RAAV-based compositions and methods for treating alpha-1 anti-trypsin deficiencies
The invention relates to isolated nucleic acids and rAAV-based compositions, methods and kits useful for treating genetic diseases (e.g., alpha-1 antitrypsin deficiency).
1. A recombinant adeno-associated viral associated vector (rAAV vector) comprising:
(a) a first region that encodes one or more miRNAs comprising a nucleic acid having sufficient sequence complementarity with an endogenous mRNA of a subject to hybridize with and inhibit expression of the endogenous mRNA, wherein the one or more miRNAs comprises the sequence set forth in SEQ ID NO: 17, 18, 19, 21, 22, or 23, wherein the endogenous mRNA encodes a mutant alpha-antitrypsin (AAT) protein; and
(b) a second region encoding an exogenous mRNA that encodes a second protein, wherein the second protein is a wild-type AAT protein, wherein the exogenous mRNA has one or more silent mutations as compared with the endogenous mRNA,
wherein the one or more miRNAs do not comprise a nucleic acid having sufficient sequence complementary to hybridize with and inhibit expression of the exogenous mRNA.
2. The rAAV vector of claim 1 , wherein the first region is positioned within an untranslated portion of the second region.
3. The rAAV vector of claim 1 , wherein the first region is between the first codon of the exogenous mRNA and 1000 nucleotides upstream of the first codon.
4. The rAAV vector of claim 1 , wherein the first region encodes two miRNAs, or three miRNAs.
5. The rAAV vector of claim 1 , wherein the AAT protein is a human AAT protein.
6. The rAAV vector of claim 1 , wherein the one or more miRNAs comprises the sequence set forth in SEQ ID NO: 17.
7. A composition comprising the rAAV vector of claim 1 .
8. A kit comprising a container housing the composition of claim 7 .
9. A method of expressing Alpha 1-Antitrypsin (AAT) protein in a subject, the method comprising:
administering to a subject an effective amount of a rAAV vector of claim 1 .
10. The method of claim 9 , wherein the rAAV vector is administered to the muscle tissue, liver or lung of the subject.
11. A method of expressing Alpha 1-Antitrypsin (AAT) protein in a subject, the method comprising:
isolating cells or tissue from a subject;
contacting the cells or tissue with an effective amount of a rAAV vector of claim 1 , thereby producing transfected cells or tissue; and
administering the transfected cells or tissue to the subject.
12. The method of claim 9 , wherein the administration occurs by intravenous, intramuscular, subcutaneous, or intraperitoneal administration.
13. A recombinant adeno-associated virus (rAAV) comprising:
(i) a rAAV vector of claim 1 ; and,
(ii) an adeno-associated virus (AAV) capsid protein.
14. The rAAV of claim 13 , wherein the capsid protein is a AAV2, AAV3, AAV8, or AAV9 capsid protein, or a variant thereof.
15. The rAAV vector of claim 2 , wherein the untranslated portion is an intron.
16. The rAAV vector of claim 1 , wherein the one or more miRNAs comprises the sequence set forth in SEQ ID NO: 18.
17. The rAAV vector of claim 1 , wherein the one or more miRNAs comprises the sequence set forth in SEQ ID NO: 19.
18. The rAAV vector of claim 1 , wherein the one or more miRNAs comprises the sequence set forth in SEQ ID NO: 21.
19. The rAAV vector of claim 1 , wherein the one or more miRNAs comprises the sequence set forth in SEQ ID NO: 22.
20. The rAAV vector of claim 1 , wherein the one or more miRNAs comprises the sequence set forth in SEQ ID NO: 23.