any one of claims 24 to 30, wherein the open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus. A further portion of Cβ contains nucleotides containing less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRBC locus. 32. A genetically engineered T cell according to any one of claims 25, 30, and 31 encoded by the sequence. 33. According to any one of claims 25 and 30-32, the further portion of Cβ is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length. Genetically engineered T cells as described. 34. A further portion of Cβ is encoded by a portion of exon 1 of the TRBC locus, said portion of exon 1 being shorter than the full length of exon 1 of the open reading frame of the TRBC locus. Genetically engineered T cells according to any one of the preceding paragraphs. 35. The transgene sequence of claims 24-34, wherein the transgene sequence is integrated downstream of the 5'-most nucleotide of exon 1 and upstream of the 3'-most nucleotide of exon 1 of the open reading frame of the endogenous TRBC locus. Genetically engineered T cells according to any one of the preceding paragraphs. The genetically engineered T according to any one of claims 24 to 35, wherein at least a part of Cβ is encoded by at least a part of exon 1 and exons 2 to 4 of the open reading frame of the endogenous TRBC locus. cell. Genetically engineered T cell according to any one of claims 24 to 36, wherein the encoded TCRβ chain is capable of dimerizing with the TCRα chain. A sequence in which the encoded Cβ is selected from any one of SEQ ID NO: 16, 17, 21, and 25, or for any one of SEQ ID NO: 16, 17, 21, and 25 At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more Genetically engineered T cells according to any one of claims 24 to 37, comprising sequences exhibiting high sequence identity, or subsequences thereof. A sequence in which the encoded Cβ is selected from any one of SEQ ID NO: 20, 21, 25, and 253 to 258, or any one of SEQ ID NO: 20, 21, 25, and 253 to 258 at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% for one; 39. A genetically engineered T cell according to any one of claims 24 to 38, which comprises a sequence exhibiting a sequence identity of 0.001 or higher, or a subsequence thereof. At least a portion of Cβ is
The sequence of nucleotides starting from residue 3 to residue 1445 of the sequence set forth in SEQ ID NO:2, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:2. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1445 or one or more exons thereof; Sequences, or subsequences thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity; or
The sequence of nucleotides starting from residue 3 to residue 1486 of the sequence set forth in SEQ ID NO:3, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:3. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1486 or one or more exons thereof; Claims 25 and 30 to 39, encoded by sequences, or subsequences thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity. genetically engineered T cells. The further portion of Cβ and/or the Cα region encoded by the nucleic acid sequence of (a) has one or more modifications, optionally one or more amino acids, compared to the native Cβ region and/or the native Cα region. one or more substitutions, deletions, or insertions in which the one or more modifications are capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. 40. The genetically engineered T cell according to any one of claims 25 and 30-39, which introduces multiple cysteine residues. 42. The genetically engineered T cell of claims 25 and 41, wherein the introduction of one or more cysteine residues comprises replacement of non-cysteine residues with cysteine residues. The encoded Cα region contains a cysteine at position corresponding to position 48 with numbering as shown in SEQ ID NO:24; and/or the encoded Cβ region contains a cysteine at position 48 as shown in SEQ ID NO:20; 43. The genetically engineered T cell of any one of claims 25, 41, and 42, comprising a cysteine at a position corresponding to position 57, numbered as follows. Genetic manipulation according to any one of claims 25 and 41 to 43, wherein the encoded Cβ comprises a sequence selected from SEQ ID NO: 253 and any one of 256 to 258, or a partial sequence thereof. T cells. 45. Genetically engineered T cell according to any one of claims 24 to 44, further comprising gene disruption at the TRAC locus. Genetically engineered T cell according to any one of claims 1 to 45, wherein the transgene sequence comprises one or more multicistronic elements. the multicistronic element is located between a nucleic acid sequence encoding TCRα or a portion thereof and a nucleic acid sequence encoding TCRβ or a portion thereof, and/or upstream of a nucleic acid sequence encoding TCR or a portion thereof; 47. The genetically engineered T cell of claim 46. 48. The genetically engineered T cell of claim 46 or claim 47, wherein the multicistronic element is or comprises a ribosome skipping sequence, optionally T2A, P2A, E2A, or F2A. the transgene sequence comprises one or more heterologous or regulatable regulatory elements operably linked to modulate expression of the TCR upon expression from the transfected cell in the genetically engineered T cell; , the genetically engineered T cell according to any one of claims 1 to 48. 50. The genetically engineered T cell of claim 49, wherein the heterologous control or regulatory element comprises a heterologous promoter. 51. The genetically engineered T cell of claim 50, wherein the heterologous promoter is selected among constitutive promoters, inducible promoters, repressible promoters, and/or tissue-specific promoters. 52. The genetically engineered T cell of claim 50 or claim 51, wherein the heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof. said recombinant TCR is capable of binding to an antigen associated with, specific for, and/or expressed on cells or tissues associated with a disease, disorder, or condition; , the genetically engineered T cell according to any one of claims 1 to 52. 54. The genetically engineered T cell of claim 53, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer. 55. A genetically engineered T cell according to any one of claims 1 to 54, wherein said T cell is a primary T cell derived from a subject, optionally said subject being human. Genetically engineered T cell according to any one of claims 1 to 55, wherein the T cell is a CD8+ T cell or a subtype thereof. Genetically engineered cells according to any one of claims 1 to 55, wherein the T cells are CD4+ T cells or subtypes thereof. Genetically engineered cells according to any one of claims 1 to 57, wherein said T cells are derived from multipotent or pluripotent cells, optionally iPSCs. A composition comprising a plurality of genetically engineered T cells according to any one of claims 1 to 58. A composition comprising a plurality of genetically engineered T cells comprising an altered T cell receptor alpha constant (TRAC) locus, the altered TRAC locus containing a recombinant TCR or a portion thereof. comprising a nucleic acid encoding a recombinant TCR, or a portion thereof, comprising: (i) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant alpha (Cα) domain; and (ii) a variable beta (Vβ) domain. and a TCR beta (TCRβ) chain comprising a constant beta (Cβ) domain, the nucleic acid sequence comprising: (a) a transgene sequence encoding a TCRβ and Vα domain; and (b) at least a portion of the Cα domain of the recombinant TCR. and the recombinant TCR is associated with, specific to, a cell or tissue associated with a disease, disorder, or condition. capable of binding to the antigen that is targeted and/or expressed thereon;
At least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition comprising gene disruption in or of the endogenous T cell receptor alpha constant region (TRAC) gene and/or the T cell receptor beta constant region (TRBC) gene;
At least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition have an endogenous TRAC or TRBC gene. does not express or does not express the gene product at a detectable level; and/or at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the cells in the composition. , 75%, 80% or 90% or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or 90% expresses a recombinant TCR and/or exhibits binding to the antigen;
Said composition. A composition comprising a plurality of genetically engineered T cells comprising an altered T cell receptor alpha constant (TRAC) locus, the altered TRAC locus containing a recombinant TCR or a portion thereof. comprising a nucleic acid encoding a recombinant TCR, or a portion thereof, comprising: (i) a TCR alpha (TCRα) chain comprising a variable alpha (Vα) domain and a constant alpha (Cα) domain; and (ii) a variable beta (Vβ) domain. and a TCR beta (TCRβ) chain comprising a constant beta (Cβ) domain, the nucleic acid sequence comprising (a) a transgene sequence encoding a TCRβ and Vα domain, and (b) at least a portion of the Cα domain of the recombinant TCR. an open reading frame of the endogenous TRAC locus encoding, or a partial sequence thereof;
a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is greater than the full length of native Cα. short; and
The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to the native Cα region and/or the native Cβ region, the one or more modifications introduces one or more cysteine residues capable of forming one or more non-natural disulfide bridges between the alpha and beta chains,
Said composition. A composition comprising a plurality of genetically engineered T cells comprising a modified T cell receptor beta constant (TRBC) locus, wherein the modified TRBC locus carries a recombinant TCR or a portion thereof. comprising a nucleic acid encoding a recombinant TCR, or a portion thereof, comprising: (i) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant beta (Cβ) domain; and (ii) a variable alpha (Vα) domain. and a TCR alpha (TCRα) chain comprising a constant alpha (Cα) domain, the nucleic acid sequence comprising: (a) a transgene sequence encoding a TCRα and Vβ domain; and (b) at least a portion of a Cβ domain of a recombinant TCR. and the recombinant TCR is associated with, specific to, a cell or tissue associated with a disease, disorder, or condition. capable of binding to the antigen that is targeted and/or expressed thereon;
At least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition comprising gene disruption in or of the endogenous T cell receptor alpha constant region (TRAC) gene and/or the T cell receptor beta constant region (TRBC) gene;
At least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition have an endogenous TRAC or TRBC gene. does not express or does not express the gene product at a detectable level; and/or at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the cells in the composition. , 75%, 80%, or 90%, or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% , expresses a recombinant TCR and/or exhibits binding to an antigen,
Said composition. A composition comprising a plurality of genetically engineered T cells comprising a modified T cell receptor beta constant (TRBC) locus, wherein the modified TRBC locus carries a recombinant TCR or a portion thereof. comprising a nucleic acid encoding a recombinant TCR, or a portion thereof, comprising: (i) a TCR beta (TCRβ) chain comprising a variable beta (Vβ) domain and a constant beta (Cβ) domain; and (ii) a variable alpha (Vα) domain. and a TCR alpha (TCRα) chain comprising a constant alpha (Cα) domain, the nucleic acid sequence comprising: (a) a transgene sequence encoding a TCRα and Vβ domain; and (b) at least a portion of a Cβ domain of a recombinant TCR. an open reading frame of the endogenous TRBC locus encoding, or a partial sequence thereof;
a portion of Cβ is encoded by an open reading frame of the endogenous TRBC locus or a subsequence thereof, and a further portion of Cβ is encoded by a transgene sequence, the further portion of Cβ being greater than the full length of native Cβ. short; and
The further portion of Cβ and/or the Cα region encoded by the nucleic acid sequence of (a) has one or more modifications, optionally one or more amino acids, compared to the native Cβ region and/or the native Cα region. one or more substitutions, deletions, or insertions in which the one or more modifications are capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. Introducing multiple cysteine residues,
Said composition. said recombinant TCR is capable of binding to an antigen associated with, specific for, and/or expressed on cells or tissues associated with a disease, disorder, or condition; , claim 61 or claim 63. 65. A composition according to any one of claims 59 to 64, comprising CD4+ T cells and/or CD8+ T cells. Claims 59 to 59, comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CD4+ T cells to CD8+ T cells is from 1:3 to 3:1 or about 1:3 to 3:1, optionally 1:1. 65. The composition according to any one of 65. At least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition comprising a genetic disruption in or to the endogenous T cell receptor alpha constant region (TRAC) gene and/or T cell receptor beta constant region (TRBC) gene; and/or at least 80% of the engineered cells in the composition; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% express no or detectable gene product of the endogenous TRAC or TRBC gene not expressed at the level,
A composition according to any one of claims 59, 61, and 63-66. at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition, or 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% expresses the recombinant TCR and/or exhibits binding to the antigen, 61, and the composition according to any one of 63 to 67. (a) (i) the T cell receptor beta (TCRβ) chain, which includes a variable beta (Vβ) domain and a constant beta (Cβ) domain, and (ii) the full length of the native T cell receptor alpha (TCRα) chain. a nucleic acid sequence encoding a portion of a recombinant T-cell receptor (TCR), encoding a portion of a short TCRα chain;
(b) a polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRAC locus, the polynucleotide comprising a viral vector; The polynucleotide contained therein. (a) (i) the T cell receptor beta (TCRβ) chain, which includes a variable beta (Vβ) domain and a constant beta (Cβ) domain; and (ii) the full length of the native T cell receptor alpha (TCRα) chain. a nucleic acid sequence encoding a portion of a recombinant T-cell receptor (TCR), encoding a portion of a short TCRα chain;
(b) a polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRAC locus, the polynucleotide comprising:
When the TCR or antigen-binding fragment thereof is expressed from cells into which the polynucleotide has been introduced:
a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is greater than the full length of native Cα. short; and
The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to the native Cα region and/or the native Cβ region, the one or more modifications introduces one or more cysteine residues that can form one or more non-natural disulfide bridges between the alpha and beta chains, and/or Cα and/or Cβ contains one or more non-natural cysteines,
The polynucleotide. (a) (i) the T cell receptor beta (TCRβ) chain, which includes a variable beta (Vβ) domain and a constant beta (Cβ) domain, and (ii) the full length of the native T cell receptor alpha (TCRα) chain. a nucleic acid sequence encoding a portion of a recombinant T-cell receptor (TCR), encoding a portion of a short TCRα chain;
(b) a polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRAC locus, the polynucleotide comprising:
one or more heterologous or regulatable regulators, including a heterologous promoter, to which the transgene sequence is operably linked to regulate expression of the TCR upon expression from the transfected cell in the genetically engineered T cell; containing elements,
The polynucleotide. The TCRα chain contains a constant alpha region (Cα), and when the TCR or antigen-binding fragment thereof is expressed from a cell into which the polynucleotide has been introduced, at least a portion of the Cα is an open reading of the endogenous TRAC locus. encoded by a frame or a subsequence thereof; and/or
The nucleic acid sequence of (a) and one of the one or more homology arms together include a sequence of nucleotides encoding a Cα that is shorter than the full length of native Cα, and the TCR or antigen-binding fragment thereof is a polynucleotide is expressed from cells into which Cα is introduced, at least a portion of the Cα is encoded by the open reading frame of the endogenous TRAC locus or a partial sequence thereof;
A polynucleotide according to any one of claims 69 to 71. 73. The polynucleotide of any one of claims 69 to 72, wherein the nucleic acid sequence encoding the TCR β chain is upstream of the nucleic acid sequence encoding a portion of the TCR α chain. 74. The polynucleotide of any one of claims 69 to 73, wherein the nucleic acid sequence of (a) does not contain a 3' untranslated region (3' UTR) or an intron-encoding sequence. 75. The nucleic acid sequence of (a) is in frame with one or more exons or subsequences thereof of the open reading frame of the TRAC locus comprised in one or more homology arms. Polynucleotide according to any one of the above. a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a partial sequence thereof, and a further portion of Cα is encoded by the nucleic acid sequence of (a), and the further portion of Cα is 76. The polynucleotide of any one of claims 69 and 71-75, wherein the polynucleotide is shorter than the length of the polynucleotide. 77. The polynucleotide of any one of claims 69 to 76, wherein the open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous TRAC locus. A further portion of Cα contains nucleotides containing less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRAC locus. 78. The polynucleotide of any one of claims 70, 76, and 77 encoded by a sequence. 79. Any one of claims 70 and 76-78, wherein the further portion of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length. The described polynucleotide. 79. A further portion of Cα is encoded by a portion of exon 1 of the TRAC locus, said portion of exon 1 being shorter than the full length of exon 1 of the open reading frame of the TRAC locus. Polynucleotide according to any one of the above. 81. The polynucleotide of any one of claims 69 to 80, wherein the TCRα chain is capable of dimerizing with the TCRβ chain upon production from a cell into which the polynucleotide has been introduced. A sequence in which, when produced from a cell into which the polynucleotide has been introduced, the encoded Cα is selected from any one of SEQ ID NO: 19, 24, and 243 to 252, or SEQ ID NO: 19, 24, and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for any one of 243-252 82. A polynucleotide according to any one of claims 69 to 81, comprising sequences, or subsequences thereof, exhibiting %, 98%, 99%, or higher sequence identity. At least a portion of Cα is
The sequence of nucleotides starting from residue 3 to residue 3155 of the sequence set forth in SEQ ID NO:1, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:1. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 3155 or one or more exons thereof; Claims 70 and 76-82, encoded by sequences, or subsequences thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity. polynucleotide. a further portion of Cα is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% relative to the sequence shown in SEQ ID NO: 142, or SEQ ID NO: 142; Any of claims 70 and 76-83, comprising sequences or subsequences thereof showing sequence identity of 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. The polynucleotide according to item 1. The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one or more amino acids, compared to the native Cα region and/or the native Cβ region. one or more substitutions, deletions, or insertions in which the one or more modifications are capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. 85. A polynucleotide according to any one of claims 76 to 84, which introduces a plurality of cysteine residues. 86. The polynucleotide of claims 70 and 85, wherein the introduction of one or more cysteine residues comprises substitution of a non-cysteine residue by a cysteine residue. upon production from a cell into which the polynucleotide has been introduced, the encoded Cα region contains a cysteine at a position corresponding to position 48 in the numbering as shown in either SEQ ID NO:24; and/or 87. The polynucleotide of any one of claims 70, 85, and 86, wherein the Cβ region comprises a cysteine at a position corresponding to position 57 in numbering as shown in SEQ ID NO:20. Claims 70 and 85-87, wherein when produced from a cell into which the polynucleotide has been introduced, the encoded Cα comprises a sequence selected from any one of SEQ ID NO: 248-252, or a partial sequence thereof. The polynucleotide according to any one of . 89. The polynucleotide of any one of claims 69-88, wherein a portion of the TCRα chain comprises a variable alpha (Vα) domain. 90. The polynucleotide of any one of claims 69-89, wherein the one or more homology arms comprises a 5' homology arm and/or a 3' homology arm. 91. The polynucleotide of claim 90, wherein the 5' homology arm and the 3' homology arm contain nucleic acid sequences homologous to nucleic acid sequences surrounding a target site, the target site being within the TRAC locus. 92. The polynucleotide of claim 91, wherein the target site is within exon 1 of the TRAC locus. The 5' homology arm is
a) At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% to the sequence shown in SEQ ID NO:124 , 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides for sequences exhibiting 97%, 98%, 99%, or higher sequence identity, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides; A sequence containing consecutive nucleotides;
b) 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides, or at least 150, 200, 250, 300, 350 of the sequence set forth in SEQ ID NO:124; , 400, 450, 500, 550, or 600 contiguous nucleotides, or a sequence comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides;
c) A polynucleotide according to any one of claims 90 to 92, comprising the sequence shown in SEQ ID NO:124. The 3' homology arm is
a) At least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% to the sequence shown in SEQ ID NO:125 , 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides for sequences exhibiting 97%, 98%, 99%, or higher sequence identity, or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides; A sequence containing consecutive nucleotides;
b) 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides, or at least 150, 200, 250, 300, 350 of the sequence set forth in SEQ ID NO:125; , 400, 450, 500, 550, or 600 contiguous nucleotides, or a sequence comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides;
c) A polynucleotide according to any one of claims 90 to 93, comprising the sequence shown in SEQ ID NO:125. (a) (i) the T cell receptor alpha (TCRα) chain, which includes a variable alpha (Vα) domain and a constant alpha (Cα) domain; and (ii) the full length of the native T cell receptor beta (TCRβ) chain. a nucleic acid sequence encoding a portion of a recombinant T cell receptor (TCR), encoding a portion of a short TCR β chain;
(b) A polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRBC locus. When the TCR β chain contains constant beta (Cβ) and the TCR or antigen-binding fragment thereof is expressed from cells into which the polynucleotide has been introduced, at least a portion of the Cβ is in the open reading frame of the endogenous TRAC locus. or a subsequence thereof, and/or a sequence of nucleotides encoding Cβ in which the nucleic acid sequence of (a) and one or more homology arms are shorter than the full length of native Cβ. and when the TCR or antigen-binding fragment thereof is expressed from cells into which the polynucleotide has been introduced, at least a portion of the Cβ is encoded by the open reading frame of the endogenous TRBC gene locus or a partial sequence thereof. 96. The polynucleotide of claim 95. 97. The polynucleotide of claim 95 or claim 96, wherein the TRBC locus is one or more of TRBC1 or TRBC2. 98. The polynucleotide of any one of claims 95 to 97, wherein the nucleic acid sequence encoding the TCRα chain is upstream of the nucleic acid sequence encoding a portion of the TCRβ chain. 99. The polynucleotide of any one of claims 95 to 98, wherein the nucleic acid sequence of (a) does not contain a 3' untranslated region (3' UTR) or an intron encoding sequence. 99. The nucleic acid sequence of (a) is in frame with one or more exons or subsequences thereof of the open reading frame of the TRAC locus comprised in one or more homology arms. Polynucleotide according to any one of the above. a portion of Cβ is encoded by the open reading frame of the endogenous TRBC locus or a partial sequence thereof, and a further portion of Cβ is encoded by the nucleic acid sequence of (a), and the further portion of Cβ is encoded by the complete sequence of native Cβ. 101. A polynucleotide according to any one of claims 95-100, which is shorter than the length of the polynucleotide. 102. The polynucleotide of any one of claims 95-101, wherein the open reading frame or subsequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus. Nucleotides in which the additional portion of Cβ encodes less than 4 exons, less than 3 exons, less than 2 exons, 1 exon, or less than 1 complete exon of the open reading frame of the TRBC locus 103. The polynucleotide of claim 101 or claim 102, encoded by the sequence of. 104. The further portion of Cβ is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, or less than 150 base pairs in length. polynucleotide. Any of claims 101 to 104, wherein the further portion of Cβ is encoded by a portion of exon 1 of the TRBC locus, said portion of exon 1 being shorter than the full length of exon 1 of the open reading frame of the TRBC locus. The polynucleotide according to item 1. 106. The polynucleotide of any one of claims 95-105, wherein the TCRβ chain is capable of dimerizing with the TCRα chain upon production from a cell into which the polynucleotide has been introduced. A sequence in which the encoded Cβ is selected from any one of SEQ ID NO: 20, 21, 25, and 253 to 258 when produced from a cell into which the polynucleotide has been introduced, or SEQ ID NO: 20, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% for any one of 21, 25, and 253-258; 107. A polynucleotide according to any one of claims 69 to 106, comprising sequences, or subsequences thereof, exhibiting 96%, 97%, 98%, 99% or higher sequence identity. At least a portion of Cβ is
The sequence of nucleotides starting from residue 3 to residue 1445 of the sequence set forth in SEQ ID NO:2, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:2. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1445 or one or more exons thereof; Sequences, or subsequences thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity; or
The sequence of nucleotides starting from residue 3 to residue 1486 of the sequence set forth in SEQ ID NO:3, or one or more exons thereof, or starting from residue 3 of the sequence set forth in SEQ ID NO:3. at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% for the sequence of nucleotides up to residue 1486 or one or more exons thereof; 108. A polypeptide according to any one of claims 101 to 107 encoded by a sequence, or subsequence thereof, exhibiting 95%, 96%, 97%, 98%, 99% or higher sequence identity. nucleotide. The further portion of Cβ and/or the Cα region encoded by the nucleic acid sequence of (a) has one or more modifications, optionally one or more amino acids, compared to the native Cβ region and/or the native Cα region. one or more substitutions, deletions, or insertions in which the one or more modifications are capable of forming one or more non-natural disulfide bridges between the alpha and beta chains. 109. A polynucleotide according to any one of claims 101 to 108, which introduces a plurality of cysteine residues. 110. The polynucleotide of claim 109, wherein the introduction of one or more cysteine residues comprises replacement of a non-cysteine residue with a cysteine residue. The encoded Cα region contains a cysteine at position corresponding to position 48 with numbering as shown in SEQ ID NO:24; and/or the encoded Cβ region contains a cysteine at position 48 as shown in SEQ ID NO:20; 111. The polynucleotide of claim 109 or claim 110, comprising a cysteine at a position corresponding to position 57 as numbered. 112. The polynucleotide of any one of claims 109-111, wherein the encoded Cβ comprises a sequence selected from SEQ ID NO: 253 and any one of 256-258, or a subsequence thereof. 113. The polynucleotide of any one of claims 95-112, wherein a portion of the TCR β chain comprises a variable beta (Vβ) domain. 114. The polynucleotide of any one of claims 95-113, wherein the one or more homology arms comprises a 5' homology arm and/or a 3' homology arm. 115. The 5' homology arm and the 3' homology arm contain nucleic acid sequences homologous to surrounding nucleic acid sequences of a target site, the target site being within an open reading frame of the TRBC locus. polynucleotide. 116. The polynucleotide of claim 115, wherein the target site is within exon 1 of the open reading frame of the TRBC locus. 117. The nucleic acid sequence of (a) is a sequence that is foreign or heterologous to the open reading frame of the endogenous genomic TRAC locus of a T cell, optionally a human T cell. polynucleotide. The 5' homology arm and the 3' homology arm are independently 50 or about 50, 100 or about 100 nucleotides in length, 100 or about 100, 250 or about 250 nucleotides in length, 250 or about 250 to 500 or about 500 nucleotides in length; 500 to or about 500 to 750 to 750 nucleotides; 750 to 750 to 1000 or about 1000 nucleotides in length; or about 2000 nucleotides in length. The 5' homology arm and the 3' homology arm are independently 100 or about 100, 1000 or about 1000 nucleotides, 100-750 nucleotides, 100-600 nucleotides, 100-400 nucleotides, 100-300 nucleotides, 100 ~200 nucleotides, 200-1000 nucleotides, 200-750 nucleotides, 200-600 nucleotides, 200-400 nucleotides, 200-300 nucleotides, 300-1000 nucleotides, 300-750 nucleotides, 300-600 nucleotides, 300-400 nucleotides, 400 The length of any one of claims 90-94 and 114-118 is between ~1000 nucleotides, 400-750 nucleotides, 400-600 nucleotides, 600-1000 nucleotides, 600-750 nucleotides, or 750-1000 nucleotides. The described polynucleotide. The 5' homology arm and the 3' homology arm are independently 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or about 200, 300, 400, 500, 600, 700, or 800 nucleotides in length, or any value between any of the foregoing. The 5' homology arm and the 3' homology arm are independently longer than 300 or about 300 nucleotides, optionally the 5' homology arm and the 3' homology arm are independently longer than 400, Any one of claims 90-94 and 114-120 that is 500, or 600 nucleotides long, or about 400, 500, or 600 nucleotides long, or any value between any of the foregoing. The polynucleotide described in. 90. The 5' homology arm and the 3' homology arm are independently 600 nucleotides in length, about 600 nucleotides in length, or less than about 600 nucleotides in length. -94 and the polynucleotide according to any one of 114-121. 123. The polynucleotide of any one of claims 90-94 and 114-122, wherein the 5' homology arm and the 3' homology arm are independently greater than 300 or about 300 nucleotides in length. 124. The polynucleotide of any one of claims 69-123, wherein the nucleic acid sequence of (a) comprises one or more multicistronic elements. The multicistronic element is located between a nucleic acid sequence encoding TCRα or a portion thereof and a nucleic acid sequence encoding TCRβ or a portion thereof, and/or upstream of a nucleic acid sequence encoding TCR or a portion thereof. 125. The polynucleotide of claim 124. 126. The polynucleotide of claim 124 or claim 125, wherein the multicistronic element is or comprises a ribosome skipping sequence, optionally T2A, P2A, E2A, or F2A. Claims wherein the nucleic acid sequence of (a) comprises one or more heterologous or regulatory regulatory elements operably linked to modulate expression of the TCR upon expression from a cell into which the polynucleotide has been introduced. The polynucleotide according to any one of paragraphs 69 to 126. 128. The polynucleotide of claim 127, wherein the heterologous control or regulatory element comprises a heterologous promoter. 129. The polynucleotide of claim 128, wherein the heterologous promoter is selected among constitutive promoters, inducible promoters, repressible promoters, and/or tissue-specific promoters. 130. The polynucleotide of claim 128 or claim 129, wherein the heterologous promoter is or comprises the human elongation factor 1 alpha (EF1α) promoter or the MND promoter or a variant thereof. 131. A polynucleotide according to any one of claims 69 to 130, comprised in a viral vector. 132. The polynucleotide of claim 131, wherein the viral vector is an AAV vector, optionally selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vectors. 133. The polynucleotide of claim 132, wherein the AAV vector is an AAV2 or AAV6 vector. 134. The polynucleotide according to any one of claims 69 to 133, comprising the structure: [5' homology arm]-[nucleic acid sequence of (a)]-[3' homology arm]. 135. The polypeptide according to any one of claims 124 to 134, comprising structure: [5' homology arm] - [multicistronic element] - [nucleic acid sequence of (a)] - [3' homology arm]. nucleotide. 135. The polynucleotide according to any one of claims 128 to 134, comprising the structure: [5' homology arm]-[heterologous promoter]-[nucleic acid sequence of (a)]-[3' homology arm]. that the encoded recombinant TCR binds to an antigen that is associated with, specific for, and/or expressed on cells or tissues associated with a disease, disorder, or condition; 137. The polynucleotide according to any one of claims 69 to 136, capable of 138. The polynucleotide of claim 137, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer. at least 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10000 nucleotides in length, or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10000 nucleotides in length, or between any of the foregoing 139. The polynucleotide of any one of claims 69-138, which is any value of. 139. Any of claims 69-139 that is 2500 or about 2500, 5000 or about 5000 nucleotides, 3500 or about 3500, 4500 or about 4500 nucleotides, or 3750 or about 3750, 4250 or about 4250 nucleotides in length. The polynucleotide according to item 1. A genetically engineered T cell comprising a modified TRAC locus comprising the step of introducing a polynucleotide according to any one of claims 69-94 or 117-140 into a T cell comprising a gene disruption at the TRAC locus. A method of generating T cells. (a) introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the endogenous T cell receptor alpha constant (TRAC) locus of the T cell; and
(b) introducing into the T cell a polynucleotide comprising a transgene sequence encoding a T cell receptor beta (TCRβ) chain and a portion of a TCRα chain that is shorter than the full-length native T cell receptor alpha (TCRα) chain; 1. A method of producing a genetically engineered T cell containing an altered TRAC locus, the method comprising:
introduction of the template polynucleotide occurs after introduction of one or more agents capable of inducing gene disruption; and generating genetically engineered cells targeted for integration of the TRAC locus, thereby containing a modified TRAC locus,
Said method. The T cell receptor beta (TCRβ) chain and a portion of the T cell receptor alpha (TCRα) chain are present in T cells with gene disruption within the endogenous T cell receptor alpha constant (TRAC) locus. 1. A method of producing a genetically engineered T cell comprising a modified TRAC locus, the method comprising the step of introducing a polynucleotide comprising a transgene sequence encoding a transgene, the method comprising: introducing a polynucleotide comprising a transgene sequence encoding said method, wherein said method generates a genetically engineered cell targeted for integration within the endogenous TRAC locus via , thereby generating a genetically engineered cell comprising an altered TRAC locus. (a) introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the endogenous T cell receptor alpha constant (TRAC) locus of the T cell; and
(b) introducing into the T cell a polynucleotide comprising a transgene sequence encoding a T cell receptor beta (TCRβ) chain and a portion of a TCRα chain that is shorter than the full-length native T cell receptor alpha (TCRα) chain; 1. A method of producing a genetically engineered T cell containing an altered TRAC locus, the method comprising:
The transgene is targeted for integration within the endogenous TRAC locus via homologous recombination repair (HDR), thereby creating genetically engineered cells containing a modified TRAC locus, and Upon targeted integration of the transgene:
a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is greater than the full length of native Cα. short; and
The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to the native Cα region and/or the native Cβ region, the one or more modifications introduces one or more cysteine residues that can form one or more non-natural disulfide bridges between the alpha and beta chains, and/or Cα and/or Cβ contains one or more non-natural cysteines,
Said method. The T cell receptor beta (TCRβ) chain and a portion of the T cell receptor alpha (TCRα) chain are present in T cells with gene disruption within the endogenous T cell receptor alpha constant (TRAC) locus. A method of producing a genetically engineered T cell comprising an altered TRAC locus, the method comprising the step of introducing a polynucleotide comprising a transgene sequence encoding the
The transgene is targeted for integration within the endogenous TRAC locus via homologous recombination repair (HDR), thereby creating genetically engineered cells containing a modified TRAC locus, and Upon targeted integration of the transgene:
a portion of Cα is encoded by the open reading frame of the endogenous TRAC locus or a subsequence thereof, and a further portion of Cα is encoded by the transgene sequence, and the further portion of Cα is greater than the full length of native Cα. short; and
The further portion of Cα and/or Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to the native Cα region and/or the native Cβ region, the one or more modifications introduces one or more cysteine residues that can form one or more non-natural disulfide bridges between the alpha and beta chains, and/or Cα and/or Cβ contains one or more non-natural cysteines,
Said method. of claims 141, 143, and 145, wherein said gene disruption is induced by one or more agents capable of inducing gene disruption of one or more target sites within the endogenous TRAC locus. The method described in any one of the above. 147. The method according to any one of claims 141-146, wherein the polynucleotide is a polynucleotide according to any one of claims 69-94 or 117-140. The modified TRAC locus comprises a nucleic acid sequence encoding a recombinant TCR or a portion thereof, the nucleic acid sequence comprising: (i) the transgene sequence; and (ii) the open reading frame of the endogenous TRAC locus or a portion thereof. 141-147, wherein the transgene sequence is in frame with one or more exons of the open reading frame of the endogenous TRAC locus or a subsequence thereof. The method described in any one of the above. 149. The method of any one of claims 141-148, wherein the engineered T cell further comprises inducing gene disruption at the TRBC locus. introducing a polynucleotide according to any one of claims 95 to 140 into a T cell containing a gene disruption at the TRBC locus, comprising the step of: How to make it. (a) introducing into the T cell one or more agents capable of inducing gene disruption at a target site within the endogenous T cell receptor beta constant (TRBC) locus of the T cell: and
(b) introducing into the T cell a polynucleotide comprising a transgene sequence encoding a T cell receptor alpha (TCRα) chain and a portion of a TCRβ chain that is shorter than the full-length native T cell receptor beta (TCRβ) chain; 1. A method of producing a genetically engineered T cell comprising a modified TRBC locus, the method comprising:
the transgene is targeted for integration within the endogenous TRBC locus via homologous recombination repair (HDR), thereby creating a genetically engineered cell containing an altered TRBC locus;
Said method. The T cell receptor alpha (TCRα) chain and a portion of the T cell receptor beta (TCRβ) chain are present in T cells with gene disruption within the endogenous T cell receptor beta constant (TRBC) locus. A method of producing a genetically engineered T cell comprising a modified TRBC locus, the method comprising the step of introducing a polynucleotide comprising a transgene sequence encoding, wherein the transgene is capable of homologous recombination repair (HDR). said method, wherein said method generates a genetically engineered cell targeted for integration within the endogenous TRBC locus via , thereby generating a genetically engineered cell comprising an altered TRBC locus. Claims 150 and 152, wherein the gene disruption is induced by one or more agents capable of inducing gene disruption of one or more target sites within the endogenous TRBC locus. the method of. 154. The method according to any one of claims 150 to 153, wherein the TRBC locus is the TRBC1 locus and/or the TRBC2 locus. 155. The method according to any one of claims 151-154, wherein the polynucleotide is a polynucleotide according to any one of claims 95-140. The modified TRBC locus comprises a nucleic acid sequence encoding a recombinant TCR or a portion thereof, which nucleic acid sequence comprises (i) a transgene sequence and (ii) an open reading frame of the endogenous TRBC locus or a portion thereof. 151-155, wherein the transgene sequence is in frame with one or more exons of the open reading frame of the endogenous TRBC locus or a subsequence thereof. The method described in any one of the above. 157. The method of any one of claims 151-156, wherein the engineered T cell further comprises inducing gene disruption at the TRAC locus. Any of claims 142 and 144-157, wherein the one or more agents capable of inducing gene disruption comprise a DNA binding protein or DNA binding nucleic acid that specifically binds or hybridizes to a target site. The method described in paragraph (1). The one or more agents are target site-specific zinc finger proteins (ZFPs), TAL proteins, or clustered regularly interspaced short palindromic nucleic acids ( CRISPR) associated nuclease (Cas). Claims 142 and 144, wherein the one or more agents comprises a CRISPR-Cas9 combination, and a CRISPR-Cas9 combination, a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. The method described in any one of ~159. 161. The method of claim 160, wherein the one or more agents are introduced as a ribonucleoprotein (RNP) complex comprising gRNA and Cas9 protein. 162. The method of claim 161, wherein the concentration of RNP is from or about 1 μM to 5 μM or about 5 μM; optionally, the concentration of RNP is 2 μM or about 2 μM. 163. The method of claim 161 or claim 162, wherein the RNP is introduced via electroporation. gRNA is
is complementary to the target site at the TRAC locus, and

164. The method of any one of claims 160-163, wherein the targeting domain comprises a sequence selected from the group consisting of: gRNA is sequenced

165. The method of claim 164, having a targeting domain comprising: gRNA is
is complementary to a target site in one or both of the TRBC1 gene and TRBC2 gene, and

164. The method of any one of claims 160-163, wherein the targeting domain comprises a sequence selected from the group consisting of: gRNA is sequenced

167. The method of claim 166, having a targeting domain comprising: 168. The method of any one of claims 141-167, wherein the T cells are primary T cells derived from the subject. 169. The method of claim 168, wherein the subject has or is suspected of having the disease or disorder condition. 169. The method of claim 168, wherein the subject is or is suspected of being healthy. 171. The method of any one of claims 141-170, wherein the T cells are CD8+ T cells or subtypes thereof. 171. The method of any one of claims 141-170, wherein the T cells are CD4+ T cells or subtypes thereof. 168. The method of any one of claims 141-167, wherein the T cells are derived from multipotent or pluripotent cells, optionally iPSCs. 174. The method of any one of claims 168-173, wherein the T cells comprise T cells that are autologous to the subject. 174. The method of any one of claims 168-173, wherein the T cells comprise T cells that are allogeneic to the subject. 176. The method of any one of claims 141-175, wherein said polynucleotide is comprised in a viral vector. 177. The method of claim 176, wherein the vector is an AAV vector, optionally selected from among AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, or AAV8 vectors. 178. The method of claim 177, wherein the AAV vector is an AAV2 or AAV6 vector. said recombinant TCR is capable of binding to an antigen associated with, specific for, and/or expressed on cells or tissues associated with a disease, disorder, or condition; , the method of any one of claims 141-178. 179. According to any one of claims 144-179, the introduction of one or more agents capable of inducing gene disruption and the introduction of the template polynucleotide are performed in any order, simultaneously or sequentially. Method described. 181. The method of any one of claims 144-180, wherein the introduction of the template polynucleotide is performed after the introduction of one or more agents capable of inducing gene disruption. 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes immediately after or after the introduction of one or more agents capable of inducing gene disruption. within minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, or 4 hours, or about 30 seconds thereafter; 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes 182. The method of claim 181, wherein the method is introduced within 2 hours, 3 hours, or 4 hours, optionally at or about 2 hours after introduction of the one or more agents. 183. The method of any one of claims 141-182, which is performed in a plurality of T cells. 184. The method of claim 183, wherein the plurality of T cells comprises CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells. Optionally, the plurality of T cells comprises CD4+ T cells and CD8+ T cells, and the ratio of CD4+ T cells to CD8+ T cells is from or about 1:3 to 3:1 or about 3:1. , 1:2 to 2:1 or about 2:1, optionally 1:1 or about 1:1. 10. A claim comprising incubating the cells with a stimulatory agent in vitro under conditions for stimulating or activating the one or more T cells prior to introduction of the one or more agents. 142 and any one of 144-185. Claims wherein the stimulant comprises anti-CD3 and/or anti-CD28 antibodies, optionally anti-CD3/anti-CD28 beads, optionally with a bead to cell ratio of 1:1 or about 1:1. The method described in paragraph 186. 188. The method of claim 186 or claim 187, comprising removing the stimulant from the one or more immune cells prior to introducing the one or more agents. Optionally, incubating the cells with one or more recombinant cytokines before, during, or after introduction of one or more agents and/or introduction of template polynucleotide; and wherein the one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15. The method described in. The one or more recombinant cytokines may be 10 U/mL or about 10 U/mL, 200 U/mL or about 200 U/mL, optionally 50 IU/mL or about 50 IU/mL, 100 U/mL. IL-2 concentration of 0.5 ng/mL to 50 ng/mL, optionally 5 ng/mL or about 5 ng/mL to 10 ng/mL or about 10 ng/mL; concentration of IL-7, and/or from 0.1 ng/mL to 20 ng/mL, optionally from 0.5 ng/mL or about 0.5 ng/mL, and from a concentration of IL-15 of 5 ng/mL or about 5 ng/mL. 190. The method of claim 189, wherein the method is added at a selected concentration. Incubation is up to 24 hours, 36 hours, 48 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, after introduction of one or more agents and introduction of template polynucleotide; 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or approximately 24 hours, 36 hours, 48 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, optionally for up to or about 7 days. the polynucleotide is at least 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10000 nucleotides in length; , or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000, or 10000 nucleotides in length, or the foregoing. 192. A method according to any one of claims 141 to 191, wherein the method is any value between any of the following. 141 . The polynucleotide is 2500 or about 2500 nucleotides to 5000 or about 5000 nucleotides, 3500 or about 3500 nucleotides to 4500 or about 4500 nucleotides, or 3750 or about 3750 nucleotides to 4250 or about 4250 nucleotides in length. The method described in any one of ~192. An engineered T cell or a plurality of engineered T cells produced using the method of any one of claims 141-193. 195. A composition comprising the engineered T cell or engineered cells of claim 194. 196. A composition according to claim 195, comprising CD4+ T cells and/or CD8+ T cells. 195 or 195, comprising CD4+ T cells and CD8+ T cells, wherein the ratio of CD4+ T cells to CD8+ T cells is from 1:3 to 3:1 or about 1:3 to 3:1, optionally 1:1; or 197. The composition of claim 196. At least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the engineered cells in the composition comprising a genetic disruption in or to the endogenous T cell receptor alpha constant region (TRAC) gene and/or the T cell receptor beta constant region (TRBC) gene; and/or at least 80% of the engineered cells in the composition; 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% express no or detectable gene product of the endogenous TRAC or TRBC gene not expressed at the level,
A composition according to any one of claims 195-197. at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition, or 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% expresses a recombinant TCR and/or exhibits binding to an antigen. 198. The composition according to any one of 198. 200. A method of treatment comprising administering to a subject an engineered cell, a plurality of engineered cells, or a composition according to any one of claims 194-199. Use of an engineered cell, a plurality of engineered cells, or a composition according to any one of claims 194 to 199 for the treatment of a disease or disorder. Use of an engineered cell, a plurality of engineered cells, or a composition according to any one of claims 194 to 199 in the manufacture of a medicament for treating a disease or disorder. 200. An engineered cell, a plurality of engineered cells, or a composition according to any one of claims 194-199 for use in the treatment of a disease or disorder. The polynucleotide according to any one of claims 69-94 or 117-140,
one or more agents capable of inducing gene disruption at a target site within the TRAC locus. (a) a nucleic acid sequence encoding a T cell receptor beta (TCRβ) chain and a portion of a TCRα chain that is shorter than the full-length native T cell receptor alpha (TCRα) chain, and
(b) a polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRAC locus encoding the TCR alpha chain;
one or more agents capable of inducing gene disruption at a target site within the TRAC locus. 206. The article of manufacture of claim 205, wherein the polynucleotide comprises a polynucleotide according to any one of claims 69-94 or 117-140. A polynucleotide according to any one of claims 95 to 140,
one or more agents capable of inducing gene disruption at a target site within the TRBC locus. (a) a nucleic acid sequence encoding a T-cell receptor alpha (TCRα) chain and a portion of a TCRβ chain that is shorter than the full-length native T-cell receptor beta (TCRβ) chain, and
(b) a polynucleotide comprising one or more homology arms linked to a nucleic acid sequence comprising sequences homologous to one or more regions of the open reading frame of the TRBC locus encoding the TCR β chain;
one or more agents capable of inducing gene disruption at a target site within the TRAC locus. 209. The article of manufacture of claim 207 or 208, wherein the polynucleotide comprises the polynucleotide of any one of claims 95-140. A kit comprising an article of manufacture according to any one of claims 204-209 and instructions for use.