TAL-effector nuclease (TALEN)-modified allogenic cells suitable for therapy
The invention relates to the fields of immunotherapy, molecular biology and recombinant nucleic acid technology. In particular, the invention relates to a TALEN-modified human primary cell comprising in its genome, a modified human T cell receptor alpha gene with an insertion comprising at least, from 5′ to 3′, a polynucleotide encoding a self-cleaving peptide, a chimeric antigen receptor, wherein the cell has undetectable cell-surface expression of the endogenous alpha beta T cell receptor as compared to a TCR positive control cell and expresses a receptor to target a pathological cell, use of said cell for treating a disease, including cancer. The invention further relates to methods for producing such a TALEN-modified cell, and to means for detecting such an engineered human primary cell or other genetically modified human primary cell obtained using alternative and/or additional rare cutting endonucleases.
1 . 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.
2 . The method of claim 1 , further comprising detecting endonuclease cleavage at a site other than at said recognition site by PCR.
3 . The method of claim 1 , wherein said engineered nuclease is a TALE-Nuclease.
4 . The method of claim 1 , wherein said second nucleic acid molecule is introduced into said cell by contacting said cell with a recombinant adeno-associated virus (AAV) vector comprising said second nucleic acid sequence.
5 . The method of claim 4 , wherein said recombinant AAV vector is a self-complementary AAV vector.
6 . The method of claim 3 , wherein said TALE-Nuclease recognizes the sequence TTGTCCCACAGATATC (SEQ ID NO: 36) in the endogenous genomic human TCR alpha constant region gene.
7 . The method of claim 1 , wherein said endonuclease is a Zinc finger nuclease that recognizes from 5′ to 3′ TGCTGTGGCCTGGAGCAAC (SEQ ID NO: 157) and GACTTTGCATGTGCA (SEQ ID NO: 158) and cleaves within ATATC (aa 12-16 of SEQ ID NO: 36), or a Crispr/Cas 9 that recognizes a complementary sequence to any one of the following sequences:
(SEQ ID NO: 39)
AGAGTCTCTCAGCTGGTACA,
(SEQ ID NO: 40)
GCACCAAAGCTGCCCTTACC,
(SEQ ID NO: 41)
AAGTTCCTGTGATGTCAAGC,
(SEQ ID NO: 42)
TTCGGAACCCAATCACTGAC,
(SEQ ID NO: 43)
GATTAAACCCGGCCACTTTTC,
(SEQ ID NO: 44)
CGTCATGAGCAGATTAAACC,
(SEQ ID NO: 45)
CTCAAGGTTCAGATCAGAAG,
(SEQ ID NO: 46)
TAGGCAGACAGACTTGTCAC,
(SEQ ID NO: 47)
AACAAATGTGTCACAAAGTA,
(SEQ ID NO: 48)
CACCAAAGCTGCCCTTACCT,
(SEQ ID NO: 49)
CTGACAGGTTTTGAAAGTTT,
(SEQ ID NO: 50)
TTCAAAACCTGTCAGTGATT,
(SEQ ID NO: 51)
CCGAATCCTCCTCCTGAAAG,
(SEQ ID NO: 52)
CCACTTTCAGGAGGAGGATT,
(SEQ ID NO: 53)
TAAACCCGGCCACTTTCAGG,
(SEQ ID NO: 54)
TCTCAAACAAATGTGTCACAAAGTA,
(SEQ ID NO: 55)
CTTACAATCTTGCAGATCTGGAATG,
(SEQ ID NO: 56)
TTAATCTGCTCATGACGCTG,
(SEQ ID NO: 57)
GGAGAAGAGGGGCAATGCAG,
(SEQ ID NO: 58)
TCTTCTCCCTCTCCAAACAG,
(SEQ ID NO: 59)
AGCAGCTTTCACCTCCTTGG,
(SEQ ID NO: 60)
GTAGCAGCTTTCACCTCCTT,
(SEQ ID NO: 61)
AGTTGGTGGCATTGCCGGGG,
(SEQ ID NO: 62)
TCTGTGATATACACATCAGAATC,
(SEQ ID NO: 63)
TCTGTGATATACACATCAGAATCC,
(SEQ ID NO: 64)
GAGTCTCTCAGCTGGTACACGGC,
(SEQ ID NO: 65)
GAGTCTCTCAGCTGGTACACGGCA,
(SEQ ID NO: 66)
ATTCTCAAACAAATGTGTCACAA,
(SEQ ID NO: 67)
ATTCTCAAACAAATGTGTCACAAA,
(SEQ ID NO: 68)
GTCTGTGATATACACATCAGAAT,
(SEQ ID NO: 69)
GTCTGTGATATACACATCAGAATC,
(SEQ ID NO: 70)
GAGAATCAAAATCGGTGAATAGG,
(SEQ ID NO: 71)
TGTGCTAGACATGAGGTCTATGG,
(SEQ ID NO: 72)
TCAGGGTTCTGGATATCTGTGGG,
(SEQ ID NO: 73)
GTCAGGGTTCTGGATATCTGTGG,
(SEQ ID NO: 74)
AAAGTCAGATTTGTTGCTCCAGG,
(SEQ ID NO: 75)
AACAAATGTGTCACAAAGTAAGG,
(SEQ ID NO: 76)
TGGATTTAGAGTCTCTCAGCTGG,
(SEQ ID NO: 77)
TAGGCAGACAGACTTGTCACTGG,
(SEQ ID NO: 78)
AGCTGGTACACGGCAGGGTCAGG,
(SEQ ID NO: 79)
GCTGGTACACGGCAGGGTCAGGG,
(SEQ ID NO: 80)
TCTCTCAGCTGGTACACGGCAGG,
(SEQ ID NO: 81)
AGAGTCTCTCAGCTGGTACACGG,
(SEQ ID NO: 82)
CTCTCAGCTGGTACACGGCAGGG,
(SEQ ID NO: 83)
ACAAAACTGTGCTAGACATGAGG,
(SEQ ID NO: 84)
ATTTGTTTGAGAATCAAAATCGG,
(SEQ ID NO: 85)
TGGAATAATGCTGTTGTTGAAGG,
(SEQ ID NO: 86)
AGAGCAACAGTGCTGTGGCCTGG,
(SEQ ID NO: 87)
CTTCTTCCCCAGCCCAGGTAAGG,
(SEQ ID NO: 88)
ACACGGCAGGGTCAGGGTTCTGG,
(SEQ ID NO: 89)
CTTCAAGAGCAACAGTGCTGTGG,
(SEQ ID NO: 90)
CTGGGGAAGAAGGTGTCTTCTGG,
(SEQ ID NO: 91)
TTCTTCCCCAGCCCAGGTAAGGG,
(SEQ ID NO: 92)
CTTACCTGGGCTGGGGAAGAAGG,
(SEQ ID NO: 93)
GACACCTTCTTCCCCAGCCCAGG,
(SEQ ID NO: 94)
TTCAAAACCTGTCAGTGATTGGG,
(SEQ ID NO: 95)
CGTCATGAGCAGATTAAACCCGG,
(SEQ ID NO: 96)
TTCGGAACCCAATCACTGACAGG,
(SEQ ID NO: 97)
TAAACCCGGCCACTTTCAGGAGG,
(SEQ ID NO: 98)
TTTCAAAACCTGTCAGTGATTGG,
(SEQ ID NO: 99)
GATTAAACCCGGCCACTTTCAGG,
(SEQ ID NO: 100)
CTCGACCAGCTTGACATCACAGG,
(SEQ ID NO: 101)
AAGTTCCTGTGATGTCAAGCTGG,
(SEQ ID NO: 102)
ATCCTCCTCCTGAAAGTGGCCGG,
(SEQ ID NO: 103)
TGCTCATGACGCTGCGGCTGTGG,
(SEQ ID NO: 104)
CATCACAGGAACTTTCTAAAAGG,
(SEQ ID NO: 105)
GTCGAGAAAAGCTTTGAAACAGG,
(SEQ ID NO: 106)
CCACTTTCAGGAGGAGGATTCGG,
(SEQ ID NO: 107)
CTGACAGGTTTTGAAAGTTTAGG,
(SEQ ID NO: 108)
AGCTTTGAAACAGGTAAGACAGG,
(SEQ ID NO: 109)
CTGTGGTCCAGCTGAGGTGAGGG,
(SEQ ID NO: 110)
CTGCGGCTGTGGTCCAGCTGAGG,
(SEQ ID NO: 111)
TGTGGTCCAGCTGAGGTGAGGGG,
(SEQ ID NO: 112)
TCCTCCTCCTGAAAGTGGCCGGG,
(SEQ ID NO: 113)
TTAATCTGCTCATGACGCTGCGG,
(SEQ ID NO: 114)
ACCCGGCCACTTTCAGGAGGAGG,
(SEQ ID NO: 115)
GCTGTGGTCCAGCTGAGGTGAGG,
(SEQ ID NO: 116)
CCGAATCCTCCTCCTGAAAGTGG
a MegaTAL, a meganuclease that recognizes and cleaves a recognition sequence within residues 93-208 of the wild-type human TCR alpha constant region, wherein said recombinant meganuclease comprises a first subunit and a second subunit, wherein said first subunit binds to a first recognition half-site of said recognition sequence and comprises a first hypervariable (HVR1) region, and wherein said second subunit binds to a second recognition half-site of said recognition sequence and comprises a second hypervariable (HVR2) region.
8 . The method of claim 7 , wherein said meganuclease is a single-chain meganuclease comprising a linker, wherein said linker covalently joins said first subunit and said second subunit.
9 . An endonuclease-modified endogenous αβ-TCR negative human cell obtained by the method of claim 1 .
10 . An endonuclease-modified endogenous αβ-TCR negative human cell obtained by the method of claim 3 .
11 . The endonuclease-modified endogenous αβ-TCR negative human cell according to claim 9 ,
wherein said CAR is an anti-CD22 CAR, which comprises the amino acid sequence of to SEQ ID NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11.
12 . The endonuclease-modified endogenous αβ-TCR negative human cell, according to claim 9 ,
wherein said exogenous polynucleotide comprises a sequence encoding anti-CD123 CAR, which is at least 80% identical to SEQ ID NO: 12.
13 . The method of claim 1 , wherein said engineered nuclease produces a cleavage at a recognition site within the nucleotide sequence TTGTCCCACAGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGA (SEQ ID NO: 38).