RNA INTERFERENCE TECHNOLOGY FOR THE MANAGEMENT OF A HEPATITIS B VIRUS INFECTION: POTENTIAL AS A CLINICAL THERAPEUTIC
Open Access
- Author:
- Starkey, Jason Leonard
- Graduate Program:
- Cell and Molecular Biology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- January 29, 2009
- Committee Members:
- Harriet C Isom, Dissertation Advisor/Co-Advisor
Harriet C Isom, Committee Chair/Co-Chair
Richard James Courtney, Committee Member
Jianming Hu, Committee Member
Neil David Christensen, Committee Member
Victor Alan Canfield, Committee Member - Keywords:
- CCC DNA
RNA Interference
Hepatitis B Virus
HepG2 Cells
shRNA - Abstract:
- Chronic infection with Hepatitis B virus (HBV) is a debilitating disease that can result in serious liver injury and the development of hepatocellular carcinoma (HCC). An estimated 350 million individuals suffer from chronic HBV infection worldwide, and although several Food and Drug Administration approved therapeutics exist for the treatment of chronic HBV, many are effective only over short administration periods due to their adverse side effect of selecting for drug resistant mutants. This characteristic of current HBV therapies can have long-term debilitating consequences for the patient. Long-term treatment with existing therapeutic agents can select for drug resistant mutants, many of which are cross-resistant to multiple drugs. As a result, the infection is even more difficult to clear due to continued HBV replication driven by the stable nuclear viral covalently closed circular (CCC) DNA, which would otherwise be suppressed by pharmacologic management. This ongoing viral replication can cause continued liver injury, thereby increasing the patient’s risk for developing HCC. Therefore, novel potent inhibitors of HBV replication capable of targeting multiple aspects of the viral life cycle, and thereby decreasing the chance for the development of resistance, need to be developed and evaluated in models of persistent HBV infection in which their effect on HBV CCC DNA can be monitored. RNA interference (RNAi)-based therapy thereby represents a potential treatment for the control and inhibition of multiple diseases. RNAi is the process by which gene expression is silenced through the sequence-specific degradation of mRNA. The unique arrangement of the HBV transcripts and the fact that replication occurs through an RNA intermediate make HBV an attractive target for RNAi-based therapy. The aim of this work was to evaluate the efficacy of RNAi technology as a potential anti-HBV therapeutic agent. To this end, a short hairpin RNA (shRNA) sequence to a specific, critical region of the HBV genome was tested as follows: 1) for its ability to prevent establishment of HBV infection (pre-HBV exposure system); and 2) for its ability to silence expression of the HBV genome in a model of persistent infection (subculture system). Lastly, liposomes containing anti-HBV small interfering RNA (siRNA) were assessed as a potential delivery system of RNAi to cells of hepatic origin. To accomplish these goals, HBV replication was initiated in HepG2 cells by transduction with HBV–expressing recombinant baculovirus. Subculture of HBV-expressing HepG2 cells at 10 days post-transduction generates a system in which HBV replication is ongoing and HBV is expressed largely from CCC DNA thus simulating chronic HBV infection. HepG2 cells were transduced with shRNA expressing baculovirus prior to initiation of HBV replication or during chronic HBV replication and the levels of HBV RNA, HBsAg, replicative intermediates (RI), extracellular (EC) and CCC DNA species were measured. HBsAg, HBV RNA and DNA levels were markedly reduced through day 8 whether cells were transduced with shRNA prior to or during a chronic infection; however, the CCC DNA species were only affected when shRNA was administered prior to initiation of infection. These data suggest that RNAi may have therapeutic value for controlling HBV replication at the level of RI and EC DNA and for reducing establishment of CCC DNA during HBV infection. To assess the utility of novel cationic liposomes for the delivery of siRNA to cell lines of hepatic origin, three different liposome formulations were generated: 1) Li-100, 2) Li-75, and 3) LiC-75. Liposome binding efficiency was tested using rhodamine-tagged fluorescent liposomes. For the HepG2 and C3A cells, all three liposome formulations displayed a strong rhodamine signal suggesting 100% binding efficiencies, while only Li-100 and Li-75 displayed strong signals for Huh7.5 cells. The ability of cationic liposomes to deliver siRNA to cells of hepatic origin was evaluated using GAPDH-specific siRNA. Of the liposome formulations tested, only Li-75 was able to deliver anti-GAPDH siRNA to HepG2 and C3A cells, as indicated by a subsequent decrease in GAPDH mRNA. The ability of anti-HBV siRNA containing Li-75 cationic liposomes to inhibit a persistent HBV infection was evaluated. Subcultured HBV-infected HepG2 cells were treated with anti-HBV siRNA containing Li-75 cationic liposomes. Total mRNA was extracted and a marked decrease in HBV mRNA was observed in liposome-treated cells compared to mock-treated cells. These data suggest that specific cationic liposomes are potential useful delivery vehicles for RNA-based therapeutics to cells of hepatic origin. Chronic HBV infections will surely continue to prove difficult to treat without the development of novel and effective therapeutics capable of inhibiting multiple aspects of the viral life cycle. The continued investigation of RNAi-based therapeutics and novel methods of their delivery, both alone and in combination with existing nucleos(t)ide analogues, for the treatment of chronic HBV infections represents a principal area of study to further pharmacotherapeutic management of HBV infections worldwide.