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. A method of providing a protein to a subject in need thereof, the method comprising administering to the liver of 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.
2. The method of claim 1 , wherein the transgene is integrated into the genome of a liver cell.
3. The method of claim 2 , 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, wherein the nuclease is a Zinc finger nuclease (ZFN), a TAL-effector nuclease (TALEN), or a CRISPR/Cas nuclease system, that is provided in an expression vector, a liposome, or a nanoparticle.
4. A method of providing a protein to a subject in need thereof, the method comprising administering to the liver of 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 a liver-specific enhancer sequence, a promoter sequence, an intron sequence as shown in any one of SEQ ID NO:15, 16, 17, or nucleotides 340-432 of SEQ ID NO:37 and a transgene, wherein the transgene encodes the protein and the protein is produced in the subject.
5. The method of claim 4 , wherein the transgene is integrated into the genome of a liver cell.
6. The method of claim 5 , 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, wherein the nuclease is a ZFN a TALEN, or a CRISPR/Cas nuclease system, that is provided in an expression vector, a liposome, or a nanoparticle.
7. The method of claim 1 , wherein the enhancer sequence has mutations at positions 1, 5, 14, 32 and/or 39 of any of SEQ ID NOs:1 13.
8. The method of claim 7 , wherein the transgene is integrated into the genome of a liver cell.
9. The method of claim 8 , 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, wherein the nuclease is a ZFN, a TALEN, or a CRISPR/Cas nuclease system, that is provided in an expression vector, a liposome, or a nanoparticle.
10. The method of claim 1 , wherein the AAV vector further comprises an intron sequence, wherein the enhancer sequence comprises a wild-type or mutated Serpin 1 enhancer sequence; and/or the promoter sequence comprises a wild-type or mutated transthyretin (TTR) promoter sequence; and/or the intron sequence comprises a wild-type or mutated minute virus of mice (MVM) intron sequence.
11. The method of claim 10 , wherein the AAV vector comprises two insulator sequences flanking the enhancer sequence, the promoter sequence, the intron sequence, and the transgene.
12. The method of claim 11 , wherein the first insulator sequence comprises SEQ ID NO: 28 and the second insulator sequence comprises SEQ ID NO: 30.
13. The method of claim 1 , wherein the transgene encodes a protein lacking or deficient in a hemophilia or a lysosomal storage disease.
14. The method of claim 13 , wherein the transgene further encodes a nuclease, wherein the nuclease is a ZFN, a TALEN, or a CRISPR/Cas nuclease system.
15. The method of claim 1 , wherein the polynucleotide expression construct comprises the sequence of SEQ ID NO: 34.
16. The method of claim 1 , wherein the polynucleotide expression construct comprises the sequence of SEQ ID NO: 37.
17. The method of claim 16 , wherein the ITRs are AAV2 ITRs.
18. The method of claim 17 , wherein the vector further comprises an AAV serotype 6 (AAV6) capsid.
19. A method of providing replacement Factor VIII to a subject with hemophilia A in need thereof, the method comprising administering to the liver of 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 from AAV2, wherein the polynucleotide expression construct comprises the sequence of SEQ ID NO: 37, and wherein the AAV vector further comprises an AAV6 capsid.
20. The method of claim 4 , wherein the enhancer sequence comprises a wild-type or mutated Serpin 1 enhancer sequence; and/or the promoter sequence comprises a wild-type or mutated the transthyretin (TTR) promoter sequence.
21. The method of claim 4 , wherein the polynucleotide expression construct further comprises at least one insulator sequence and the polynucleotide expression construct optionally comprises a polyadenylation signal.
22. The method of claim 4 , wherein the transgene encodes a protein lacking or deficient in a hemophilia or a lysosomal storage disease.
23. The method of claim 22 , wherein the transgene further encodes a nuclease, wherein the nuclease is a ZFN, a TALEN, or a CRISPR/Cas nuclease system.
24. The method of claim 1 , wherein the transgene encodes replacement Factor VIII.
25. The method of claim 4 , wherein the transgene encodes replacement Factor VIII.
26. The method of claim 19 , wherein the AAV vector is administered intravenously into a peripheral vein of the subject.