Claims
- 1. A recombinant pseudotyped adeno-associated virion for use in neural cells comprising:
a transgene flanked 5′ and 3′ by inverted terminal repeat sequences, wherein the inverted terminal repeat sequences are derived from a first adeno-associated virus; and a non-native capsid derived from a second adeno-associated virus that is different from the first adeno-associated virus, such that the transgene is packaged within the non-native capsid, and wherein the non-native capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and upon entering a cell has a transduction rate that is about 2-fold to about 30-fold higher than the transduction rate of the corresponding wild type adeno-associated virion.
- 2. The recombinant virion of claim 1, wherein the transduction rate is determined by densiometry analysis.
- 3. The recombinant virion of claim 1, wherein the first adeno-associated virus type is selected from the group consisting of AAV 1, AAV2, AAV3, AAV4, AAV5, and AAV6.
- 4. The recombinant virion of claim 1, wherein second adeno-associated virus type is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6 and is different from the first AAV.
- 5. The recombinant virion of claim 1, wherein the neural cell is present in a region of a brain or spinal cord.
- 6. The recombinant virion of claim 5, wherein the region of the brain is the stratium.
- 7. The recombinant virion of claim 5, wherein the region of the brain is the hippocampus.
- 8. A recombinant pseudotyped adeno-associated virus type-1 virion for use in neural cells comprising:
a transgene flanked 5′ and 3′ by inverted terminal repeat sequences, wherein the inverted terminal repeat sequences are derived from adeno-associated virus type-2 (AAV2); and a non-native capsid derived from adeno-associated virus type-1 (AAV1), such that the transgene is packaged within the AAV1 capsid, wherein the AAV1 capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and upon entry into a cell has a transduction rate that is about 8-fold higher than the transduction rate of the corresponding wild type adeno-associated virion.
- 9. The recombinant virion of claim 8, wherein the neural cell is present in a region of a brain or spinal cord.
- 10. The recombinant virion of claim 9, wherein the region of the brain is the stratium.
- 11. The recombinant virion of claim 9, wherein the region of the brain is the hippocampus.
- 12. A recombinant pseudotyped adeno-associated virus type-5 virion for use in neural cells comprising:
a transgene flanked 5′ and 3′ by inverted terminal repeat sequences, wherein the inverted terminal repeat sequences are derived from adeno-associated virus-2 (AAV2); and a non-native capsid derived from adeno-associated virus-5 (AAV5), such that the transgene is packaged within the AAV5 capsid, wherein the AAV5 capsid provides a modified tropism and can bind to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and upon entry into a cell has a transduction rate that is about 2-fold higher than the transduction rate of the corresponding wild type adeno-associated virion.
- 13. The recombinant virion of claim 12, wherein the neural cell is present in a region of a brain or spinal cord.
- 14. The recombinant virion of claim 13, wherein the region of the brain is the stratium.
- 15. The recombinant virion of claim 13, wherein the region of the brain is the hippocampus.
- 16. A method of making a recombinant pseudotyped adeno-associated virion with a modified tropism for use in neural cells comprising:
providing a first construct comprising a transgene flanked 5′ and 3′ with inverted terminal repeat sequences derived from a first adeno-associated virus type, wherein at least one inverted terminal repeat sequence comprises a packaging signal, and a second helper construct comprising a rep coding region derived from the first adeno-associated virus type and a cap coding region derived from a second adeno-associated virus type, wherein the cap coding region encodes for a non-native capsid; and contacting a population of cells with the first and second constructs, such that the population of cells allows assembly of a recombinant virion comprising a non-native capsid, to thereby produce a recombinant pseudotyped virion with a modified tropism, wherein the recombinant pseudotyped virion can bind to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and wherein the recombinant pseudotyped virion has a transduction rate that is about 2-fold to about 30-fold higher than the transduction rate of the corresponding wild type adeno-associated virion.
- 17. The method of claim 16, wherein the first construct comprises inverted terminal repeat sequences derived from an adeno-associated virus type selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.
- 18. The method of claim 16, wherein the first construct comprises inverted terminal repeat sequences derived from adeno-associated virus type-2 (AAV2).
- 19. The method of claim 16, wherein the second construct comprises a nucleic acid sequence encoding a non-native capsid derived from an adeno-associated virus type that is different from the first adeno-associated virus type.
- 20. The method of claim 16, wherein the step of contacting the population of cells further comprises contacting a population of 293 cells.
- 21. An isolated nucleic acid molecule encoding an adeno-associated virus (AAV) helper function, said nucleic acid molecule comprising:
a rep coding region derived from a first adeno-associated virus type; and a cap coding region derived from a second adeno-associated virus type encoding a non-native capsid that provides a modified tropism which permits binding to an attachment site present on a cell surface of a neural cell, with a higher affinity than a corresponding adeno-associated virion with a wild type capsid, and which upon entry into a cell has a transduction rate that is about 2-fold to about 30-fold higher than the transduction rate of the corresponding wild type adeno-associated virion.
- 22. The nucleic acid molecule of claim 21, wherein the rep coding region is derived from a first adeno-associated virus (AAV) type selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.
- 23. The nucleic acid molecule of claim 21, wherein the cap coding region encoding a non-native capsid is derived from a second adeno-associated virus (AAV) type selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6, wherein the second AAV is different from the first AAV.
- 24. The nucleic acid molecule of claim 21, wherein the rep coding region is derived from adeno-associated virus type-2 (AAV2) and the non-native cap coding region is derived from adeno-associated virus type-1 (AAV1).
- 25. The nucleic acid molecule of claim 21, wherein the rep coding region is derived from adeno-associated virus type-2 (AAV2) and the non-native cap coding region is derived from adeno-associated virus type-5 (AAV5).
- 26. An adeno-associated virus (AAV) helper function vector comprising the nucleic acid molecule of claim 1.
- 27. The adeno-associated virus (AAV) helper function vector of claim 26, wherein the vector is a plasmid.
- 28. A host cell transfected with the nucleic acid molecule of claim 27.
- 29. A pharmaceutical composition comprising a recombinant pseudotyped viral vector of claim 1; and a pharmaceutically acceptable carrier.
- 30. A pharmaceutical composition comprising a recombinant pseudotyped viral vector of claim 8; and a pharmaceutically acceptable carrier.
- 31. A pharmaceutical composition comprising a recombinant pseudotyped viral vector of claim 12; and a pharmaceutically acceptable carrier.
RELATED CASE INFORMATION
[0001] This application claims priority to U.S. Provisional Patent Application No. 60/189,110, filed Mar. 14, 2000 and is a continuation-in-part of U.S. patent application Ser. No. 09/804,898 filed: Mar. 13, 2001.
Provisional Applications (1)
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Number |
Date |
Country |
|
60189110 |
Mar 2000 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
09804898 |
Mar 2001 |
US |
Child |
10427129 |
May 2003 |
US |