HCV Replicon Technology


		HCV Replicon Technology

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).

HCV Genome Organization

Structure of HCV Replicons

(modified from Lohmann et al. Science 285:110, 1999)

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

hvc_3.gif (6696 bytes)

transfect Huh7 hepatoma cells (electroporation)

select G418-resistant cell clones (v. low frequency)

persistently replicating HCV RNAs

copy #: 500-1000 RNAs/cell

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:

	Evaluating lead compounds which biochemical analyses suggest have anti-HCV activity
	Screening libraries to find compounds with anti-HCV activity
	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:

	The replicon contains the ORF of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B
	Genetic evidence has shown that NS5B is required (GDD mutant)
	The replicon contains both the 5' NTR and 3' NTR
	Replicon RNA replication is resistant to actinomycin D
	Expression of non-structural proteins is resistant to actinomycin D
	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).

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:

	Assay set up
	Total RNA extraction
	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:

	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.)
	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.
	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.
	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)