# US 11,390,853 B2 · Freeze dried viral nanoparticle constructs

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- Number: US 11,390,853 B2
- Document ID: US11390853B2
- Status: Granted
- Kind: B2
- Application number: 16/859,400
- Filed: 2020-04-27
- Published: 2020-10-29
- Granted: 2022-07-19
- Earliest claimed: 2019-04-26
- Assignees: Case Western Reserve University
- Applicants: Case Western Reserve University
- Inventors: Jonathan Pokorski; Nicole Steinmetz; Parker Lee
- Web page: https://patentgrep.com/patent/US11390853B2

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2770/18023: Virus like particles [VLP]
- C12N 2770/18051: Methods of production or purification of viral material
- C12N 2770/32023: Virus like particles [VLP]
- C12N 2770/32051: Methods of production or purification of viral material
- C12Y 301/27005: Pancreatic ribonuclease (3.1.27.5)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

A method of producing plant virus-like particles includes freeze drying an aqueous solution of plant virus particles to produce a substantially RNA-free plant virus-like particles.

## Claims

### Claim 1

A method of producing plant virus-like particles, the method comprising:
freeze drying an aqueous solution of plant virus particles to produce substantially RNA-free plant virus-like particles, wherein the plant virus particles have an icosahedral structure.

### Claim 2 (depends on 1)

The method of claim 1, the aqueous solution of plant virus particles includes plant virus particles at a concentration of less than about 10 mg/ml.

### Claim 3 (depends on 1)

The method of claim 1, wherein the freeze drying includes freezing an aqueous solution of the plant virus particles at a temperature and for an amount of time that allows the plant virus particles to eject encapsulated genomic RNA while maintaining an intact capsid conformation.

### Claim 4 (depends on 3)

The method of claim 3, wherein the freeze drying includes heating the frozen aqueous solution at a temperature above freezing and at a pressure less than about 100 millibar to sublimate frozen water.

### Claim 5 (depends on 1)

The method of claim 1, further comprising contacting the freeze dried plant virus particles with RNase to degrade nucleic acid.

### Claim 6 (depends on 1)

The method of claim 1, wherein the plant virus particles comprise picornavirus particles.

### Claim 7 (depends on 6)

The method of claim 6, wherein the plant picornavirus particles are of the Comovirus genus.

### Claim 8 (depends on 6)

The method of claim 6, wherein the picornavirus comprising cowpea mosaic virus (CPMV).

### Claim 9

A method of producing empty plant virus-like particles, the method comprising:
freezing an aqueous solution of plant virus particles at a temperature and for an amount of time that allows the plant virus particles to eject the encapsulated genomic RNA while maintaining an intact capsid conformation, wherein the plant virus particles have an icosahedral structure; and
sublimating the frozen aqueous solution of plant virus particles to remove water and RNA in the plant virus capsid.

### Claim 10 (depends on 9)

The method of claim 9, the aqueous solution of plant virus particles includes plant virus particles at a concentration of less than about 10 mg/ml.

### Claim 11 (depends on 9)

The method of claim 9, wherein the aqueous solution of the plant virus particles is frozen at a temperature of about −5° C. to about −40° C.

### Claim 12 (depends on 9)

The method of claim 9, wherein the step of sublimating the frozen aqueous solution includes heating the frozen aqueous solution at a temperature above freezing and at a pressure less than about 100 millibar.

### Claim 13 (depends on 9)

The method of claim 9, further comprising contacting the freeze dried plant virus particles with RNase to degrade nucleic acid.

### Claim 14 (depends on 9)

The method of claim 9, wherein the plant virus particles comprise picornavirus particles.

### Claim 15 (depends on 14)

The method of claim 14, wherein the plant picornavirus particles are of the Comovirus genus.

### Claim 16 (depends on 14)

The method of claim 14, the picornavirus comprising cowpea mosaic virus (CPMV).

### Claim 17 (depends on 1)

A composition comprising a plurality of freeze dried, endoribonuclease treated plant virus-like particles prepared by the method of claim 1; wherein the plant virus-like particles have an icosahedral structure.

### Claim 18 (depends on 17)

The composition of claim 17, wherein during freeze drying an aqueous solution of icosahedral plant virus particles is frozen at a temperature and for an amount of time that allows the virus to eject encapsulated genomic RNA while maintaining an intact capsid conformation.

### Claim 19 (depends on 17)

The composition of claim 17, wherein freeze drying includes heating a frozen aqueous solution of icosahedral plant virus particles at a temperature above freezing and at a pressure less than about 100 millibar.

### Claim 20 (depends on 17)

The composition of claim 17, wherein the endoribonuclease comprises RNaseA.

### Claim 21 (depends on 17)

The composition of claim 17, wherein the plant virus-like particles comprise cowpea mosaic virus-like particles.