Claims
- 1. A method of expressing an RNA molecule within a cell, the method comprising:
transfecting a packaging cell line with a retroviral construct; recovering a recombinant retrovirus from the packaging cell line; and infecting a target cell with the recombinant retrovirus, wherein the recombinant retrovirus construct comprises a first RNA polymerase III promoter region, a first RNA coding region, and a first termination sequence, and wherein the RNA coding region comprises a sequence that is at least about 90% identical to a target region of a pathogenic virus genome or genome transcript or a target cell gene involved in the pathogenic virus life cycle.
- 2. The method of claim 1, wherein the retroviral construct further comprises the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR) and a self-inactivating lentiviral 3′ LTR.
- 3. The method of claim 1, wherein the RNA coding region encodes an RNA molecule having a sense region, an antisense region and a loop region, and wherein the sense region is substantially complementary to the antisense region.
- 4. The method of claim 3, wherein the loop region is about 2 to about 15 nucleotides in length.
- 5. The method of claim 1, wherein the target region is between about 15 and about 30 nucleotides in length.
- 6. The method of claim 5, wherein the target region is from 18 to 23 nucleotides in length.
- 7. The method of claim 1, wherein the pathogenic virus is human immunodeficiency virus.
- 8. The method of claim 6, wherein the target region is a region of a human gene transcript selected from the group consisting of CCR5 and CXCR4.
- 9. The method of claim 6, wherein the target region is a region of the human immunodeficiency virus transcript.
- 10. The method of claim 1, wherein the pathogenic virus is selected from the group consisting of hepatitis C virus (HCV), cytomegalovirus (CMV), herpes simplex virus (HSV), influenza virus, an adenovirus and poliovirus.
- 11. The method of claim 1, wherein the first RNA coding region encodes a first RNA molecule, and the retroviral construct further comprises a second RNA polymerase III promoter and a second RNA coding region operably linked to the second RNA polymerase III promoter, wherein the second RNA coding region encodes a second RNA molecule substantially complementary to the first RNA molecule at the target region.
- 12. The method of claim 1, wherein the retroviral construct further comprises a second RNA polymerase III promoter region operably linked to the first RNA coding region, such that expression of the first RNA coding region from the first RNA polymerase III promoter results in a synthesis of a first RNA molecule and expression of the first RNA coding region from the second RNA polymerase III promoter results in synthesis of a second RNA molecule substantially complementary to the first RNA molecule at the target region.
- 13. The method of claim 1 wherein said packaging cell line is an HEK293 cell line.
- 14. The method of claim 1 wherein the 5′ LTR sequences are from HIV.
- 15. The method of claim 1 wherein the self-inactivating 3′ LTR comprises a U3 element with a deletion of its enhancer sequence.
- 16. The method of claim 15, wherein the self-inactivating 3′ LTR is a modified HIV 3′ LTR.
- 17. The method of claim 1, wherein the recombinant retrovirus is pseudotyped.
- 18. The method of claim 17, wherein the recombinant retrovirus is pseudotyped with the vesicular stomatitits virus envelope glycoprotein.
- 19. The method of claim 1 wherein the 5′ LTR sequences are from Moloney Murine Leukemia Virus.
- 20. The method of claim 1 wherein the 5′LTR sequences are from murine stem cell virus (MSCV).
- 21. The method of claim 1, wherein the target cell is a human cell.
- 22. The method of claim 21, wherein the target cell is a hematopoietic cell.
- 23. The method of claim 22, wherein the target cell is a CD34-positive hematopoietic cell.
- 24. The method of claim 23, further comprising isolating the target CD34-positive hematopoietic cells from a patient.
- 25. The method of claim 24, further comprising reintroducing the infected CD34-positive hematopoietic into the patient.
- 26. The method of claim 1, wherein the cell is a cultured cell.
- 27. The method of claim 1, wherein the cell is a human cell in vivo.
- 28. A method of treating a patient infected with HIV, the method comprising:
isolating a CD34-positive target cell from a patient; infecting the target cell with a recombinant retrovirus recovered from a packaging cell line transfected with a retroviral construct, wherein the recombinant retroviral construct comprises a first RNA polymerase III promoter region, at least one RNA coding region, and at least one termination sequence, and wherein the RNA coding region has a sequence that is at least about 90% identical to a target region of the HIV genome, an HIV genome transcript or another gene involved in the virus life cycle.
- 29. The method of claim 28, wherein the retroviral construct further comprises the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR) and a self-inactivating lentiviral 3′ LTR.
- 30. The method of claim 28, wherein the RNA coding region encodes an RNA molecule having a sense region, an antisense region and a loop region, wherein the sense region is substantially complementary to the antisense region.
- 31. The method of claim 28, wherein the RNA coding region encodes a first RNA molecule, and the retroviral construct further comprises a second RNA polymerase III promoter and a second RNA coding region operably linked to the second RNA polymerase III promoter, wherein the second RNA coding region encodes a second RNA molecule substantially complementary to the first RNA molecule at the target region.
- 32. The method of claim 28, wherein the retroviral construct further comprises a second RNA polymerase III promoter region operably linked to the RNA coding region, such that expression of the RNA coding region from the first RNA polymerase III promoter results in a synthesis of a first RNA molecule and expression of the RNA coding region from the second RNA polymerase III promoter results in synthesis of a second RNA molecule substantially complementary to the first RNA molecule at the target region.
- 33. The method of claim 28, wherein the target region is from 18 to 23 nucleotides in length.
- 34. The method of claim 28, wherein the gene is selected from the group consisting of CCR5 and CXCR4.
REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/322,031, filed Sep. 13, 2001, U.S. Provisional Application No. 60/347,782, filed Jan. 9, 2002, U.S. Provisional Application No. 60/389,592, filed Jun. 18, 2002, and U.S. Provisional Application No., attorney docket number CALTE.011PR, filed Aug. 27, 2002.
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant Number GM39458 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.
Provisional Applications (4)
|
Number |
Date |
Country |
|
60322031 |
Sep 2001 |
US |
|
60347782 |
Jan 2002 |
US |
|
60389592 |
Jun 2002 |
US |
|
60406436 |
Aug 2002 |
US |