Modified MGLUR6 promoter and methods of use
The invention provides nucleic acids and nucleic acid expression vectors containing optimized mGluR6 promoters for expression of transgenes in the retina. The compositions and methods of the invention are useful for expression of gene products to preserve, improve, or restore phototransduction or vision.
1. An isolated nucleic acid molecule comprising
a. an mGluR6 enhancer or a variant thereof at least 70% identical to SEQ ID NO: 1 or SEQ ID NO: 2;
b. an mGluR6 promoter or a variant thereof at least 70% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6;
c. an intron 4 of the mGluR6 gene or a variant thereof at least 70% identical to SEQ ID NO: 7 or SEQ ID NO. 8; and
d. an intron 3 of the mGluR6 gene or a variant thereof at least 70% identical to SEQ ID NO: 9 or SEQ ID NO. 10.
2. The isolated nucleic acid molecule of claim 1 , wherein said mGluR6 enhancer comprises the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein said mGluR6 promoter comprises the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein said intron 4 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and wherein said intron 3 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.
3. The isolated nucleic acid molecule of claim 1 , wherein the intron 4 of the mGluR6 gene is upstream of the intron 3 of the mGluR6 gene, wherein the intron 3 of the mGluR6 gene is upstream of the mGluR6 enhancer, and wherein the mGluR6 enhancer is upstream of the mGluR6 promoter.
4. A nucleic acid expression vector comprising the nucleic acid molecule of claim 1 operably linked to at least one transgene.
5. The nucleic acid expression vector of claim 4 , wherein said mGluR6 enhancer comprises the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein said mGluR6 promoter comprises the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein said intron 4 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and wherein said intron 3 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.
6. The nucleic acid expression vector of claim 4 , wherein the intron 4 of the mGluR6 gene is upstream of the intron 3 of the mGluR6 gene, wherein the intron 3 of the mGluR6 gene is upstream of the mGluR6 enhancer, and wherein the mGluR6 enhancer is upstream of the mGluR6 promoter.
7. The nucleic acid expression vector of claim 4 , wherein the vector is an adeno-associated virus vector or a recombinant adeno-associated virus (rAAV) vector.
8. The nucleic acid expression vector of claim 7 , wherein the vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 vector.
9. The nucleic acid expression vector of claim 4 , wherein the vector comprises a capsid protein comprising at least one mutation.
10. The nucleic acid expression vector of claim 9 , wherein said at least one mutation is selected from the group consisting of:
a tyrosine (Y) to phenylalanine (F) at amino acid position 444;
a tyrosine (Y) to phenylalanine (F) at amino acid position 730;
a tyrosine (Y) to phenylalanine (F) at amino acid positions 272, 444, 500, and 730;
a threonine (T) to valine (V) at amino acid position 491; and
a tyrosine (Y) to phenylalanine (F) at amino acid positions 272, 444, 500, 730, and a threonine (T) to valine (V) at amino acid position 491,
wherein the amino acid positions correspond to amino acid positions of the AAV capsid protein.
11. The nucleic acid expression vector of claim 9 , wherein the capsid protein comprises a peptide insert selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37.
12. The nucleic acid expression vector of claim 4 , wherein the transgene is an opsin gene.
13. The nucleic acid expression vector of claim 12 , wherein said opsin gene is selected from the group consisting of channelrhodopsin, melanopsin, pineal opsin, photopsins, halorhodopsin, bacteriorhodopsin and proteorhodopsin.
14. The nucleic acid expression vector of claim 4 , wherein the transgene encodes a gene product that increases light sensitivity, increases light detection, increases photosensitivity, increases visual evoked potential, or restores vision in a retina.
15. A pharmaceutical composition comprising the nucleic acid expression vector of claim 4 and a pharmaceutically acceptable excipient.
16. A method for expressing a transgene in the eye comprising introducing into the eye the nucleic acid expression vector of any one of claims 4 - 14 .
17. A method for expressing a transgene in the eye comprising introducing into the eye the pharmaceutical composition of claim 15 .