Adeno-associated virus vectors
The invention provides an isolated and purified DNA molecule comprising at least one DNA segment, a biologically active subunit or variant thereof, of a circular intermediate of adeno-associated virus, which DNA segment confers increased episomal stability, persistence or abundance of the isolated DNA molecule in a host cell. The invention also provides a composition comprising at least two adeno-associated virus vectors.
1. A composition comprising:
a) a first adeno-associated virus vector comprising linked:
i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus;
ii) a second DNA segment comprising a promoter operably linked to a DNA fragment comprising an exon of a gene and a splice donor site, wherein the second DNA segment does not encode a full-length polypeptide; and
iii) a third DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus; and
b) a second adeno-associated virus vector comprising linked:
i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus;
ii) a second DNA segment comprising a splice acceptor site and a DNA fragment with at least one other exon which together with the DNA segment of (a)(ii) encodes a full-length polypeptide; and
iii) a third DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus.
2. The composition of claim 1 further comprising a delivery vehicle.
3. A method to transfer and express a polypeptide in a host cell comprising contacting the host cell with the composition of claim 1 .
4. A method to transfer at least a portion of an open reading frame to a host cell comprising contacting the host cell with a first adeno-associated virus vector comprising linked:
i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus;
ii) a second DNA segment comprising a promoter operably linked to a DNA fragment comprising an exon of a gene and a splice donor site, wherein the second DNA segment does not encode a full-length polypeptide; and
iii) a third DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus.
5. The method of claim 4 wherein the host cell is further contacted with a second adeno-associated virus vector comprising linked:
i) a first DNA segment comprising a 5′-inverted terminal repeat of adeno-associated virus;
ii) a second DNA segment comprising a splice acceptor site and a DNA fragment with at least one other exon which together with the DNA segment of (a)(ii) encodes a full-length polypeptide; and
iii) a third DNA segment comprising a 3′-inverted terminal repeat of adeno-associated virus.
6. The method of claim 3 or 4 wherein the host cell is a lung epithelial cell, a muscle cell or a neuron.
7. The method of claim 3 or 5 wherein the polypeptide is the CFTR polypeptide.
8. A method of expressing a gene product in the muscle tissue of an animal, comprising contacting the muscle tissue with the composition of claim 1 in an amount effective to express the polypeptide.
9. A method of expressing a gene product in the airway epithelia of an animal, comprising contacting the airway epithelia with the composition of claim 1 in an amount effective to express the polypeptide.
10. A method of expressing a gene product in the neurons of an animal, comprising contacting the neurons with the composition of claim 1 in an amount effective to express the polypeptide.
11. The composition of claim 1 wherein the exon in the second DNA segment of the first adeno-associated virus vector is a first exon of a gene for the full-length polypeptide.
12. The method of claim 4 wherein the exon in the second DNA segment of the first adeno-associated virus vector is a first exon of a gene for the full-length polypeptide.