TAL-effector nuclease (TALEN)-modified allogenic cells suitable for therapy

A method for producing an endonuclease-modified endogenous αβ-TCR negative human T cell said method comprising introducing into a human T cell:

• (i) an engineered nuclease or a first nucleic acid molecule encoding the engineered nuclease, wherein said engineered nuclease is a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nuclease (TALE-Nuclease), or a CRISPR/Cas nuclease, or a megaTAL nuclease;
• wherein said engineered nuclease produces a cleavage at a recognition site within an endogenous genomic human T cell receptor (TCR) alpha constant region gene in the human T cell;
• and
• (ii) a second nucleic acid molecule comprising an exogenous polynucleotide encoding a chimeric antigen receptor (CAR) comprising an scFv, a transmembrane domain from CD8 alpha, and one or more intracellular signaling domains from CD3 zeta and costimulatory domain from 4-1BBor a recombinant TCR,
• wherein said exogenous polynucleotide is inserted into said endogenous genomic human TCR alpha constant region gene at said cleavage at said recognition site by homologous recombination to generate a genetically-modified T cell;
• wherein insertion of said exogenous polynucleotide into said endogenous genomic human TCR alpha constant region gene results in a recombinant genomic nucleic acid molecule comprising the sequence:
• gctggggttt tgaagaagat cctattaaat aaaagaataa gcagtattat taagtagccc tgcatttcag gtttccttga gtggcaggcc aggcctggcc gtgaacgttc actgaaatca tggcctcttg gccaagattg atagcttgtg cctgtccctg agtcccagtc catcacgagc agctggtttc taagatgcta tttcccgtat aaagcatgag accgtgactt gccagcccca cagagccccg cccttgtcca tcactggcat ctggactcca gcctgggttg gggcaaagag ggaaatgaga tcatgtccta accctgatcc tottgtccca cagatatcca gtccggtgag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atccgggccc c (SEQ ID NO:
• 8),
• wherein said genetically-modified T cell has no cell-surface expression of the endogenous TCR.