# US 10,040,830 B2 · Methods and compositions for protein delivery

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- Number: US 10,040,830 B2
- Document ID: US10040830B2
- Status: Granted
- Kind: B2
- Application number: 15/082,401
- Filed: 2016-03-28
- Published: 2016-07-14
- Granted: 2018-08-07
- Earliest claimed: 2008-10-03
- Assignees: The United States of America, as represented by the Secretary, Dept. of Health and Human Services
- Applicants: The United States of America, as represented by the Secretary, Dept. of Health and Human Services
- Inventors: Deb Chatterjee; Stanislaw Jan Kaczmarczyk
- Web page: https://patentgrep.com/patent/US10040830B2

## CPC

- C07K 14/005: from viruses
- A61K 47/6901: {Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors}
- A61P 35/00: Antineoplastic agents
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/245: Escherichia (G)
- 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 2319/09: containing a nuclear localisation signal
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C07K 2319/50: containing protease site
- C07K 2319/705: containing a protein-A fusion
- C07K 2319/735: containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
- C12N 2740/13043: viral genome or elements thereof as genetic vector
- C12N 2760/20222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/20223: Virus like particles [VLP]
- C12N 2810/6072: negative strand RNA viruses
- C12N 2810/6081: rhabdoviridae, e.g. VSV
- 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)
- 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
- 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 methods and compositions for protein delivery. The invention features virus like particles, methods of making virus like particles and methods of using virus like particles to deliver proteins to a cell, to provide protein therapy and to treat diseases or disorders. The invention also features methods of targeting a protein to a cell, methods of protein therapy and methods of treating diseases or disorders using a TUS protein, a NLS or NES identified from full length TUS.

## Claims

### Claim 1

A VLP comprising:
a Gag fusion protein comprising a matrix protein, a capsid protein, a nucleocapsid protein, covalently linked to a protein of interest selected from the group consisting of a cytotoxic enzyme, an interferon, a tumor suppressor, a recombinase, a hormone, and a stem cell transcription factor;
a fusogenic protein having reduced ligand-binding activity; and
a nuclear localization signal (NLS) comprising at least a portion of SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 2 (depends on 1)

The VLP of claim 1, wherein the fusogenic protein is selected from the group consisting of: an influenza haemagglutinin, a respiratory syncytial virus fusion protein, a tick borne encephalitis virus or dengue fever virus E protein, a Semliki Forest virus E1 protein, a rabies virus or vesicular stomatitis virus (VSV) G protein, a baculovirus gp64, and fragments thereof.

### Claim 3 (depends on 1)

The VLP of claim 1, wherein the NLS consists of SEQ ID NO: 1.

### Claim 4 (depends on 1)

The VLP of claim 1, wherein the NLS consists of SEQ ID NO: 2.

### Claim 5 (depends on 1)

The VLP of claim 1, wherein the Gag fusion protein does not comprise a reverse transcriptase, a protease, or an integrase.

### Claim 6 (depends on 1)

The VLP of claim 1, further comprising a second Gag fusion protein comprising a protease.

### Claim 7 (depends on 1)

The VLP of claim 1, wherein the fusogenic protein is an envelope glycoprotein or fragment thereof.

### Claim 8 (depends on 7)

The VLP of claim 7, wherein the envelope glycoprotein is from a RNA virus or a retrovirus.

### Claim 9 (depends on 1)

The VLP of claim 1, wherein the fusogenic protein is a VSV-G glycoprotein comprising a substitution at the second amino acid of (phenylalanine (F)) for cysteine (C) of the mature VSV-G protein.

### Claim 10

A method of targeting one or more proteins to a cell, comprising contacting a cell with a VLP comprising:
a Gag fusion protein comprising a matrix protein, a capsid protein, a nucleocapsid protein, covalently linked to a protein of interest selected from the group consisting of a cytotoxic enzyme, an interferon, a tumor suppressor, a recombinase, a hormone, and a stem cell transcription factor;
a fusogenic protein having reduced ligand-binding activity; and,
a nuclear localization signal comprising at least a portion of SEQ ID NO: 1 or SEQ ID NO: 2,
thereby targeting one or more proteins to the cell.

### Claim 11 (depends on 10)

The method of claim 10, wherein the fusogenic protein is selected from the group consisting of: an influenza haemagglutinin, a respiratory syncytial virus fusion protein, a tick borne encephalitis virus or dengue fever virus E protein, a Semliki Forest virus E1 protein, a rabies virus or vesicular stomatitis virus (VSV) G protein, a baculovirus gp64, and fragments thereof.

### Claim 12 (depends on 10)

The method of claim 10, wherein the nuclear localization signal consists of SEQ ID NO: 1.

### Claim 13 (depends on 10)

The method of claim 10, wherein the nuclear localization signal consists of SEQ ID NO: 2.

### Claim 14 (depends on 10)

The method of claim 10, wherein the Gag fusion protein does not comprise a reverse transcriptase, a protease, or an integrase.

### Claim 15 (depends on 10)

The method of claim 10, further comprising a second Gag fusion protein comprising a protease.

### Claim 16

A method of providing protein therapy to a target cell, comprising contacting the cell with a VLP comprising:
a Gag fusion protein comprising a matrix protein, a capsid protein, a nucleocapsid protein, covalently linked to a protein of interest selected from the group consisting of a cytotoxic enzyme, an interferon, a tumor suppressor, a recombinase, a hormone, and a stem cell transcription factor;
a fusogenic protein having reduced ligand-binding activity; and,
a nuclear localization signal comprising at least a portion of SEQ ID NO: 1 or SEQ ID NO: 2,
thereby providing protein therapy to the target cell.

### Claim 17 (depends on 16)

The method of claim 16, wherein the nuclear localization signal consists of SEQ ID NO: 1.

### Claim 18 (depends on 16)

The method of claim 16, wherein the nuclear localization signal consists of SEQ ID NO: 2.

### Claim 19 (depends on 16)

The method of claim 16, wherein the Gag fusion protein does not comprise a reverse transcriptase, a protease, or an integrase.

### Claim 20 (depends on 16)

The method of claim 16, further comprising a second Gag fusion protein comprising a protease.