# US 9,518,262 B2 · Methods and compositions for the specific inhibition of gene expression by double-stranded RNA

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- Number: US 9,518,262 B2
- Document ID: US9518262B2
- Status: Granted
- Kind: B2
- Application number: 14/463,427
- Filed: 2014-08-19
- Published: 2014-12-04
- Granted: 2016-12-13
- Earliest claimed: 2004-03-15
- Assignees: City of Hope; Integrated Dna Technologies, Inc.
- Applicants: City of Hope; Integrated Dna Technologies, Inc.
- Inventors: John J. Rossi; Mark A. Behlke; Dongho Kim
- Web page: https://patentgrep.com/patent/US9518262B2

## 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)}
- A61P 1/00: Drugs for disorders of the alimentary tract or the digestive system
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 5/14: of the thyroid hormones, e.g. T3, T4
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 7/06: Antianaemics
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 11/06: Antiasthmatics
- A61P 15/02: for disorders of the vagina
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 27/02: Ophthalmic agents
- A61P 27/16: Otologicals
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/08: for leprosy
- A61P 31/14: for RNA viruses
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/33: of the base
- C12N 2310/50: Physical structure
- C12N 2310/51: in polymeric form, e.g. multimers, concatemers
- C12N 2320/30: Special therapeutic applications
- C12N 2320/50: Methods for regulating/modulating their activity
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- C12N 2330/30: chemically synthesised
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

## Claims

### Claim 1

A nucleic acid molecule capable of reducing expression of a target gene comprising:
a first oligonucleotide strand 25-30 nucleotides in length comprising RNA, a 3′ terminus and a 5′ terminus, and
a second oligonucleotide strand at least 26 nucleotides in length comprising RNA, a 3′ terminus, and a 5′ terminus,
wherein said second strand is annealed to said first strand under biological conditions to form a duplex region of at least 25 nucleotides in length,
wherein the 5′ terminus of said second strand and the 3′ terminus of said first strand form a blunt end,
wherein the 3′ terminus of said second strand comprises 1-4 single-stranded nucleotides,
wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA encoded by said target gene to reduce expression of said target gene when said nucleic acid molecule is introduced to a mammalian cell; and wherein said nucleic acid molecule reduces expression of said target gene.

### Claim 2 (depends on 1)

The nucleic acid molecule of claim 1, wherein said second oligonucleotide strand is complementary to said target mRNA along at least 19 nucleotides of said second oligonucleotide strand length.

### Claim 3 (depends on 1)

The nucleic acid molecule of claim 1, wherein said second oligonucleotide strand is sufficiently complementary to said target mRNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid molecule is introduced into a mammalian cell.

### Claim 4 (depends on 1)

The nucleic acid molecule of claim 1, wherein said first strand has a length which is at least 26 nucleotides.

### Claim 5 (depends on 1)

The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a duplex region of at least 26 nucleotides in length.

### Claim 6 (depends on 1)

The nucleic acid molecule of claim 1, wherein said second strand is two nucleotides longer at its 3′ terminus than said first strand.

### Claim 7 (depends on 1)

The nucleic acid molecule of claim 1, wherein said first strand of said nucleic acid molecule is 25 nucleotides in length and said second strand is 27 nucleotides in length.

### Claim 8 (depends on 1)

The nucleic acid molecule of claim 1, wherein said blunt end formed by said 3′ terminus of said first strand and said 5′ terminus of said second strand is a base-paired blunt end.

### Claim 9 (depends on 1)

The nucleic acid molecule of claim 1, wherein said second strand is fully complementary to the target mRNA.

### Claim 10 (depends on 1)

The nucleic acid molecule of claim 1, wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate.

### Claim 11 (depends on 1)

The nucleic acid molecule of claim 1, wherein the relative length in nucleotide residues of said second and first strands is selected from the group consisting of: second strand 26-29 nucleotide residues in length and said first strand 25 nucleotide residues in length, second strand 27-30 nucleotide residues in length and said first strand 26 nucleotide residues in length, second strand 28-30 nucleotide residues in length and first strand 27 nucleotide residues in length, second strand 29-30 nucleotide residues in length and first strand 28 nucleotide residues in length, and second strand 30 nucleotide residues in length and first strand 29 nucleotide residues in length.

### Claim 12 (depends on 1)

The nucleic acid molecule of claim 1 comprising a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, and a locked nucleic acid.

### Claim 13 (depends on 1)

The nucleic acid molecule of claim 1 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

### Claim 14 (depends on 1)

The nucleic acid molecule of claim 1, wherein said nucleic acid molecule reduces target gene expression when introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80%, as compared to the level in an untreated cell.

### Claim 15 (depends on 1)

The nucleic acid molecule of claim 1, wherein said nucleic acid molecule possesses greater potency than an isolated 21mer siRNA directed to the identical nucleotide sequence of said target mRNA of said target gene in reducing target gene expression when suitably formulated at a concentration of 1 nanomolar or less.

### Claim 16 (depends on 1)

The nucleic acid molecule of claim 1, wherein said nucleic acid molecule does not induce in a mammalian cell at least one response selected from the group consisting of interferon-alpha induction, interferon-beta induction and PKR activation, when suitably formulated at a concentration of 20 nM or less.

### Claim 17 (depends on 1)

The nucleic acid molecule of claim 1, wherein said mammalian cell is a HEK 293 cell.

### Claim 18 (depends on 1)

The nucleic acid molecule of claim 1, wherein said nucleic acid molecule reduces target gene expression by at least 50% in said mammalian cell at least 6, 8 or 10 days post-administration, when suitably formulated at a concentration of 5 nM.

### Claim 19

A nucleic acid molecule capable of reducing expression of a target gene comprising:
a first oligonucleotide strand 25-29 nucleotides in length comprising RNA, a 3′ terminus and a 5′ terminus, and
a second oligonucleotide strand at most 30 nucleotides in length comprising RNA, a 3′ terminus, and a 5′ terminus,
wherein said second strand is annealed to said first strand under biological conditions to form a duplex region of at least 25 nucleotides in length,
wherein the 5′ terminus of said second strand and the 3′ terminus of said first strand form a blunt end,
wherein the 3′ terminus of said second strand comprises 1-4 single-stranded nucleotides;
wherein said second strand is sufficiently complementary to a target mRNA encoded by said target gene to reduce target gene expression when said nucleic acid molecule is introduced to a mammalian cell; and
wherein said nucleic acid molecule reduces expression of said target gene.

### Claim 20 (depends on 19)

The nucleic acid molecule of claim 19, wherein said second oligonucleotide strand is complementary to said target mRNA along at least 19 nucleotides of said second oligonucleotide strand length.

### Claim 21 (depends on 19)

The nucleic acid molecule of claim 19, wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA of said target gene along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid molecule is introduced into a mammalian cell.

### Claim 22 (depends on 19)

The nucleic acid molecule of claim 19, wherein said first strand has a length which is at least 26 nucleotides.

### Claim 23 (depends on 19)

The nucleic acid molecule of claim 19, wherein said nucleic acid molecule comprises a duplex region of at least 26 nucleotides in length.

### Claim 24 (depends on 19)

The nucleic acid molecule of claim 19, wherein said second strand is two nucleotides longer at its 3′ terminus than said first strand.

### Claim 25 (depends on 19)

The nucleic acid molecule of claim 19, wherein said first strand of said nucleic acid molecule is 25 nucleotides in length and said second strand is 27 nucleotides in length.

### Claim 26 (depends on 19)

The nucleic acid molecule of claim 19, wherein said blunt end formed by said 3′ terminus of said first strand and said 5′ terminus of said second strand is a base-paired blunt end.

### Claim 27 (depends on 19)

The nucleic acid molecule of claim 19, wherein said second strand is fully complementary to the target mRNA.

### Claim 28 (depends on 19)

The nucleic acid molecule of claim 19, wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate.

### Claim 29 (depends on 19)

The nucleic acid molecule of claim 19, wherein the relative length in nucleotide residues of said second and first strands is selected from the group consisting of: second strand 26-29 nucleotide residues in length and said first strand 25 nucleotide residues in length, second strand 27-30 nucleotide residues in length and said first strand 26 nucleotide residues in length, second strand 28-30 nucleotide residues in length and first strand 27 nucleotide residues in length, second strand 29-30 nucleotide residues in length and first strand 28 nucleotide residues in length, and second strand 30 nucleotide residues in length and first strand 29 nucleotide residues in length.

### Claim 30 (depends on 19)

The nucleic acid molecule of claim 19 comprising a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-0-alkyl ribonucleotide, a 2′-0-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, and a locked nucleic acid.

### Claim 31 (depends on 19)

The nucleic acid molecule of claim 19 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

### Claim 32 (depends on 19)

The nucleic acid molecule of claim 19, wherein said nucleic acid molecule reduces target gene expression when introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80%, as compared to the level in an untreated cell.

### Claim 33 (depends on 19)

The nucleic acid molecule of claim 19, wherein said nucleic acid molecule possesses greater potency than an isolated 21mer siRNA directed to the identical nucleotide sequence of said target mRNA of said target gene in reducing target gene expression when suitably formulated at a concentration of 1 nanomolar or less.

### Claim 34 (depends on 19)

The nucleic acid molecule of claim 19, wherein said nucleic acid molecule does not induce in a mammalian cell at least one response selected from the group consisting of interferon-alpha induction, interferon-beta induction and PKR activation, when suitably formulated at a concentration of 20 nM or less.

### Claim 35 (depends on 19)

The nucleic acid molecule of claim 19, wherein said mammalian cell is a HEK 293 cell.

### Claim 36 (depends on 19)

The nucleic acid molecule of claim 19, wherein said nucleic acid molecule reduces target gene expression by at least 50% in said mammalian cell at least 6, 8 or 10 days post-administration, when suitably formulated at a concentration of 5 nM.

### Claim 37

A nucleic acid molecule capable of reducing expression of a target gene comprising:
a first oligonucleotide strand 25 nucleotides in length comprising RNA, a 3′ terminus and a 5′ terminus, and
a second oligonucleotide strand 27 nucleotides in length comprising RNA, a 3′ terminus and a 5′ terminus,
wherein said second strand is annealed to said first strand under biological conditions to form a duplex region of 25 nucleotides in length,
wherein the 5′ terminus of said second strand and the 3′ terminus of said first strand form a blunt end,
wherein the 3′ terminus of said second strand comprises two single-stranded nucleotides,
wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA to reduce target gene expression when said nucleic acid molecule is introduced in a mammalian cell; and
wherein said nucleic acid molecule reduces expression of said target gene.

### Claim 38 (depends on 37)

The nucleic acid molecule of claim 37, wherein said second oligonucleotide strand is complementary to said target mRNA along at least 19 nucleotides of said second oligonucleotide strand length.

### Claim 39 (depends on 37)

The nucleic acid molecule of claim 37, wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA of said target gene along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said nucleic acid molecule is introduced into a mammalian cell.

### Claim 40 (depends on 37)

The nucleic acid molecule of claim 37, wherein said blunt end formed by said 3′ terminus of said first strand and said 5′ terminus of said second strand is a base-paired blunt end.

### Claim 41 (depends on 37)

The nucleic acid molecule of claim 37, wherein said second strand is fully complementary to the target mRNA.

### Claim 42 (depends on 37)

The nucleic acid molecule of claim 37, wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate.

### Claim 43 (depends on 37)

The nucleic acid molecule of claim 37 comprising a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-0-alkyl ribonucleotide, a 2′-0-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, and a locked nucleic acid.

### Claim 44 (depends on 37)

The nucleic acid molecule of claim 37 comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

### Claim 45 (depends on 37)

The nucleic acid molecule of claim 37, wherein said nucleic acid molecule reduces target gene expression when introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80%, as compared to the level in an untreated cell.

### Claim 46 (depends on 37)

The nucleic acid molecule of claim 37, wherein said nucleic acid molecule possesses greater potency than an isolated 21mer siRNA directed to the identical nucleotide sequence of said target mRNA of said target gene in reducing target gene expression when suitably formulated at a concentration of 1 nanomolar or less.

### Claim 47 (depends on 37)

The nucleic acid molecule of claim 37, wherein said nucleic acid molecule does not induce in a mammalian cell at least one response selected from the group consisting of interferon-alpha induction, interferon-beta induction and PKR activation, when suitably formulated at a concentration of 20 nM or less.

### Claim 48 (depends on 37)

The nucleic acid molecule of claim 37, wherein said mammalian cell is a HEK 293 cell.

### Claim 49 (depends on 37)

The nucleic acid molecule of claim 37, wherein said nucleic acid molecule reduces target gene expression by at least 50% in said mammalian cell at least 6, 8 or 10 days post-administration, when suitably formulated at a concentration of 5 nM.

### Claim 50 (depends on 1)

The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is an isolated, synthetic or an isolated and synthetic nucleic acid molecule.

### Claim 51 (depends on 1)

The nucleic acid molecule of claim 1, wherein the duplex region comprises a mismatch.

### Claim 52 (depends on 51)

The nucleic acid molecule of claim 51, wherein said duplex region comprises a modifier selected from the group consisting of: deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides, 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddl), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) the monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), the monophosphate nucleotide of 2′,3′-dideoxy-3′-thiacytidine (3TC) and the monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), and deoxynucleotides.

### Claim 53

A method for treating a disease in a subject comprising providing a nucleic acid molecule capable of reducing expression of a target gene comprising:
a first oligonucleotide strand 25-30 nucleotides in length comprising RNA, a 3′ terminus and a 5′ terminus, and
a second oligonucleotide strand at least 26 nucleotides in length comprising RNA, a 3′ terminus, and a 5′ terminus,
wherein said second strand is annealed to said first strand under biological conditions to form a duplex region of at least 25 nucleotides in length,
wherein the 5′ terminus of said second strand and the 3′ terminus of said first strand form a blunt end,
wherein the 3′ terminus of said second strand comprises 1-4 single-stranded nucleotides,
wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA encoded by said target gene to reduce expression of said target gene when said nucleic acid molecule is introduced to a mammalian cell; and wherein said nucleic acid molecule reduces expression of said target gene, and
wherein said nucleic acid molecule treats said disease in said subject.

### Claim 54 (depends on 1)

A method for preparing the nucleic acid molecule of claim 1 comprising:
selecting a sequence of a target mRNA,
synthesizing a second oligonucleotide strand at least 26 nucleotides in length having a nucleotide sequence that is complementary to the selected sequence of a target mRNA; and
synthesizing a first oligonucleotide strand comprising 25 to 30 nucleotides that is capable of annealing to the second oligonucleotide strand under biological conditions, combining said first and second oligonucleotide under conditions wherein the first and second strands anneal.