# US 8,148,345 B2 · Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA

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- Number: US 8,148,345 B2
- Document ID: US8148345B2
- Status: Granted
- Kind: B2
- Application number: 12/422,640
- Filed: 2009-04-13
- Published: 2009-08-27
- Granted: 2012-04-03
- Earliest claimed: 1999-01-28
- Assignees: Georgia Health Sciences University Research Institute, Inc.
- Inventors: Yin-Xiong Li; Michael J. Farrell; Margaret J. Kirby
- Web page: https://patentgrep.com/patent/US8148345B2

## 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)}
- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 2310/13: Decoys
- C12N 2310/14: interfering nucleic acids [NA]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

Introduction of double stranded RNA into cells, cell culture, organs and tissues, and whole organisms, particularly vertebrates, specifically attenuates gene expression.

## Claims

### Claim 1

A method for attenuating the expression of a target gene in a cell, the method comprising contacting an embryonic zebrafish, embryonic chick or fibroblast murine cell with a double stranded RNA (dsRNA) in an amount sufficient to specifically attenuate expression of the target gene, wherein one of the strands of the double stranded RNA is capable of hybridizing to the target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 50° C.

### Claim 2 (depends on 1)

The method of claim 1, wherein said dsRNA has a length of less than about 200 bases.

### Claim 3 (depends on 1)

The method of claim 1, wherein said dsRNA has a length of at least about 25 bases.

### Claim 4 (depends on 1)

The method of claim 1, wherein the target gene is an endogenous gene.

### Claim 5 (depends on 1)

The method of claim 1, wherein the target gene is a foreign gene.

### Claim 6 (depends on 1)

The method of claim 1, wherein the target gene is a chromosomal gene.

### Claim 7 (depends on 1)

The method of claim 1, wherein the target gene is an extrachromosomal gene.

### Claim 8 (depends on 1)

The method of claim 1, wherein the double stranded RNA comprises a nucleotide sequence that is complementary to the nucleotide sequence of at least a portion of the target gene.

### Claim 9 (depends on 1)

The method of claim 1, wherein the double stranded RNA is supplied in an amount sufficient to completely inhibit expression of the target gene.

### Claim 10 (depends on 1)

The method of claim 1, wherein the double stranded RNA comprises a single strand comprising self-complementary portions.

### Claim 11 (depends on 1)

The method of claim 1, wherein the double stranded RNA comprises two separate complementary strands.

### Claim 12 (depends on 1)

The method of claim 1, wherein the double stranded RNA is supplied to the cell by delivery to the cell of the double stranded RNA.

### Claim 13 (depends on 12)

The method of claim 12, wherein the double stranded RNA is purified in the absence of phenol or chloroform.

### Claim 14 (depends on 1)

The method of claim 1, wherein the double stranded RNA is supplied to the cell by delivering to the cell a DNA encoding the double stranded RNA.

### Claim 15 (depends on 1)

The method of claim 1, wherein the double stranded RNA is treated with RNAse prior to delivery to the cell.

### Claim 16 (depends on 1)

The method of claim 1, wherein supplying the double stranded RNA to the cell comprises delivering double stranded RNA to the cell, the method further comprising, prior to delivering the double stranded RNA to the cell, annealing two complementary single stranded RNAs to yield the double stranded RNA.

### Claim 17 (depends on 1)

The method of claim 1, wherein the single stranded RNAs are annealed in the presence of potassium chloride.

### Claim 18 (depends on 1)

The method of claim 1, wherein the function of the target gene is unknown.

### Claim 19 (depends on 1)

The method of claim 1 further comprising supplying the cell with a second double stranded RNA in an amount sufficient to specifically attenuate expression of a second target gene, wherein one of the strands of the second double stranded RNA is capable of hybridizing to the second target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 50° C.

### Claim 20 (depends on 1)

The method of claim 1 further comprising supplying the cell with multiple double stranded RNAs in an amount sufficient to specifically attenuate expression of multiple target genes, wherein one strand of each double stranded RNA is capable of hybridizing to the corresponding target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 50° C.

### Claim 21 (depends on 1)

The method of claim 1 further comprising identifying a phenotypic change in the cell associated with attenuated expression of the target gene.

### Claim 22 (depends on 1)

The method of claim 1, wherein the target gene is associated with a disease.

### Claim 23 (depends on 1)

The method of claim 1, wherein the murine cell is a rat cell.

### Claim 24 (depends on 1)

The method of claim 1, wherein one of the strands of the double stranded RNA is capable of hybridizing to the target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 70° C.

### Claim 25 (depends on 1)

The method of claim 1, wherein said cell is an embryonic zebrafish cell.

### Claim 26 (depends on 1)

The method of claim 1, wherein said cell is an embryonic chick cell.

### Claim 27 (depends on 1)

The method of claim 1, wherein said cell is a fibroblast murine cell.