Introduction
Apath is pleased to announce the
availability of an HCV replicon system for use in antiviral discovery
research programs. Apath possesses both a highly robust
replication system and a critical intellectual property position with
respect to HCV replicons. Apath and
various other labs have conducted numerous experiments which validate
important attributes of Apath's replicon system, including the presence
of bona fide replication, and usefulness in bioassays to test for
anti-HCV activity. Apath is making these assets available to the
pharmaceutical research field via non-exclusive
licenses.
Background
Identification of a well-defined
and robust cell-culture system for hepatitis C virus (HCV) is critical
to efforts to identify and evaluate antivirals against HCV. Until
recently, none of the published HCV cell-culture replication systems was
sufficiently robust to be used effectively in an antiviral screening
assay. In 1999, Lohmann et al. described a subgenomic HCV replicon
constructed from genotype 1b RNA, and isolated Huh7 cell lines which
contain this autonomously replicating replicon (
Lohmannet al. Science. 285:110-3).
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Structure
of HCV
Replicons
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Cell lines
containing a similar HCV replicon were made in the laboratory of
Charles M. Rice, PhD at Washington University, St. Louis, MO and are
licensed exclusively to Apath. The cells have been analyzed extensively
and have been demonstrated to contain an HCV replicon which exhibits
autonomous HCV RNA replication.
Selectable
HCV RNA replicons
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- transfect Huh7 hepatoma
cells (electroporation)
- select G418-resistant cell
clones (v. low frequency)
- persistently replicating
HCV RNAs
- copy #: 500-1000 RNAs/cell
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The Rice laboratory
has also produced a series of second-generation replicons, known as
Blazing Blight, which exhibit similar characteristics in cell
culture, but have a transfection efficiency several orders of magnitude
higher than the first generation.
(see
Science 290(5498):1972-1975;Dec 8, 2000)
Value
of Replicon Cell Lines
This replication
system represents the best available system for identifying and testing
inhibitors of HCV and thus could be an essential tool for the discovery
of new drugs.
Apath's
replicon system has the potential to serve as a valuable tool for:
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Evaluating lead
compounds which biochemical analyses suggest have anti-HCV activity
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Screening
libraries to find compounds with anti-HCV activity
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Identifying
drug-resistant mutations
This system may also
represent a major step toward development of complete virus replication
in cell culture.
Apath's
Replicon System
Dr. Keril
Blight in the laboratory of Dr. Charles Rice constructed the replicon
and isolated the replicon-expressing cell lines. Two
independently-derived Huh7 cell lines, known as Huh7/Ava.5 and
Huh7/Clone A, contain functioning replicons.
Evidence that the Apath replicon represents bona fide HCV replication
Several lines
of evidence obtained by both the Rice and Apath labs demonstrate these
cells contain a valid model of HCV RNA replication and protein
expression:
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The replicon
contains the ORF of the nonstructural proteins NS3, NS4A, NS4B, NS5A,
and NS5B
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Genetic evidence
has shown that NS5B is required (GDD mutant)
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The replicon
contains both the 5' NTR and 3' NTR
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Replicon RNA
replication is resistant to actinomycin D
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Expression of
non-structural proteins is resistant to actinomycin D
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Southern blots are
negative for any replicon nucleic acid integrated into the host
chromosome
Validation of Bona Fide Replication
Dr. Blight has
extensive data verifying that expression of the replicon represents bona
fide HCV replication. The replicon cells have also been sent to two
independent laboratories within the pharmaceutical industry. Both
laboratories verified that HCV replication is ongoing in the cells and
that expression of HCV RNA and proteins is independent of cellular
DNA-dependent transcription (actinomycin D-resistant).
Click here to view validation reports from third parties
Validation as an Anti-Viral Screening Tool
Toxicity
Profiles
Apath and two
independent groups have shown that the replicon cells have toxicity
profiles (e.g. to DMSO and ethanol) that are compatible with drug
testing.
Testing of
Inhibitors
Apath has
tested the usefulness of the replicon cell lines in a bioassay to test
for anti-HCV activity. In these experiments, various compounds were
evaluated for their impact on replication, as measured by replicon RNA
levels in the cells. The first-generation assay has three steps:
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Assay set up
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Total RNA
extraction
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Replicon RNA
quantitation by qRT-PCR or TaqMan
Responsiveness to Non-Specific Inhibitors
Apath has treated
the Huh7/Ava.5 and Huh7/Clone A cells with interferon
a
and observed dramatic reductions in replicon RNA levels within 24 hours.
These results demonstrate that the rate of replicon turnover is
sufficiently robust that inhibitors of HCV can be assessed in such an assay based on measurement of
total replicon levels following treatment. This in turn provides
evidence that specific inhibitors of HCV could be evaluated in
this system.
Responsiveness to Specific Inhibitors
Apath has been in
contact with several large and small pharmaceutical companies who have
indicated interest in having Apath test their lead compounds for
anti-HCV activity in a replicon-based assay. These compounds have been
screened for activity against HCV enzymes such as the NS3 protease or
the NS5B RNA polymerase in biochemical assays and now require evaluation
in a cell-based antiviral assay.
Apath has performed
several rounds of blind testing on a group of such lead compounds
together with inactive compounds and negative controls. Two compounds
were identified that exhibited significant inhibitory activity in our
assay. After testing, it was later learned that these compound were ones
that displayed potent anti-HCV activity in an in vitro assay.
These results validate that for compounds that have inhibitory activity
against a process that would be expected to affect replicon replication,
this system can be a very useful secondary bioassay.
Limitations as a Drug-Screening Tool
Although the
replicon systems can be an excellent tool in the anti-HCV drug
development process, several limitations and caveats must be considered:
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The present system
has limited usefulness for reverse genetics because of the low
transfection efficiency. (The latest version, BB7, is close to
overcoming this limitation.)
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The replicon as
presently formatted is a system of steady-state RNA replication as
opposed to the burst of RNA replication that occurs in a
virus-infected cell.
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The replicon cell
lines have been selected for and are adapted for non-cytopathic
replication in Huh7 cells. Thus the replicon may exhibit differences
in response to certain agents compared to wild type virus.
-
Because the
replicon represents subgenomic RNA replication, it is not useful for
certain anti-HCV agents that target certain events in the HCV life
cycle, i.e., the early and late stages of virus infection.
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Because the
existing replicon was generated using genotype 1b HCV RNA, the present
replicon system may not be used to detect responses that are genotype
and subtype-dependent.
Apath is actively
working to make improvements in the replicon and address these
limitations (see below).
Improvements in the system: Second-Generation Replicons
One of the
limitations of the replicon system described in Lohmann et al. was the
poor transfection efficiency (<10-6 colonies/ug). Dr. Keril
Blight isolated replicons which have dramatically increased transfection
efficiency. Dr. Blight isolated
Blazing Blight 7 which has a transfection efficiency of 10-1
(i.e., 10% of the cell transfection display replicon-dependent G418
resistance). This level of transfection efficiency may be sufficient to
enable reverse genetic analyses. In fact, consistent with this,
experiments have shown that the level of RNA replication following
transfection of BB7 is sufficiently robust to allow first cycle RNA
replication measurement.
(see
Science 290(5498):1972-1975;Dec 8, 2000)
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