Liver-specific constructs, factor viii expression cassettes and methods of use thereof

Abstract

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.