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 (PD) and Gaucher disease. In some embodiments, the disclosure provides expression constructs comprising a transgene encoding beta-Glucocerebrosidase (GBA) or a portion thereof alone or in combination with one or more PD-associated genes. 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 TREM2 protein, wherein the TREM2 protein is encoded by the nucleic acid sequence in SEQ ID NO: 58; and (ii) two adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences flanking the expression construct.
2. The isolated nucleic acid of claim 1 , wherein the transgene is operably linked to a promoter, optionally wherein the promoter comprises a chicken beta-actin (CBA) promoter.
3. The isolated nucleic acid of claim 1 , wherein each ITR sequence is a wild-type AAV2 ITR sequence.
4. A recombinant adeno-associated virus (rAAV) vector comprising a nucleic acid comprising an expression construct comprising a transgene encoding a TREM2 protein flanked by two adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein the TREM2 protein is encoded by the nucleic acid sequence set forth in SEQ ID NO: 58.
5. The rAAV vector of claim 4 , wherein the transgene is operably linked to a promoter, optionally wherein the promoter comprises a chicken beta-actin (CBA) promoter.
6. The rAAV vector of claim 4 , wherein each ITR sequence is a wild-type AAV2 ITR sequence.
7. A recombinant adeno-associated virus (rAAV) comprising:
(i) an AAV capsid protein; and
(ii) the rAAV vector of claim 4 .
8. The rAAV of claim 7 , wherein the AAV capsid protein is AAV9 capsid protein.
9. A recombinant adeno-associated virus (AAV)(rAAV) vector comprising a nucleic acid comprising, in 5′ to 3′ order:
(a) a 5′ AAV inverted terminal repeat (ITR);
(b) a cytomegalovirus (CMV) enhancer;
(c) a chicken beta-actin (CBA) promoter;
(d) a transgene encoding a TREM2 protein, wherein the TREM2 protein is encoded by the nucleic acid sequence in SEQ ID NO: 58;
(e) a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE);
(f) a Bovine Growth Hormone polyA signal tail; and
(g) a 3′ AAV ITR.
10. A recombinant adeno-associated virus (rAAV) comprising:
(i) an AAV capsid protein; and
(ii) the rAAV vector of claim 9 .
11. The rAAV of claim 10 , wherein the AAV capsid protein is AAV9 capsid protein.
12. A plasmid comprising the rAAV vector of claim 9 .
13. A Baculovirus vector comprising the isolated nucleic acid of claim 1 .
14. A cell comprising:
(i) a first vector encoding one or more AAV rep protein and/or one or more AAV cap protein; and
(ii) a second vector comprising the isolated nucleic acid of claim 1 .
15. The cell of claim 14 , wherein the first vector is a plasmid and the second vector is a plasmid.
16. The cell of claim 14 , wherein the first vector is a Baculovirus vector and the second vector is a Baculovirus vector.
17. A method of producing an rAAV, the method comprising:
(i) delivering to a cell a first vector encoding one or more AAV rep protein and/or one or more AAV cap protein, and the rAAV vector of claim 9 ;
(ii) culturing the cells under conditions allowing for packaging the rAAV; and
(iii) harvesting the cultured host cell or culture medium for collection of the rAAV.
18. A method for treating a subject having or suspected of having Alzheimer's disease, the method comprising administering to the subject the rAAV of claim 7 .
19. The method of claim 18 , wherein the administration comprises direct injection to the CNS of the subject, optionally wherein the direct injection is intracerebral injection, intraparenchymal injection, intrathecal injection, intra-cisterna magna (ICM) injection or any combination thereof.
20. The method of claim 18 , wherein the administration comprises peripheral injection, optionally wherein the peripheral injection is intravenous injection.