LIVER-SPECIFIC CONSTRUCTS, FACTOR VIII EXPRESSION CASSETTES AND METHODS OF USE THEREOF
Described herein are constructs used for liver-specific expression of a transgene.
1 - 10 . (canceled)
11 . A method of providing a protein to a subject in need thereof, the method comprising administering to the subject an AAV vector, wherein the AAV vector comprises a polynucleotide expression construct between a 5′ inverted terminal repeat (ITR) and a 3′ ITR, the polynucleotide expression construct comprising at least one insulator sequence selected from SEQ ID NOs: 28, 29, 30, and 38, a liver-specific enhancer sequence, a promoter sequence, and a transgene, wherein the transgene encodes the protein and the protein is produced in the subject.
12 - 15 . (canceled)
16 . The method of claim 11 , wherein the polynucleotide expression construct further comprises an intron sequence comprising a wild-type or mutated minute virus of mice (MVM) intron sequence.
17 . The method of claim 11 , wherein the enhancer sequence comprises a wild-type or mutated Serpin 1 enhancer; and/or the promoter sequence comprises a wild-type or mutated transthyretin (TTR) promoter.
18 . The method of claim 11 , wherein the polynucleotide expression construct further comprises a polyadenylation signal.
19 - 27 . (canceled)
28 . The method of claim 11 , wherein the enhancer sequence has mutations at positions 1, 5, 14, 32 and/or 39 of any of SEQ ID NOs:1-13.
29 . The method of claim 16 , wherein the intron sequence is as shown in any one of SEQ ID NO:15, 16, 17, or nucleotides 340-432 of SEQ ID NO:37.
30 . (canceled)
31 . The method of claim 11 , wherein the transgene encodes a protein lacking or deficient in a hemophilia or a lysosomal storage disease.
32 . The method of claim 31 , wherein the transgene further encodes one or more nucleases.
33 - 35 . (canceled)
36 . The method of claim 11 , wherein the transgene is integrated into the genome of a liver cell.
37 . The method of claim 36 , further comprising administering one or more nucleases to the subject, wherein the nuclease cleaves an endogenous albumin gene and the transgene is integrated into the endogenous albumin gene.
38 . The method of claim 11 , further comprising inducing tolerance in the subject to the protein by treating the subject with one or more steroids and/or B-cell inhibitors.
39 . (canceled)
40 . The method of claim 16 , wherein the AAV vector comprises two insulator sequences flanking the enhancer sequence, the promoter sequence, the intron sequence, and the transgene.
41 . The method of claim 40 , wherein the first insulator sequence comprises SEQ ID NO: 28 and the second insulator sequence comprises SEQ ID NO: 30.
42 . The method of claim 11 , wherein the AAV vector is administered intravenously into a peripheral vein of the subject.
43 . The method of claim 11 , wherein the polynucleotide expression construct comprises the sequence of SEQ ID NO: 34.
44 . The method of claim 11 , wherein the polynucleotide expression construct comprises the sequence of SEQ ID NO: 37.
45 . The method of claim 44 , wherein the AAV vector comprises an AAV6 capsid and the ITRs are AAV2 ITRs.
46 . The method of claim 11 , wherein the transgene encodes replacement Factor VIII.
47 . The method of claim 38 , wherein the inducing step is performed after the subject shows elevated levels of aspartate aminotransferase (AST) and/or alanine aminotransferase (ALT).
48 . A method of providing replacement Factor VIII to a hemophilia A patient in need thereof, the method comprising administering into a peripheral vein of the patient an AAV vector, wherein the AAV vector comprises a polynucleotide expression construct between a 5′ inverted terminal repeat (ITR) and a 3′ ITR from AAV2, wherein the polynucleotide expression construct comprises SEQ ID NO: 37, and wherein the AAV vector comprises an AAV6 capsid.