# US 10,174,317 B2 · Recombinant RNA particles and methods of use

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- Number: US 10,174,317 B2
- Document ID: US10174317B2
- Status: Granted
- Kind: B2
- Application number: 14/622,671
- Filed: 2015-02-13
- Published: 2015-08-27
- Granted: 2019-01-08
- Earliest claimed: 2014-02-13
- Assignees: Synthetic Genomics, Inc.
- Applicants: Synthetic Genomics, Inc.
- Inventors: Robert C. Brown; Kurt I. Kamrud
- Web page: https://patentgrep.com/patent/US10174317B2

## CPC

- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/86: Viral vectors
- C07K 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2720/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2720/00043: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

The present invention provides compositions and methods for the production and delivery of recombinant double-stranded RNA molecules (dsRNA) targeting pathogen sequences, which can be useful as an RNA vaccine. The compositions contain engineered double-stranded RNA particles (dsRPs) that can contain a double-stranded RNA molecule that can be a genome or portion of a genome, which can be enclosed in a capsid or coat protein. The dsRNA molecule also comprises an RNA sub-sequence that binds to a target sequence of a pathogenic organism. The dsRPs can be derived from wild-type viral organisms. The delivery of the dsRPs of the invention to an organism provides a protection to the organism from the pathogen.

## Claims

### Claim 1

A double-stranded RNA particle (dsRP) comprising,
a recombinant double-stranded RNA molecule (dsRNA) comprising
at least one heterologous sub-sequence of RNA that binds to a target sequence, and
at least 90% sequence identity to a wild type L-A virus genome, not counting the sub-sequence of RNA that binds to the target sequence, wherein the wild type L-A virus genome encodes a gag protein and an RNA-dependent RNA polymerase, and is sufficient for autonomous replication of the dsRP in a host cell, and
encapsidated in a capsid.

### Claim 2 (depends on 1)

The dsRNA particle of claim 1 wherein the host cell is a yeast cell, and the dsRNA particle is less than 100 nm in diameter.

### Claim 3 (depends on 2)

The dsRNA particle of claim 2 wherein the dsRNA molecule is less than about 6 kb.

### Claim 4 (depends on 2)

The dsRNA particle of claim 2 wherein the double-stranded RNA molecule comprises at least 98% of an L-A virus genome.

### Claim 5 (depends on 1)

The dsRNA particle of claim 1 wherein the gag protein and the RNA-dependent RNA polymerase comprise a Gag-Pol fusion protein.

### Claim 6 (depends on 1)

The dsRP of claim 1 wherein the target sequence is an RNA sequence coded for by a pathogen genome.

### Claim 7 (depends on 6)

The dsRP of claim 6 wherein the target sequence is a critical gene of the pathogen or a portion thereof.

### Claim 8 (depends on 6)

The dsRNA particle of claim 6 wherein the target sequence is a regulatory element of a gene that codes for a critical pathogen protein.

### Claim 9 (depends on 8)

The dsRNA particle of claim 8 wherein the dsRNA particle provides an at least partial immunity in the salmonid fish against IPN for at least 60 days.

### Claim 10 (depends on 9)

The dsRNA particle of claim 9 wherein the dsRNA particle provides an at least partial immunity in the salmonid fish against IPN for at least 75 days.

### Claim 11 (depends on 8)

The dsRNA particle of claim 8 wherein the dsRNA particle provides an at least partial immunity in the penaeid shrimp against WSS for at least 7 days.

### Claim 12 (depends on 11)

The dsRNA particle of claim 11 wherein the dsRNA particle provides an at least partial immunity in the penaeid shrimp against WSS for at least 90 days.

### Claim 13 (depends on 1)

The dsRNA particle of claim 1 wherein the target sequence is an RNA sequence of a pathogen that causes Infectious Pancreatic Necrosis (IPN) disease in salmonid fish.

### Claim 14 (depends on 13)

The dsRNA particle of claim 13 wherein the target sequence is an RNA sequence coded for by a sequence selected from the group consisting of: an at least 10 bp portion of any one or more of the nucleotide sequences found in SEQ ID NOs: 2-103.

### Claim 15 (depends on 1)

The dsRNA particle of claim 1 wherein the target sequence is an RNA sequence of a pathogen that causes white spot syndrome (WSS) in penaeid shrimp.

### Claim 16 (depends on 1)

The dsRNA particle of claim 1 wherein the target sequence is an RNA sequence of a pathogen that causes disease in an animal of the genus Sus.

### Claim 17 (depends on 16)

The dsRNA particle of claim 16 wherein the dsRNA particle provides an at least partial immunity in the animal of the genus Sus against said disease for at least 60 days.

### Claim 18 (depends on 1)

The dsRNA particle of claim 1 wherein the sub-sequence of RNA binds to an RNA target sequence and disrupts a critical function of a viral or bacterial pathogen.

### Claim 19 (depends on 1)

The dsRNA particle of claim 1 wherein the sub-sequence of RNA binds to an RNA target that is a unique sequence in a pathogen genome.

### Claim 20 (depends on 1)

The dsRNA particle of claim 1 wherein the RNA molecule further comprises a sequence complementary to the at least one heterologous sub-sequence of RNA, and a separator sequence located between the RNA sub-sequence and the complementary sequence so that the RNA sub-sequence and complementary sequence can bind and form a hairpin structure.

### Claim 21 (depends on 1)

The dsRNA particle of claim 1 wherein the double-stranded RNA particle further encodes a viral particle binding site (VBS) and an internal replication enhancer.

### Claim 22 (depends on 1)

The dsRNA particle of claim 1 wherein the RNA sub-sequence has a length of up to 2 kb.

### Claim 23 (depends on 1)

A formulation comprising a dsRP of claim 1.

### Claim 24 (depends on 13)

A formulation comprising a dsRP of claim 13.

### Claim 25 (depends on 15)

A formulation comprising a dsRP of claim 15.

### Claim 26 (depends on 14)

A formulation comprising a dsRP of claim 14.

### Claim 27 (depends on 16)

A formulation comprising a dsRP of claim 16.

### Claim 28 (depends on 1)

A method of treating an animal disease comprising administering to an animal to be treated a double-stranded RNA particle (dsRP) of claim 1.

### Claim 29 (depends on 28)

The method of claim 28 wherein the dsRP is derived from L-A virus and is less than 100 nm in diameter.

### Claim 30 (depends on 29)

The method of claim 29 wherein the animal is selected from the group consisting of: a peneid shrimp, a salmonid fish, or an animal of the genus Sus.

### Claim 31 (depends on 30)

The method of claim 30 wherein the animal is a peneid shrimp and the disease is white spot disease.