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 beta-Glucocerebrosidase (GBA) or a portion thereof, Lysosomal Membrane Protein 2 (LIMP2), Prosaposin, 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. A recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15, which is a codon optimized version of wild type GBA1 nucleotide sequence, wherein the codon optimized sequence eliminates a predicted donor splice site that begins at nucleotide 49 in the wild type GBA1 nucleotide sequence.
2. The rAAV vector of claim 1 , wherein the promoter is a chicken beta actin (CBA) promoter.
3. The rAAV vector of claim 1 , further comprising a CMV enhancer.
4. The rAAV vector of claim 1 , further comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
5. The rAAV vector of claim 1 , further comprising a Bovine Growth Hormone polyA signal tail.
6. The rAAV vector of claim 1 , wherein the nucleic acid comprises two adeno-associated virus inverted terminal repeats (ITR) sequences flanking the expression construct.
7. The rAAV vector of claim 6 , wherein each ITR sequence is a wild-type AAV2 ITR sequence.
8. The rAAV vector of claim 6 , wherein each ITR sequence comprises a “D” region (SEQ ID NO: 27) that is proximal to the expression construct.
9. The rAAV vector of claim 6 , wherein at least one of the ITR sequences comprises a “D” region (SEQ ID NO: 27) positioned on the outside of the ITR sequence relative to the expression construct.
10. The rAAV vector of claim 6 , wherein the ITR sequence positioned 5′ relative to the expression construct comprises a “D” region (SEQ ID NO: 27) that is proximal to the expression construct, and the ITR sequence positioned 3′ relative to the expression construct comprises a “D” region (SEQ ID NO: 27) positioned on the outside of the ITR sequence relative to the expression construct.
11. The rAAV vector of claim 10 , wherein the nucleic acid sequence of the 5′ ITR is nucleotides 1 145 of SEQ ID NO: 1 and the nucleic acid sequence of the 3′ ITR is nucleic nucleotides 3867 4011 of SEQ ID NO: 1.
12. The rAAV vector of claim 10 , 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: 28.
13. An rAAV comprising:
(i) an AAV capsid protein; and
(ii) the rAAV vector of claim 1 .
14. The rAAV of claim 13 , wherein the AAV capsid protein is AAV9 capsid protein.
15. An 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 insert encoding a Gcase protein, wherein the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15;
(e) a WPRE;
(f) a Bovine Growth Hormone polyA signal tail; and
(g) a 3′ AAV ITR.
16. An rAAV comprising:
(i) an AAV capsid protein; and
(ii) the rAAV vector of claim 15 .
17. The rAAV of claim 16 , wherein the AAV capsid protein is AAV9 capsid protein.