HCV causes chronic liver infection in 130-170 million people worldwide and leads to more than 350,000 deaths each year. No vaccine is available and, without treatment, about 15-30 percent of infected individuals develop liver failure or cancer. Until the work of Bartenschlager, Rice, and Sofia, therapy included toxic drugs that many people can’t tolerate and that often don’t cure the disease.
To study a virus and develop drugs against it, scientists need a way to grow it in the lab. With the isolation of HCV in 1989, Michael Houghton (Albert Lasker Clinical Medical Research Award, 2000) opened a new avenue toward that end. Investigators could use standard recombinant DNA techniques to produce the virus’s RNA. After putting it into cells, they expected that the host machinery would use this genetic code to construct infectious HCV.
The approach flopped.
As researchers tried to prod HCV to replicate in cells, they began wondering whether they had captured its entire sequence. Knowing that viral-preparation methods could introduce errors, Rice (then at Washington University) and postdoctoral fellow Alexander Kolykhalov deployed a method for defining the complete HCV RNA that would avoid these technical pitfalls.
In 1996, their strategy revealed an unanticipated structure at the end of the virus. This finding, also reported by Kunitada Shimotohno (then at the National Cancer Center Research Institute, Tokyo), implied that all of the constructed genomes had failed to replicate in the lab because they lacked this crucial feature.
With high hopes, Rice made a series of HCV sequences that included the bona fide end and injected them into chimpanzees. Unfortunately, these RNAs produced no hepatitis or other signs of infection.
Undaunted, he performed further analysis and found that the RNAs had acquired sequence changes—either while being amplified in the lab or before that, when the virus was replicating inside an infected human. These genetic errors, he reasoned, weakened HCV’s ability to propagate. He created a “consensus” genome—one that, at every position, held the most common RNA letter rather than a deleterious one.
The resulting HCV RNA infected chimps and gave them hepatitis, Rice and Jens Bukh (National Institutes of Health) independently reported in 1997. Researchers had designed and produced a functional virus in the lab. Surely the consensus RNAs would generate HCV in lab-grown cells and thus allow investigators to dissect its detailed biology.
In the meantime, Bartenschlager (then at the University of Mainz) had made a different consensus HCV sequence. He introduced it into various host liver cells, but never detected replication. Rice’s consensus RNA also flunked this test.
Bartenschlager had an idea. He knew that relatives of HCV could lose a chunk of their genomes—the region that encodes viral-packaging proteins—yet still multiply inside host cells. He wondered whether he could replace these dispensable sequences with a genetic marker that would expose cells that contain replicating HCV.
In the late 1990’s, he and postdoctoral fellow Volker Lohmann gutted their consensus RNA and inserted a gene whose product confers resistance to a lethal drug. If the HCV RNA-copying machinery did its job, it would amplify not only viral RNA, but also that of the drug-resistance gene. Consequently, host cells that carried this “replicon” would withstand the otherwise deadly toxin.
The scheme worked. The ability of the replicons to bestow a survival advantage upon host liver cells had thus allowed researchers to detect HCV replication in the lab. Still, as they clinked in celebration, their brows wrinkled. Surviving cells contained thousands of RNAs, but only about one in a million cells that received the input RNA survived.
The replicons that flourished, it turned out, had picked up adaptive sequence changes inside cells, Rice and Bartenschlager independently showed. After the investigators engineered these replication-enhancing mutations back into the starting RNA, production of infected cells jumped 500- to 10,000-fold.
For the first time, researchers had generated efficient HCV replication in the lab. Because the replicons did not produce infectious virus particles, the system could be used safely without high-level precautions. Furthermore, the mini-genomes encoded proteins critical for HCV multiplication, important drug targets.
Unmasking a champion
The pharmaceutical industry now had a manageable way to test whether candidate agents thwart HCV inside living liver cells. New medicines were sorely needed. Standard regimens required weekly injections of interferon, which delivers severe side effects. Furthermore, treatment took 24-72 weeks and frequently failed.
In their quest to improve therapy, scientists at Pharmasset, a small biotech company founded by Raymond Schinazi, focused on HCV’s RNA-copying enzyme. Its active site was similar among disparate types of HCV and the enzyme has no human counterpart, so perhaps the team could develop an inhibitor that would combat multiple HCV genotypes without disrupting host physiology.
The investigators set their sights on chemicals that resemble normal RNA building blocks, but differ in a crucial way. These so-called nucleoside analogs attach to a growing RNA chain, but when the copying enzyme tries to add the next subunit, it can’t. The analogs’ extra chemical groups interfere, and RNA elongation stops.
In 2005, Jeremy L. Clark, a member of the Pharmasset team, identified a nucleoside analog that blocks HCV replication in the replicon system. In people, its safety profile looked promising, but most of it broke down into an inactive form.
By modifying its chemical properties, the Pharmasset scientists, now under the direction of Michael Sofia, solved these problems. In 2010, they reported that their improved nucleoside analog slashes viral load when combined with a different class of drug that inhibits a different HCV enzyme, but large and frequent doses of the analog were required.
Aiming to boost its potency, Sofia hit on an unconventional idea. The team’s studies had revealed that the original analog might not be a dead-end after all. Through a series of reactions, enzymes in the body convert a small proportion of it into a different compound that foils HCV replication and persists intact in liver cells.
To harness the potential of this powerful and stable inhibitor, Sofia had to overcome a substantial challenge. He needed to supply not the original analog, which is transformed inefficiently into the desired chemical, but a specific molecular relative. This relative carries a phosphate chemical group, whose negative charge renders it unable to traverse oily cell membranes.
Sofia and his colleagues aimed to mask the phosphate so the compound would slip into cells. In his dream scenario, liver enzymes would then disrobe the agent and, with its charged portion revealed, it would be stuck. Conversion enzymes would set upon it and turn it into the active drug. His vision also promised to minimize harmful effects that might result from delivery to other parts of the body. Only liver cells possess the natural metabolic capabilities that trap the compound inside.
With intense tweaking and evaluation, Sofia designed a chemical candidate that performed well in the replicon system and passed other tests. Early clinical trials showed extremely promising results, and Gilead Sciences acquired Pharmasset in early 2012. In January 2013, the Pharmasset group, Gilead, and clinical collaborators from New Zealand published the dramatic findings. In combination with ribavirin, a toxic but non-interferon-based antiviral agent, the new drug, sofosbuvir, eradicated HCV long term. In people infected with some viral genotypes, no virus could be detected 24 weeks after a 12-week treatment period ended. The absence of interferon in this regimen launched a new era in curative HCV therapy.
On December 6, 2013, a sofosbuvir (Sovaldi®) regimen was approved by the US Federal Drug Administration (FDA) for some HCV genotypes. Finally, people with chronic HCV infections had an interferon-free therapy. Subsequently, sofosbuvir proved effective in diverse patient populations and across viral genotypes.
In the meantime, the replicon assay was fueling other scientists’ discovery of new drugs. A Bristol-Myers Squibb group led by Min Gao identified a compound that targets an HCV protein of unknown function. This achievement showcased another strength of the replicon system—its ability to let the cell reveal what is important for replication, regardless whether scientists understand the underpinnings.
Gilead rapidly developed a derivative of the compound Gao and colleagues had pioneered. The combination of Gilead’s new agent, ledipasvir, plus sofosbuvir (Figure 1A) rapidly quashes the virus. This regimen boasts cure rates of 94-99% in only 8-12 weeks of therapy (Figure 1B), even among difficult-to-treat patients. The ledipasvir/sofosbuvir combination, Harvoni®, is now FDA-approved for numerous types of HCV infection, including the most common form in the US and Europe. It is the first HCV treatment that avoids both interferon and ribavirin. Four other such medications have since been approved. Sofosbuvir serves as the backbone of two, and the other two are based on different compounds.
Bartenschlager, Rice, and Sofia surmounted numerous hurdles as they devised innovative solutions to the biological and chemical obstacles that confronted them. Their victories culminated in a safe, effective, oral therapy for HCV that set a new standard and transformed the treatment of a devastating illness.
by Evelyn Strauss
Key Publications of Ralf F.W. Bartenschlager
Lohmann, V., Körner, F., Koch, J.O., Herian, U., Theilmann, L., and Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 285, 110-113.
Bartenschlager, R. and Lohmann, V. (2000). Replication of hepatitis C virus. J. Gen. Virol. 81, 1631-1648.
Krieger, N., Lohmann, V., and Bartenschlager, R. (2001). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J. Virol. 75, 4614-4624.
Wakita, T., Pietschmann, T., Kato, T., Date, T., Miyamoto, M., Zhao, Z., Murthy, K., Habermann, A., Kräusslich, H.-G., Mizokami, M., Bartenschlager, R., and Liang, T.J. (2005). Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat. Med. 11, 791-796.
Pietschmann, T., Kaul, A., Koutsoudakis, G., Shavinskaya, A., Kallis, S., Steinmann, E., Abid, K., Negro, F., Dreux, M., Cosset, F.L., and Bartenschlager, R. (2006). Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimeras. Proc. Natl. Acad. Sci. USA. 103, 7408-7413.
Key Publications of Charles M. Rice
Grakoui, A., Lin, C., Wychowski, C., Feinstone, S., and Rice, C.M. (1993). Expression and identification of hepatitis C virus polyprotein cleavage products. J. Virol. 67, 1385-1395.
Kolykhalov, A.A., Feinstone, S.M., and Rice, C.M. (1996). Identification of a highly conserved sequence element at the 3′ terminus of hepatitis C virus genome RNA. J. Virol. 70, 3363-3371.
Kolykhalov, A.A., Agapov, E.V., Blight, K.J., Mihalik, K., Feinstone, S.M., and Rice, C.M. (1997). Transmission of hepatitis C by intrahepatic inoculation with transcribed RNA. Science. 277, 570-574.
Blight, K.J., Kolykhalov, A.A., and Rice, C.M. (2000). Efficient initiation of HCV RNA replication in cell culture. Science. 290, 1972-1974.
Lindenbach, B.D., Evans, M.J., Syder, A.J., Wölk, B., Tellinghuisen, T.L., Liu, C.C., Maruyama, T., Hynes, R.O., Burton, D.R., McKeating, J.A., and Rice, C.M. (2005). Complete replication of hepatitis C virus in cell culture. Science. 309, 623-626.
Key Publications of Michael J. Sofia
Sofia, M.J., Bao, D., Chang, W., Du, J., Nagarathnam, D., Rachakonda, S., Reddy, P.G., Ross, B.S., Wang, P., Zhang, H.R., Bansal, S., Espiritu, C., Keilman, M., Lam, A.M., Steuer, H.M., Niu, C., Otto, M.J., and Furman, P.A. (2010). Discovery of a β-ᴅ-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus. J. Med. Chem. 53, 7202-7218.
Sofia, M.J., Furman, P.A., and Symonds, W.T. (2010). 2′-F-2′-C-methyl nucleosides and nucleotides for the treatment of hepatitis C virus: from discovery to the clinic. In: Accounts in Drug Discovery: Case Studies in Medicinal Chemistry. Chapter 11. Royal Society of Chemistry, London, pp. 238-266.
Sofia, M.J. (2013). Nucleotide prodrugs for the treatment of HCV infection. Adv. Pharmacol. 67, 39-73.
Sofia, M.J. (2014). Beyond sofosbuvir: what opportunity exists for a better nucleoside/nucleotide to treat hepatitis C? Antiviral Res. 107, 119-124.
Sofia, M.J. (2015). Sofosbuvir: a breakthrough curative therapy for the treatment of HCV infection. In: Medicinal Chemistry Reviews. Vol. 50. Med. Chem. Div., Am. Chem. Soc., Foster City, CA. pp. 397-416.
Editorial Comments on Therapy for Hepatitis C
Hoofnagle, J.H. and Sherker, A.H. (2014). Therapy for Hepatitis C – the costs of success. New Engl. J. Med. 370, 1552-1553.
Liang, T.J. and Ghany, M.G. (2014). Therapy of Hepatitis C – back to the future. New Engl. J. Med. 370, 2043-2047.