# US 11,873,507 B2 · Compositions and methods for expression of IL-12 and IL-1RA

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- Number: US 11,873,507 B2
- Document ID: US11873507B2
- Status: Granted
- Kind: B2
- Application number: 17/537,211
- Filed: 2021-11-29
- Published: 2023-06-01
- Granted: 2024-01-16
- Earliest claimed: 2021-11-29
- Assignees: Replicate Bioscience, Inc.
- Applicants: Replicate Bioscience, Inc.
- Inventors: Nathaniel Stephen Wang; Shigeki Joseph Miyake-Stoner; Parinaz Aliahmad
- Web page: https://patentgrep.com/patent/US11873507B2

## CPC

- C12N 15/86: Viral vectors
- A61K 9/5123: {Organic compounds, e.g. fats, sugars}
- C07K 14/54: Interleukins [IL]
- C07K 14/5434: {IL-12}
- C12N 2770/36143: 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)
- 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)

## Abstract

The present disclosure relates to the field of molecular virology, and particularly relates to nucleic acid molecules encoding a modified alphavirus virus viral genome or self-replicating RNA (srRNA) construct, pharmaceutical compositions containing the same, and the use of such nucleic acid molecules and compositions for production of desired products in cell cultures or in a living body. Also provided are methods for eliciting a pharmacodynamics effect in a subject.

## Claims

### Claim 1

A nucleic acid construct comprising a nucleic acid sequence encoding a modified alphavirus genome or self-replicating RNA (srRNA) obtained from an alphavirus genome or srRNA, wherein said modified alphavirus genome or srRNA is produced by replacing at least a portion of the nucleic acid sequence encoding the viral structural proteins of the alphavirus genome or srRNA with a coding sequence for a polypeptide construct comprising: a) a coding sequence for a p35 subunit of interleukin 12 (p35 or IL-12A) or a functional variant thereof; b) a coding sequence for a p40 subunit of interleukin 12 (p40 or IL-12B) or functional variant thereof; and c) a coding sequence for an interleukin-1 receptor antagonist (IL-1RA) or a functional variant thereof, wherein the coding sequences for IL-12A, IL-12B, and IL-1RA, or functional variants thereof, are operably linked to one another, and wherein the coding sequences for IL-12A, IL-12B, and IL-1RA express protein that is functional in a bioactivity assay, and wherein the nucleic acid sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 10.

### Claim 2 (depends on 1)

The nucleic acid construct of claim 1, wherein the modified alphavirus genome or srRNA comprises no nucleic acid sequence encoding viral structural proteins.

### Claim 3 (depends on 1)

The nucleic acid construct of claim 1, wherein the coding sequences of (a) through (c) are operably linked to one another within a single open reading frame or a polycistronic ORF.

### Claim 4 (depends on 1)

The nucleic acid construct of claim 1, wherein the coding sequence encoding the polypeptide construct is operably linked to a promoter sequence.

### Claim 5 (depends on 4)

The nucleic acid construct of claim 4, wherein the promoter sequence is a 26S subgenomic (sg) promoter.

### Claim 6 (depends on 1)

The nucleic acid construct of claim 1, wherein the coding sequences of (a) through (c) are operably linked to one another by connector sequences comprising sequences encoding at least one of an autoproteolytic peptide and an internal ribosomal entry site (IRES).

### Claim 7 (depends on 6)

The nucleic acid construct of claim 6, wherein the autoproteolytic peptide comprises one or more autoproteolytic cleavage sequences from a calcium-dependent serine endoprotease (furin), a porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.

### Claim 8 (depends on 6)

The nucleic acid construct of claim 6, wherein the IRES is a Kaposi's sarcoma-associated herpesvirus (KSHV) IRES, a hepatitis virus IRES, a Pestivirus IRES, a Cripavirus IRES, a Rhopalosiphum padi virus IRES, a fibroblast growth factor IRES, a platelet-derived growth factor IRES, a vascular endothelial growth factor IRES, an insulin-like growth factor IRES, a picornavirus IRES, an encephalomyocarditis virus (EMCV) IRES, a Pim-1 IRES, a p53 IRES, an Apaf-1 IRES, a TDP2 IRES, an L-myc IRES, or a c-myc IRES.

### Claim 9 (depends on 1)

The nucleic acid construct of claim 1, wherein the alphavirus genome or srRNA is of an alphavirus belonging to the Venezuelan equine encephalitis virus/Eastern Equine Encephalitis virus (WEEV/EEEV) group, or the Semliki Forest virus (SFV) group, or the Sindbis virus (SINV) group.

### Claim 10 (depends on 9)

The nucleic acid of claim 9, wherein the alphavirus is VEEV, EEEV, CHIKV, or SINV.

### Claim 11 (depends on 1)

The nucleic acid construct of claim 1, wherein the polypeptide construct comprises, in N-terminus to C-terminus direction:
a) an IL-12A polypeptide, an IL-12B polypeptide, and an IL-1RA polypeptide; or;
b) an IL-1RA polypeptide, an IL-12B polypeptide, and an IL-12A polypeptide;
wherein the IL-12A, IL-12B, and IL-1RA polypeptides are operably linked to one another by one or more autoproteolytic cleavage sequences or IRES.

### Claim 12 (depends on 1)

A recombinant cell comprising the nucleic acid construct according to claim 1.

### Claim 13 (depends on 12)

The recombinant cell of claim 12, wherein the recombinant cell is a mammalian cell or an insect cell.

### Claim 14 (depends on 1)

A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the nucleic acid construct of claim 1.

### Claim 15 (depends on 14)

The pharmaceutical composition of claim 14, wherein the composition is formulated with a delivery vehicle into a delivery system, wherein the delivery system comprises a liposome, a viral replicon particle (VRP), a lipid-based nanoparticle (LNP), a polymer nanoparticle, a physiologic buffer, a microsphere, an immune stimulating complex (ISCOM), a conjugate of bioactive ligand, or a combination of any thereof.

### Claim 16 (depends on 15)

The pharmaceutical composition of claim 15, wherein the LNP delivery system comprises a cationic lipid, an ionizable cationic lipid, an anionic lipid, or a neutral lipid.

### Claim 17 (depends on 15)

The pharmaceutical composition of claim 15, wherein the lipid is present in mass ratio of lipid to RNA from about 100:1 to about 4:1.

### Claim 18 (depends on 15)

The pharmaceutical composition of claim 15, wherein the LNP have an average diameter of about 25 nm to about 1000 nm.

### Claim 19 (depends on 15)

The pharmaceutical composition of claim 15, wherein the composition is formulated as a biotherapeutic.