บทความเรื่อง Hepatitis C Virus – From Discovery to Cure

1. Copyright 2016 American Medical Association. All rights reserved.
Hepatitis C Virus—From Discovery to Cure
The 2016 Lasker-DeBakey
Clinical Medical Research Award
The 2016 Lasker-DeBakey Clinical Medical Research Award
has been presented to Ralf F. W. Bartenschlager, Charles M.
Rice, and Michael J. Sofia for the development of a system
to study the replication of the virus that causes hepatitis C
virus and for use of this system to revolutionize the treat-
ment of this chronic, often lethal disease.
The liver is the largest organ in the human body and is
central for metabolism and many other functions. Sev-
eralvirusesspecializeininfectingtheliverandarecalled
hepatitis viruses. Five such viruses are known, includ-
ing hepatitis C virus (HCV), which was originally recog-
nizedasanagentofposttransfusionnon-A,non-Bhepa-
titis. Given that about 6% of patients receiving blood
transfusionsdevelopednon-A,non-Bhepatitis,tremen-
dous efforts were mounted to isolate and molecularly
clone this filterable agent, likely a virus.
Inalandmarkpaperin1989,Houghtonandhisteam
isolated the first molecular clone of HCV and provided
aglimpseoftheHCVgenome:apositive-strandRNAvi-
rus with a genome length of around 9500 nucleotides
encoding a long polyprotein that was likely cleaved co-
translationally and posttranslationally into 8 to 10
products.1
Work in many laboratories, subsequently
identified 10 HCV proteins generated by the action of
host cell and viral proteases, including 2 viral enzymes,
thenonstructuralproteins(NS)3-4Aserineproteaseand
the NS5B RNA-dependent RNA polymerase, highly at-
tractive HCV drug targets. Subsequent research by
Drs Bartenschlager, Rice, and Sofia led to the develop-
ment of new and effective treatments for HCV.2-7
The bottleneck in drug development was the lack
of cell culture systems for HCV, but the availability of
molecular HCV clones raised hope because the RNA
genome of positive-strand RNA viruses is infectious.
Introducing genome RNA or a genome RNA equivalent
transcribed from a plasmid into permissive cells can ini-
tiate an entire viral life cycle. The genome RNA is recog-
nized by cellular ribosomes, translated to produce the
viral proteins, and, in concert with additional factors
from the host cell, amplified and used to make infec-
tious virus. However, this approach, which had suc-
ceeded for many other viruses, failed for HCV. One rea-
son was a missing piece at the 3′ end of viral genome
finally discovered by the laboratories of Kunitada
Shimotohno and Charles M. Rice. With the HCV
genome now likely complete, making a functional
complementary DNA (cDNA) clone should be easy, but
how would this be tested without a cell culture system?
In 1997, clones reflecting a “consensus” sequence were
used to filter out possible lethal mutations present in
the patient-derived HCV population or acquired during
cDNA cloning in the laboratory. Injection of this syn-
thetic, naked genome RNA into the liver of chimpan-
zees gave rise to a productive HCV infection and pro-
vided the first genetic system for proving that possible
HCV-specific drug targets were essential for the virus.
With virtually unlimited quantities of HCV ge-
nome RNA, validated as infectious in vivo, it might be
expected that finding a suitable cell culture system
would quickly follow, but that was not the case. The
solution came from work in the laboratory of Ralf
Bartenschlager that used another HCV consensus
genome cloned from the liver of a chronically infected
patient. With the aim to isolate rare cells supporting ro-
bust HCV replication, “selectable minigenomes,” called
replicons,wereengineered.Theserepliconsencodethe
minimal set of viral proteins assumed to be required for
autonomousreplicationand,inaddition,ageneconfer-
ringresistanceagainstthecytotoxicdrugG418.Byusing
drug selection, cells supporting efficient and long-term
HCV replication could be isolated. This first robust HCV
cellculturemodelrecapitulatedalltheintracellularsteps
of the HCV replication cycle and because replication of
these HCV RNAs relied on the viral enzymes, most no-
tably the NS3 protease and the NS5B polymerase, the
replicon system was suitable for drug development.
Subsequent studies conducted in the Rice and
Bartenschlager laboratories unveiled the reasons for
such high replication efficiency. First, the most HCV-
permissive individual cells in a given cell pool had been
selected; second, the replicons present in selected cell
clones harbored mutations that enhanced HCV RNA
replication by orders of magnitude. Insertion of these
mutations into the parental replicon allowed direct
measurement of HCV replication. Thus, the first widely
useful genetic systems for studying HCV biology were
created. Subsequent work by Bartenschlager, Rice, and
others refined this approach.
With the robust HCV replicon whole-cell system
availableforscreeningofsmallmolecules,thesearchfor
inhibitors of HCV became a major focus of pharmaceu-
tical and biotech companies. This was propelled by the
desire to identify direct-acting antivirals with the ulti-
mategoalofreplacingthethencurrentstandardofcare
for treating HCV infection, the combination of inject-
able interferon plus ribavirin, which was limited by se-
vere adverse effects, modest cure rates, and limited
genotype coverage.
In 2005 efforts in HCV drug discovery were fo-
cused on several key viral targets, including the NS5B
RNA-dependent RNA polymerase (NS5B RdRp). The
Ralf F. W.
Bartenschlager, PhD
Heidelberg University
Hospital, Heidelberg,
Germany.
Charles M. Rice, PhD
Laboratory of Virology
and Infectious Disease
and Center for the
Study of Hepatitis C,
Rockefeller University,
New York, New York.
Michael J. Sofia, PhD
Arbutus Biopharma,
Doylestown,
Pennsylvania.
Viewpoint
pages 1252 and 1256
Supplemental
content
Corresponding
Author: Michael J.
Sofia, PhD, Arbutus
Biopharma, 3805 Old
Easton Rd, Doylestown,
PA 18902 (msofia
@arbutusbio.com).
VIEWPOINT
Opinion
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search for nucleoside inhibitors of HCV replication at Pharmasset
identified a unique 2′-α-F-2′-β-C-methylcytidine derivative
PSI-6130 (an inhibitor of HCV). Targeting of the HCV RdRp with a
nucleoside drug held particular promise for providing broad geno-
type coverage and for preventing formation of resistant virus. This
nucleoside was shown to be active as an inhibitor of HCV using the
repliconsystemanddemonstratedhighselectivityforHCVvsother
viruses,exhibitedapangenotypicprofile,anddemonstratedahigh
barrier to resistance. However, PSI-6130 lacked sufficient oral bio-
availabilityandwasmetabolizedtoformasubstantialamountofan
inactiveuridinenucleoside—thuslimitingtheamountofactivedrug
available to target HCV.
An initial attempt to improve on PSI-6130 led to the develop-
ment of a PSI-6130 prodrug. In a clinical study, PSI-6130 prodrug
demonstrated proof of concept that the 2′-α-F-2′-β-C-methylcyti-
dinenucleosideinhibitedHCVinpatientsandthat,forthefirsttime,
a direct-acting antiviral was effective in non–genotype 1 patients.
However, the limitations of this drug candidate were potency, the
requirement for a large amount of drug given twice daily to show
efficacy, and the inactive uridine metabolite still persisted.
To address the deficiencies of the PSI-6130 prodrug a radical
redesign of this nucleoside inhibitor was developed by Sofia and
colleagues. Studies showed that the triphosphate of the uridine
nucleoside metabolite was a potent inhibitor of the HCV RdRp
and also exhibited a long half-life in primary human hepatocytes.
A long triphosphate half-life would lead to high active drug con-
centrations inside cells. It was determined that a block in the
metabolic conversion of the uridine nucleoside to the active tri-
phosphate was the reason for its inactivity. The block was specifi-
cally in the conversion of the uridine nucleoside to the mono-
phosphate intermediate.
To address the problem of delivering a highly charged, un-
stable, and membrane-impermeable uridine monophosphate into
cells and ultimately into the body, a “Trojan horse” strategy was de-
veloped that masked the uridine monophosphate in such a way as
toimpartstabilityandfacilitatetransportintothebodyandintoliver
cells. Once inside the liver cell, the “mask” would need to fall off re-
vealing the monophosphate that would then be converted by the
cell to the triphosphate inhibitor. At the same time, the mask was
designedwiththeintentionoffacilitatinglivertargetingofthedrug
byleveragingliverfirst-passmetabolism—usingliverenzymestose-
lectively remove the mask and ultimately trap the drug inside liver
cells. This would increase the concentration of the drug in the liver
and reduce drug exposure to the rest of the body.
Afterextensiveinvitroandinvivopreclinicaldevelopmentthat
evaluated numerous versions of masked uridine nucleoside mono-
phosphates, the clinical candidate PSI-7851 was selected. Subse-
quently, PSI-7851 was shown to be highly efficacious in HCV pa-
tientswhendosedorallyoncedailyinashort-durationstudywithout
any observed drug-related adverse events or drug resistance. This
resultprovidedproofofconceptforaliver-targetednucleosidemono-
phosphate prodrug. In a landmark clinical study named “Electron,”
theinterferon-freecombinationofPSI-7977(thesingle-isomericver-
sion of PSI-7851) plus ribavirin dosed for 12 weeks resulted in a
100% cure rate (ie, defined as sustained virologic response at the
completion of therapy) in patients with HCV genotype 2 and 3. This
result established a new approach for how clinicians could treat pa-
tientsinfectedwithHCV.Nolongerwasinjectableinterferonneeded
as part of the drug combination. Phase 3 clinical studies with
PSI-7977(nowcalledsofosbuvir)plusribavirindemonstratedhighcure
rates, and, subsequently, sofosbuvir plus ribavirin was approved by
theUSFoodandDrugAdministrationasthefirstinterferon-freetreat-
mentregimenforpatientswithHCVgenotype2and3.However,so-
fosbuvir plus ribavirin for 12 weeks was not quite good enough to
achieve high cure rates in patients with HCV genotype 1.
With the development of reporter replicons containing both a
selection marker and a reporter gene, high-throughput screens
against millions of compounds were enabled, and this led to iden-
tification of a new class of very potent HCV inhibitors, the NS5A in-
hibitors.Thisdevelopmentdemonstratedtheversatilityoftherepli-
con system and the identification of a peculiar drug target lacking
enzymaticactivity.WithfurtherdevelopmentofNS5Ainhibitorsand
next-generation NS3-4 protease inhibitors, new and potent drug
combinations with sofosbuvir as the backbone were now available
for clinical study. This led to the approval of multiple sofosbuvir-
based drug combinations for treating HCV, and the approval of the
firstsingle-pill,fixed-dosecombinationofsofosbuvirplustheNS5A
inhibitor ledipasvir that provided more than 95% cure rates for pa-
tients infected with HCV genotype 1.
The journey from the identification of a virus that causes de-
bilitatingliverdamagetoasafeandhighlyeffectivecureforHCVhas
taken more than 24 years. The possibility now exists that with time
and commitment, HCV infection can one day be given the designa-
tion of a rare disease.
ARTICLE INFORMATION
Published Online: September 13, 2016.
doi:10.1001/jama.2016.13713
Conflict of Interest Disclosures: All authors have
completed and submitted the ICMJE Form for
Disclosure of Potential Conflicts of Interest. For a
full list of disclosures for each author, see the
Supplement.
REFERENCES
1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley
DW, Houghton M. Isolation of a cDNA clone derived
from a blood-borne non-A, non-B viral hepatitis
genome. Science. 1989;244(4902):359-362.
2. Lohmann V, Körner F, Koch J, Herian U,
Theilmann L, Bartenschlager R. Replication of
subgenomic hepatitis C virus RNAs in a hepatoma
cell line. Science. 1999;285(5424):110-113.
3. Lohmann V, Körner F, Dobierzewska A,
Bartenschlager R. Mutations in hepatitis C virus
RNAs conferring cell culture adaptation. J Virol.
2001;75(3):1437-1449.
4. Blight KJ, Kolykhalov AA, Rice CM. Efficient
initiation of HCV RNA replication in cell culture.
Science. 2000;290(5498):1972-1974.
5. Kolykhalov AA, Agapov EV, Blight KJ, Mihalik K,
Feinstone SM, Rice CM. Transmission of hepatitis C
by intrahepatic inoculation with transcribed RNA.
Science. 1997;277(5325):570-574.
6. Sofia MJ, Bao D, Chang W, et al. Discovery of a
β-D-2′-deoxy-2′-α-fluoro-2′-β-C-methyluridine
nucleotide prodrug (PSI-7977) for the treatment of
hepatitis C virus. J Med Chem. 2010;53(19):7202-
7218.
7. Lam AM, Murakami E, Espiritu C, et al. PSI-7851,
a pronucleotide of beta-D-2′-deoxy-2′-fluoro-2′-C-
methyluridine monophosphate, is a potent and
pan-genotype inhibitor of hepatitis C virus
replication. Antimicrob Agents Chemother. 2010;54
(8):3187-3196.
Viewpoint Opinion
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