Gene therapies for lysosomal disorders
The disclosure relates, in some aspects, to compositions and methods for treatment of diseases associated with aberrant lysosomal function, for example Parkinson's disease and Gaucher disease. In some embodiments, the disclosure provides expression constructs comprising a transgene encoding one or more inhibitory nucleic acids targeting SCNA or a portion thereof, TMEM106B or a portion thereof, or any combination of the foregoing. In some embodiments, the disclosure provides methods of Parkinson's disease by administering such expression constructs to a subject in need thereof.
1. An isolated nucleic acid comprising
(i) an expression construct comprising a transgene encoding a single gene product, wherein the gene product is an inhibitory nucleic acid targeting α-Synuclein, wherein the inhibitory nucleic acid is encoded by the sequence set forth in SEQ ID NO: 3; and
(ii) two adeno-associated virus (AAV) inverted terminal repeats (ITR) sequences flanking the expression construct, wherein the first ITR sequence is a 5′ ITR, and the second ITR sequence is a 3′ ITR.
2. The isolated nucleic acid of claim 1 , wherein the transgene is operably linked to a promoter.
3. The isolated nucleic acid of claim 2 , wherein the promoter is a chicken beta actin (CBA) promoter.
4. The isolated nucleic acid of claim 3 , further comprising a CMV enhancer.
5. The isolated nucleic acid of claim 1 , further comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
6. The isolated nucleic acid of claim 1 , further comprising a Bovine Growth Hormone polyA signal tail.
7. The isolated nucleic acid of claim 1 , wherein each of the two ITR sequences is a wild-type AAV2 ITR sequence.
8. The isolated nucleic acid of claim 1 , wherein each of the two ITR sequences comprises a “D” region comprising SEQ ID NO: 14 that is proximal to the expression construct.
9. The isolated nucleic acid of claim 1 , wherein at least one of the ITR sequences comprises a “D” region comprising SEQ ID NO: 14 positioned on the outside of the ITR sequence relative to the expression construct.
10. The isolated nucleic acid of claim 1 , wherein the ITR sequence positioned 5′ relative to the expression construct comprises a “D” region comprising SEQ ID NO: 14 that is proximal to the expression construct, and the ITR sequence positioned 3′ relative to the expression construct comprises a “D” region comprising SEQ ID NO: 14 positioned on the outside of the ITR sequence relative to the expression construct.
11. The isolated nucleic acid of claim 1 , wherein the nucleic acid sequence of the 5′ ITR is nucleotides 1-145 of SEQ ID NO: 25 and the nucleic acid sequence of the 3′ ITR is nucleotides 4018-4162 of SEQ ID NO: 25.
12. The isolated nucleic acid of claim 1 , further comprising a TRY region between the 5′ ITR and the expression construct, wherein the TRY region has the sequence set forth in SEQ ID NO: 15.
13. The isolated nucleic acid of claim 1 , wherein the expression construct comprises the sequence set forth in nucleotides 1105-1367 of SEQ ID NO: 23.
14. A recombinant adeno-associated virus (rAAV) vector comprising
(i) an expression construct comprising a transgene encoding a single gene product, wherein the gene product is an inhibitory nucleic acid targeting α-Synuclein, wherein the inhibitory nucleic acid is encoded by the sequence set forth in SEQ ID NO: 3; and
(ii) two AAV ITR sequences flanking the expression construct, wherein the first ITR sequence is a 5′ ITR, and the second ITR sequence is a 3′ ITR.
15. The rAAV vector of claim 14 , wherein the transgene is operably linked to a promoter.
16. The rAAV vector of claim 15 , wherein the promoter is a CBA promoter.
17. The rAAV vector of claim 16 , further comprising a CMV enhancer.
18. The rAAV vector of claim 14 , further comprising a WPRE.
19. The rAAV vector of claim 14 , further comprising a Bovine Growth Hormone polyA signal tail.
20. The rAAV vector of claim 14 , wherein each of the two ITR sequences is a wild-type AAV2 ITR sequence.
21. The rAAV vector of claim 14 , wherein each of the two ITR sequences comprises a “D” region comprising SEQ ID NO: 14 that is proximal to the expression construct.
22. The rAAV vector of claim 14 , wherein at least one of the ITR sequences comprises a “D” region comprising SEQ ID NO: 14 positioned on the outside of the ITR sequence relative to the expression construct.
23. The rAAV vector of claim 14 , wherein the ITR sequence positioned 5′ relative to the expression construct comprises a “D” region comprising SEQ ID NO: 14 that is proximal to the expression construct, and the ITR sequence positioned 3′ relative to the expression construct comprises a “D” region comprising SEQ ID NO: 14 positioned on the outside of the ITR sequence relative to the expression construct.
24. The rAAV vector of claim 14 , wherein the nucleic acid sequence of the 5′ ITR is nucleotides 1-145 of SEQ ID NO: 25 and the nucleic acid sequence of the 3′ ITR is nucleotides 4018-4162 of SEQ ID NO: 25.
25. The rAAV vector of claim 14 , further comprising a TRY region between the 5′ ITR and the expression construct, wherein the TRY region has the sequence set forth in SEQ ID NO: 15.
26. The rAAV vector of claim 14 , wherein the expression construct comprises the sequence set forth in nucleotides 1105-1367 of SEQ ID NO: 23.
27. A rAAV comprising:
(i) an AAV capsid protein; and
(ii) the rAAV vector of claim 14 .
28. The rAAV of claim 27 , wherein the AAV capsid protein is AAV9 capsid protein.
29. A rAAV vector comprising a nucleic acid comprising, in 5′ to 3′ order:
(a) a 5′ AAV ITR;
(b) a CMV enhancer;
(c) a CBA promoter;
(d) a transgene encoding a single gene product, wherein the gene product is an inhibitory nucleic acid targeting α-Synuclein, wherein the inhibitory nucleic acid is encoded by the sequence set forth in SEQ ID NO: 3;
(e) a WPRE;
(f) a Bovine Growth Hormone polyA signal tail; and
(g) a 3′ AAV ITR.
30. The rAAV vector of claim 29 , wherein the expression construct comprises the sequence set forth in nucleotides 1105-1367 of SEQ ID NO: 23.
31. A rAAV comprising:
(i) an AAV capsid protein; and
(ii) the rAAV vector of claim 29 .
32. The rAAV of claim 31 , wherein the AAV capsid protein is AAV9 capsid protein.
33. A plasmid comprising the rAAV vector of claim 14 .
34. A Baculovirus vector comprising a transgene encoding a single gene product, wherein the gene product is an inhibitory nucleic acid coding sequence comprising the sequence set forth in SEQ ID NO: 3, and two AAV ITR sequences flanking the sequence set forth in SEQ ID NO: 3.
35. An isolated cell comprising:
(i) a first vector encoding one or more AAV rep proteins and/or one or more AAV cap proteins; and
(ii) a second vector comprising a transgene encoding a single gene product, wherein the gene product is an inhibitory nucleic acid coding sequence comprising the sequence set forth in SEQ ID NO: 3, and two AAV ITR sequences flanking the sequence set forth in SEQ ID NO: 3.
36. The cell of claim 35 , wherein the first vector is a plasmid and the second vector is a plasmid.
37. The cell of claim 35 , wherein the cell is a mammalian cell.
38. The cell of claim 37 , wherein the mammalian cell is a HEK293 cell.
39. The cell of claim 35 , wherein the first vector is a Baculovirus vector and the second vector is a Baculovirus vector.
40. The cell of claim 39 , wherein the cell is an insect cell.
41. A method of producing the rAAV of claim 27 , the method comprising:
(i) delivering to a cell a first vector encoding one or more AAV rep proteins and/or one or more AAV cap proteins, and the rAAV vector;
(ii) culturing the cell of (i) under conditions allowing for packaging the rAAV; and
(iii) harvesting the cultured cells or culture medium for collection of the rAAV.