Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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HCV NS3 PROTEASE REPLICON SHUTTLE VECTORS
This invention pertains to novel HCV NS3 protease replicon shuttle vectors
which are useful
for screening, testing and evaluating HCV and other Flavivirus protease
inhibitors.
Hepatitis C virus is a major health problem and the leading cause of chronic
liver disease
throughout the world. (Boyer, N. et al. J. Hepatol. 2000 32:98-112). Patients
infected with
HCV are at risk of developing cirrhosis of the liver and subsequent
hepatocellular carcinoma
and hence HCV is the major indication for liver transplantation.
According to the World Health Organization, there are more than 200 million
infected
individuals worldwide, with at least 3 to 4 million people being infected each
year. Once
infected, about 20% of people clear the virus, but the rest can harbor HCV the
rest of their
lives. Ten to twenty percent of chronically infected individuals eventually
develop liver-
destroying cirrhosis or cancer. The viral disease is transmitted parenterally
by contaminated
blood and blood products, contaminated needles, or sexually and vertically
from infected
mothers or carrier mothers to their offspring. Current treatments for HCV
infection, which
are restricted to immunotherapy with recombinant interferon-a alone or in
combination with
the nucleoside analog ribavirin, are of limited clinical benefit particularly
for genotype 1.
There is an urgent need for improved therapeutic agents that effectively
combat chronic HCV
infection
HCV has been classified as a member of the virus family Flaviviridae that
includes the
genera flaviviruses, pestiviruses, and hepaciviruses which includes hepatitis
C viruses (Rice,
C. M., Flaviviridae: The viruses and their replication, in: Fields Virology,
Editors: Fields, B.
N., Knipe, D. M., and Howley, P. M., Lippincott-Raven Publishers,
Philadelphia, Pa.,
Chapter 30, 931-959, 1996). HCV is an enveloped virus containing a positive-
sense single-
stranded RNA genome of approximately 9.4 kb. The viral genome consists of a 5'-
untranslated region (UTR), a long open reading frame encoding a polyprotein
precursor of-
approximately 3011 amino acids, and a short 3' UTR. The 5' UTR is the most
highly
conserved part of the HCV genome and is important for the initiation and
control of
polyprotein translation.
Genetic analysis of HCV has identified six main genotypes showing a >30%
divergence in
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the DNA sequence. Each genotype contains a series of more closely related
subtypes which
show a 20-25 % divergence in nucleotide sequences (Simmonds, P. 2004 J. Gen.
Virol.
85:3173-88) . More than 30 subtypes have been distinguished. In the US
approximately 70%
of infected individuals have type la and lb infection. Type lb is the most
prevalent subtype
in Asia. (X. Forns and J. Bukh, Clinics in Liver Disease 1999 3:693-716; J.
Bukh et at.,
Semin. Liv. Dis. 1995 15:41-63). Unfortunately Type 1 infections are less
responsive to the
current therapy than either type 2 or 3 genotypes (N. N. Zein, Clin.
Microbiol. Rev., 2000
13:223-235).
The genetic organization and polyprotein processing of the nonstructural
protein portion of
the ORF of pestiviruses and hepaciviruses is very similar. These positive
stranded RNA
viruses possess a single large open reading frame (ORF) encoding all the viral
proteins
necessary for virus replication. These proteins are expressed as a polyprotein
that is co- and
post-translationally processed by both cellular and virus-encoded proteinases
to yield the
mature viral proteins. The viral proteins responsible for the replication of
the viral genome
RNA are located towards the carboxy-terminal. Two-thirds of the ORF are termed
nonstructural (NS) proteins. For both the pestiviruses and hepaciviruses, the
mature
nonstructural (NS) proteins, in sequential order from the amino-terminus of
the nonstructural
protein coding region to the carboxy-terminus of the ORF, consist of p7, NS2,
NS3, NS4A,
NS4B, NS5A, and NS5B.
The NS proteins of pestiviruses and hepaciviruses share sequence domains that
are
characteristic of specific protein functions. For example, the NS3 proteins of
viruses in both
groups possess amino acid sequence motifs characteristic of serine proteinases
and of
helicases (Gorbalenya et at. Nature 1988 333:22; Bazan and Fletterick Virology
1989
171:637-639; Gorbalenya et at. Nucleic Acid Res. 1989 17.3889-3897).
Similarly, the NS5B
proteins of pestiviruses and hepaciviruses have the motifs characteristic of
RNA-directed
RNA polymerases (Koonin, E. V. and Do1ja, V. V. Crit. Rev. Biochem. Molec.
Biol. 1993
28:375-430).
The actual roles and functions of the NS proteins of pestiviruses and
hepaciviruses in the
lifecycle of the viruses are directly analogous. In both cases, the NS3 serine
proteinase is
responsible for all proteolytic processing of polyprotein precursors
downstream of its position
in the ORF (Wiskerchen and Collett Virology 1991 184:341-350; Bartenschlager
et at. J.
Virol. 1993 67:3835-3844; Eckart et at. Biochem. Biophys. Res. Comm. 1993
192:399-406;
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Grakoui et at. J. Virol. 1993 67:2832-2843; Grakoui et at. Proc. Natl. Acad.
Sci. USA 1993
90:10583-10587; Ilijikata et at. J. Virol. 1993 67:4665-4675; Tome et at. J.
Virol. 1993
67:4017-4026). The NS4A protein, in both cases, acts as a cofactor with the
NS3 serine
protease (Bartenschlager et at. J. Virol. 1994 68:5045-5055; Failla et at. J.
Virol. 1994 68:
3753-3760; Xu et at. J Virol. 1997 71:53 12-5322). The NS3 protein of both
viruses also
functions as a helicase (Kim et at. Biochem. Biophys. Res. Comm. 1995 215: 160-
166; Jin and
Peterson Arch. Biochem. Biophys. 1995, 323:47-53; Warrener and Collett J.
Virol. 1995
69:1720-1726). Finally, the NS5B proteins of pestiviruses and hepaciviruses
have the
predicted RNA-dependent RNA polymerase activity (Behrens et al. EMBO 1996
15:12-22;
Lechmann et at. J. Virol. 1997 71:8416-8428; Yuan et at. Biochem. Biophys.
Res. Comm.
1997 232:231-235; Hagedorn, PCT WO 97/12033; Zhong et at. J. Virol. 1998
72:9365-
9369).
The HCV NS protein 3 (NS3) contains a serine protease activity that helps
process the
majority of the viral enzymes, and is thus considered essential for viral
replication and
infectivity. It is known that mutations in the yellow fever virus NS3 protease
decreases viral
infectivity (Chambers et. at., Proc. Natl. Acad. Sci. USA, 1990, 87:8898-
8902). The first 181
amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been
shown to contain
the serine protease domain of NS3 that processes all four downstream sites of
the HCV
polyprotein (Lin et at., J. Virol. 1994 68:8147-8157). The HCV NS3 serine
protease and its
associated cofactor, NS4A, helps process all of the viral enzymes, and is thus
considered
essential for viral replication. This processing appears to be analogous to
that carried out by
the human immunodeficiency virus aspartyl protease, which is also involved in
viral enzyme
processing HIV protease inhibitors, which inhibit viral protein processing are
potent antiviral
agents in man, indicating that interrupting this stage of the viral life cycle
results in
therapeutically active agents. Consequently it is an attractive target for
drug discovery.
Currently there are a limited number of approved therapies are currently
available for the
treatment of HCV infection. New and existing therapeutic approaches to
treating HCV and
inhibition of HCV NS5B polymerase have been reviewed: R. G. Gish, Sem. Liver.
Dis., 1999
19:5; Di Besceglie, A. M. and Bacon, B. R., Scientific American, October: 1999
80-85; G.
Lake-Bakaar, Current and Future Therapy for Chronic Hepatitis C Virus Liver
Disease, Curr.
Drug Targ. Infect Dis. 2003 3(3):247-253; P. Hoffmann et at., Recent patents
on
experimental therapy for hepatitis C virus infection (1999-2002), Exp. Opin.
Ther. Patents
2003 13(11):1707-1723; F. F. Poordad et at. Developments in Hepatitis C
therapy during
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2000-2002, Exp. Opin. Emerging Drugs 2003 8(1):9-25; M. P. Walker et at.,
Promising
Candidates for the treatment of chronic hepatitis C, Exp. Opin. Investig.
Drugs 2003
12(8):1269-1280; S.-L. Tan et at., Hepatitis C Therapeutics: Current Status
and Emerging
Strategies, Nature Rev. Drug Discov. 2002 1:867-88 1; R. De Francesco et at.
Approaching a
new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine
protease and the NS5B
RNA-dependent RNA polymerase, Antiviral Res. 2003 58:1-16; Q. M. Wang et at.
Hepatitis
C virus encoded proteins: targets for antiviral therapy, Drugs of the Future
2000 25(9):933-8-
944; J. A. Wu and Z. Hong, Targeting NS5B-Dependent RNA Polymerase for Anti-
HCV
Chemotherapy Cur. Drug Targ.-Inf. Dis.2003 3:207-219.
Despite advances in understanding the genomic organization of the virus and
the functions of
viral proteins, fundamental aspects of HCV replication and pathogenesis remain
unknown. A
major challenge in gaining experimental access to HCV replication is the lack
of an efficient
cell culture system that allows production of infectious virus particles.
Although infection of
primary cell cultures and certain human cell lines has been reported, the
amounts of virus
produced in those systems and the levels of HCV replication have been too low
to permit
detailed analyses.
The construction of selectable subgenomic HCV RNAs that replicate with minimal
efficiency
in the human hepatoma cell line Huh-7 has been reported. Lohman et al.
reported the
construction of a replicon (I 377/NS3-3') derived from a cloned full-length
HCV consensus
genome (genotype lb) by deleting the C-p7 or C-NS2 region of the protein-
coding region
(Lohman et al., Science 1999 285: 110-113). The replicon contained the
following elements:
(i) the HCV 5'-UTR fused to 12 amino acids of the capsid encoding region; (ii)
the neomycin
phosphotransferace gene (NPTII); (iii) the IRES from encephalomyocarditis
virus (EMCV),
inserted downstream of the NPTII gene and which directs translation of HCV
proteins NS2 or
NS3 to NS5B; and (iv) the 3'-UTR. After transfection of Huh-7 cells, only
those cells
supporting HCV RNA replication expressed the NPTII protein and developed
resistance
against the drug G418. While the cell lines derived from such G418 resistant
colonies
contained substantial levels of replicon RNAs and viral proteins, only 1 in
106 transfected
Huh-7 cells supported HCV replication.
Similar selectable HCV replicons were constructed based on an HCV-H genotype 1
a
infectious clone (Blight et al., Science 2000 290:1972-74). The HCV-H derived
replicons
were unable to establish efficient HCV replication, suggesting that the
earlier-constructed
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replicons of Lohmann (1999), supra, were dependent on the particular genotype
1 b
consensus cDNA clone used in those experiments. Blight et al. (2000), supra,
reproduced the
construction of the replicon made by Lohmann et al. (1999), supra, by carrying
out a PCR-
based gene assembly procedure and obtained G418-resistant Huh-7 cell colonies.
Independent G418-resistant cell clones were sequenced to determine whether
high-level HCV
replication required adaptation of the replicon to the host cell. Multiple
independent adaptive
mutations that cluster in the HCV nonstructural protein NS5A were identified.
The mutations
conferred increased replicative ability in vitro, with transduction efficiency
ranging from 0.2
to 10% of transfected cells as compared to earlier- constructed replicons in
the art, e.g., the I
377/NS3-3' replicon had a 0.0001% transduction efficiency.
The present invention features the development of a novel HCV replicon shuttle
vector in
which unique restriction enzyme sites are introduced at the 5' and 3' ends of
the protease
domain of the NS3 gene such that NS3 protease sequences derived from the
samples of
HCV-infected patients can be cloned in the shuttle vector and the resulting
replicons be
evaluated for replication fitness and susceptibility to HCV NS3 protease
inhibitors. Since an
individual HCV-infected patient typically contains a genetically diverse virus
population due
to the high error rate of the NS5B RNA polymerase, the use of the shuttle
vector of the
present invention would allow the characterization of specific patient-derived
NS3 protease
variants and the sensitivity or resistance of these variants to drug
treatment.
Accordingly, the present invention provides an HCV replicon shuttle vector
comprising an
HCV polynucleotide sequence comprising, in order, a unique restriction enzyme
sequence
placed between 10 nucleotides 5' and 10 nucleotides 3' from the 5' end of a
polynucleotide
sequence encoding a NS3 protein; a polynucleotide sequence encoding the
protease domain
of the NS3 protein; a unique restriction enzyme sequence placed between 10
nucleotides 5'
and 10 nucleotides 3' from the 3' end of the polynucleotide sequence encoding
the protease
domain of the NS3 protein; a polynucleotide sequence encoding the helicase
domain of the
NS3 protein;
a polynucleotide sequence encoding a NS4A protein; a polynucleotide sequence
encoding a
NS4B protein; a polynucleotide sequence encoding a NS5A protein; and a
polynucleotide
sequence encoding a NS5B protein. In one embodiment of the invention, the
polynucleotide
sequence encoding the protease domain of the NS3 protein has been modified or
deleted such
that the protease domain of the NS3 protein is non-functional.
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In another embodiment of the invention, the unique restriction enzyme sequence
at the 5' end
of the polynucleotide sequence encoding the NS3 protein recognizes EcoRV and
the unique
restriction enzyme sequence at the 3' end of the polynucleotide sequence
encoding the
protease domain of the NS3 protein recognizes AsiSI. In still another
embodiment of the
invention, the HCV replicon shuttle vector comprises an HCV polynucleotide
sequence
selected from SEQ ID NO:3 or SEQ ID NO:6.
A further embodiment of the present invention provides a method for assessing
the
effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in
a subject
comprising the steps of providing a sample from the subject infected with HCV,
PCR-
amplifying polynucleotide sequences encoding the protease domain of the NS3
protein from
a plurarity of HCV quasispecies present in the sample with the use of a sense-
strand primer
which comprises a unique restriction enzyme sequence, and an anti-sense strand
primer
which comprises a different unique restriction enzyme sequence, cloning said
PCR-amplifed
polynucleotide sequences into an HCV replicon shuttle vector to produce
chimeric HCV
replicon plasmids, linearizing said chimeric HCV replicon plasmids and
subjecting said
linearized plasmids to in vitro transcription to produce chimeric HCV replicon
RNAs, and
transfecting a Huh7 cell line with said HCV replicon RNAs and measuring
replication level
of said HCV replicon RNAs in the presence or absence of the HCV NS3 protease
inhibitor.
A still further embodiment of the present invention provides a method for
assessing the
effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in
a subject
comprising the steps of providing a sample from the subject infected with HCV,
PCR-
amplifying polynucleotide sequences encoding the protease domain of the NS3
protein from
a plurarity of HCV quasispecies present in the sample with the use of a sense-
strand primer
which comprises a unique restriction enzyme sequence, and an anti-sense strand
primer
which comprises a different unique restriction enzyme sequence, cloning said
PCR-amplifed
polynucleotide sequences into an HCV replicon shuttle vector to produce
chimeric HCV
replicon plasmids, transforming said plasmids into cells to generate a
plurarity of colonies of
transformed cells, pooling said colonies and isolating chimeric HCV replicon
plasmids from
the pooled colonies, linearizing said chimeric HCV replicon plasmids,
subjecting said
linearized plasmids to in vitro transcription to produce chimeric HCV replicon
RNAs, and
transfecting Huh7 cell line with said HCV replicon RNAs and measuring
replication level of
said HCV replicon RNAs in the presence or absence of the HCV NS3 protease
inhibitor.
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The foregoing and other advantages and features of the invention, and the
manner in which
the same are accomplished, will become more readily apparent upon
consideration of the
following detailed description of the invention taken in conjunction with the
accompanying
examples, which illustrate exemplary embodiments.
Brief description of the drawings
Figure 1 is a schematic representation of the components of the HCV replicon
shuttle vectors.
Figure 2 shows the plasmid maps of the NS3 protease replicon shuttle vectors
(A)
pSC_lb_NS3/Protease_EcoRV_AsiSI (SEQ ID NO:3); (B)
pSC_lb_NS3/Protease/LacZ_EcoRV_AsiSI (SEQ ID NO:6).
Figure 3 shows the replication capability ofpSC_lb_NS3/Protease_EcoRV_AsiSI as
well as
replicon that contain patient-derived NS3 proteases from HCV genotype-la or
genotype lb.
RLU represents level of firefly luciferase signal observed after 96 hours
following
tranfection.
The term used in the present invention are defined below.
The term "HCV replicon" refers to a nucleic acid from the Hepatitis C virus
that is capable of
directing the generation of copies of itself. As used herein, the term
"replicon" includes RNA
as well as DNA, and hybrids thereof. For example, double-stranded DNA versions
of HCV
genomes can be used to generate a single-stranded RNA transcript that
constitutes an HCV
replicon. The HCV replicons can include full length HCV genome or HCV
subgenomic
contructs also referred as a "subgenomic replicon". For example, the
subgenomic replicons
of HCV described herein contain most of the genes for the non-structural
proteins of the
virus, but are missing most of the genes coding for the structural proteins.
Subgenomic
replicons are capable of directing the expression of all of the viral genes
necessary for the
replication of the viral subgenome, replication of the sub-genomic replicon,
without the
production of viral particles.
A basic HCV replicon is a subgenomic construct containing an HCV 5'-
untranslated (UTR)
region, an HCV NS3-NS5B polyprotein encoding region, and a HCV 3'-UTR. Other
nucleic
acid regions can be present such as those providing for HCV NS2, structural
HCV protein(s)
and non-HCV sequences.
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The HCV 5'-UTR region provides an internal ribosome entry site (IRES) for
protein
translation and elements needed for replication. The HCV 5'-UTR region
includes naturally
occurring HCV 5'-UTR extending about 36 nucleotides into a HCV core encoding
region,
and functional derivatives thereof. The 5'-UTR region can be present in
different locations
such as site downstream from a sequence encoding a selection protein, a
reporter, protein, or
an HCV polyprotein.
In addition to the HCV 5'-UTR-PC region, non-HCV IRES elements can also be
present in
the replicon. The non-HCV IRES elements can be present in different locations
including
immediately upstream the region encoding for an HCV polyprotein. Examples of
non-HCV
IRES elements that can be used are the EMCV IRES, poliovirus IRES, and bovine
viral
diarrhea virus IRES.
The HCV 3'-UTR assists HCV replication. HCV 3' UTR includes naturally
occurring HCV
3'-UTR and functional derivatives thereof. Naturally occurring 3'-UTRs include
a poly U
tract and an additional region of about 100 nucleotides.
The NS3-NS5B polyprotein encoding region provides for a polyprotein that can
be processed
in a cell into different proteins. Suitable NS3-NS5B polyprotein sequences
that may be part
of a replicon include those present in different HCV strains and functional
equivalents thereof
resulting in the processing of NS3-NS5B to produce a functional replication
machinery.
Proper processing can be measured for by assaying, for example, NS5B RNA
dependent
RNA polymerase.
A "vector" is a piece of DNA, such as a plasmid, phage or cosmid, to which
another piece of
DNA segment may be attached so as to bring about the replication, expression
or integration
of the attached DNA segment. A "shuttle vector" refers to a vector in which a
DNA segment
can be inserted into or excised from a vector at specific restriction enzyme
sites. The
segment of DNA that is inserted into shuttle vector generally encodes a
polypeptide or RNA
of interest and the restriction enzyme sites are designed to ensure insertion
of the DNA
segment in the proper reading frame for transcription and translation.
A variety of vectors can be used to express a nucleic acid molecule. Such
vectors include
chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from
bacterial
plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal
elements,
including yeast artificial chromosomes, from viruses such as baculoviruses,
papovaviruses
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such as SV40, vaccinia viruses, adenoviruses, poxviruses, pseudorabies
viruses, herpes
viruses, and retroviruses. Vectors may also be derived from combinations of
these sources,
such as those derived from plasmid and bacteriophage genetic elements, e.g.,
cosmids and
phagemids. Appropriate cloning and expression vectors for prokaryotic and
eukaryotic hosts
are described in Sambrook et al., (1989) Molecular Cloning: A Laboratory
Manual. 2nd edn.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
A vector containing the appropriate nucleic acid molecule can be introduced
into an
appropriate host cell for propagation or expression using known techniques.
Host cells can
include bacterial cells including, but not limited to, E. coli, Streptomyces,
and Salmonella
typhimurium, eukaryotic cells including, but not limited to, yeast, insect
cells, such as
Drosophila, animal cells, such as Huh-7, HeLa, COS, HEK 293, MT- 2T, CEM-SS,
and
CHO cells, and plant cells.
Vectors generally include selectable markers that enable the selection of a
subpopulation of
cells that contain the recombinant vector constructs. The marker can be
contained in the same
vector that contains the nucleic acid molecules described herein or may be on
a separate
vector. Markers include tetracycline- or ampicillin-resistance genes for
prokaryotic host cells
and dihydrofolate reductase or neomycin resistance for eukaryotic host cells.
However, any
marker that provides selection for a phenotypic trait will be effective.
A "polynucleotide" or "nucleic acid molecule" generally refers to any
polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or
DNA.
"Polynucleotides" include, without limitation single- and double-stranded DNA,
DNA that is
a mixture of single- and double-stranded regions, single- and double-stranded
RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid molecules
comprising
DNA and RNA that may be single-stranded or, more typically, double-stranded or
a mixture
of single- and double-stranded regions. In addition, "polynucleotide" refers
to triple-stranded
regions comprising RNA or DNA or both RNA and DNA. "Polynucleotide" also
embraces
relatively short polynucleotides, often referred to as oligonucleotides.
In addition, the term "DNA molecule" refers only to the primary and secondary
structure of
the molecule, and does not limit it to any particular tertiary forms. Thus,
the term includes
double-stranded DNA found, inter alia, in linear DNA molecules (e.g.,
restriction fragments),
viruses, plasmids, and chromosomes. In discussing the structure of particular
double-stranded
DNA molecules, sequences may be described herein according to the normal
convention of
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giving only the sequence in the 5' to 3' direction along the nontranscribed
strand of DNA
(i.e., the strand having a sequence homologous to the mRNA).
An "RNA molecule" refers to the polymeric form of ribonucleotides in its
either single-
stranded form or a double-stranded helix form. In discussing the structure of
particular RNA
molecules, sequence may be described herein according to the normal convention
of giving
the sequence in the 5' to 3' direction.
The term "restriction enzyme sequence" refers to a specific double stranded-
DNA sequence
which is recognized and cut by bacterial enzymes, each of which cut double-
stranded DNA at
or near a specific nucleotide sequence. The restriction enzyme "EcoRV"
recognizes the sequence 5' GAT'ATC 3' and cuts the double-stranded DNA at the
indicated
3' CTAATAG 5'
nucleotide position (shown with ' A). The restriction enzyme "AsiSI"
recognizes the
sequence
5' GCGAT'CGC 3' and cuts after the T residue on the recognition sequence.
3' CGCATAGCG 5'
The term "primer" as used herein refers to an oligonucleotide, either RNA or
DNA, either
single-stranded or double-stranded, either derived from a biological system,
generated by
restriction enzyme digestion, or produced synthetically which, when placed in
the proper
environment, is able to functionally act as an initiator of template-dependent
nucleic acid
synthesis. When presented with an appropriate nucleic acid template, suitable
nucleoside
triphosphate precursors of nucleic acids, a polymerase enzyme, suitable
cofactors and
conditions such as a suitable temperature and pH, the primer may be extended
at its 3'
terminus by the addition of nucleotides by the action of a polymerase or
similar activity to
yield a primer extension product. The primer may vary in length depending on
the particular
conditions and requirement of the application. For example, in PCR reactions,
the primer is
typically 15-25 nucleotides or longer in length. The primer must be of
sufficient
complementarity to the desired template to prime the synthesis of the desired
extension
product, i.e. to be able to anneal with the desired template strand in a
manner sufficient to
provide the 3'-hydroxyl moiety of the primer in appropriate juxtaposition for
use in the
initiation of synthesis by a polymerase or similar enzyme. It is not required
that the primer
sequence represent an exact complement of the desired template. For example, a
non-
complementary nucleotide sequence (e.g. a restriction enzyme recognition
sequence) may be
attached to the 5'-end of an otherwise complementary primer. Alternatively,
non-
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complementary bases may be interspersed within the oligonucleotide primer
sequence,
provided that the primer sequence has sufficient complementarity with the
sequence of the
desired template strand to functionally provide a template-primer complex for
the synthesis
of the extension product.
The term "chimeric" as used herein means a molecule of DNA that has resulted
from DNA
from two or more different sources that have been fused or spliced together.
As used herein, the term "quasispecies" means a collection of microvariants of
a predominant
HCV genome sequence (i.e. genotype), said microvariants being formed in a
single infected
subject or even in a single cell clone or even in a single cell clone as a
result of high mutation
rate during HCV replication.
The term "subject" as used herein refers to vertebrates, particular members of
the mammalian
species and includes, but not limited to, rodents, rabbits, shrews, and
primates, the latter
including humans.
The term "sample" refers to a sample of tissue or fluid isolated from a
subject, including but
not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the
external sections of
the skin, respiratory, intestinal and genitourinary tracts, tears, saliva,
milk, blood cells,
tumors, organs, and also samples of in vitro cell culture constituents
(including but not
limited to, conditioned medium resulting from the growth of cultured cells,
putatively viral
infected cells, recombinant cells, and cell components).
A cell has been "transformed" or "transfected" by exogenous or heterologous
DNA or RNA
when such DNA or RNA has been introduced inside the cell. The transforming or
transfecting DNA or RNA may or may not be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell. For example, in prokaryotes,
yeast,
and mammalian cells, the transforming DNA may be maintained on an episomal
element
such as a plasmid. With respect to eukaryotic cells, a stably transformed cell
is one in which
the transforming DNA has become integrated into a chromosome so that it is
inherited by
daughter cells through chromosome replication. This stability is demonstrated
by the ability
of the eukaryotic cell to establish cell lines or clones comprised of a
population of daughter
cells containing the transforming DNA. In the case of an HCV replicon that
transforms a
mammalian cell as described in the present invention, the RNA molecule, e.g.,
an HCV RNA
molecule, has the ability to replicate semi-autonomously. Huh-7 cells carrying
the HCV
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replicons are detected either by the presence of a selection marker or a
reporter gene present
on the replicon.
A "clone" refers to a population of cells derived from a single cell or common
ancestor
generally by the process of mitosis.
EXAMPLES
The following preparations and examples are given to enable those skilled in
the art to more
clearly understand and to practice the present invention. They should not be
considered as
limiting the scope of the invention, but merely as being illustrative and
representative thereof.
Example 1
Construction of Plasmids
The NS3 protease replicon shuttle vectors were derived from the HCV replicon
shuttle
vector, pSC_lb NS3_EcoRV which is an intermediate vector used to generate the
HCV NS3
replicon shuttle vector, pSC_lbNS3_EcoRV Xbal, and is disclosed in US patent
application,
USSN 60/995,558, filed on September 27, 2007 by Chua et al., entitled "HCV NS3
Replicon
Shuttle Vectors", which is incorporated by reference in full herein. The
components of the
replicon shuttle vectors are shown in Figure 1 and contain the HCV
polynucleotide sequence
from the 5'-UTR, NS3 through NS5B proteins and the 3'-UTR. The vectors also
include the
poliovirus internal ribosome entry site (IRES), which controls the translation
of a firefly
luciferase gene. Downstream of the firefly luciferase gene, the IRES from the
encephalomyocarditis virus (EMCV) controls the translation of the HCV non-
structural genes
(NS3, NS4A, NS4B, NS5A and NS5B).
Mutations were introduced into pSC_lb_NS3_EcoRV_Xbal using the QuickChange
site-
directed mutagenesis kit following the manufacturers' instructions
(Stratagene, La Jolla, CA,
USA). To introduce an AsiSI restriction enzyme sequence at the 3' end of the
protease
domain of the NS3 gene (corresponding to amino acid position Ser 181), the
following
primers were used:
Sense primer 5'-GAGTCTATGGGAACCACTATGCGATCGCCGGTCTTCAC
GGACAACTCGTC-3' (SEQ ID NO:1)
Anti-sense primer 5'-GACGAGTTGTCCGTGAAGACCGGCGATCGCATAGTGG
TTCCCATAGACTC-3' (SEQ ID NO:2)
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These mutations did not change the amino acid coding sequence of the NS3
protein and
resulted in the generation of the NS3 protease shuttle vector,
pSC_lb_NS3/Protease_EcoRV_AsiSl (SEQ ID NO:3, Figure 2A). Another NS3 protease
replicon shuttle vector was generated whereby the sequence encoding the
protease domain of
the NS3 gene was replaced by the beta-galactosidase (lacZ) coding sequence
from pUC19
(GenBank Accession Number M77789). An EcoRV restriction site at the 5' end and
an
AsiSI restriction site at the 3' end of the lacZ gene were introduced by PCR
amplification
using the following primers
5'-ATCATCATC GATATC ACC GCGTTGGCCGATTCATTAATG-3' (SEQ ID NO:4)
(EcoRV) (LacZ)
5'-GATGATGAT GCGATCGC CAGCTCCCGGAGACGGTCAC-3' (SEQ ID NO:5)
(AsiSI) (LacZ)
to generate the vector, pSC_lbNS3/Protease/LacZ_EcoRV_AsiSI (SEQ ID NO:6,
Figure
2B).
Replication capability of the NS3 protease replicon shuttle vectors was
assessed using the
phenotypic assay described in Example 3 (and shown in Figure 3).
Example 2
Cloning of the NS3 protease domain PCR samples amplified from infected
patients into the
NS3 protease replicon shuttle vectors
DNA sequences encoding the protease domain of the NS3 gene were generated by
reverse
transcription-polymerase chain reaction (RT-PCR) of RNA from plasma obtained
from
patients infected with HCV genotype-la and genotype 1-b using the SuperScript
III system
(Invitrogen) according to the manufacturer's protocol. The primers used for
this RT-PCR
step were the following.
Genotype 1 a:
Sense primer (NS2) 5'-CGTGCGGTGACATCATCAACGG-3' (SEQ ID NO:7)
Antisense primer (NS3/helicase) 5' -CTCGCCCCCGCAGTCTCTGC-3' (SEQ ID NO:8)
Genotype lb:
Sense primer (NS2) 5' -GAGACCAAGATCATCACCTGG-3' (SEQ ID NO:9)
Antisense primer (NS3/helicase) 5' -GTCCAGGACTGTGCCGATGCC-3' (SEQ ID NO:l0)
Annealing was first done at 50 C and was followed by PCR cycles of
denaturation at 94 C,
annealing at 53 C and extension at 68 C. The amplified products were then
subjected to a
second round of PCR with the PhusionTM High-Fidelity DNA Polymerase system
(New
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England Biolabs) using primers that introduce unique restriction enzyme
sequences at the 5'
and 3' ends of the protease domain of the NS3 protein. Sense primers used to
introduce the
EcoRV restriction enzyme sequence near the 5' end of the patient NS3 gene
sequence were
as follows:
Genotype 1 a
5'-CTGTCTGTCTGATATCACCATGGCGCCCATCACGGCGTACGCC-3'
(SEQ ID NO:11)
Genotype lb
5'-CTGTCTGTCTGATATCACCATGGCGCCTATTACGGCCTACTC-3'
(SEQ ID NO: 12)
Antisense primers used to introduce the AsiSI restriction enzyme sequence near
the 3' end
(corresponding to amino acid position Serl8l) of the protease domain of the
NS3 gene
sequence were as follows:
Genotype 1 a
5' -TAGTAGTAGGCGATCGCATGGTTGTCCCTAGGTTCTC-3' (SEQ ID NO:13)
Genotype lb
5' -TAGTAGTAGGCGATCGCATAGTGGTTCCCATAGACTCG-3' (SEQ ID NO:14)
Amplification was carried out for PCR cycles with denaturation at 98 C,
annealing at 53 C
and extension at 72 C. The amplified patient NS3 protease DNA were then
purified using
Qiagen PCR Purification Columns and digested with EcoRV and Sgfl (isoschizomer
of
AsiSI).
The NS3 protease shuttle replicon vectors, pSC_lbNS3/protease_EcoRV_AsiSI or
pSC_lbNS3/protease/LacZ_EcoRV_AsiSI were prepared by double digestion with
restriction endonucleaseas, EcoRV and Sgfl. Twenty-five ng of shuttle vector
were ligated
with the digested patient amplicons using MightMix ligation kit (Takara Bio
Inc.) for 1.5
hours at 16 C. Vector to insert ratio of 1:2 to 1:4 were routinely used. 5 gl
of the reaction
were transformed into 50 gl of Topl O Cells (Invitrogen) and plated after 1
hour of phenotypic
expression at 37 C.
96 individual colonies were picked to inoculate 200 gl of Terrific Broth (TB)
supplemented
with 50 gg/ml ampicillin. This "stock" 96-well plate was incubated overnight
at 37 C. The
next day, this plate was used to prepare a replica plate. A 48 pin stamp was
used to replica
plate onto two LB plates supplemented with 100 gg/ml carbenicillin. The 96
individual 200
gl cultures were also transferred into 1.5 ml TB cultures supplemented with 50
gg/ml
ampicillin to be used for DNA preparation. After an overnight incubation at 37
C, the replica
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plate was spread with 6 mis of LB to obtain a heterogeneous pool of 96 clones,
which was
then used for mini-DNA preps. This DNA patient pool was then used for the
subsequent
Replicon Phenotypic Assay. After overnight shaking at 37 C, the 96 individual
1.5 ml TB
cultures were spun down, decanted and plasmid DNA was extracted using Qiagen's
Qiaprep
96 Turbo Mini-DNA Kit. These individual molecular clones, which represent the
composite
96 pool used for the Replicon Phenotypic Assay, were used for sequencing
reactions.
Heterogeneous clone pool plasmid DNAs or individual molecular clones were
submitted to
sequencing to confirm the identity of patient samples and to examine
sensitivity to inhibitors
in the Phenotypic Replicon Assay.
Example 3
Phenotypic Replicon Assay
A. Preparation of in vitro transcribed RNA
Five micrograms of DNA plasmids were linearized by Sea I restriction enzyme
(Roche).
After overnight digestion at 37 C, the DNA was purified using Qiagen PCR
purification kit.
One microgram of linearized DNA was used for the in vitro transcription using
T7 RiboMAX
Express (Promega) following manufacturer's protocol. After 2 hours of
incubation at 37 C,
DNase treatment was performed for 30 minutes at 37 C to remove the DNA
template. In vitro
transcribed RNA was then purified using RNeasy spin column (Qiagen) following
manufacturer's protocol.
B. Hepatoma cell line
The hepatoma Lunet Huh-7 cell line were cultured at 37 C in a humidified
atmosphere with 5
% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with GlutamaxTM
and
100 mg/ml sodium pyruvate. The medium was further supplemented with 10 % (v/v)
FBS
and 1 % (v/v) penicillin/streptomycin. All reagents were from
Invitrogen/Gibco.
C. Determination of transient replicons replication level
Four million Lunet Huh7 cells were transfected with 5 .tg of in vitro
transcribed RNA using
electroporation. Cells were then resuspended in 7.2 ml of DMEM containing 5 %
FBS and
plated in 96-well plate at 50000 cells/well (in 90 1 final volume). Inhibitors
(when used) were
added 24 hours post-transfection in 3 fold dilutions at a final DMSO
concentration of 1 %
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and firefly luciferase reporter signal was read 72 hours after addition of
inhibitors using the
Luciferase Assay system (Promega). The IC50 values were assessed as the
inhibitor
concentration at which a 50 % reduction in the level of firefly luciferase
reporter was
observed as compared to the level of firefly luciferase signal without the
addition of
compounds.
The replication capacity of the replicon shuttle vector,
pSC_lb_NS3/Protease_EcoRV_AsiSI,
as well as those of replicons that contain HCV genotype-la and genotype-lb
patient-derived
NS3 protease domains were tested with the luciferase signal as the readout and
the results are
shown on Figure 3. The inhibitory effects of the HCV protease inhibitors,
BILN2061, VX-
950 and NMI 07 were also tested on these replicons and the results are shown
on Table 1.
TABLE 1
Genotype Replicon Mean IC50
Control or Patient Sample Number BILN2061 VX-950 NM107
nM nM M
pSC_1 b_NS3/Protease_EcoRV_AsiSl 0.489 0.160 0.283
GT-1a RO-191 0.798 0.057 0.539
GT-1a RO-192 0.340 0.068 0.554
GT-1a RO-193 0.298 0.132 0.640
GT-1a RO-194 0.665 0.052 0.509
GT-1a RO-195 0.629 0.028 0.312
GT-1a RO-207 0.386 0.083 1.162
GT-1 b PC232 0.468 0.166 0.449
GT-1 b 206 0.643 0.175 0.279
GT-1 b 301 0.378 0.162 0.279
