Self-regulating AAV vectors for safe expression of MeCP2 in rett syndrome
In some aspects, the disclosure relates to compositions and methods of engineering a transgene. In some embodiments, the disclosure provides self-regulating recombinant nucleic acids, viral vectors and pharmaceutical compositions comprising a MeCP2 transgene. In some embodiments, compositions and methods described by the disclosure are useful for treating diseases and disorders associated with a loss of function mutation, for example Rett syndrome.
1. A recombinant nucleic acid encoding a transcript having
(a) a coding region encoding human MeCP2 or a functional fragment thereof; and,
(b) a 3′-non-coding region comprising one or more miRNA binding sites, wherein transcript is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
2. The recombinant nucleic acid of claim 1 further comprising:
(c) a coding region encoding brain-derived neurotrophic factor (BDNF).
3. The recombinant nucleic acid of claim 1 , wherein the one or more miRNA binding sites comprise a binding site for miR-132.
4. The recombinant nucleic acid of claim 3 , wherein the miR-132 binding site comprises the sequence set forth in SEQ ID NO: 6.
5. The recombinant nucleic acid of claim 3 , wherein further comprising one or more binding sites for miR-122 or miR-1.
6. The recombinant nucleic acid of claim 5 , wherein the one or more binding sites comprises the nucleotide sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 5.
7. The recombinant nucleic acid of claim 1 , wherein the one or more miRNA binding sites comprise a binding site for miR-22.
8. The recombinant nucleic acid of claim 1 , wherein the one or more miRNA binding sites comprise a binding site for miR-19.
9. The recombinant nucleic acid of claim 1 , wherein the one or more miRNA binding sites comprise:
(a) at least one miRNA binding site specific for a miRNA that negatively regulates expression of the transcript; and
(b) at least one second miRNA binding site specific for a miRNA that inhibits expression of the transcript in a cell of a non-target tissue.
10. The recombinant nucleic acid of claim 1 , wherein the one or more miRNA binding sites comprise:
(a) at least one first miRNA binding site specific for a first miRNA that is positively regulated by expression of MeCP2 in a cell of a target tissue; and
(b) at least one second miRNA binding site specific for a second miRNA that is expressed, independent of expression of MeCP2, in cells of a non-target tissue.
11. The recombinant nucleic acid of claim 1 , wherein the coding region encodes MeCP2 isoform e1.
12. The recombinant nucleic acid of claim 1 , wherein the human MeCP2 comprises the sequence set forth in SEQ ID NO:1.
13. The recombinant nucleic acid of claim 1 , wherein the coding region encodes MeCP2 isoform e2.
14. The recombinant nucleic acid of claim 1 , wherein the human MeCP2 comprises the sequence set forth in SEQ ID NO: 2.
15. A recombinant adeno-associated virus (rAAV) comprising at least one AAV capsid protein and the recombinant nucleic acid of claim 1 .
16. The rAAV of claim 15 , wherein the AAV ITRs are AAV2 ITRs.
17. The rAAV of claim 15 , wherein the AAV capsid protein:
(i) has a serotype selected from the group consisting of AAV-PHP.B, AAV1, AAV2, AAV2i8, AAV2.5, AAV5, AAV6, AAV8, AAVrh8, AAV9, AAVrh10, AAV-B1, AAV9.45A-String, AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-CAM130, and AAV9HR.
18. The rAAV of claim 17 , wherein the AAV9 capsid protein comprises the sequence set forth in SEQ ID NO: 14.
19. A recombinant adeno-associated virus (rAAV) comprising:
(i) a nucleic acid encoding a transcript comprising a coding region encoding human MeCP2 or a functional fragment thereof; and, a 3′-non-coding region comprising one or more miR-132 miRNA binding sites, wherein transcript is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs); and
(ii) an AAV9 capsid protein.
20. The rAAV of claim 19 , further comprising one or more miR-19 miRNA binding sites in the 3′ non-coding region.
21. The rAAV of claim 20 , further comprising one or more miR-22 miRNA binding sites in the 3′ non-coding region.
22. The rAAV of claim 19 , wherein the AAV9 capsid protein comprises the sequence set forth in SEQ ID NO: 14.