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 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, and
wherein the RNA coding region is substantially complementary to a target region that is from about 10 to about 50 nucleotides in length.
2. The retroviral construct of claim 1 , wherein the RNA coding region consists of SEQ ID NO: 17.
3. The retroviral construct of claim 1 , wherein the RNA polymerase III promoter and the RNA coding region are located between the 5′ LTR and the 3′ LTR.
4. A 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;
at least one RNA coding region which encodes SEQ ID NO: 16; and
wherein the RNA coding region is substantially complementary to a target region that is from about 10 to about 50 nucleotides in length.
5. The retroviral construct of claim 4 , wherein the RNA coding region consists of SEQ ID NO: 17.
6. The retroviral construct of claim 4 additionally comprising an RNA polymerase III promoter, wherein the RNA polymerase III promoter and the RNA coding region are located between the 5′ LTR and the 3′ LTR.
7. The retroviral construct of claim 1 , wherein the RNA Polymerase III promoter is inducible.
8. The retroviral construct of claim 7 , wherein the inducible promoter can be activated with tetracycline.
9. The retroviral construct of claim 1 , wherein the RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
10. The retroviral construct of claim 9 , wherein the loop region is about 2 to about 10 nucleotides in length.
11. The retroviral construct of claim 9 , wherein the sense region and the antisense region are between about 15 and about 30 nucleotides in length.
12. The retroviral construct of claim 1 , wherein the 5′ LTR sequences are from HIV.
13. The retroviral construct of claim 1 , wherein the retroviral construct comprises the woodchuck hepatitis virus enhancer element sequence.
14. The retroviral construct of claim 1 , wherein the retroviral construct comprises a tRNA amber suppressor sequence.
15. The retroviral construct of claim 1 , wherein the self-inactivating 3′ LTR comprises a U3 element with a deletion of its enhancer sequence.
16. The retroviral construct of claim 1 , wherein the self-inactivating 3′ LTR is a modified HIV 3′ LTR.
17. The retroviral construct of claim 1 , wherein the retroviral construct is pseudotyped.
18. The retroviral construct of claim 17 , wherein the retroviral construct is pseudotyped with the vesicular stomatitis virus envelope glycoprotein.