# US 2016/0250267 A1 · Oncolytic hsv vector

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- Number: US 2016/0250267 A1
- Document ID: US20160250267A1
- Status: Published application
- Kind: A1
- Application number: 15/032,958
- Filed: 2014-10-28
- Published: 2016-09-01
- Earliest claimed: 2013-10-28
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Applicants: University of Pittsburgh - of the Commonwealth System of Higher Education
- Inventors: Hiroaki UCHIDA; Justus B. COHEN; Joseph C. GLORIOSO, III; Paola GRANDI
- Web page: https://patentgrep.com/patent/US20160250267A1

## CPC

- A61K 35/763: Herpes virus
- A61P 35/00: Antineoplastic agents
- C07K 14/005: from viruses
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2710/16621: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/16622: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/16632: Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
- C12N 2710/16641: Use of virus, viral particle or viral elements as a vector
- C12N 2710/16643: viral genome or elements thereof as genetic vector
- C12N 2710/16645: Special targeting system for viral vectors
- C12N 2710/16662: by genetic engineering
- C12N 2710/16671: Demonstrated in vivo effect
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides a recombinant oncolytic Herpes Simplex Virus (oHSV) comprising a non-HSV ligand specific for a molecule (protein, lipid, or carbohydrate determinant) present on the surface of a cell (such as a cancer cell) and one or more copies of one or more microRNA target sequences inserted into one or more HSV gene loci, preferably one or more HSV gene(s) required for replication of HSV in normal (i.e., non-cancerous) cells. The invention further provides stocks and pharmaceutical compositions comprising the inventive oHSV and methods for killing tumor cells employing the inventive oHSV.

## Claims

### Claim 1

A recombinant oncolytic Herpes Simplex Virus (oHSV), comprising:
(a) a non-HSV ligand displayed on the surface of the oHSV capsid, which is specific for a molecule present on the surface of a cancer cell and;
(b) a plurality of copies of one or more microRNA target sequences inserted into a locus of an HSV gene required for HSV replication in normal (non-cancerous) cells.

### Claim 2 (depends on 1)

The oHSV of claim 1, wherein the ligand is incorporated into a glycoprotein exposed on the HSV surface.

### Claim 3 (depends on 1)

The oHSV of claim 1, wherein the viral envelope protein is gD or gC.

### Claim 4 (depends on 1)

The oHSV of claim 1, wherein the ligand is incorporated between residues 1 and 25 of gD.

### Claim 5 (depends on 1)

The oHSV of claim 1, wherein the ligand is capable of specifically binding EGFR or EGFRvIII.

### Claim 6 (depends on 1)

The oHSV of claim 1, wherein the ligand is a single-chain antibody (scFv) or a peptide or non-peptide hormone or growth factor that binds a cellular receptor.

### Claim 7 (depends on 1)

The oHSV of claim 1, wherein said microRNA target sequence is the reverse complement of a microRNA.

### Claim 8 (depends on 1)

The oHSV of claim 1, which comprises two or more (2, 3, 4, 5, or 6 in tandem) of said microRNA target sequences inserted into a locus of said HSV gene.

### Claim 9 (depends on 8)

The oHSV of claim 8, which comprises 4 tandem copies of said microRNA target sequences inserted into a locus of said HSV gene.

### Claim 10 (depends on 8)

The oHSV of claim 8, wherein the multiple copies of said microRNA target sequences are separated by spacers of four or more nucleotides within the oHSV genome.

### Claim 11 (depends on 1)

The oHSV of claim 1, wherein said HSV gene into which said microRNA target sequence(s) are inserted is ICP4.

### Claim 12 (depends on 1)

The oHSV of claim 1, wherein said microRNA target sequence(s) are inserted in the 3′ untranslated region (3′ UTR) of said HSV gene.

### Claim 13 (depends on 1)

The oHSV of claim 1, wherein said microRNA is miR-124.

### Claim 14 (depends on 1)

The oHSV of claim 1, wherein said microRNA is miR-122, miR-124, miR-128, miR-137, and/or miR-199, or a combination of two or more thereof.

### Claim 15

A recombinant oHSV, comprising
(a) a non-HSV ligand specific for a protein present on the surface of a cancer cell, which is a scFv specifically binding EGFR or EGFRvIII, and which is inserted between residues 1 and 25 of the oHSV gD glycoprotein, and
(b) 4 copies of the reverse complement of microRNA miR-124, each of said copies separated by spacers of eight nucleotides, inserted into the 3′ UTR of ICP4 of the oHSV genome.

### Claim 16 (depends on 1)

The oHSV of claim 1, comprising a deletion of the internal repeat (joint) region comprising one copy each of the diploid genes ICP0, ICP34.5, LAT and ICP4 along with the promoter for the ICP47 gene.

### Claim 17 (depends on 1)

The oHSV of claim 1, further comprising a mutation of the gB or gH gene that facilitates vector entry though non-canonical receptors.

### Claim 18 (depends on 1)

The oHSV of claim 1, further comprising a transgene.

### Claim 19 (depends on 18)

The oHSV of claim 18, wherein the transgene encodes an oncolytic factor.

### Claim 20 (depends on 18)

The oHSV of claim 18, wherein the transgene encodes a protein or polypeptide that enhances the lateral spread of the oHSV.

### Claim 21 (depends on 20)

The oHSV of claim 20, wherein the transgene encodes metalloproteinase 9 (“MMP9”).

### Claim 21

(canceled)

### Claim 22 (depends on 18)

The oHSV of claim 18, wherein the transgene encodes a protein or polypeptide that catalyzes the conversion of a prodrug.

### Claim 23 (depends on 22)

The oHSV of claim 22, wherein the transgene encodes cytosine deaminase or thymidine kinase.

### Claim 24 (depends on 18)

The oHSV of claim 18, wherein the transgene encodes purine nucleoside phosphorylase (PNP).

### Claim 25 (depends on 1)

The oHSV of claim 1, which is KGE-4:T124.

### Claim 26 (depends on 1)

A nucleic acid encoding the oHSV of claim 1.

### Claim 27 (depends on 26)

The nucleic acid of claim 26, which is a bacterial artificial chromosome (BAC).

### Claim 28 (depends on 1)

A viral stock comprising the oHSV vector of claim 1.

### Claim 29 (depends on 1)

A composition comprising the oHSV of claim 1 and a pharmaceutically-acceptable carrier.

### Claim 30 (depends on 28)

A composition comprising the viral stock of claim 28 and a pharmaceutically-acceptable carrier.

### Claim 31 (depends on 1)

A method of killing a cancerous cell, comprising exposing the cell to the oHSV of claim 1 under conditions sufficient for said oHSV to infect said cancerous cell, whereby replication of the oHSV within the cancerous cell results in cell death.

### Claim 32 (depends on 31)

The method of claim 31 wherein the cell is in vivo.

### Claim 33 (depends on 31)

The method of claim 31, wherein the cell is within a tumor.

### Claim 34 (depends on 33)

The method of claim 33, wherein the tumor is glioblastoma multiforme.

### Claim 35 (depends on 31)

The method of claim 31, wherein the cell is human.

### Claim 36 (depends on 33)

The method of claim 33, wherein the tumor is within the brain of an animal.

### Claim 37 (depends on 36)

The method of claim 36, wherein the oHSV is exposed to the cell by intracranially injecting the oHSV, stock, or composition to the animal.

### Claim 38 (depends on 37)

The method of claim 37, wherein the animal is human.

### Claim 39 (depends on 18)

The oHSV of claim 18, wherein the transgene encodes a protein or polypeptide that induces patient immune response against the cancer.