Frataxin expression constructs
The invention provides polynucleotides, vectors and viruses expressing frataxin and methods of treating Friedreich's Ataxia.
1. A polynucleotide comprising a nucleic acid molecule encoding a human frataxin polypeptide having an amino acid sequence of SEQ ID NO:1, wherein the nucleic acid molecule is operably linked to a 5′UTR and a 3′UTR, wherein:
the 5′UTR is RPL6-5′Splice and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7; or
the 5′UTR is 5U2 and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7,
wherein the encoded human frataxin polypeptide is a functional frataxin protein.
2. The polynucleotide of claim 1 , wherein said nucleic acid molecule encoding a human frataxin polypeptide is operably connected to a promoter.
3. The polynucleotide of claim 2 wherein said promoter is an inducible promoter.
4. The polynucleotide of claim 3 further comprising a gene switch that regulates expression from said inducible promoter.
5. The polynucleotide of claim 4 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch.
6. The polynucleotide of claim 2 wherein said promoter is a CMV promoter, a UBC promoter, an EF1α promoter, a PGK1 promoter or a minimal frataxin promoter.
7. The polynucleotide of claim 2 wherein the promoter is a UBC promoter, the 5′UTR is 5U2, and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7.
8. A viral vector comprising the polynucleotide of claim 1 .
9. The viral vector of claim 8 wherein said vector is an adeno-associated viral vector.
10. The viral vector of claim 9 wherein said adeno-associated viral vector is AAV5.
11. A recombinant virion which comprises a viral vector, wherein said vector comprises a nucleic acid molecule encoding human frataxin operably linked to a 5′UTR, a 3′UTR, and a control element that directs transcription and/or translation, wherein:
the 5′UTR is RPL6-5′Splice and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7; or
the 5′UTR is 5U2 and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7.
12. The virion of claim 11 wherein said vector is an adeno-associated viral vector.
13. The virion of claim 12 wherein said adeno-associated viral vector is AAV5.
14. The virion of claim 11 , wherein said nucleic acid encodes a protein with the amino acid sequence of SEQ ID NO:1.
15. The virion of claim 11 , wherein said nucleic acid encodes a protein with the amino acid sequence of SEQ ID NO:1, the control element is a UBC promoter, the 5′UTR is 5U2, and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7.
16. A composition comprising:
(a) a viral vector, wherein said vector comprises a nucleic acid molecule encoding human frataxin operably linked to a 5′UTR, a 3′UTR, and a control element that directs transcription and/or translation, wherein:
the 5′UTR is RPL6-5′Splice and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7; or
the 5′UTR is 5U2 and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7; and
(b) a pharmaceutically acceptable excipient.
17. The composition of claim 16 wherein said vector is an adeno-associated viral vector.
18. The composition of claim 17 wherein said adeno-associated viral vector is AAV5.
19. The composition of claim 16 , wherein said nucleic acid encodes a protein with the amino acid sequence of SEQ ID NO:1.
20. The composition of claim 16 , wherein said nucleic acid encodes a protein with the amino acid sequence of SEQ ID NO:1, the control element is a UBC promoter, the 5′UTR is 5U2, and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7.
21. The polynucleotide of claim 2 , wherein the promoter is a UBC promoter, the 5′UTR is RPL6-5′ Splice, and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7.
22. The virion of claim 11 , wherein said nucleic acid encodes a protein with the amino acid sequence of SEQ ID NO:1, the control element is a UBC promoter, the 5′UTR is RPL6-5′ Splice, and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7.
23. The composition of claim 16 , wherein said nucleic acid encodes a protein with the amino acid sequence of SEQ ID NO:1, the control element is a UBC promoter, the 5′UTR is RPL6-5′ Splice, and the 3′UTR is a synthetic 3′ regulatory element having a nucleic acid sequence of SEQ ID NO:7.