# US 10,010,594 B2 · Methods and compositions for transgene expression in a herpesvirus vector system

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- Number: US 10,010,594 B2
- Document ID: US10010594B2
- Status: Granted
- Kind: B2
- Application number: 15/118,421
- Filed: 2015-02-11
- Published: 2017-06-15
- Granted: 2018-07-03
- Earliest claimed: 2014-02-11
- Assignees: University of Miami; President and Fellows of Harvard College
- Applicants: University of Miami; President and Fellows of Harvard College
- Inventors: Ronald Desrosiers; Young Shin
- Web page: https://patentgrep.com/patent/US10010594B2

## CPC

- A61K 39/12: Viral antigens
- A61K 39/0011: {Cancer antigens}
- A61K 39/001104: {Epidermal growth factor receptors [EGFR]}
- A61K 39/001106: {Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4}
- A61K 39/001109: {Vascular endothelial growth factor receptors [VEGFR]}
- A61K 39/001122: {Ephrin Receptors [Eph]}
- A61K 39/001124: {CD20}
- A61K 39/00115: {Apoptosis related proteins, e.g. survivin or livin}
- A61K 39/001151: {p53}
- A61K 39/001153: {Wilms tumor 1 [WT1]}
- A61K 39/001156: {Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]}
- A61K 39/001157: {Telomerase or TERT [telomerase reverse transcriptase]}
- A61K 39/001162: {Kinases, e.g. Raf or Src}
- A61K 39/001164: {GTPases, e.g. Ras or Rho}
- A61K 39/00117: {Mucins, e.g. MUC-1}
- A61K 39/001171: {Gangliosides, e.g. GM2, GD2 or GD3}
- A61K 39/001172: {Sialyl-Thomson-nouvelle antigen [sTn]}
- A61K 39/001173: {Globo-H}
- A61K 39/001176: {Heat shock proteins}
- A61K 39/001182: {Carcinoembryonic antigen [CEA]}
- A61K 39/001184: {Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE}
- A61K 39/001186: {MAGE}
- A61K 39/001188: {NY-ESO}
- A61K 39/001191: {Melan-A/MART}
- A61K 39/001192: {Glycoprotein 100 [Gp100]}
- A61K 39/001194: {Prostate specific antigen [PSA]}
- A61K 39/001195: {Prostate specific membrane antigen [PSMA]}
- A61K 39/015: Hemosporidia antigens, e.g. Plasmodium antigens
- A61K 39/04: Mycobacterium, e.g. Mycobacterium tuberculosis
- A61K 39/21: Retroviridae, e.g. equine infectious anemia virus
- C12N 15/86: Viral vectors
- C12N 15/869: Herpesviral vectors
- A61K 2039/5256: {expressing foreign proteins}
- A61K 2039/53: {DNA (RNA) vaccination}
- C12N 2710/16443: viral genome or elements thereof as genetic vector
- C12N 2740/15034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2740/16034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Compositions are provided comprising a replication-competent herpesvirus vector system and/or a herpesvirus vector particle comprising a heterologous recombinant transgene of interest encoding an antigen of interest (e.g., a tumor-associated antigen, a microbial antigen, etc.), wherein the recombinant transgene has been modified to comprise a codon usage signature of a herpesvirus late gene operably linked to an active promoter. Such compositions find use in a variety of methods including, for example, methods of generating an immune response against an antigen of interest in a subject in need thereof, as well as methods for treating or preventing cancer or a microbial infection.

## Claims

### Claim 1

A replication-competent herpesvirus vector system comprising (a) a polynucleotide encoding a trans-inducer polypeptide that enhances expression of a herpesvirus late gene, and (b) a polynucleotide comprising a heterologous recombinant transgene of interest operably linked to a promoter, wherein said heterologous recombinant transgene of interest comprises a codon usage signature of the herpesvirus late gene.

### Claim 2 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein said herpesvirus vector system is derived from a Gammaherpes virus.

### Claim 3 (depends on 2)

The replication-competent herpesvirus vector system of claim 2, wherein said Gammaherpesvirus is a gamma-2 herpesvirus.

### Claim 4 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein said herpesvirus vector system is derived from a herpesvirus of the Betaherpesvirinae subfamily.

### Claim 5 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein said herpesvirus vector system is derived from a herpesvirus of the Alphaherpesvirinae subfamily.

### Claim 6 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein said heterologous recombinant transgene of interest encodes an antigen.

### Claim 7 (depends on 6)

The replication-competent herpesvirus vector system of claim 6, wherein said antigen is a microbial antigen or a tumor-associated antigen.

### Claim 8 (depends on 7)

The replication-competent herpesvirus vector system of claim 7, wherein said microbial antigen comprises a viral antigen, a parasitic antigen, a mycobacteria antigen, or a bacterial antigen.

### Claim 9 (depends on 8)

The replication-competent herpesvirus vector system of claim 8, wherein said viral antigen comprises a viral antigen from Retroviridae or Flaviviridae.

### Claim 10 (depends on 9)

The replication-competent herpesvirus vector system of claim 9, wherein said Retroviridae or Flaviviridae viral antigen comprises a viral antigen from a dengue virus, a lentivirus, a human immunodeficiency virus (HIV) or a simian immunodeficiency virus (SIV).

### Claim 11 (depends on 8)

The replication-competent herpesvirus vector system of claim 8, wherein said viral antigen comprises an envelope polypeptide.

### Claim 12 (depends on 11)

The replication-competent herpesvirus vector system of claim 11, wherein said envelope polypeptide is from HIV or SIV.

### Claim 13 (depends on 12)

The replication-competent herpesvirus vector system of claim 12, wherein said recombinant transgene comprises the polynucleotide set forth in SEQ ID NO: 1 or a polynucleotide having at least 90% sequence identity to SEQ ID NO: I, wherein said polynucleotide provides an improved immune response when compared to a recombinant transgene that encodes the envelope polypeptide from HIV or SIV but that lacks the codon usage signature.

### Claim 14 (depends on 8)

The replication-competent herpesvirus vector system of claim 8, wherein said bacterial antigen is from Mycobacterium tuberculosis.

### Claim 15 (depends on 7)

The replication-competent herpesvirus vector system of claim 7, wherein said microbial antigen is from Plasmodium.

### Claim 16 (depends on 7)

The replication-competent herpesvirus vector system of claim 7, wherein said tumor-associated antigen (a) is associated with melanoma, lymphoma, leukemia, lung cancer, bladder cancer, colon cancer, breast cancer, prostate cancer, esophageal cancer, liver cancer, pancreatic cancer, ovarian cancer, non-small cell lung cancer, renal cancer, neuroblastoma, colorectal cancer, uterine cancer, acute myelocytic leukemia, sarcoma, brain cancer or bone cancer or (b) comprises BAGE, GAGE, MAGE, NY-ESO-1, SSX, gp100, Melan-A/Mart-1, Tyrosinase, PSA, CEA, Mammaglobin-A, p53, HER-2/neu, livin, survivin, B-catenin-m, B-Actin/4/m, Myosin/m, HSP70-2/m, HLA-A2-R170J, GM2, GD2, GD3, MUC-1, sTn, globo-H, WT1, PR1, E75, ras, AFP, URLC10, VEGFR1 and 2, mutant p53, NY-ESO-1, HPV16 E7, B-catenin, CDK4, CDC27, a actinin-4, TRPI/gp75, TRP2, gangliosides, PSMA, HER2, WT1, EphA3, EGFR, CD20, telomerase, MART-1, or an antigenic portion thereof.

### Claim 17 (depends on 1)

A host cell or cell line comprising the replication-competent herpesvirus vector system of claim 1.

### Claim 18

A replication-competent herpesvirus vector particle comprising within its genome a polynucleotide comprising a sequence encoding a trans-inducer polypeptide that enhances expression of a herpesvirus late gene and a heterologous recombinant transgene of interest operably linked to a promoter, wherein said heterologous recombinant transgene comprises a codon usage signature of the herpesvirus late gene.

### Claim 19 (depends on 18)

The replication-competent herpesvirus vector particle of claim 18, wherein said herpesvirus particle is derived from a Gammaherpes virus.

### Claim 20 (depends on 19)

The replication-competent herpesvirus vector particle of claim 19, wherein said Gammaherpes virus is a gamma-2 herpesvirus.

### Claim 21 (depends on 18)

The replication-competent herpesvirus vector particle of claim 18, wherein said herpesvirus particle is derived from a herpesvirus of the Betaherpesvirinae subfamily.

### Claim 22 (depends on 18)

The replication-competent herpesvirus vector particle of claim 18, wherein said herpesvirus particle is derived from a herpesvirus of the Alphaherpesvirinae subfamily.

### Claim 23 (depends on 18)

The replication-competent herpesvirus vector particle of claim 18, wherein said heterologous recombinant transgene of interest encodes an antigen.

### Claim 24 (depends on 23)

The replication-competent herpesvirus vector particle of claim 23, wherein said antigen is a microbial antigen or a tumor-associated antigen.

### Claim 25 (depends on 24)

The replication-competent herpesvirus vector particle of claim 24, wherein said microbial antigen comprises a viral antigen, a parasitic antigen, a mycobacteria antigen or a bacterial antigen.

### Claim 26 (depends on 18)

A pharmaceutical composition comprising the replication-competent herpesvirus vector particle of claim 18.

### Claim 27 (depends on 18)

A method for delivering a transgene of interest to a cell comprising contacting the cell with a replication-competent herpesvirus vector particle of claim 18.

### Claim 28 (depends on 18)

A method of generating an immune response against a microbial antigen in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the replication competent herpesvirus vector particle of claim 18, wherein the heterologous recombinant transgene encodes the microbial antigen, and wherein an immune response against said microbial antigen is generated in said subject.

### Claim 29 (depends on 18)

A method for treating or inhibiting a microbial infection or cancer in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the replication-competent herpesvirus vector particle of claim 18, wherein the heterologous recombinant transgene encodes a microbial antigen or a tumor-associated antigen, and wherein an immune response against said microbial antigen or tumor-associated antibody is generated in said subject and thereby treats or inhibits said microbial infection or cancer.

### Claim 30 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein the herpesvirus late gene comprises a polynucleotide encoding a capsid protein, a tegument protein, or an envelope protein.

### Claim 31 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein the herpesvirus late gene comprises a polynucleotide encoding a glycoprotein.

### Claim 32 (depends on 31)

The replication-competent herpesvirus vector system of claim 31, wherein the glycoprotein is selected from the group consisting of glycoprotein B (gB), glycoprotein C (gC), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL).

### Claim 33 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein the trans-inducer polypeptide is ORF57.

### Claim 34

A method of increasing expression of a transgene by a host cell, comprising (a) altering codons of the transgene to reflect the codon usage signature of a herpesvirus late gene, (b) inserting the transgene with the altered codons into a replication-competent herpesvirus vector, (c) introducing into the host cell the replication-competent herpesvirus vector, and (d) culturing the host cell comprising the replication-competent herpesvirus vector in the presence of a herpesvirus trans-inducer polypeptide that enhances expression of the herpesvirus late gene.

### Claim 35 (depends on 34)

The method of claim 34, wherein the replication-competent herpesvirus vector is derived from a Gammaherpes virus, a Betaherpes virus, or an Alphaherpes virus.

### Claim 36 (depends on 34)

The method of claim 34, wherein the herpesvirus late gene comprises a polynucleotide encoding a capsid protein, a tegument protein, or an envelope protein.

### Claim 37 (depends on 34)

The method of claim 34, wherein the herpesvirus late gene comprises a polynucleotide encoding a glycoprotein.

### Claim 38 (depends on 37)

The method of claim 37, wherein the glycoprotein is selected from the group consisting of glycoprotein B (gB), glycoprotein C (gC), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL).

### Claim 39 (depends on 38)

The method of claim 38, wherein the trans-inducer polypeptide is ORF57.

### Claim 40 (depends on 34)

The method of claim 34, comprising altering codons of the transgene to codons that are present at high frequency in the coding sequence of the herpesvirus late gene.

### Claim 41 (depends on 34)

The method of claim 34, wherein the altered codons are codons that are present at high frequency in the coding sequence of the herpesvirus late gene.

### Claim 42 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein said heterologous recombinant transgene of interest comprises codons used at high frequency in the coding sequence of the herpesvirus late gene.

### Claim 43 (depends on 1)

The replication-competent herpesvirus vector system of claim 1, wherein said heterologous recombinant transgene of interest has been modified to comprise one or more codons present at high frequency in the coding sequence of the herpesvirus late gene.