Back to results
US 2010/0173281 A1Published application
HCV NS3 protease replicon shuttle vectors
AssigneeRoche Palo Alto LLC
InventorsSamir Ali, Wen-Rong Jiang
Life of this patent
Estimating the term…
Abstract
The present invention provides for novel HCV NS3 protease replicon shuttle vectors useful for cloning in HCV polynucleotide sequences from samples of HCV-infected patients and testing the resulting replicons for drug susceptibility.
Claims34
An HCV replicon shuttle vector comprising an HCV polynucleotide sequence that comprises, in order:
- (a) 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;
- (b) a polynucleotide sequence encoding the protease domain of the NS3 protein;
- (c) 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;
- (d) a polynucleotide sequence encoding the helicase domain of the NS3 protein;
- (e) a polynucleotide sequence encoding a NS4A protein;
- (f) a polynucleotide sequence encoding a NS4B protein;
- (g) a polynucleotide sequence encoding a NS5A protein;
- and
- (h) a polynucleotide sequence encoding a NS5B protein.
6 more elements
An HCV replicon shuttle vector comprising an HCV polynucleotide sequence selected from SEQ ID NO:3 or SEQ ID NO:6.
A method for assessing the effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in a subject comprising the steps of:
- (a) providing a sample from the subject infected with HCV;
- (b) 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;
- (c) cloning said PCR-amplified polynucleotide sequences into an HCV replicon shuttle vector to produce chimeric HCV replicon plasmids;
- (d) linearizing said chimeric HCV replicon plasmids and subjecting said linearized plasmids to in vitro transcription to produce chimeric HCV replicon RNAs;
- and
- (e) 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.
3 more elements
A method for assessing the effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in a subject comprising the steps of:
- (a) providing a sample from the subject infected with HCV;
- (b) 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 restriction enzyme sequence that recognizes EcoRV, and an anti-sense strand primer which comprises a restriction enzyme sequence that recognizes AsiSI;
- (c) cloning said PCR-amplified polynucleotide sequences into an HCV replicon shuttle vector to produce chimeric HCV replicon plasmids;
- (d) linearizing said chimeric HCV replicon plasmids and subjecting said linearized plasmids to in vitro transcription to produce chimeric HCV replicon RNAs;
- and
- (e) 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.
3 more elements
A method for assessing the effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in a subject comprising the steps of:
- (a) providing a sample from the subject infected with HCV;
- (b) 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 comprising a nucleotide sequence selected from SEQ ID NO:11 or SEQ ID NO:12, and an anti-sense strand primer comprising a nucleotide selected from SEQ ID NO:13 or SEQ ID NO:14;
- (c) cloning said PCR-amplified polynucleotide sequences into an HCV replicon shuttle vector to produce chimeric HCV replicon plasmids;
- (d) linearizing said chimeric HCV replicon plasmids and subjecting said linearized plasmids to in vitro transcription to produce chimeric HCV replicon RNAs;
- and
- (e) 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.
3 more elements
A method for assessing the effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in a subject comprising the steps of:
- (a) providing a sample from the subject infected with HCV;
- (b) 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;
- (c) cloning said PCR-amplified polynucleotide sequences into an HCV replicon shuttle vector to produce chimeric HCV replicon plasmids;
- (d) transforming said plasmids into cells to generate a plurarity of colonies of transformed cells;
- (e) pooling said colonies and isolating chimeric HCV replicon plasmids from the pooled colonies;
- (f) linearizing said chimeric HCV replicon plasmids from step (e) and subjecting said linearized plasmids to in vitro transcription to produce chimeric HCV replicon RNAs;
- and
- (g) 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.
5 more elements
A method for assessing the effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in a subject comprising the steps of:
- (a) providing a sample from the subject infected with HCV;
- (b) 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 restriction enzyme sequence that recognizes EcoRV, and an anti-sense strand primer which comprises a restriction enzyme sequence that recognizes AsiSI;
- (c) cloning said PCR-amplified polynucleotide sequences into an HCV replicon shuttle vector to produce chimeric HCV replicon plasmids;
- (d) transforming said plasmids into cells to generate a plurarity of colonies of transformed cells;
- (e) pooling said colonies and isolating chimeric HCV replicon plasmids from the pooled colonies;
- (f) linearizing said chimeric HCV replicon plasmids from step (e) and subjecting said linearized plasmids to in vitro transcription to produce chimeric HCV replicon RNAs;
- and
- (g) 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.
5 more elements
A method for assessing the effectiveness of an HCV NS3 protease inhibitor to control an HCV infection in a subject comprising the steps of:
- (a) providing a sample from the subject infected with HCV;
- (b) 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 comprising a nucleotide sequence selected from SEQ ID NO:11 or SEQ ID NO:12, and an anti-sense strand primer comprising a nucleotide selected from SEQ ID NO:13 or SEQ ID NO:14;
- (c) cloning said PCR-amplified polynucleotide sequences into an HCV replicon shuttle vector to produce chimeric HCV replicon plasmids;
- (d) transforming said plasmids into cells to generate a plurarity of colonies of transformed cells;
- (e) pooling said colonies and isolating chimeric HCV replicon plasmids from the pooled colonies;
- (f) linearizing said chimeric HCV replicon plasmids from step (e) and subjecting said linearized plasmids to in vitro transcription to produce chimeric HCV replicon RNAs;
- and
- (g) 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.
5 more elements
Dependencies are inferred from the claim text.