Optimized RPE65 promoter and coding sequences
The present invention relates to the prevention and/or treatment of retinal dystrophyin a patient, including Leber congenital amaurosis (LCA).
1. An expression construct comprising a promoter and an operably linked polynucleotide sequence, wherein the promoter consists of:
(a) a sequence of no more than 800 contiguous nucleotides from SEQ ID NO:1 comprising nucleotides 12-761 of SEQ ID NO:2, or
(b) a sequence having at least 90% sequence identity to said sequence of (a); and
wherein the operably linked polynucleotide sequence comprises the sequence of SEQ ID NO: 4.
2. The expression construct of claim 1 , wherein the promoter:
(a) comprises the sequence of SEQ ID NO: 2 , or
(b) consists of a sequence with at least 90% sequence identity to said sequence of SEQ ID NO: 2, wherein the sequence of the promoter is no longer than 800 nucleotides.
3. A vector comprising an expression construct according to claim 1 .
4. The vector according to claim 3 , wherein the vector is a viral vector.
5. The vector according to claim 4 , wherein the vector is an adeno-associated virus (AAV) vector or comprises an AAV genome or a derivative thereof.
6. The vector according to claim 5 , wherein said derivative is a chimeric, shuffled or capsid modified derivative.
7. The vector according to claim 5 , wherein said AAV genome is from a naturally derived serotype or isolate or clade of AAV.
8. The vector according to claim 7 , wherein said AAV genome is from AAV serotype 2 (AAV2), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5) or AAV serotype 8 (AAV8) and/or wherein the capsid is derived from AAV5 or AAV8.
9. The vector according to claim 8 , wherein the genome is derived from AAV2 and the capsid is derived from AAV5 or AAV8.
10. A host cell that contains a vector of claim 3 .
11. The cell according to claim 10 that is a HEK293 or HEK293T cell.
12. A pharmaceutical composition comprising a vector of claim 3 and a pharmaceutically acceptable carrier.
13. The expression construct of claim 1 , wherein the promoter consists of a sequence of no more than 800 contiguous nucleotides from SEQ ID NO:1 comprising nucleotides 12-761 of SEQ ID NO:2.
14. The expression construct of claim 2 , wherein the promoter comprises the sequence of SEQ ID NO: 2.
15. A promoter which: (a) comprises the sequence of SEQ ID NO: 2, or (b) consists of a sequence having at least 90% sequence identity to said sequence of SEQ ID NO: 2, wherein the sequence of the promoter is no longer than 800 nucleotides.
16. A vector comprising a promoter according to claim 15 .
17. The vector according to claim 16 , wherein the vector which is a viral vector.
18. An expression construct comprising the promoter of claim 15 , operably linked to a polynucleotide sequence.
19. The expression construct according to claim 18 , wherein the operably linked polynucleotide sequence is an RPE65, MERTK, LRAT, TYR, GRP143 or MY07A coding sequence, or a sequence encoding a neurotrophic factor, an anti-angiogenic polypeptide or a polypeptide with anti-apoptotic effects in the RPE.
20. The expression construct according to claim 19 , wherein the operably linked polynucleotide sequence comprises a sequence selected from SEQ ID NOs: 3 to 8, or that has at least 80% sequence identity to any one of SEQ ID NOs: 3 to 8.
21. A promoter consisting of:
(a) a sequence of no more than 800 contiguous nucleotides from SEQ ID NO:1 comprising nucleotides 12-761 of SEQ ID NO:2, or
(b) a sequence having at least 90% sequence identity to said sequence of (a).