Method for expression of small antiviral RNA molecules with reduced cytotoxicity within a cell
View Patent ↗In one aspect, the invention provides methods and compositions for the expression of small RNA molecules within a cell using a retroviral vector (FIG. 1 A). Small interfering RNA (siRNA) can be expressed using the methods of the invention within a cell. In a further aspect, the invention provides methods for producing siRNA encoding lentivirus where the siRNA activity may interfere with the lentiviral life cycle. In yet a further aspect, the invention provides methods for expression of a small RNA molecule within a cell, such as an siRNA capable of downregulating CCR5, wherein expression of the small RNA molecule is relatively non-cytotoxic to the cell. The invention also includes small RNA molecules, such as an siRNA capable of downregulating CCR5, that are relatively non-cytotoxic to cells.
1. A method of expressing an siRNA 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 ex vivo 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 expression of the RNA coding region results in the down regulation of a target gene or genome transcript; and
wherein the first RNA coding region encodes an siRNA comprising SEQ ID NO: 16,
wherein the RNA coding region is substantially complementary to a target region that is from 15 to 30 nucleotides in length.
2. The method of claim 1 , wherein the RNA coding region consists of SEQ ID NO: 17.
3. The method of claim 1 , wherein the RNA coding region comprises a hairpin region.
4. The method of claim 1 , wherein expression of the RNA coding region within the target cell does not alter the growth kinetics of the target cell for at least about 10 days.
5. 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.
6. 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.
7. The method of claim 6 , wherein the loop region is about 2 to about 15 nucleotides in length.
8. The method of claim 1 , wherein the target region is from 18 to 23 nucleotides in length.
9. The method of claim 1 wherein said packaging cell line is an HEK293 cell line.
10. The method of claim 1 wherein the 5′ LTR sequences are from HIV.
11. The method of claim 1 wherein the self-inactivating 3′ LTR comprises a U3 element with a deletion of its enhancer sequence.
12. The method of claim 5 , wherein the self-inactivating 3′ LTR is a modified HIV 3′ LTR.
13. The method of claim 1 , wherein the recombinant retrovirus is pseudotyped.
14. The method of claim 1 , wherein the recombinant retrovirus is pseudotyped with the vesicular stomatitis virus envelope glycoprotein.
15. The method of claim 1 , wherein the 5′ LTR sequences are from Moloney Murine Leukemia Virus.
16. The method of claim 1 , wherein the 5′ LTR sequences are from murine stem cell virus (MSCV).
17. The method of claim 1 , wherein the target cell is a human cell.
18. The method of claim 1 , wherein the target cell is a hematopoietic cell.
19. The method of claim 18 , wherein the target cell is a CD34-positive hematopoietic cell.
20. The method of claim 19 , further comprising isolating the target CD34-positive hematopoietic cells from a patient.
21. The method of claim 20 , further comprising reintroducing the infected CD34-positive hematopoietic cell into the patient.
22. The method of claim 1 , wherein the cell is a cultured cell.
23. The method of claim 1 , wherein the cell is a human cell.
24. 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 retrovirus construct comprises a first RNA polymerase III promoter region, a first RNA coding region encoding SEQ ID NO: 17, and a first termination sequence; and
wherein expression of the RNA coding region results in the down regulation of CCR5.
25. The method of claim 24 , wherein expression of the RNA coding region within the target cell does not alter the growth kinetics of the target cell for at least about 10 days.
26. The method of claim 24 , 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.
27. A method of expressing an siRNA within a cell, the method comprising:
infecting a target cell in vitro with a recombinant retrovirus comprising a first RNA polymerase III promoter region, a first RNA coding region encoding SEQ ID NO: 16 operably connected to the RNA polymerase III promoter region, and a first termination sequence, wherein the RNA coding region is substantially complementary to a target region that is from 15 to 30 nucleotides in length.
28. A method of downregulating CCR5 in a cell, comprising transfecting a cell in vitro with a recombinant lentivirus comprising a first RNA polymerase III promoter region, a first RNA coding region, and a first termination sequence; wherein the RNA coding region encodes SEQ ID NO: 16, wherein the RNA coding region is substantially complementary to a target region that is from 15 to 30 nucleotides in length.
29. The method of claim 28 , wherein the RNA coding region encodes SEQ ID NO: 17.
30. A retroviral construct for the expression of an siRNA within a cell, the retroviral construct comprising:
a nucleic acid having the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR);
a self-inactivating lentiviral 3′ LTR;
an RNA Polymerase III promoter; and
at least one RNA coding region operably linked to the RNA Polymerase III promoter;
wherein the RNA coding region encodes SEQ ID NO: 16, wherein the RNA coding region is substantially complementary to a target region that is from 15 to 30 nucleotides in length.
31. The retroviral construct of claim 30 , wherein the RNA polymerase III promoter and the RNA coding region are located between the 5′ LTR and the 3′ LTR.
32. The retroviral construct of claim 31 , further comprising at least one termination sequence.
33. The retroviral construct of claim 31 , wherein the RNA Polymerase III promoter is inducible.
34. The retroviral construct of claim 33 , wherein the inducible promoter is activated with tetracycline.
35. The retroviral construct of claim 31 , wherein the RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
36. The retroviral construct of claim 35 , wherein the loop region is about 2 to about 10 nucleotides in length.
37. The retroviral construct of claim 35 , wherein the sense region and the antisense region are between about 15 and about 30 nucleotides in length.
38. The retroviral construct of claim 31 , wherein the 5′ LTR sequences are from HIV.
39. The retroviral construct of claim 31 , wherein the viral construct comprises the woodchuck hepatitis virus enhancer element sequence.
40. The retroviral construct of claim 31 , wherein the viral construct comprises a tRNA amber suppressor sequence.
41. The retroviral construct of claim 31 , wherein the self-inactivating 3′ LTR comprises a U3 element with a deletion of its enhancer sequence.
42. The retroviral construct of claim 31 , wherein the self-inactivating 3′ LTR is a modified HIV 3′ LTR.
43. The retroviral construct of claim 31 , wherein the retroviral construct is pseudotyped.
44. The retroviral construct of claim 43 , wherein the retroviral construct is pseudotyped with the vesicular stomatitis virus envelope glycoprotein.
45. 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 retrovirus construct comprises a first RNA polymerase III promoter region, a first RNA coding region encoding SEQ ID NO. 16, and a first termination sequence, wherein the RNA coding region is substantially complementary to a target region that is from 15 to 30 nucleotides in length.
46. A retroviral construct for the expression of an siRNA within a cell, the retroviral construct comprising:
a nucleic acid having the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR);
a self-inactivating lentiviral 3′ LTR;
an RNA Polymerase III promoter; and
at least one RNA coding region operably linked to the RNA polymerase III promoter;
wherein the RNA coding region encodes SEQ ID NO: 17, wherein the RNA coding region is substantially complementary to a target region that is from 15 to 30 nucleotides in length.