# US 11,555,205 B2 · Recombinant arterivirus replicon systems and uses thereof

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

- Number: US 11,555,205 B2
- Document ID: US11555205B2
- Status: Granted
- Kind: B2
- Application number: 16/703,359
- Filed: 2019-12-04
- Published: 2020-04-23
- Granted: 2023-01-17
- Earliest claimed: 2016-04-13
- Assignees: Janssen Pharmaceuticals, Inc.
- Applicants: Janssen Pharmaceuticals, Inc.
- Inventors: Kurt Iver Kamrud; Nathaniel Stephen Wang; Martina Felderman; Nancy C. Carrico
- Web page: https://patentgrep.com/patent/US11555205B2

## CPC

- C12N 15/86: Viral vectors
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 37/04: Immunostimulants
- C07K 14/005: from viruses
- C07K 16/32: against translation products of oncogenes
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2319/00: Fusion polypeptide
- C12N 2760/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/18522: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2770/10021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2770/10043: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present disclosure generally relates to viral-based expression systems suitable for the production of molecule of interests in recombinant host cells. The disclosure particularly relates to nucleic acid constructs, such as expression vectors, containing a modified arterivirus genome or replicon RNA in which at least some of its original viral sequence has been deleted. Also included in the disclosure are viral-based expression vectors including one or more expression cassettes encoding heterologous polypeptides. In some embodiments, the expression cassettes are configured and positioned at defined locations on the viral genome so as to enable expression of the heterologous polypeptides in a tunable manner.

## Claims

### Claim 1

A method for producing a polypeptide of interest, comprising culturing a host cell comprising a nucleic acid, wherein the nucleic acid comprises a gene encoding the polypeptide of interest and a nucleotide sequence encoding a modified Equine arteritis virus (EAV) genome or replicon RNA, wherein the modified EAV genome or replicon RNA comprises a sequence fragment exhibiting at least 80% sequence identity to the sequence encoding open reading frame ORF7 of the wild-type sequence of EAV, and wherein the modified genome or replicon RNA is devoid of the sequence encoding ORF2a, and further wherein the polypeptide of interest is expressed.

### Claim 2

A method for producing a polypeptide of interest in a subject, comprising administering to the subject a nucleic acid, wherein the nucleic acid comprises:
i. a nucleotide sequence encoding a modified Equine arteritis virus (EAV) genome or replicon RNA, wherein the modified EAV genome or replicon RNA comprises a sequence fragment exhibiting at least 80% sequence identity to the sequence encoding open reading frame ORF7 of the wild-type sequence of EAV, and wherein the modified EAV genome or replicon RNA is devoid of the sequence encoding ORF2a; and
ii. one or more expression cassettes, wherein each of the one or more expression cassettes comprises a subgenomic (sg) promoter operably linked to a heterologous nucleotide sequence encoding the polypeptide of interest;
and wherein the polypeptide of interest is expressed.

### Claim 3

A composition comprising a nucleic acid, wherein the nucleic acid comprises a nucleotide sequence encoding a modified Equine arteritis virus (EAV) genome or replicon RNA, further wherein the nucleic acid comprises a gene encoding a polypeptide of interest, wherein the modified EAV genome or replicon RNA comprises a sequence fragment exhibiting at least 80% sequence identity to the sequence encoding open reading frame ORF7 of the wild-type sequence of EAV, and wherein the modified EAV genome or replicon RNA is devoid of the sequence encoding ORF2a; and a pharmaceutically acceptable carrier.

### Claim 4

A composition comprising a recombinant cell comprising a nucleic acid, wherein the nucleic acid comprises a nucleotide sequence encoding a modified Equine arteritis virus (EAV) genome or replicon RNA, further wherein the nucleic acid comprises a sequence encoding a gene of interest (GOI), wherein the modified EAV genome or replicon RNA comprises a sequence fragment exhibiting at least 80% sequence identity to the sequence encoding open reading frame ORF7 of the wild-type sequence of EAV and wherein the modified EAV genome or replicon RNA is devoid of the sequence encoding ORF2a, and a pharmaceutically acceptable carrier.

### Claim 5 (depends on 1)

The method of claim 1, wherein the modified EAV genome or replicon RNA is devoid of an ATG start codon of ORF7 and/or ORF6, or comprises an inactivated ATG start codon of ORF7 and/or ORF6.

### Claim 6 (depends on 2)

The method of claim 2, wherein the modified EAV genome or replicon RNA is devoid of an ATG start codon of ORF7 and/or ORF6, or comprises an inactivated ATG start codon of ORF7 and/or ORF6.

### Claim 7 (depends on 3)

The composition of claim 3, wherein the modified EAV genome or replicon RNA is devoid of an ATG start codon of ORF7 and/or ORF6, or comprises an inactivated ATG start codon of ORF7 and/or ORF6.

### Claim 8 (depends on 4)

The composition of claim 4, wherein the modified EAV genome or replicon RNA is devoid of an ATG start codon of ORF7 and/or ORF6, or comprises an inactivated ATG start codon of ORF7 and/or ORF6.

### Claim 9 (depends on 2)

The method of claim 2, wherein the modified EAV genome or replicon RNA comprises a 5′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 1 and a 3′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 2.

### Claim 10 (depends on 9)

The method of claim 9, wherein the 5′ region comprises SEQ ID NO: 1 and the 3′ region comprises SEQ ID NO: 2.

### Claim 11 (depends on 2)

The method of claim 2, wherein the modified EAV genome or replicon RNA comprises a 5′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 40 and a 3′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 41.

### Claim 12 (depends on 11)

The method of claim 11, wherein the 5′ region comprises SEQ ID NO: 40 and the 3′ region comprises SEQ ID NO: 41.

### Claim 13 (depends on 2)

The method of claim 2, wherein the sg promoter comprises a sequence exhibiting at least 80% sequence identity to a sequence selected from the group consisting of sg promoter 1, sg promoter 2, sg promoter 3, sg promoter 4, sg promoter 5, sg promoter 6, and sg promoter 7.

### Claim 14 (depends on 2)

The method of claim 2, wherein the nucleic acid further comprises one or more spacer regions operably positioned adjacent to the sg promoter.

### Claim 15

A method for producing a polypeptide of interest, comprising culturing a host cell comprising a nucleic acid, wherein the nucleic acid comprises a gene encoding the polypeptide of interest and a modified Equine arteritis virus (EAV) sub-genomic replicon RNA, wherein the modified EAV sub-genomic replicon RNA comprises a sequence fragment exhibiting at least 80% sequence identity to the sequence encoding open reading frame ORF7 of the wild-type sequence of EAV and further comprises a nucleotide sequence encoding at least one of (a) ORF1a and ORF1b; (b) a 5′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 1 and a 3′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 2; and (c) a 5′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 40 and a 3′ region comprising a nucleotide sequence exhibiting at least 80% sequence identity to SEQ ID NO: 41; and wherein the modified sub-genomic replicon RNA is devoid of the sequence encoding ORF2a, and further wherein the polypeptide of interest is expressed.

### Claim 16 (depends on 15)

The method of claim 15, wherein the 5′ region comprises SEQ ID NO: 1 and the 3′ region comprises SEQ ID NO: 2.

### Claim 17 (depends on 15)

The method of claim 15, wherein the 5′ region comprises SEQ ID NO: 40 and the 3′ region comprises SEQ ID NO: 41.