Modulation of liver genes
Described herein are compositions and methods for modulation of gene expression in the liver including modulation of PCSK9, TTR, SERPINA1, KLKB1 and/or HAO1.
1. An isolated liver cell in which expression of an endogenous PCSK9 gene is altered as compared to wild-type, wherein the liver cell comprises a zinc finger protein comprising 5 or 6 zinc finger domains, each zinc finger domain comprising a recognition helix region sequence ordered F1 to F5 or F1 to F6, wherein the zinc finger protein comprises recognition helix regions ordered as shown in a single row of the following Table:
F1
F2
F3
QSSHLTR (SEQ
QSGNLAR (SEQ
QS SDLSR (SEQ
ID NO:13)
ID NO:14)
ID NO:15)
RSDVLSE (SEQ
TRNGLKY (SEQ
TSGHLSR (SEQ
ID NO:19)
ID NO:20)
ID NO:21)
QSGALAR (SEQ
RSDVLSE (SEQ
RSAHLSR (SEQ
ID NO:25)
ID NO:19)
ID NO:26)
RSDHLSR (SEQ
QSGHLSR (SEQ
HKQHRDA(SEQ
ID NO:27)
ID NO:22)
ID NO:18)
RSDHLSR (SEQ
QQWDRKQ (SEQ
TPSYLPT (SEQ
ID NO:27)
ID NO:32)
ID NO:33)
QSGNLAR (SEQ
TSGNLTR (SEQ
RSDDLTR (SEQ
ID NO:14)
ID NO:36)
ID NO:37)
QSGDLTR (SEQ
RSDNLTR (SEQ
QSGALAR (SEQ
ID NO:39)
ID NO:40)
ID NO:25)
RSAHLSR (SEQ
DSSDRKK (SEQ
QHQVLVR (SEQ
ID NO:26)
ID NO:43)
ID NO:44)
DGYYLPT (SEQ
DRSALAR (SEQ
ERQTLIK (SEQ
ID NO:48)
ID NO:34)
ID NO:49)
RSDNLAR (SEQ
DRSVLHR (SEQ
RSDTLSA (SEQ
ID NO:52)
ID NO:53)
ID NO:54)
RSDVLSE (SEQ
QKCCLRS (SEQ
DRSHLTR (SEQ
ID NO:19)
ID NO:58)
ID NO:59)
HRQRLEE (SEQ
RNASRTR (SEQ
DRSHLTR (SEQ
ID NO:62)
ID NO:63)
ID NO:59)
F4
F5
F6
WHSSLHQ (SEQ
LRHHLTR (SEQ
HKQHRDA (SEQ
ID NO:16)
ID NO:17)
ID NO:18)
QSGHLSR (SEQ
NNRDLIN (SEQ
TSSNLSR (SEQ
ID NO:22)
ID NO:23)
ID NO:24)
RSDHLSR (SEQ
DRSVLAR (SEQ
N/A
ID NO:27)
ID NO:28)
DNSNRIK (SEQ
RSDHLSE (SEQ
HSRTRTK (SEQ
ID NO:29)
ID NO:30)
ID NO:31)
DRSALAR (SEQ
RSDHLSE (SEQ
RKDARIT (SEQ
ID NO:34)
ID NO:30)
ID NO:35)
RSDHLSE (SEQ
DKSNRKK (SEQ
N/A
ID NO:30)
ID NO:38)
QSGHLSR (SEQ
RSDHLSQ (SEQ
DSSHRTR (SEQ
ID NO:22)
ID NO:41)
ID NO:42)
QNATRTK (SEQ
RSDTLSE (SEQ
RSPGRMG (SEQ
ID NO:45)
ID NO:46)
ID NO:47)
QSGHLSR (SEQ
HRWHLQT (SEQ
AQCCLFH (SEQ
ID NO:22)
ID NO:50)
ID NO:51)
DKSTRTK (SEQ
PCRYRLD (SEQ
RSANLTR (SEQ
ID NO:55)
ID NO:56)
ID NO:57)
RSDDLTR (SEQ
RSDTLSN (SEQ
TNSDRTK (SEQ
ID NO:37)
ID NO:60)
ID NO:61)
RSDDLTR (SEQ
RSDTLSE (SEQ
KPYNLQQ (SEQ
ID NO:37)
ID NO:46)
ID NO:64).
2. The liver cell of claim 1 , wherein the sequence of the endogenous PCSK9 gene is altered by cleaving the gene using a pair of zinc finger nucleases (ZFNs), wherein at least one of the zinc finger nucleases of the pair comprises the zinc finger protein of claim 1 and a cleavage domain.
3. The liver cell of claim 2 , wherein the pair of ZFNs binds to paired target sites within SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4 SEQ ID NO:5 and SEQ ID NO:6; SEQ ID NO:7 and SEQ ID NO:8; SEQ ID NO:9 and SEQ ID NO:10; or SEQ ID NO:11 and SEQ ID NO:12.
4. The liver cell of claim 2 , further comprising an exogenous sequence integrated into the cleaved endogenous PCSK9 gene.
5. The liver cell of claim 4 , wherein the exogenous sequence comprises a transgene; introduces a mutation into the gene, or corrects a mutation in the endogenous gene.
6. The liver cell of claim 1 , wherein the liver cell further comprises an artificial transcription factor comprising the zinc finger protein of claim 1 and a transcriptional regulatory domain, wherein the artificial transcription factor alters expression of the endogenous gene.
7. The liver cell of claim 6 , wherein the artificial transcription factor activates or represses expression of the endogenous gene.
8. A pharmaceutical composition comprising the liver cell of claim 1 .
9. A method of producing the liver cell of claim 1 , the method comprising administering an artificial transcription factor or artificial nuclease comprising the zinc finger protein of claim 1 to the liver cell.
10. A fusion molecule comprising a zinc finger protein that binds to a target site in an endogenous PCSK9 gene and a functional domain, wherein the zinc finger protein comprises 5 or 6 zinc finger domains, each zinc finger domain comprising a recognition helix region sequence ordered F1 to F5 or F1 to F6, wherein the zinc finger protein comprises recognition helix regions ordered as shown in a single row of the following Table:
F1
F2
F3
QSSHLTR (SEQ
QSGNLAR (SEQ
QS SDLSR (SEQ
ID NO:13)
ID NO:14)
ID NO:15)
RSDVLSE (SEQ
TRNGLKY (SEQ
TSGHLSR (SEQ
ID NO:19)
ID NO:20)
ID NO:21)
QSGALAR (SEQ
RSDVLSE (SEQ
RSAHLSR (SEQ
ID NO:25)
ID NO:19)
ID NO:26)
RSDHLSR (SEQ
QSGHLSR (SEQ
HKQHRDA(SEQ
ID NO:27)
ID NO:22)
ID NO:18)
RSDHLSR (SEQ
QQWDRKQ (SEQ
TPSYLPT (SEQ
ID NO:27)
ID NO:32)
ID NO:33)
QSGNLAR (SEQ
TSGNLTR (SEQ
RSDDLTR (SEQ
ID NO:14)
ID NO:36)
ID NO:37)
QSGDLTR (SEQ
RSDNLTR (SEQ
QSGALAR (SEQ
ID NO:39)
ID NO:40)
ID NO:25)
RSAHLSR (SEQ
DSSDRKK (SEQ
QHQVLVR (SEQ
ID NO:26)
ID NO:43)
ID NO:44)
DGYYLPT (SEQ
DRSALAR (SEQ
ERQTLIK (SEQ
ID NO:48)
ID NO:34)
ID NO:49)
RSDNLAR (SEQ
DRSVLHR (SEQ
RSDTLSA (SEQ
ID NO:52)
ID NO:53)
ID NO:54)
RSDVLSE (SEQ
QKCCLRS (SEQ
DRSHLTR (SEQ
ID NO:19)
ID NO:58)
ID NO:59)
HRQRLEE (SEQ
RNASRTR (SEQ
DRSHLTR (SEQ
ID NO:62)
ID NO:63)
ID NO:59)
F4
F5
F6
WHSSLHQ (SEQ
LRHHLTR (SEQ
HKQHRDA (SEQ
ID NO:16)
ID NO:17)
ID NO:18)
QSGHLSR (SEQ
NNRDLIN (SEQ
TS SNLSR (SEQ
ID NO:22)
ID NO:23)
ID NO:24)
RSDHLSR (SEQ
DRSVLAR (SEQ
N/A
ID NO:27)
ID NO:28)
DNSNRIK (SEQ
RSDHLSE (SEQ
HSRTRTK (SEQ
ID NO:29)
ID NO:30)
ID NO:31)
DRSALAR (SEQ
RSDHLSE (SEQ
RKDARIT (SEQ
ID NO:34)
ID NO:30)
ID NO:35)
RSDHLSE (SEQ
DKSNRKK (SEQ
N/A
ID NO:30)
ID NO:38)
QSGHLSR (SEQ
RSDHLSQ (SEQ
DSSHRTR (SEQ
ID NO:22)
ID NO:41)
ID NO:42)
QNATRTK (SEQ
RSDTLSE (SEQ
RSPGRMG (SEQ
ID NO:45)
ID NO:46)
ID NO:47)
QSGHLSR (SEQ
HRWHLQT (SEQ
AQCCLFH (SEQ
ID NO:22)
ID NO:50)
ID NO:51)
DKSTRTK (SEQ
PCRYRLD (SEQ
RSANLTR (SEQ
ID NO:55)
ID NO:56)
ID NO:57)
RSDDLTR (SEQ
RSDTLSN (SEQ
TNSDRTK (SEQ
ID NO:37)
ID NO:60)
ID NO:61)
RSDDLTR (SEQ
RSDTLSE (SEQ
KPYNLQQ (SEQ
ID NO:37)
ID NO:46)
ID NO:64).
11. The fusion molecule of claim 10 , wherein the functional domain comprises a transcriptional regulatory domain or a cleavage domain.
12. A polynucleotide encoding the fusion molecule of claim 10 .
13. The polynucleotide of claim 12 , wherein the polynucleotide is mRNA form.
14. A viral or non-viral expression vector comprising one or more polynucleotides of claim 12 .
15. A pharmaceutical composition comprising the polynucleotide of claim 12 .
16. A method of altering expression of a PCSK9 gene in a liver cell, the method comprising introducing the polynucleotide of claim 12 into the cell under conditions such that the expression of the PCSK9 gene is altered.
17. The method of claim 16 , wherein the fusion molecule comprises an artificial zinc finger nuclease that alters expression of the PCSK9 gene by introducing one or more insertions and/or deletions into the gene.
18. A method of treating Familial Hypercholesterolemia/Static resistant hypercholesterolemia, the method comprising administering a pharmaceutical composition of claim 15 to a subject in need thereof.
19. A kit comprising a fusion molecule of claim 10 .