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 expressing an RNA within a cell, comprising:
a nucleic acid having the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR);
a self-inactivating lentiviral 3′ LTR; and
a first promoter configured to be operably linked to a first RNA coding region encoding a first RNA,
wherein the first promoter is located between the 5′ LTR and the 3′ LTR, and wherein the first RNA comprises a RNA duplex.
2. The retroviral construct of claim 1 , wherein the first RNA comprises a sequence that is at least about 90% complementary to a target region of a pathogenic virus genome or genome transcript.
3. The retroviral construct of claim 2 , wherein the pathogenic virus is human immunodeficiency virus (HIV), hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), cytomegalovirus (CMV), herpes simplex virus (HSV), influenza virus, adeno virus, human papillomavirus, Coxsackieviruses, Measles virus, or poliovirus.
4. The retroviral construct of claim 3 , wherein the pathogenic virus is HIV-1 or HIV-2.
5. The retroviral construct of claim 2 , wherein the target region of the pathogenic virus genome or genome transcript is a region in HIV vif gene, HIV nef gene, HIV rev gene, HIV gag gene, HIV pol gene, HIV LTRs, HIV trans-activation response (TAR) element, HIV Rev response element (RRE), or a transcript thereof.
6. The retroviral construct of claim 2 , wherein the target region comprises a DNA cis-regulatory element, a RNA cis-regulatory element, or a combination thereof.
7. The retroviral construct of claim 1 , wherein the first RNA comprises a sequence that is at least about 90% complementary to a cellular target sequence that encodes a protein involved in viral life cycle.
8. The retroviral construct of claim 7 , wherein the protein involved in viral life cycle is selected from the group consisting of a cell surface receptors involved in viral entry, a transcription factor involved in the transcription of a viral genome, a protein involved in viral integration into a host chromosome, a protein involved in translation of viral gene expression, and a combination thereof.
9. The retroviral construct of claim 7 , wherein the protein involved in viral life cycle is a protein selected from the group consisting of CAR, an Integrins, MHC I, Heparan sulfate glycoaminoglycan, Siliac Acid, Heparan sulfate glycosaminoglycan, ICAM-1, murine-like class I integral membrane glycoprotein, CD81, Low density lipoprotein receptor, CD4, PVR, HveB, HveC, CD46, and CD55.
10. The retroviral construct of claim 7 , wherein the protein involved in viral life cycle is a protein selected from the group consisting of CC chemokine receptor 5 (CCR5), CXC chemokine receptor 4 (CXCR4), cyclophilin, CRM-1, importin-β, and HP68.
11. The retroviral construct of claim 7 , wherein the protein involved in viral life cycle is a cellular receptor involved in recognition by a virus selected from the group consisting of adenoviruses, cytomegalovirus, coxsackieviruses, hepatitis A virus, hepatitis C virus, HIV, HSV, influenza virus, measles virus, poliovirus, and human papillomavirus.
12. The retroviral construct of claim 7 , wherein the protein involved in viral life cycle is a co-receptor necessary for viral replication.
13. The retroviral construct of claim 1 , wherein the RNA duplex is at least about 15 nucleotides in length.
14. The retroviral construct of claim 1 , wherein the first RNA is a single stranded RNA comprising a stem-loop.
15. The retroviral construct of claim 1 , wherein the RNA duplex is flanked by a single stranded region on one side or both sides.
16. The retroviral construct of claim 1 , wherein the first promoter is a RNA polymerase III promoter.
17. The retroviral construct of claim 1 , wherein the first RNA coding region is followed by at least one termination sequence.
18. The retroviral construct of claim 1 , wherein the first RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
19. The retroviral construct of claim 18 , wherein the sense region and the antisense region are about 15 and about 30 nucleotides in length.
20. The retroviral construct of claim 1 , wherein the target region is about 15 to about 30 nucleotides in length.