Targeted disruption of the T cell receptor
Disclosed herein are methods and compositions for inactivating TCR genes, using engineered nucleases comprising at least one DNA binding domain and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding nucleases, vectors comprising polynucleotides encoding nucleases and cells comprising polynucleotides encoding nucleases and/or cells comprising nucleases are also provided. Disclosed herein are also methods and compositions for expressing a functional exogenous TCR in the absence of endogenous TCR expression in T lymphocytes, including lymphocytes with a central memory phenotype. Polynucleotides encoding exogenous TCR, vectors comprising polynucleotides encoding exogenous TCR and cells comprising polynucleotides encoding exogenous TCR and/or cells comprising exogenous TCR are also provided.
1. A fusion molecule comprising a DNA-binding domain that binds to exon c1, c2 or c3 of the TCRA gene and a transcriptional regulatory domain or a nuclease domain, wherein the DNA-binding domain comprises:
a zinc finger protein (ZFP) comprising 5 or 6 zinc finger domains designated F1 to F5 or F1 to F6, each zinc finger domain comprising a recognition helix region as shown in a single row of the following Table:
ZFN Name
F1
F2
F3
F4
F5
F6
SBS55204
DRSNLSR
QKVTLAA
DRSALSR
TSGNLTR
YRSSLKE
TSGNLTR
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 22)
NO: 23)
NO: 24)
NO: 25)
NO: 26)
NO: 25)
SBS53759
QQNVLIN
QNATRTK
QSGHLAR
NRYDLMT
RSDSLLR
QSSDLTR
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 27)
NO: 28)
NO: 29)
NO: 30)
NO: 31)
NO: 32)
SBS55229
DRSALAR
QSGNLAR
HRSTLQG
QSGDLTR
TSGSLTR
NA
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 33)
NO: 34)
NO: 35)
NO: 36)
NO: 37)
SBS53785
QHQVLVR
QNATRTK
QSGHLSR
DRSDLSR
RSDALAR
NA
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 38)
NO: 28)
NO: 39)
NO: 40)
NO: 41)
SBS53810
DQSNLRA
TSSNRKT
DSSTRKT
QSGNLAR
RSDDLSE
TNSNRKR
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 42)
NO: 43)
NO: 44)
NO: 34)
NO: 45)
NO: 46)
SBS55255
RSDHLST
DRSHLAR
LKQHLNE
TSGNLTR
HRTSLTD
NA
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 47)
NO: 48)
NO: 49)
NO: 25)
NO: 50)
SBS55248
DQSNLRA
TSSNRKT
LQQTLAD
QSGNLAR
RREDLIT
TSSNLSR
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 42)
NO: 43)
NO: 51)
NO: 34)
NO: 52)
NO: 53)
SBS55254
RSDHLST
DRSHLAR
LKQHLNE
QSGNLAR
HNSSLKD
NA
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 47)
NO: 48)
NO: 49)
NO: 34)
NO: 54)
SBS55260
RSDHLST
DRSHLAR
LNHHLQQ
QSGNLAR
HKTSLKD
NA
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 47)
NO: 48)
NO: 55)
NO: 34)
NO: 56)
SBS55266
QSSDLSR
QSGNRTT
RSANLAR
DRSALAR
RSDVLSE
KHSTRRV
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 57)
NO: 58)
NO: 59)
NO: 33)
NO: 60)
NO: 61)
SBS53853
TMHQRVE
TSGHLSR
RSDHLTQ
DSANLSR
QSGSLTR
AKWNLDA
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 62)
NO: 63)
NO: 64)
NO: 65)
NO: 66)
NO: 67)
SBS53860
TMHQRVE
TSGHLSR
RNDSLKT
DSSNLSR
QKATRTT
RNASRTR
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 62)
NO: 63)
NO: 68)
NO: 69)
NO: 70)
NO: 72)
SBS53863
RSDSLLR
QSSDLRR
RSDNLSE
ERANRNS
RSDNLAR
QKVNLMS
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 31)
NO: 73)
NO: 74)
NO: 75)
NO: 76)
NO: 77)
SBS55287
RSDSLLR
QSSDLRR
RSDNLSE
ERANRNS
RSDNLAR
QKVNLRE
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 31)
NO: 73)
NO: 74)
NO: 75)
NO: 76)
NO: 78)
SBS53855
TMHQRVE
TSGHLSR
RSDTLSQ
DRSDLSR
QKATRTT
RNASRTR
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 62)
NO: 63)
NO: 79)
NO: 40)
NO: 70)
NO: 72)
SBS52774
RKQTRTT
HRSSLRR
RSDHLST
TSANLSR
RSDNLSE
WHSSLRV
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 80)
NO: 81)
NO: 47)
NO: 82)
NO: 74)
NO: 83)
SBS53909
RSAHLSR
DRSDLSR
RSDVLSV
QNNHRIT
RSDVLSE
SPSSRRT
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 84)
NO: 40)
NO: 85)
NO: 86)
NO: 60)
NO: 87)
SBS52742
RSAHLSR
DRSDLSR
RSDSLSV
QNANRKT
RSDVLSE
SPSSRRT
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 84)
NO: 40)
NO: 88)
NO: 89)
NO: 60)
NO: 87)
SBS53856
TMHQRVE
TSGHLSR
RSDSLST
DRANRIK
QKATRTT
RNASRTR
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 62)
NO: 63)
NO: 90)
NO: 91)
NO: 70)
NO: 72).
2. An isolated cell comprising the fusion molecule of claim 1 .
3. The cell of claim 2 , further comprising an inactivated beta 2 microglobulin (B2M), PD1 and/or CTLA4 gene.
4. The cell of claim 2 , further comprising a transgene encoding a chimeric antigen receptor (CAR), a transgene encoding an Antibody-coupled T-cell Receptor (ACTR) and/or a transgene encoding an engineered TCR.
5. The cell of claim 2 , wherein the cell is a lymphoid cell, a stem cell, or a progenitor cell.
6. The cell of claim 4 , wherein the cell is a T-cell, an induced pluripotent stem cell (iPSC), an embryonic stem cell, a mesenchymal stem cell (MSC), or a hematopoietic stem cell (HSC).
7. A pharmaceutical composition comprising the cell according to claim 2 .
8. A polynucleotide encoding the fusion molecule of claim 1 .
9. The polynucleotide of claim 8 , wherein the polynucleotide is a viral vector, a plasmid or mRNA.
10. A method of treating a cancer, the method comprising introducing the cell according to claim 2 to a subject with cancer.
11. The pharmaceutical composition of claim 7 , further comprising a genetically modified cell descended from the cell of claim 1 .