# US 10,870,849 B2 · Extended dicer substrate agents and methods for the specific inhibition of gene expression

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- Number: US 10,870,849 B2
- Document ID: US10870849B2
- Status: Granted
- Kind: B2
- Application number: 14/518,379
- Filed: 2014-10-20
- Published: 2015-02-05
- Granted: 2020-12-22
- Earliest claimed: 2008-12-18
- Assignees: Dicerna Pharmaceuticals, Inc.
- Applicants: Dicerna Pharmaceuticals, Inc.
- Inventors: Bob Dale Brown
- Web page: https://patentgrep.com/patent/US10870849B2

## 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 31/713: Double-stranded nucleic acids or oligonucleotides
- A61P 31/14: for RNA viruses
- A61P 31/20: for DNA viruses
- A61P 31/22: for herpes viruses
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/51: in polymeric form, e.g. multimers, concatemers
- C12N 2310/533: having a mismatch or nick in at least one of the strands
- C12N 2320/53: reducing unwanted side-effects
- 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

The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a single stranded extension (in most embodiments, the single stranded extension comprises at least one modified nucleotide and/or phosphate back bone modification). Such single stranded extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be effective RNA inhibitory agents compared to corresponding double stranded DsiRNAs.

## Claims

### Claim 1

A double stranded nucleic acid capable of reducing expression of a target gene comprising:
a first oligonucleotide strand of 33 to 49 nucleotides in length comprising a 3′ terminus and a 5 terminus, and
a second oligonucleotide strand of 19-39 nucleotides in length, comprising a 3′ terminus and a 5′ terminus,
wherein said second strand is annealed to said first strand to form a duplex region of 18 to 30 nucleotides in length,
wherein the 3′ terminus of said first strand consists of 10-30 unpaired nucleotides that overhang the 5′ terminus of the second strand, wherein at least one of the 10-30 unpaired nucleotides is a modified nucleotide, wherein the modified nucleotide comprises a 2′-O-methyl and/or a 2′-fluoro modified sugar moiety; and
wherein said second strand is sufficiently complementary to a target RNA along at least 19 nucleotides of said second strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell.

### Claim 2 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the 3′ terminus of said first strand consists of 10 to 15 unpaired nucleotides that overhang the 5′ terminus of the second strand.

### Claim 3 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the 3′ and/or 5′ terminus of said second strand comprises at least one single-stranded nucleotide.

### Claim 4 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the 5′ terminus of said first strand comprises at least one single-stranded nucleotide.

### Claim 5 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the 5′ or 3′ terminus of said first strand is conjugated to a non-nucleic acid moiety.

### Claim 6 (depends on 5)

The double stranded nucleic acid of claim 5, wherein the non-nucleic acid moiety is a peptide or an organic compound.

### Claim 7 (depends on 6)

The double stranded nucleic acid of claim 6, wherein the organic compound is a dye.

### Claim 8 (depends on 6)

The double stranded nucleic acid of claim 6, wherein the organic compound is cholesterol.

### Claim 9 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the double stranded nucleic acid possesses enhanced pharmacokinetics when compared to an appropriate control nucleic acid molecule.

### Claim 10 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the double stranded nucleic acid possesses enhanced pharmacodynamics when compared to an appropriate control nucleic acid molecule.

### Claim 11 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the double stranded nucleic acid possesses reduced toxicity when compared to an appropriate control nucleic acid molecule.

### Claim 12 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the double stranded nucleic acid possesses enhanced intracellular uptake when compared to an appropriate control nucleic acid molecule.

### Claim 13 (depends on 5)

The double stranded nucleic acid of claim 5, wherein the non-nucleic acid moiety increases cellular uptake.

### Claim 14 (depends on 5)

The double stranded nucleic acid of claim 5, wherein the non-nucleic acid moiety possesses enhanced cellular targeting of the double stranded nucleic acid, increases stability of the double stranded nucleic acid, facilitates tracking of the double stranded nucleic acid, increases the binding affinity of the double stranded nucleic acid, or decreases the immunogenicity of the double stranded nucleic acid.

### Claim 15 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the double stranded nucleic acid comprises a modification that decreases the immunogenicity of the double stranded nucleic acid.

### Claim 16 (depends on 1)

The double stranded nucleic acid of claim 1, wherein said double stranded nucleic acid is enzymatically synthesized.

### Claim 17 (depends on 1)

The double stranded nucleic acid of claim 1, wherein said double stranded nucleic acid is cleaved endogenously to produce a double stranded nucleic acid that reduces target gene expression.

### Claim 18 (depends on 17)

The double stranded nucleic acid of claim 17, wherein said double stranded nucleic acid is cleaved in a mammalian cell.

### Claim 19 (depends on 1)

The double stranded nucleic acid of claim 1, wherein said double stranded nucleic acid is cleaved by dicer.

### Claim 20 (depends on 1)

The double stranded nucleic acid of claim 1, wherein each of the first and second strands comprise at least one modified nucleotide.

### Claim 21 (depends on 1)

The double stranded nucleic acid of claim 1, wherein all of the nucleotides of the double stranded nucleic acid are modified.

### Claim 22 (depends on 20)

The double stranded nucleic acid of claim 20, wherein the at least one modified nucleotide is a phosphorothioate-modified nucleotide residue (PS-NA).

### Claim 23 (depends on 20)

The double stranded nucleic acid of claim 20, wherein the at least one modified nucleotide is a peptide nucleic acid (PNA).

### Claim 24 (depends on 20)

The double stranded nucleic acid of claim 20, wherein the at least one modified nucleotide comprises a modified base and/or a modified sugar moiety.

### Claim 25 (depends on 20)

The double stranded nucleic acid of claim 20, wherein the at least one modified nucleotide is selected from the group consisting of: dideoxyribonucleotides, acyclonucleotides 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), the monophosphate nucleotides of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) and 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T).

### Claim 26 (depends on 24)

The double stranded nucleic acid of claim 24, wherein the modified sugar moiety is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O-(N-methylcarbamate).

### Claim 27 (depends on 20)

The double stranded nucleic acid of claim 20, wherein at least one internucleoside linkage is modified.

### Claim 28 (depends on 27)

The double stranded nucleic acid of claim 27, wherein the internucleoside linkage modification is selected from the group consisting of: methylphosphonate, phosphorothioate, and phosphotriester modifications.

### Claim 29 (depends on 20)

The double stranded nucleic acid of claim 20, further comprising a fluorescein.

### Claim 30 (depends on 20)

The double stranded nucleic acid of claim 20, wherein the at least one modified nucleotide comprises an LNA modification.

### Claim 31 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the modified phosphate backbone is selected from the group consisting of: a phosphonate, a phosphorothioate and a phosphotriester.

### Claim 32 (depends on 20)

The double stranded nucleic acid of claim 20, wherein the at least one modified nucleotide comprises a 2′-O-methyl ribonucleotide.

### Claim 33 (depends on 1)

The double stranded nucleic acid of claim 1, wherein all of the unpaired nucleotides at the 3′ terminus of the first strand are modified nucleotides.

### Claim 34 (depends on 3)

The double stranded nucleic acid of claim 3, wherein the at least one single stranded nucleotide of the 3′ terminus and/or the 5′ terminus of the second strand comprises a modified nucleotide.

### Claim 35 (depends on 1)

The double stranded nucleic acid of claim 1, wherein one or both of said first and second strands comprises a 5′ phosphate.

### Claim 36 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the first strand has a nucleotide sequence that is at least 80%, 90%, 95% or 100% complementary to the second strand nucleotide sequence.

### Claim 37 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the 5′ terminus of said first strand and the 3′ terminus of said second strand are joined by a chemical linker.

### Claim 38 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the nucleic acid reduces target gene expression in 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 80-90%.

### Claim 39 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the double stranded nucleic acid, when introduced into a mammalian cell, reduces target gene expression by at least 70% when transfected into said cell at a concentration selected from the group consisting of 1 nM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less and 10 pM or less.

### Claim 40 (depends on 1)

The double stranded nucleic acid of claim 1, wherein at least 50% of the ribonucleotide residues of said double stranded nucleic acid are unmodified ribonucleotides.

### Claim 41 (depends on 1)

The double stranded nucleic acid of claim 1, wherein at least 50% of the ribonucleotide residues of said first strand are unmodified ribonucleotides.

### Claim 42 (depends on 1)

The double stranded nucleic acid of claim 1, wherein at least 50% of the ribonucleotide residues of said second strand are unmodified ribonucleotides.

### Claim 43 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the second oligonucleotide strand, starting from the nucleotide residue of said second strand that is complementary to the 5′ terminal nucleotide residue of said first oligonucleotide strand and toward the 5′ end of said second strand, comprises alternating modified and unmodified nucleotide residues.

### Claim 44 (depends on 1)

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

### Claim 45 (depends on 44)

The double stranded nucleic acid of claim 44, wherein the duplex region comprises a base analog selected from the group consisting of: hypoxanthine (I), xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds), pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl and pentacenyl.

### Claim 46 (depends on 45)

The double stranded nucleic acid of claim 45, wherein the base analog comprises a universal base.

### Claim 47 (depends on 46)

The double stranded nucleic acid of claim 46, wherein the universal base does not form a hydrogen bond with the nucleotide opposite to it on the opposite strand of the duplex.

### Claim 48 (depends on 1)

A method for reducing expression of a target gene in a cell, comprising: contacting a cell with the double stranded nucleic acid of claim 1 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference nucleic acid.

### Claim 49 (depends on 1)

A method for reducing expression of a target gene in an animal, comprising: treating an animal with the double stranded nucleic acid of claim 1 in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference nucleic acid.

### Claim 50 (depends on 48)

The method of claim 48, wherein the double stranded nucleic acid possesses enhanced pharmacokinetics, enhanced pharmacodynamics, reduced toxicity, or enhanced intracellular uptake when compared to an appropriate control nucleic acid.

### Claim 51 (depends on 1)

A pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising the double stranded nucleic acid of claim 1 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA and a pharmaceutically acceptable carrier.

### Claim 52 (depends on 1)

A method of synthesizing the double stranded nucleic acid of claim 1, comprising chemically or enzymatically synthesizing said double stranded nucleic acid.

### Claim 53 (depends on 1)

A kit comprising the double stranded nucleic acid of claim 1 and instructions for its use.

### Claim 54 (depends on 1)

The nucleic acid molecule of claim 1 wherein the double stranded nucleic acid is isolated, synthetic, or isolated and synthetic.

### Claim 55 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the first strand is 35 to 49 nucleotides in length.

### Claim 56 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the first strand is 37 to 49 nucleotides in length.

### Claim 57 (depends on 1)

The double stranded nucleic acid of claim 1, wherein the 10-30 unpaired nucleotides comprise a modified phosphate backbone.

### Claim 58 (depends on 57)

The double stranded nucleic acid of claim 57, wherein the modified phosphate backbone comprises a phosphorothioate.

### Claim 59 (depends on 58)

The double stranded nucleic acid of claim 58, wherein the 3′ terminus of said first strand consists of 1-3 terminal deoxyribonucleotides.

### Claim 60 (depends on 1)

The double stranded nucleic acid of claim 1, wherein each of the 10-30 unpaired nucleotides is a modified nucleotide.

### Claim 61 (depends on 58)

The double stranded nucleic acid of claim 58, wherein each of the 10-30 unpaired nucleotides is a modified nucleotide.

### Claim 62 (depends on 60)

The double stranded nucleic acid of claim 60, wherein each of the 10-30 unpaired nucleotides comprises a 2′-O-methyl and/or a 2′-fluoro modified sugar moiety.

### Claim 63 (depends on 61)

The double stranded nucleic acid of claim 61, wherein each of the 10-30 unpaired nucleotides comprises a 2′-O-methyl and/or a 2′-fluoro modified sugar moiety.