Method for expression of small antiviral RNA molecules with reduced cytotoxicity within a cell
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 RNA molecule within a cell, the method comprising:
infecting a target cell ex vivo with a recombinant retrovirus;
wherein the recombinant retrovirus 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 10 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 recombinant retrovirus 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 15nucleotides in length.
8. The method of claim 1 , wherein the target region is from 15 to 30nucleotides in length.
9. The method of claim 5 , wherein the target region is from 18 to 23nucleotides in length.
10. The method of claim 5 wherein the 5′ LTR sequences are from HIV.
11. The method of claim 5 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 13 , wherein the recombinant retrovirus is pseudotyped with the vesicular stomatitis virus envelope glycoprotein.
15. The method of claim 5 , wherein the 5′ LTR sequences are from Moloney Murine Leukemia Virus.
16. The method of claim 5 , 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 1 , wherein the target cell is a CD34-positive hematopoietic cell.