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 method for treating Parkinson's disease characterized by a mutation in a GBA1 gene in a subject, the method comprising administering directly to the central nervous system of the subject a recombinant adeno-associated virus (rAAV) comprising:
(i) an AAV9 capsid protein; and
(ii) a 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 the 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, and wherein the rAAV vector comprises two adeno-associated virus inverted terminal repeats (ITR) sequences flanking the expression construct:
thereby mediating the treatment of the Parkinson's disease characterized by the mutation in the GBA1 gene in the subject.
2. The method of claim 1 , wherein the promoter is a chicken beta actin (CBA) promoter.
3. The method of claim 1 , wherein the rAAV vector further comprises a CMV enhancer.
4. The method of claim 1 , wherein the rAAV vector further comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE).
5. The method of claim 1 , wherein the rAAV vector further comprises a Bovine Growth Hormone polyA signal tail.
6. The method of claim 1 , wherein each of the two ITR sequences is a wild-type AAV2 ITR sequence.
7. The method of claim 1 , wherein each of the two ITR sequences comprises a “D” region (SEQ ID NO: 27) that is proximal to the expression construct.
8. The method of claim 1 , wherein at least one of the two ITR sequences comprises a “D” region (SEQ ID NO: 27) positioned on the outside of the ITR sequence relative to the expression construct.
9. The method of claim 1 , 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.
10. The method of claim 9 , 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 nucleotides 3867-4011 of SEQ ID NO: 1.
11. The method of claim 9 , wherein the rAAV vector further comprises comprising a TRY region between the 5′ ITR and the expression construct, wherein the TRY region has the nucleotide sequence of SEQ ID NO: 28.
12. The method of claim 1 , wherein the rAAV is administered by intra-cisterna magna injection.
13. A method for treating Parkinson's disease characterized by a mutation in a GBA1 gene in a subject, the method comprising administering directly to the central nervous system of the rAAV comprising:
(i) an AAV9 capsid protein; and
(ii) 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 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,
thereby mediating the treatment of the Parkinson's disease characterized by the mutation in the GBA1 gene in the subject.
14. The method of claim 11 , wherein the rAAV is administered by intra-cisterna magna injection.
15. A method for increasing activity of a Gcase protein in a subject with Parkinson's disease characterized by a mutation in a GBA1 gene, the method comprising administering directly to the central nervous system of the subject a rAAV comprising:
(i) an AAV9 capsid protein; and
(ii) a rAAV vector comprising a nucleic acid comprising an expression construct comprising a promoter operably linked to a transgene insert encoding the Gcase protein, wherein
the transgene insert comprises the nucleotide sequence of SEQ ID NO: 15, which is a codon optimized version of the 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, and wherein the rAAV vector comprises two adeno-associated virus ITRs sequences flanking the expression construct;
thereby mediating the increase in the activity in the Gcase protein in the subject with Parkinson's disease characterized by the mutation in the GBA1 gene.