Progress toward the therapy of hepatitis with RNAi
Authors: Glenn Rendall
Journal: Hepatology (2005)

The discovery of RNA interference (RNAi) has revolutionized mammalian functional genomics. Scientists can now rapidly assess the phenotypes associated with silencing the expression of any gene. Many hope that this breakthrough will translate into the clinic, both in terms of drug discovery and RNAi-based therapeutics. In this issue of HEPATOLOGY, Morrissey et al. describe progress toward this goal.1 They take a step beyond "proof of concept" studies to investigate chemical modifications to small interfering RNAs (siRNAs) that improve their efficacy in a mouse model of hepatitis B virus replication. They demonstrate that chemically modified siRNAs have improved delivery characteristics, both in terms of stability and efficacy in vivo. Importantly, siRNAs delivered via a clinically relevant route—standard intravenous injection—reduced hepatitis B virus (HBV) replication in mice. RNAi, RNA interference; siRNAs, small interfering RNAs; HBV, hepatitis B virus. The use of nucleic acids to target RNAs for degradation has been studied for 25 years. Initial approaches used DNA oligonucleotides that hybridized with complementary RNAs, resulting in degradation by the enzyme ribonuclease H. This was popularized as "antisense technology." Catalytic RNA molecules called ribozymes were then discovered. They could be engineered to cleave any desired target sequence. While generating initial excitement, these approaches have led to limited clinical treatments. In the last 4 years, the robust and evolutionarily conserved RNAi pathway was discovered to exist in mammals.2 The mechanism of RNAi is being rapidly characterized (Fig. 1). siRNAs are generated from longer double-stranded RNAs; the antisense strand of the siRNA then hybridizes to a complementary RNA and targets it for degradation by the RNA-induced silencing complex. This pathway is active both in the response to double-stranded RNA and in the biogenesis of cellular micro-RNAs, which regulate diverse cellular pathways.3 The discovery of RNAi has generated renewed optimism in nucleic acid therapy for two reasons: first, it is a naturally occurring antisense mechanism that is highly efficient in cells; and second, it is an innate antiviral defense in plants and insects.4 Exploitation of RNA interference pathways for therapeutics. (A) In the siRNA pathway, long double-stranded RNAs are processed into siRNAs by Dicer. One strand of the siRNA associates with the RNA-induced silencing complex and binds to a complementary RNA. The siRNA–RNA duplex is cleaved by Argonaute 2, which is a component of the RNA-induced silencing complex. This pathway is activated by chemically synthesized siRNAs (green) when introduced into cells. (B) In the microRNA (miRNA) pathway, a pri-miRNA is transcribed in the nucleus and processed by Drosha to yield the pre-miRNA. This is then exported from the nucleus and cleaved by Dicer to produce the miRNA. In humans, miRNAs (red) generally have nucleotide mismatches with their target sequence. They bind the target sequence in the 3′ untranslated region of a messenger RNA and repress its translation. However, if a miRNA has perfect complementarity to its target, it can trigger RNA degradation, as in the siRNA pathway. Viruses can be engineered to express short hairpin RNAs, which mimic a pre-miRNA. The short hairpin RNA is then processed to yield an active siRNA, leading to target RNA degradation. siRNA, small interfering RNA; RISC, RNA-induced silencing complex; shRNA, short hairpin RNA. Initial studies have pointed to the potential of RNAi to treat hepatitis. RNAi inhibits the replication of many viruses in vitro, including hepatitis A, B, C, and delta viruses.5, 6 The liver was the first organ to be successfully targeted by siRNAs. Hepatitis C virus–specific siRNAs silenced the expression of a coinjected reporter plasmid containing the hepatitis C virus target sequence in mouse liver.7 RNAi was then shown to alter disease outcome in a mouse model of hepatitis triggered by Fas. siRNAs targeting Fas messenger RNA successfully prevented the onset of acute hepatitis and subsequent mortality in this model.8 Numerous studies then demonstrated in vivo activity of RNAi in a mouse model of HBV infection.9-11 While these studies provided compelling proof of principle for RNAi-based therapeutics for hepatitis, the siRNAs were not delivered in a therapeutically feasible route. They were instead injected into the mouse tail vein via high-pressure hydrodynamic injection. The volume injected is the equivalent of 8 L for a human, taken in the course of seconds. This promotes the uptake of nucleic acid by the liver. An initial disappointment was that siRNAs delivered via standard intravenous injection were ineffective as a result of poor stability in serum and inefficient uptake by the liver. Many groups are now undertaking efforts to demonstrate effective RNAi therapy in vivo using clinically relevant delivery systems. There are two main approaches. The first applies classical gene therapy methodology, wherein a virus expresses short hairpin RNAs targeting a RNA of interest. This approach has recently proven successful in an HBV-transgenic mouse replication model. Recombinant adenoviruses expressing HBV-specific siRNAs suppressed ongoing HBV replication to near undetectable levels for the course of the experiment (26 days).12 This result is promising, although it remains unclear if adenovirus would be the virus of choice for expression of short hairpin RNAs in humans. The second approach is the delivery of chemically synthesized siRNAs that have been modified for improved pharmokinetic properties. This approach faces numerous obstacles for the treatment of hepatitis. The siRNAs must be delivered systemically, thus they need to be resistant to nucleases present in the blood. They must efficiently target the liver and produce prolonged efficacy. The activity of siRNAs in dividing cells is relatively short (4-7 days), presumably because the siRNAs are diluted by cell division. Efficacy may be prolonged in hepatocytes in vivo, because hepatocytes are terminally differentiated. Given these obstacles, initial attempts to treat disease with siRNAs use direct delivery to a target tissue. The first clinical trials are assessing the safety and efficacy of siRNA therapy in treating the "wet" form of age-related macular degeneration, a leading cause of adult blindness. These siRNAs limit the production of vascular endothelial growth factor and thus limit vascularization of the eye. Because the siRNAs are delivered directly by intravitreal injection, they avoid some issues associated with systemic delivery, such as stability in blood and target organ uptake. In the meantime, efforts are underway to tackle obstacles of systemic siRNA delivery. It has been shown that certain chemical modifications promote siRNA stability in serum without affecting siRNA activity.13, 14 Morrissey et al. modify a siRNA such that all 2′-OH residues are substituted with a combination of 2′-fluoro, 2′-O-methyl, and 2′-deoxy sugars. The siRNA also contains phosphorothioate linkages and 5′ and 3′ inverted abasic end caps. These modifications produced a 3-log10 enhancement in siRNA stability, resulting in a half-life of approximately 2.5 days in human serum. They next test the efficacy of the chemically modified siRNAs in an HBV cell culture system. They identify seven chemically modified siRNAs that successfully target highly conserved HBV sequences. These sequences were chosen because the siRNAs should be resistant to viral escape, because the evolution of this sequence is constrained. When less conserved viral sequences were targeted in previous studies, siRNA-resistant mutants emerge with a number of viruses, including HBV.15 They next investigate the properties of their optimal siRNA in vivo in a mouse model of HBV replication. In this model, a plasmid encoding HBV is delivered to the mouse liver via high-pressure hydrodynamic injection into the mouse tail vein.16 Initially, the modified siRNAs were coadministered with the HBV plasmid, as was done in previous studies. Modified HBV-specific siRNAs reduced HBV serum DNA by approximately 4 logs. The modifications showed enhanced efficacy compared with the unmodified HBV-specific siRNA at higher doses. They then tested the modified HBV siRNA via standard intravenous injection 3 days after delivery of the HBV vector. While the unmodified HBV siRNA did not affect HBV replication, the stabilized HBV siRNA produced a 1-log10 reduction in serum HBV DNA. Although this was the first demonstration of anti-HBV activity in vivo with chemically synthesized siRNAs delivered via a clinically relevant route, it should be noted that extremely high doses (10 mg/kg) were required to attain an antiviral effect. Part of the reason for this is that while the chemical modifications stabilize the siRNA in serum, they are unlikely to enhance siRNA uptake by the liver. Indeed, less than 1% of the modified siRNAs reached the liver in this study. Further modifications designed to enhance liver targeting will be required. One possibility recently reported is the conjugation of a cholesterol group to the siRNA.17 This resulted in enhanced liver targeting in a mouse model for cholesterol therapy. A chemically stabilized, cholesterol-conjugated siRNA targeted apolipoprotein B, a gene whose expression directly correlates with cholesterol levels. This treatment successfully reduced apolipoprotein B expression and resulted in a corresponding decrease in mouse cholesterol levels. By comparison, chemically stabilized siRNAs without cholesterol conjugation produced no effect. It is clear that additional improvements are required to boost the in vivo efficacy of chemically stabilized siRNAs in the liver. The largest current obstacle appears to be effective liver targeting. Perhaps the addition of lipid conjugates to the siRNA stability modifications described by Morrissey et al. will produce antiviral activity at more clinically relevant doses. This will move us one step closer to siRNA therapeutics for hepatitis. I am supported in part by postdoctoral fellowship PF-02-016-01-MBC from the American Cancer Society, grants from the Public Health Service (CA57973 and AI40034) to Charles Rice (PI), and the Greenberg Medical Research Institute. I thank Catherine Murray and Shihyun You for critical review of this editorial.