IP Library › Granted Patent US 8,563,314

Granted Patent B2

US 8,563,314 · App. 12/927,557 · Granted Oct 22, 2013

Methods and compositions for modulating PD1

Inventors: Philip D. Gregory (Orinda, CA); Michael C. Holmes (Oakland, CA); Matthew C. Mendel (Richmond, CA); Xiangdong Meng (San Pablo, CA); David Paschon (Oakland, CA); Andreas Reik (Vallejo, CA); Fyodor Urnov (Point Richmond, CA)

Assignee: Sangamo BioSciences, Inc.

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Quick Facts

Patent No.: US 8,563,314
App. No.: 12/927,557
Granted: Oct 22, 2013
Kind: B2

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Abstract

Disclosed herein are methods and compositions for modulating expression of a PD 1 gene.

Claims (157)

1. 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: 11), 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),

wherein the zinc finger protein is capable of binding a target site in an endogenous human PD1 gene.

2. A fusion protein comprising a zinc finger protein according to claim 1 and a functional domain.

3. The fusion protein of claim 2 , wherein the functional domain comprises a transcriptional regulatory domain.

4. The fusion protein of claim 3 , wherein the transcriptional regulatory domain is an activation domain or a repression domain.

5. The fusion protein of claim 2 , wherein the functional domain comprises a cleavage domain or a cleavage half-domain.

6. A polynucleotide encoding 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),

wherein the zinc finger protein is capable of binding a target site in an endogenous human PD1 gene.

7. A polynucleotide encoding a fusion protein comprising

(a) 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: 11), 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),

wherein the zinc finger protein is capable of binding a target site in an endogenous human PD1 gene; and

(b) a functional domain.

8. A zinc finger protein comprising four or five zinc finger recognition regions ordered from F1 to F4 or F1 to F5, from N-terminus to C-terminus, and wherein the recognition regions comprise the following amino acid sequences:

(i) 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);

(ii) F1: QSGHLSR (SEQ ID NO: 11), 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);

(iii) 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);

(iv) 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);

(v) 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);

(vi) 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);

(vii) 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);

(viii) 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);

(ix) 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);

(x) 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);

(xi) 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);

(xii) F1: RPSTLHR (SEQ ID NO: 27), F2: RSDELTR (SEQ ID NO: 28), F3: RHSRLTT (SEQ ID NO: 40), 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: RANHRVC (SEQ ID NO: 42), 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: RSTHLLG (SEQ ID NO: 43), F4: TRPVLKR (SEQ ID NO: 25), and F5: DRSALAR (SEQ ID NO: 9);

(xv) 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);

(xvi) 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);

(xvii) 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);

(xviii) 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);

(xix) 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);

(xx) 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);

(xxi) 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);

(xxii) 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); or

(xxiii) 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),

wherein the zinc finger protein is capable of binding a target site in an endogenous human PD1 gene.

9. A fusion protein comprising a zinc finger protein according to claim 8 and a functional domain.

10. The fusion protein of claim 9 , wherein the functional domain comprises a transcriptional regulatory domain.

11. The fusion protein of claim 10 , wherein the transcriptional regulatory domain is an activation domain or a repression domain.

12. The fusion protein of claim 9 , wherein the functional domain comprises a cleavage domain or a cleavage half-domain.

13. A polynucleotide encoding a zinc finger protein comprising four or five zinc finger recognition regions ordered from F1 to F4 or F1 to F5,from N-terminus to C-terminus, and wherein the recognition regions comprise the following amino acid sequences:

(i) 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);

(ii) F1: QSGHLSR (SEQ ID NO: 11), 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);

(iii) 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);

(iv) 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);

(v) 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);

(vi) 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);

(vii) 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);