Method for transducing cells with primary cilia
View Patent ↗This invention provides methods for transducing a ciliated cell with a recombinant serotype 2 adeno-associated virus (AAV) vector. Additionally, the invention provides methods of treating diseases associated with a mutated gene by transducing a ciliated cell with a recombinant serotype 2 AAV vector containing a corrective transgene.
1. An in vivo method for transducing a human retinal pigment epithelial (RPE) cell having a mutated fibrillin-like extracellular matrix protein 1 (EFEMP1) gene comprising the step of administering subretinally a recombinant adeno-associated virus (AAV) vector comprising a capsid from an AAV of serotype AAV1, AAV2, AAV5, AAV8, or AAV9; and a wild-type EFEMP1 transgene, wherein the human RPE cell is transduced and the human RPE cell expresses a wild-type EFEMP1 gene.
2. The method of claim 1 , wherein said capsid is from an AAV of serotype AAV2.
3. The method of claim 1 , wherein said transgene is bounded by inverted terminal repeats (ITRs).
4. The method of claim 3 , wherein said ITRs are of serotype 2 AAV.
5. The method of claim 1 , wherein said transgene is under the control of an RPE specific promoter.
6. The method of claim 5 , wherein the RPE-specific promoter is the human vitelliform macular dystrophy 2 (VMD2) promoter.
7. The method of claim 1 , wherein said cell forms tight junctions with neighboring cells and forms a layer of cells having a transepithelial resistance (TER) of >200 Ω/cm 2 .
8. The method of claim 1 , wherein said transgene encodes a protein secreted from the cell apically.
9. The method of claim 1 , wherein said transgene encodes a protein secreted from the cell basolaterally.
10. The method of claim 1 , wherein said cell is a polarized RPE cell.
11. The method of claim 1 , wherein said cell is a pigmented RPE cell.
12. The method of claim 3 , wherein said ITRs are from an AAV of a serotype other than serotype 2.
13. The method of claim 2 , wherein said AAV vector comprises inverted terminal repeats (ITRs) from an AAV of a serotype other than serotype 2.
14. The method of claim 2 , wherein said AAV vector comprises inverted terminal repeats (ITRs) from an AAV of serotype 2.
15. The method of claim 1 , wherein administering the AAV vector comprises administering by injection.
16. The method of claim 1 , wherein the transgene encodes a secreted protein.
17. The method of claim 1 , wherein the method comprises detecting transduced RPE cells.
18. The method of claim 17 , wherein the detecting comprises detecting fluorescence.
19. The method of claim 1 , wherein a population of human RPE cells are contacted with the recombinant AAV, wherein 80%-90% of the human RPE cells are transduced.
20. An in vitro method for transducing a human retinal pigment epithelial (RPE) cell having a mutated fibrillin-like extracellular matrix protein 1 (EFEMP1) gene comprising the steps of:
(a) providing a human RPE cell having a mutated EFEMP1 gene; and
(b) contacting said RPE cell with a recombinant adeno-associated virus (AAV) vector comprising comprising a capsid from an AAV of serotype AAV1, AAV2, AAV5, AAV8, or AAV9; and a wild-type EFEMP1 transgene, wherein the human retinal pigment epithelial cell is transduced and the human RPE cell expresses a wild-type EFEMP1 gene.
21. The method of claim 20 , wherein said capsid is from an AAV of serotype AAV2.
22. The method of claim 20 , wherein said transgene is bounded by inverted terminal repeats (ITRs).
23. The method of claim 22 , wherein said ITRs are of serotype 2 AAV.
24. The method of claim 20 , wherein said transgene is under the control of an RPE specific promoter.
25. The method of claim 24 , wherein the RPE-specific promoter is the human vitelliform macular dystrophy 2 (VMD2) promoter.
26. The method of claim 20 , wherein said cell forms tight junctions with neighboring cells and forms a layer of cells having a transepithelial resistance (TER) of >200 Ω/cm 2 .
27. The method of claim 20 , wherein said transgene encodes a protein secreted from the cell apically.
28. The method of claim 20 , wherein said transgene encodes a protein secreted from the cell basolaterally.
29. The method of claim 20 , wherein said cell is a polarized RPE cell.
30. The method of claim 20 , wherein said cell is a pigmented RPE cell.
31. The method of claim 22 , wherein said ITRs are from an AAV of a serotype other than serotype 2.
32. The method of claim 21 , wherein said AAV vector comprises inverted terminal repeats (ITRs) from an AAV of a serotype other than serotype 2.
33. The method of claim 21 , wherein said AAV vector comprises inverted terminal repeats (ITRs) from an AAV of serotype 2.
34. The method of claim 20 , wherein contacting the cell comprises administering the AAV vector comprises administering by injection.
35. The method of claim 20 , wherein the transgene encodes a secreted protein.
36. The method of claim 20 , wherein the method comprises detecting transduced RPE cells.
37. The method of claim 36 , wherein the detecting comprises detecting fluorescence.
38. The method of claim 20 , wherein a population of human RPE cells are contacted with the recombinant AAV, wherein 80%-90% of the human RPE cells are transduced.