# US 11,279,949 B2 · Recombinant vectors comprising 2A peptide

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- Number: US 11,279,949 B2
- Document ID: US11279949B2
- Status: Granted
- Kind: B2
- Application number: 15/757,292
- Filed: 2016-09-01
- Published: 2018-09-06
- Granted: 2022-03-22
- Earliest claimed: 2015-09-04
- Assignees: Denovo Biopharma LLC
- Applicants: Denovo Biopharma LLC
- Inventors: Douglas J. Jolly; Amy H. Lin; Andrew Hofacre; Daniel J. Hogan; Derek G. Ostertag
- Web page: https://patentgrep.com/patent/US11279949B2

## 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 35/00: Antineoplastic agents
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- C07K 14/70507: {CD2}
- C07K 16/2827: {against B7 molecules, e.g. CD80, CD86}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/50: containing protease site
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2740/13021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2740/13022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2740/13043: viral genome or elements thereof as genetic vector
- C12N 2840/20: translation of more than one cistron
- C12N 2840/203: having an IRES
- 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)

## Abstract

This disclosure provides modified recombinant retroviruses comprisings containing a 2A-peptide or peptide-like coding sequence operably linked to a heterologous polynucleotide. The disclosure further relates to cells and vector expressing or comprising such vectors and methods of using such modified vectors in the treatment of disease and disorders.

## Claims

### Claim 1

A recombinant replication competent retrovirus comprising:
a retroviral GAG protein;
a retroviral POL protein;
a retroviral envelope;
a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain;
a cassette comprising a 2A peptide or 2A peptide-like coding sequence that encodes the peptide of any one of SEQ ID Nos: 55-58 and is operably linked to a heterologous polynucleotide encoding a polypeptide having cytosine deaminase activity comprising the amino acid sequence as set forth in SEQ ID NO: 29, wherein the amino acid residue at position 10 and 152 of SEQ ID NO: 29 is F, D, M, L, or R, and wherein the cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope; and
cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell.

### Claim 2 (depends on 1)

The recombinant replication competent retrovirus of claim 1, wherein the envelope is chosen from one of amphotropic, polytropic, xenotropic, 10A1, GALV, Baboon endogenous virus, RD114, rhabdovirus, alphavirus, measles or influenza virus envelopes.

### Claim 3 (depends on 1)

The retrovirus of claim 1, wherein the retroviral polynucleotide sequence is engineered from a virus selected from the group consisting of murine leukemia virus (MLV), Moloney murine leukemia virus (MoMLV), Feline leukemia virus (FeLV), Baboon endogenous retrovirus (BEV), porcine endogenous virus (PERV), the cat derived retrovirus RD 114, squirrel monkey retrovirus, Xenotropic murine leukemia virus-related virus (XMRV), avian reticuloendotheliosis virus (REV), and Gibbon ape leukemia virus (GALV) to contain the cassette.

### Claim 4 (depends on 1)

The retrovirus of claim 1, wherein the retrovirus is a gammaretrovirus.

### Claim 5 (depends on 1)

The retrovirus of claim 1, wherein the 2A peptide or 2A peptide like coding sequence encodes the peptide sequence as set forth in SEQ ID NO: 55.

### Claim 6 (depends on 1)

The retrovirus of claim 1, wherein the 2A peptide or 2A peptide-like coding sequence encodes the peptide sequence as set forth in SEQ ID NO: 56.

### Claim 7 (depends on 1)

The retrovirus of claim 1, wherein the 2A peptide or 2A peptide-like coding sequence encodes the peptide sequence as set forth in SEQ ID NO: 57.

### Claim 8 (depends on 1)

The retrovirus of claim 1, further comprising a second cassette downstream of the cassette, wherein the second cassette comprises an internal ribosome entry site (IRES) or a minipromoter or a polIII promoter or a second 2A peptide or 2A peptide-like coding sequence operably linked to a second heterologous polynucleotide.

### Claim 9

A recombinant replication competent retrovirus comprising:
a retroviral GAG protein;
a retroviral POL protein;
a retroviral envelope;
a retroviral polynucleotide comprising Long-Terminal Repeat (LTR) sequences at the 3′ end of the retroviral polynucleotide sequence, a promoter sequence at the 5′ end of the retroviral polynucleotide, said promoter being suitable for expression in a mammalian cell, a gag nucleic acid domain, a pol nucleic acid domain and an env nucleic acid domain;
a cassette comprising regulatory domain operably linked to a first heterologous polynucleotide encoding a polypeptide having cytosine deaminase activity comprising the amino acid sequence as set forth in SEQ ID NO: 29, wherein the amino acid residue at position 10 and 152 of SEQ ID NO: 29 is F, D, M, L, or R, and operably linked to at least one 2A cassette comprising a GSG linker coding sequence and a 2A peptide or 2A peptide-like coding sequence that encodes a peptide of any one of SEQ ID Nos: 55-58 and is operably linked to a second heterologous polynucleotide, wherein the cassette is positioned 5′ to the 3′ LTR and 3′ to the env nucleic acid domain encoding the retroviral envelope and wherein the 2A cassette is downstream of the first heterologous polynucleotide; and
cis-acting sequences necessary for reverse transcription, packaging and integration in a target cell.

### Claim 10 (depends on 9)

The retrovirus of claim 9, wherein the envelope is chosen from one of amphotropic, polytropic, xenotropic, 10A1, GALV, Baboon endogenous virus, RD 114, rhabdovirus, alphavirus, measles or influenza virus envelopes.

### Claim 11 (depends on 9)

The retrovirus of claim 9, wherein the retrovirus is a gammaretrovirus.

### Claim 12 (depends on 9)

The retrovirus of claim 9, wherein the 2A peptide or 2A peptide-like coding sequence encodes the peptide sequence as set forth in SEQ ID NO: 55.

### Claim 13 (depends on 9)

The retrovirus of claim 9, wherein the 2A peptide or 2A peptide-like coding sequence encodes the peptide sequence as set forth in SEQ ID NO: 56.

### Claim 14 (depends on 9)

The retrovirus of claim 9, wherein the second heterologous nucleic acid sequence encodes an immunopotentiating cytokine.

### Claim 15 (depends on 9)

The retrovirus according to claim 9, wherein the second heterologous nucleic acid sequence encodes a receptor domain, an antigen binding domain, an antibody, or antibody fragment.

### Claim 16 (depends on 8)

The retrovirus according to claim 8, wherein the second cassette comprises an inhibitory polynucleotide.

### Claim 17 (depends on 16)

The retrovirus according to claim 16, wherein the inhibitory polynucleotide comprises an miRNA, RNAi or siRNA sequence.

### Claim 18 (depends on 1)

A recombinant polynucleotide comprising a cassette comprising a 2A peptide or 2A peptide-like coding sequence that encodes the peptide of any one of SEQ ID Nos: 55-58 and is operably linked to a heterologous polynucleotide encoding a polypeptide having cytosine deaminase activity comprising the amino acid sequence as set forth in SEQ ID NO: 29, wherein the amino acid residue at position 10 and 152 of SEQ ID NO: 29 is F, D, M, L, or R, for producing the retrovirus of claim 1.

### Claim 19 (depends on 1)

The recombinant replication competent retrovirus of claim 1, wherein the retroviral polynucleotide or the heterologous polynucleotide has been engineered to remove tryptophan codons susceptible to human APOBEC hypermutations.

### Claim 20 (depends on 9)

The recombinant replication competent retrovirus of claim 9, wherein the retroviral polynucleotide or the first or the second heterologous polynucleotide has been engineered to remove tryptophan codons susceptible to human APOBEC hypermutation.

### Claim 21 (depends on 1)

The recombinant replication competent retrovirus of claim 1, wherein the recombinant replication competent retrovirus is resistant to inactivation by human APOBEC by engineering codons in a retroviral polynucleotide susceptible to APOBEC hypermutation to a non-susceptible codon.

### Claim 22 (depends on 21)

The recombinant replication competent retrovirus of claim 21, wherein the retrovirus comprises an IRES cassette, promoter cassette and/or 2A peptide cassette downstream of the env gene.