# US 8,961,995 B2 · Methods and compositions for alphavirus replicons

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- Number: US 8,961,995 B2
- Document ID: US8961995B2
- Status: Granted
- Kind: B2
- Application number: 13/789,215
- Filed: 2013-03-07
- Published: 2014-03-20
- Granted: 2015-02-24
- Earliest claimed: 2012-09-20
- Assignees: The UAB Research Foundation
- Applicants: UAB Research Foundation
- Inventors: Ilya Frolov; Elena Frolova
- Web page: https://patentgrep.com/patent/US8961995B2

## CPC

- C12N 15/86: Viral vectors
- A61K 39/12: Viral antigens
- C12N 2770/00: ssRNA viruses positive-sense
- C12N 2770/36011: Togaviridae
- C12N 2770/36111: Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N 2770/36132: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2770/36134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2770/36151: Methods of production or purification of viral material
- 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)

## Abstract

The present invention provides alphavirus replicons and methods of their use in producing heterologous protein.

## Claims

### Claim 1

A recombinant replicon nucleic acid comprising:
a) a nucleic acid sequence encoding a first alphavirus 5′ untranslated region (5′ UTR) and a 51 nucleotide conserved sequence element (51 -nt CSE);
b) a nucleic acid sequence encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3 and nsP4;
c) a nucleic acid sequence encoding an alphavirus subgenornic promoter;
d) a nucleic acid sequence encoding a second alphavirus 5′ untranslated region (5′ UTR) and 51 nucleotide conserved sequence element (51 -nt CSE);
e) a nucleic acid sequence encoding a protease;
f) a nucleic acid sequence encoding a heterologous protein; and
g) a nucleic acid sequence encoding an alphavirus 3′ untranslated region (3′ UTR).

### Claim 2 (depends on 1)

The recombinant replicon nucleic acid of claim 1, wherein the nucleic acid sequence of (d) is modified to move all AUGs in the nucleic acid sequence into the same open reading frame as the initiating AUG.

### Claim 3 (depends on 1)

The recombinant replicon nucleic acid of claim 1, wherein the protease is selected from the group consisting of a 2A protease from foot and mouth disease virus (FMDV), ubiquitin, a ubiquitin-like protein, Npro of pestiviruses and alphavirus capsid-encoded protease.

### Claim 4 (depends on 1)

The recombinant replicon nucleic acid of claim 1, further comprising a nucleic acid sequence encoding an alphavirus capsid and the first 2 or more amino acids of E3 protein upstream of the nucleic acid sequence of (f).

### Claim 5

A recombinant replicon nucleic acid comprising:
a) a nucleic acid sequence encoding a first aiphavirus 5 untranslated region (5′ UTR) and a 51 nucleotide conserved sequence element (51 -nt CSE);
b) a nucleic acid sequence encoding alphavirus nonstructural proteins nsP1, nsP2; ns P3 and nsP4;
c) a nucleic acid sequence encoding a first alphavirus subgenomic promoter;
d) a nucleic acid sequence encoding a second alphavirus 5′ untranslated region (5′ UTR) and a 51 nucleotide conserved sequence element (51 -nt CSE);
e) a nucleic acid sequence encoding a second alphavirus subgenomic promoter;
f) a nucleic acid sequence encoding a heterologous protein; and
g) a nucleic, acid sequence encoding an alphavirus 3′ untranslated region (3′ UTR).

### Claim 6

A recombinant replicon nucleic acid comprising:
a) a nucleic acid sequence encoding a first alphavirus 5′ untranslated region (5′ UTR) and a first 51 nucleotide conserved sequence element (51 -nt CSE);
b) a nucleic acid sequence encoding alphavirus nonstructural proteins nsP1, nsP2, nsP3 and nsP4;
c) a nucleic acid sequence encoding a first alphavirus subgenomic promoter;
d) a nucleic acid sequence encoding a second alphavirus 5′ untranslated region (5′ UTR) or a modified 5′ UTR comprising a cellular Asp tRNA sequence, and a second 51 nucleotide conserved sequence element (51 -nt CSE);
e) a nucleic acid sequence encoding a second alphavirus subgenomic promoter;
f) a nucleic acid sequence encoding a translational enhancer derived from an Old World alphavirus;
g) a nucleic acid sequence encoding a protease;
h) a nucleic acid sequence encoding a heterologous protein; and
i) a nucleic acid sequence encoding an alphavirus 3′ untranslated region (3′ UTR).

### Claim 7 (depends on 6)

The recombinant replicon nucleic acid of claim 6, wherein the protease is selected from the group consisting of a 2A protease from FMDV, ubiquitin, a ubiquitin-like protein, alphavirus capsid protein and Npro of pestiviruses.

### Claim 8 (depends on 1)

The recombinant replicon nucleic acid of claim 1, wherein the nucleic acid is RNA.

### Claim 9 (depends on 1)

The recombinant replicon nucleic acid of claim 1, wherein the nucleic acid is DNA cloned under control of SP6, T7 or DNA-dependent RNA polymerase I or II.

### Claim 10 (depends on 1)

An alphavirus particle comprising the recombinant replicon nucleic acid of claim 1.

### Claim 11 (depends on 10)

A population of alphavirus particles, comprising the alphavirus particle of claim 10.

### Claim 12 (depends on 11)

A composition comprising the population of claim 11 in a pharmaceutically acceptable carrier.

### Claim 13 (depends on 1)

A composition comprising the recombinant replicon nucleic acid of claim 1, in a pharmaceutically acceptable carrier,

### Claim 14 (depends on 1)

An isolated cell comprising the recombinant replicon nucleic acid of claim 1.

### Claim 15 (depends on 1)

A method of producing a heterologous protein in a cell, comprising introducing the recombinant replicon nucleic acid of claim 1 into the cell under conditions whereby the nucleic acid sequence of (f) is expressed, wherein the cell is in a cell culture thereby producing the heterologous protein in the cell.

### Claim 16 (depends on 15)

The method of claim 15, further comprising the step of harvesting the heterologons protein from the cell culture.

### Claim 17 (depends on 1)

A method eliciting an immune response to a heterologous protein in a subject, comprising administering to the subject an immunogenic amount of the recombinant replicon nucleic acid of claim 1, thereby eliciting an immune response to the heterologous protein encoded by the nucleic acid sequence of (f).

### Claim 18 (depends on 1)

A method of delivering a therapeutic heterologous protein to a subject, comprising administering to the subject a therapeutic amount of the recombinant replicon nucleic acid of claim 1, wherein the nucleic acid sequence of (f) encodes a therapeutic heterologous protein, thereby delivering a therapeutic heterologous protein to the subject.

### Claim 19 (depends on 1)

A method of making infectious, defective alphavirus particles, comprising:
a) introducing into a cell the following:
(i) the recombinant replicon nucleic acid of claim 1; and
(ii) one or more helper nucleic acids encoding alphavirus structural proteins,
wherein the one or more helper nucleic acids produce all of the alphavirus structural proteins, and
b) producing said alphavirus particles in the cell.