METHODS AND COMPOSITIONS FOR GENOME ENGINEERING
Disclosed herein are methods and compositions for insertion of transgene sequences encoding proteins that is aberrantly expressed in disease or disorder such as a lysosomal storage disease.
1 . A pharmaceutical composition comprising one or more polynucleotides encoding a pair of zinc finger nucleases (ZFNs) that cleave an endogenous albumin gene, each zinc finger nuclease comprising a cleavage domain and a zinc finger protein (ZFP) that binds to a target site in the endogenous albumin gene, wherein the zinc finger proteins of the pair are selected from the group consisting of a left ZFP designated SBS #47171, 47192, 47863, or 47864, the left ZFP comprising five recognition helix regions ordered F1 to F5 as shown in a single row of the following Table:
SBS#
F1
F2
F3
F4
F5
47171
QSGNLSR
LKQNLCM
WADNLQN
TSGNLTR
RQSHLCL
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 23)
NO: 18)
NO: 24)
NO: 20)
NO: 21)
47863
QSGNLAR
LIQYLQS
WADNLQN
TSGNLTR
RQSHLSL
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 2)
NO: 26)
NO: 24)
NO: 20)
NO: 27)
47192
QSGNLAR
LIQYLQS
WADNLQN
TSGNLTR
RQSHLCL
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 2)
NO: 26)
NO: 24)
NO: 20)
NO: 21)
47864
QSGNLAR
LIQYLQS
WQSNLQN
TSGNLTR
RQSHLCL
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 2)
NO: 26)
NO: 19)
NO: 20)
NO: 21)
and a right ZFP designated SBS #40477, 47079, 47169, 47898 or 47931, the right ZFP comprising six recognition helix regions ordered F1 to F6 as shown in a single row of the following Table:
SBS#
F1
F2
F3
F4
F5
F6
47931
TPQLLDR
LKWNLRT
DQSNLNA
RNFSLTM
LRHDLDR
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 14)
NO: 9)
NO: 25)
NO: 15)
NO: 16)
NO: 12)
47079
TPQLLDR
LKWNLRT
DQSNLRA
RNFSLTM
LRHDLDR
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 14)
NO: 9)
NO: 10)
NO: 15)
NO: 16)
NO: 12)
47898
TPQLLDR
LKHNLLT
DQSNLNA
RNFSLTM
LRHDLDR
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 14)
NO: 28
NO: 25)
NO: 15)
NO: 16)
NO: 12)
47169
TPQLLDR
LKWNLRT
DQSNLRA
RNFSLTM
LRHDLDR
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 14)
NO: 9)
NO: 10)
NO: 15)
NO: 16)
NO: 12)
40477
QSSDLSR
LKHNLLT
LKHNLLT
RPYTLRL
LRPDLER
HRSNLNK
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
NO: 8)
NO: 28)
NO: 28)
NO: 11)
NO: 41)
NO: 12)
2 . The pharmaceutical composition of claim 1 , wherein the cleavage domain is an engineered cleavage domain.
3 . The pharmaceutical composition of claim 1 , wherein the one or more polynucleotides are viral or non-viral vectors.
4 . The pharmaceutical composition of claim 3 , wherein the polynucleotides encoding the first and second zinc finger nucleases are carried on the same or on different vectors.
5 . The pharmaceutical composition of claim 3 , wherein the viral vector is an AAV vector.
6 . The pharmaceutical composition of claim 5 , wherein the AAV vector is an AAV2/8 vector.
7 . The pharmaceutical composition of claim 1 , further comprising a donor polynucleotide comprising a transgene and the transgene is integrated into the endogenous albumin gene following cleavage by the pair of ZFNs.
8 . The pharmaceutical composition of claim 7 , wherein the donor polynucleotide is carried on an AAV vector.
9 . The pharmaceutical composition of claim 7 , wherein the transgene encodes one or more proteins lacking in a lysosomal storage disease.
10 . The pharmaceutical composition of claim 9 , wherein the protein is a glucocerebrosidase, an α galactosidase, an iduronate-2-sulfatase and/or an alpha-L iduronidase protein.
11 . The pharmaceutical composition of claim 7 , wherein the transgene encodes one or more clotting factors.
12 . The pharmaceutical composition of claim 11 , wherein the clotting factor is Factor IX (F.IX) protein, a Factor VII protein and/or a Factor X protein.
13 . A method of treating a lysosomal storage disease in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 7 to the subject.
14 . A method of treating a clotting disorder in a subject in need thereof, the method comprising administering the pharmaceutical composition of claim 11 to the subject.