Compositions and methods for modulating RPGR expression
Aspects of the disclosure relate to compositions and methods for modulating expression of certain RPGR retinitis pigmentosa GTPase regulator (RPGR) proteins in a cell or subject. In some embodiments the disclosure provides expression vectors encoding one or more RPGR proteins and/or one or more inhibitory nucleic acids that target certain isoforms of RPGR, for example RPGRCONST. In some aspects, the disclosure relates to methods of treating retinitis pigmentosa (RP) in a subject by administering compositions that modulate RPGR expression or activity.
1 . An isolated nucleic acid comprising an expression cassette that comprises a first region that encodes an inhibitory nucleic acid a miRNA or an artificial miRNA (amiRNA), the first region comprising a first strand that comprises SEQ ID NO 7 and a second strand that comprises SEQ ID NO 8.
2 . The isolated nucleic acid of claim 1 , wherein the expression cassette further comprises a second region encoding an RPGR ORF15 protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 17.
3 . The isolated nucleic acid of claim 1 , wherein the expression cassette is flanked by adeno associated virus (AAV) inverted terminal repeats (ITRs).
4 . A recombinant adeno-associated virus (rAAV) comprising:
(i) the isolated nucleic acid of claim 1 ; and
(ii) at least one AAV capsid protein.
5 . The rAAV of claim 4 , wherein the capsid protein is of a serotype selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh.10.
6 . The isolated nucleic acid of claim 1 , wherein the first region encodes an amiRNA.
7 . The isolated nucleic acid of claim 1 , wherein the first region further comprises a first promoter operably linked to the sequence encoding the miRNA or the amiRNA.
8 . The isolated nucleic acid of claim 7 , wherein the first promoter is a constitutive promoter, inducible promoter, or tissue-specific promoter.
9 . The isolated nucleic acid of claim 7 or claim 8 , wherein the first promoter is an RNA polymerase III (pol III) promoter.
10 . The isolated nucleic acid of claim 9 , wherein the pol III promoter is a U6 promoter or an H1 promoter.
11 . The isolated nucleic acid of claim 2 , wherein the RPGR ORF15 protein is encoded by the nucleic acid sequence set forth in any one of SEQ ID NO: 16 or 18.
12 . The isolated nucleic acid of claim 2 or 11 , wherein the second region comprises a second promoter operably linked to the sequence encoding the RPGR ORF15 protein.
13 . The isolated nucleic acid of claim 12 , wherein the second promoter is a constitutive promoter, inducible promoter, or tissue-specific promoter.
14 . The isolated nucleic acid of claim 12 , wherein the second promoter is an RNA polymerase II (pol II) promoter.
15 . A plasmid comprising the isolated nucleic acid of claim 1 .
16 . A host cell comprising the isolated nucleic acid of claim 1 .
17 . The isolated nucleic acid of claim 13 , wherein the second promoter is an RNA polymerase Il (pol II) promoter.