Methods and compositions for modulating pd1

Abstract

Disclosed herein are methods and compositions for modulating expression of a PD1 gene.

What is claimed is: 
     
         1 . A human cell comprising a transcription factor or nuclease comprising a DNA-binding domain that binds to target site in a PD1 gene as shown in any of SEQ ID NOs:56-60 and a functional domain, wherein the functional domain of the transcription factor comprises a transcriptional activation or repression domain and the functional domain of the nuclease comprises a cleavage domain. 
     
     
         2 . The human cell of  claim 1 , wherein the DNA-binding domain comprises a zinc finger protein comprising four, five or six zinc finger recognition regions ordered from F1 to F4, F1 to F5 or F1 to F6, from N-terminus to C-terminus, and wherein the recognition regions comprise the following amino acid sequences:
 (i) F1: QSGHLSR (SEQ ID NO: 1), F2: RSDSLSV (SEQ ID NO: 2), F3: HNDSRKN (SEQ ID NO: 3), F4: RSDDLTR (SEQ ID NO: 4), and F5: RSDHLTQ (SEQ ID NO: 5);   (ii) F1: RSAALSR (SEQ ID NO: 6), F2: RSDDLTR (SEQ ID NO: 4), F3: RSDHLTT (SEQ ID NO: 7), F4: DRSALSR (SEQ ID NO: 8), and F5: DRSALAR (SEQ ID NO: 9);   (iii) F1: RSAALAR (SEQ ID NO: 10), F2: RSDDLSK (SEQ ID NO: 11), F3: RNDHRKN (SEQ ID NO: 12), F4: DRSALSR (SEQ ID NO: 8), and F5: DRSALAR (SEQ ID NO: 9);   (iv) F1: RSDHLSE (SEQ ID NO: 13), F2: TSSDRTK (SEQ ID NO: 14), F3: RSDHLSE (SEQ ID NO: 13), and F4: QSASRKN (SEQ ID NO: 15);   (v) F1: RSDVLSE (SEQ ID NO: 16), F2: RSANLTR (SEQ ID NO: 17), F3: RSDHLSQ (SEQ ID NO: 18), F4: TSSNRKT (SEQ ID NO: 19), F5: DRSNLSR (SEQ ID NO: 20), and F6: RSDALAR (SEQ ID NO: 21); or   (vi) F1: DDWNLSQ (SEQ ID NO: 22), F2: RSANLTR (SEQ ID NO: 17), F3: RSDHLSQ (SEQ ID NO: 18), F4: TSSNRKT (SEQ ID NO: 19), F5: DRSNLSR (SEQ ID NO: 20), and F6: RSDALAR (SEQ ID NO: 21);   (vii) F1: RSSALSR (SEQ ID NO: 23), F2: RPLALKH (SEQ ID NO: 24), F3: RNDHRKN (SEQ ID NO: 12), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (viii) F1: QSGHLSR (SEQ ID NO: 1), F2: RSDSLSV (SEQ ID NO: 2), F3: HNDSRKN (SEQ ID NO: 3), F4: RANSLLR (SEQ ID NO: 26), and F5: RSDHLTQ (SEQ ID NO: 5);   (ix) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RNNNLRT (SEQ ID NO: 29), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (x) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: TNWHLRT (SEQ ID NO: 30), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xi) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RTPHLTL (SEQ ID NO: 31), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xii) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RSAQLAT (SEQ ID NO: 32), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xiii) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RCTHLYL (SEQ ID NO: 33), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xiv) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RPTQRYS (SEQ ID NO: 34), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xiv) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RANHREC (SEQ ID NO: 35), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xvi) F1: RKFARPS (SEQ ID NO: 36), F2: RNFSRSD (SEQ ID NO: 37), F3: HPHHRMC (SEQ ID NO: 38), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xvii) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RMGRLST (SEQ ID NO: 39), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xviii) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RHSRLTT (SEQ ID NO: 40), F4: TRPVLMR (SEQ ID NO: 41), and F5: DRSALAR (SEQ ID NO: 9);   (xix) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RANHRVC (SEQ ID NO: 42), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xx) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RSTHLLG (SEQ ID NO: 43), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xxi) F1: RNAALTR (SEQ ID NO: 45), F2: RSDELTR (SEQ ID NO: 28) F3: RSCGLWS (SEQ ID NO: 44), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xxii) F1: CNAALTR (SEQ ID NO: 46), F2: RSDELTR (SEQ ID NO: 28), F3: REEHRAT (SEQ ID NO: 47), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xxiii) F1: RNAALTR (SEQ ID NO: 45), F2: RSDELTR (SEQ ID NO: 28), F3: RHHHLAA (SEQ ID NO: 48), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xxiv) F1: RNAALTR (SEQ ID NO: 45), F2: RSDELTR (SEQ ID NO: 28), F3: RPMHLTN (SEQ ID NO: 49), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xxv) F1: RNAALTR (SEQ ID NO: 45), F2: RSDELTR (SEQ ID NO: 28), F3: RSPHLYH (SEQ ID NO: 50), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xxvi) F1: RNAALTR (SEQ ID NO: 45), F2: RSDELTR (SEQ ID NO: 28), F3: RCEALHH (SEQ ID NO: 51), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSAQAR (SEQ ID NO: 52);   (xxvii) F1: RNAALTR (SEQ ID NO: 45), F2: RSDELTR (SEQ ID NO: 28), F3: RCEALHH (SEQ ID NO: 51), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);   (xxviii) F1: RNAALTR (SEQ ID NO: 45), F2: RSDELTR (SEQ ID NO: 28), F3: RLPALLS (SEQ ID NO: 53), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9); and   (xxix) F1: HNAALTR (SEQ ID NO: 54), F2: RSDELTR (SEQ ID NO: 28), F3: RTYNRTQ (SEQ ID NO: 55), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9).   
     
     
         3 . A human T-cell or stem cell cultured from a cell according to  claim 2 , wherein the cell comprises an insertion and/or deletion in an endogenous PD1 gene. 
     
     
         4 . The cell of  claim 3 , wherein the insertion comprises a transgene. 
     
     
         5 . A human T-cell or stem cell comprising an insertion and/or deletion in an endogenous PD1 gene, the insertion and/or deletion made by following cleavage by a nuclease that binds to target sites as shown in SEQ ID NO:56-60. 
     
     
         6 . The cell of  claim 1 , wherein the cell is a CD4+ T-cell. 
     
     
         7 . The cell of  claim 5 , further comprising one or more additional genetic modifications.