# US 2013/0157258 A1 · Hcv ns5b protease mutants

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2013/0157258 A1
- Document ID: US20130157258A1
- Status: Published application
- Kind: A1
- Application number: 13/704,107
- Filed: 2011-06-15
- Published: 2013-06-20
- Earliest claimed: 2010-06-15
- Assignees: Vertex Pharmaceuticals Incorporated
- Inventors: Douglas J. Bartels; Min Jiang; Tara Lynn Kieffer; Olivier Nicolas
- Web page: https://patentgrep.com/patent/US20130157258A1

## CPC

- C12Q 1/707: {non-A, non-B Hepatitis, excluding hepatitis D}
- C07K 14/005: from viruses
- C12N 9/127: {RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase}
- C12Y 207/07048: RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
- C12N 2770/24222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The invention provides polypeptides comprising an amino acid sequence comprising at least one variation from wild-type HCV NS5B polymerase, the at least one variation selected from the group consisting of cysteine, isoleucine, valine, or proline at amino acid position 419; alanine, valine, or asparagine at amino acid position 482; valine, isoleucine, threonine, or serine at amino acid position 486; and isoleucine at amino acid position 494, as the amino acid positions are defined in SEQ ID NO: 1, and having Hepatitis C Virus (HCV) NS5B polymerase activity. Polynucleotides encoding the polypeptide, antibodies, host cells, compositions, and methods for detecting an HCV NS5B polymerase having resistance to a polymerase inhibitor also are provided.

## Claims

### Claim 1

An isolated polypeptide comprising an amino acid sequence comprising at least one variation from SEQ ID NO: 1, the at least one variation selected from the group consisting of
cysteine, isoleucine, valine, or proline at amino acid position 419;
alanine, valine, or asparagine at amino acid position 482;
valine, isoleucine, threonine, or serine at amino acid position 486; and
isoleucine at amino acid position 494,
as the amino acid positions are defined in SEQ ID NO: 1, wherein the polypeptide has Hepatitis C Virus (HCV) NS5B polymerase activity.

### Claim at position 2

-3. (canceled)

### Claim 4 (depends on 1)

The isolated polypeptide of claim 1 comprising an amino acid sequence at least 90% identical to SEQ ID NO: 1.

### Claim at position 4

-7. (canceled)

### Claim 8 (depends on 1)

The isolated polypeptide of claim 1, wherein the amino acid residue of the polypeptide corresponding to amino acid 419 of SEQ ID NO: 1 is cysteine, isoleucine, valine, or proline.

### Claim 9 (depends on 1)

The isolated polypeptide of claim 1, wherein the amino acid residue of the polypeptide corresponding to amino acid 482 of SEQ ID NO: 1 is alanine, valine, or asparagine.

### Claim 10 (depends on 1)

The isolated polypeptide of claim 1, wherein the amino acid residue of the polypeptide corresponding to amino acid 486 of SEQ ID NO: 1 is valine, isoleucine, threonine, or serine.

### Claim 11 (depends on 1)

The isolated polypeptide of claim 1, wherein the amino acid residue of the polypeptide corresponding to amino acid 494 of SEQ ID NO: 1 is isoleucine.

### Claim at position 9

-20. (canceled)

### Claim 21

An isolated polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 516 or at least 90% identical to a portion of SEQ ID NO: 516 having Hepatitis C Virus (HCV) NS5B polymerase activity, with the proviso that at least one of the amino acids of the polypeptide that correspond to positions 419, 482, 486, and/or 494 of SEQ ID NO: 516 is identical to the corresponding amino acid(s) of SEQ ID NO: 516.

### Claim 22 (depends on 21)

The isolated polypeptide of claim 21, wherein the amino acid residue of the polypeptide corresponding to amino acid 419 of SEQ ID NO: 3 is cysteine, isoleucine, valine, or proline.

### Claim 23 (depends on 21)

The isolated polypeptide of claim 21, wherein the amino acid residue of the polypeptide corresponding to amino acid 482 of SEQ ID NO: 3 is alanine, threonine, valine, or asparagine.

### Claim 24 (depends on 21)

The isolated polypeptide of claim 21, wherein the amino acid residue of the polypeptide corresponding to amino acid 486 of SEQ ID NO: 3 is valine, isoleucine, threonine, or serine.

### Claim 25 (depends on 21)

The isolated polypeptide of claim 21, wherein the amino acid residue of the polypeptide corresponding to amino acid 494 of SEQ ID NO: 3 is isoleucine.

### Claim at position 15

-35. (canceled)

### Claim 36 (depends on 1)

The isolated polypeptide of claim 1 fused to a heterologous peptide.

### Claim 37

(canceled)

### Claim 38

An isolated peptide having no more than 50 amino acids and comprising a sequence of 6-50 amino acids that is at least 90% identical to a portion of the amino acid sequence of SEQ ID NO: 516 encompassing at least one of amino acid residues 419, 482, 486, and 494, with the proviso that, when present, at least one of the amino acids of the polypeptide that corresponds to positions 419, 482, 486, and/or 494 of SEQ ID NO: 516 is identical to the corresponding amino acid(s) of SEQ ID NO: 516.

### Claim at position 19

-41. (canceled)

### Claim 42

An isolated polypeptide comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 4-502.

### Claim 43 (depends on 1)

An isolated antibody or fragment thereof that specifically binds the polypeptide of claim 1.

### Claim at position 22

-47. (canceled)

### Claim 48 (depends on 1)

An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1.

### Claim at position 24

-53. (canceled)

### Claim 54

An isolated polynucleotide comprising a nucleotide sequence that encodes at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 4-502.

### Claim at position 26

-59. (canceled)

### Claim 60 (depends on 54)

An expression vector comprising the polynucleotide of claim 54.

### Claim at position 28

-63. (canceled)

### Claim 64 (depends on 60)

An isolated cell comprising the expression vector of claim 60.

### Claim 65

(canceled)

### Claim 66 (depends on 48)

An isolated cell transformed or transfected with a polynucleotide according to claim 48, wherein the cell expresses a polypeptide encoding by the polynucleotide, and wherein the cell is free of HCV infection.

### Claim at position 32

-68. (canceled)

### Claim 69 (depends on 1)

An assay mixture comprising an isolated polypeptide according to claim 1, an isolated RNA template, an RNA primer, nucleotide triphosphates, and buffer.

### Claim 70 (depends on 1)

A method for characterizing the HCV inhibitory activity of an agent, the method comprising performing an HCV NS5B polymerase reaction with the polypeptide of claim 1 in the presence of an agent, and comparing polymerase activity in the presence of the agent with polymerase activity of the polypeptide in the absence of the agent.

### Claim at position 35

-72. (canceled)

### Claim 73

A method for detecting the presence of drug-resistant HCV in a sample comprising HCV, wherein the method comprises determining the presence or absence of an HCV NS5B polypeptide with an amino acid sequence in which at least one amino acid that corresponds to positions 419, 482, 486, and/or 494 of SEQ ID NO: 516 is identical to the corresponding amino acid(s) of SEQ ID NO: 516, wherein the presence of the at least one amino acid in the HCV NS5B protein indicates the presence of drug-resistant HCV.

### Claim 74 (depends on 73)

The method of claim 73, wherein determining the presence or absence of the polypeptide comprises contacting the sample with an antibody or fragment thereof that specifically binds said polypeptide and detecting binding of the antibody or fragment thereof to the polypeptide.

### Claim 75

A method for detecting the presence of drug-resistant HCV in a sample, wherein the method comprises determining the presence or absence of a polynucleotide in the sample, the polynucleotide comprising a nucleic acid sequence encoding HCV NS5B polymerase containing at least one codon selected from the group consisting of
a codon encoding cysteine, isoleucine, valine, or proline at a position corresponding to codon position 419 of SEQ ID NO: 2;
a codon encoding alanine, valine, or asparagine at a position corresponding to codon position 482 of SEQ ID NO: 2;
a codon encoding valine, isoleucine, or serine at a position corresponding to codon position 486 of SEQ ID NO: 2; and
a codon encoding isoleucine at a position corresponding to codon position 494 of SEQ ID NO: 2,
wherein the presence of the at least one codon indicates the presence of drug-resistant HCV in the sample.

### Claim 76 (depends on 75)

The method of claim 75, wherein the method comprises
contacting polynucleotides in the sample with a nucleic acid probe, under conditions allowing sequence-specific hybridization of the nucleic acid probe with a target sequence, wherein hybridization of the nucleic acid probe to a polynucleotide in the sample indicates the presence of drug-resistant HCV in the sample, wherein the nucleic acid probe comprises nucleotide sequence of 14-50 nucleotides complementary to a continuous portion of a nucleotide sequence of a of nucleotide encoding a polypeptide having HCV NS5B polymerase activity, the nucleotide having at least one codon variation of SEQ ID NO: 2 selected from the group consisting of a codon encoding cysteine, isoleucine, valine, or proline at codon 419; a codon encoding alanine, valine, or asparagine at codon 482; a codon encoding valine, isoleucine, threonine, or serine at codon 486; and a codon encoding isoleucine at codon 494, as the codon positions are defined in SEQ ID NO: 2, the portion including at least one codon at a codon position selected from codon positions 419, 482, 486, and 494 of SEQ ID NO: 2.

### Claim 77

(canceled)

### Claim 78

A method for determining whether an HCV-infected patient is infected with an HCV strain that has a decreased sensitivity to VX-222, the method comprising determining the presence or absence of a polypeptide in a biological sample from the patient, the polypeptide comprising an amino acid sequence comprising at least one variation from SEQ ID NO: 1, the at least one variation selected from the group consisting of
cysteine, isoleucine, methionine, serine, valine, or proline at amino acid position 419;
lysine at amino acid position 422;
alanine, isoleucine, threonine, or valine at amino acid position 423;
alanine, leucine, threonine, valine, or asparagine at amino acid position 482;
valine, isoleucine, threonine, or serine at amino acid position 486; and
isoleucine or alanine at amino acid position 494,
as the amino acid positions are defined in SEQ ID NO: 1, wherein the presence of the polypeptide indicates infection with an HCV strain that has a decreased sensitivity to VX-222.

### Claim 79 (depends on 78)

The method of claim 78, wherein determining the presence or absence of the polypeptide comprises contacting the sample with an antibody or fragment thereof that specifically binds said polypeptide and detecting binding of the antibody or fragment thereof to the polypeptide.

### Claim 80

A method for identifying an agent able to rescue the polymerase-inhibitor activity of VX-222 against an HCV NS5B polymerase having resistance to VX-222, the method comprising:
a) performing an HCV NS5B polymerase reaction with a polypeptide comprising an amino acid sequence comprising at least one variation from SEQ ID NO: 1, the at least one variation selected from the group consisting of
cysteine, isoleucine, methionine, serine, valine, or proline at amino acid position 419;
lysine at amino acid position 422;
alanine, isoleucine, threonine, or valine at amino acid position 423;
alanine, leucine, threonine, valine, or asparagine at amino acid position 482;
valine, isoleucine, threonine, or serine at amino acid position 486; and
isoleucine or alanine at amino acid position 494,
as the amino acid positions are defined in SEQ ID NO: 1, in the presence of an agent and VX-222; and
b) comparing polymerase activity of the polypeptide in the presence of the agent with polymerase activity of the polypeptide in the absence of the agent, wherein a decrease in HCV polymerase activity in the presence of the agent is indicative of the ability to rescue the polymerase-inhibitory activity of VX-222 against an HCV NSSB polymerase having resistance to VX-222.

### Claim 81

(canceled)

### Claim 82

A method for determining whether an HCV-infected patient is infected with an HCV strain that has a decreased sensitivity to VX-222, the method comprising determining the presence or absence of a polynucleotide in a biological sample from the patient, the polynucleotide comprising a nucleic acid sequence encoding HCV NS5B polymerase containing at least one codon selected from the group consisting of a codon encoding cysteine, isoleucine, methionine, serine, valine, or proline at a position corresponding to codon position 419 of SEQ ID NO: 2;
a codon encoding lysine at a position corresponding to codon position 422 of SEQ ID NO: 2;
a codon encoding alanine, isoleucine, threonine, or valine at a position corresponding to codon position 423 of SEQ ID NO: 2;
a codon encoding alanine, leucine, threonine, valine, or asparagine at a position corresponding to codon position 482 of SEQ ID NO: 2;
a codon encoding valine, isoleucine, threonine, or serine at a position corresponding to codon position 486 of SEQ ID NO: 2; and
a codon encoding isoleucine or alanine at a position corresponding to codon position 494 of SEQ ID NO: 2,
wherein the presence of the polynucleotide indicates infection with an HCV strain having a decreased sensitivity to VX-222.

### Claim 83

(canceled)