Post-Transcriptionally Chemically Modified Double Strand RNAs

• US 20190040389A1
• Filed: 10/22/2018
• Published: 02/07/2019
• Est. Priority Date: 11/07/2016
• Status: Active Grant

• Current Assignee: Nanosur LLC
• Original Assignee: Nanosur LLC
• Inventors: Arhancet, Juan P., Cheerla, Sreevishnu, Arhancet, Graciela B., Rozema, David B.
• Granted Patent: US 10,640,769 B2
• Time in Patent Office: Days
• CPC Class Codes: A01N 57/16 containing heterocyclic radicals
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: 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
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3527 Other alkyl chain
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

Described are post transcriptionally chemically modified double strand RNAs (MdsRNAs) having more than 30 base pairs. The MdsRNAs inhibit gene expression in target organisms. Also described are methods of making and using MdsRNAs.

Claims

1. A composition, comprising a post-transcriptionally modified double strand RNA (MdsRNA), wherein the MdsRNA comprises a double strand RNA having more than 36 nucleotide base pairs wherein at least 5% of the nucleotides independently comprise Formula (I):

2. The composition of claim 1, wherein R¹ is alkyl;

substituted alkyl;

alkenyl;

substituted alkenyl;

alkynyl;

substituted alkynyl;

aryl;

substituted aryl;

C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl wherein alkyl and alkenyl can be linear, branched or cyclic;

methyl;

ethyl;

propyl;

isopropyl;

butyl;

isobutyl;

tert-butyl;

pentyl;

hexyl;

cyclohexyl;

heptyl;

octyl;

nonyl;

decyl;

vinyl;

allyl;

ethynyl;

benzyl;

cinnamyl;

C6-C14 aryl;

C6-C14 substituted aryl;

heterocyclyl;

C5-C14 heterocyclyl;

phenyl;

mono or disubstituted phenyl wherein the substituents are selected from C1-C10 alkyl, C1-C10 alkenyl, C1-C6 alkoxy, halogen, nitro, methylsulfonyl, and trifluoromethyl;

2-nitrophenyl;

4-nitrophenyl;

2,4-dinitrophenyl;

2-trifluromethylphenyl;

4-triflouromethylphenyl;

styryl;

C8-C16 substituted styryl;

2-aminophenyl;

mono or disubstituted 2-aminophenyl wherein the substituents are selected from C1-C10 alkyl, C1-C10 alkenyl, C1-C6 alkoxy, halogen, nitro, methylsulfonyl, and trifluoromethyl;

N-alkyl-2-aminophenyl or N-aryl-2-aminophenyl wherein alkyl has the formula —

C_mH_2m+1 (wherein m is an integer less than or equal to

     12) and aryl is an aromatic moiety;

2-amino-3-methyl-phenyl;

2-amino-5-chlorophenyl;

2-methyl-5-chlorophenyl;

N-methyla-2-minophenyl;

N-ethyl-2-aminophenyl;

N-propyl-2-aminophenyl;

N-butyl2-aminophenyl;

N-pentyl-2-aminophenyl;

N-methyl-2-amino-4-nitrophenyl;

2-methyl-3-furyl;

2-methylnicotyl or N-trifluoromethyl-2-aminophenyl;

silanyl;

substituted silanyl;

C1-C10 alkylsilanyl;

C3-C12 trialkylsilanyl;

C2-C12 alkoxyalkyl;

C2-C12 alkoxyalkenyl;

C2-C12 alkylthioalkyl;

alkylsulfonyl;

C1-C10 alkylsulfonyl;

C1-C10 haloalkyl;

C1-C10 haloalkenyl or C1-C10 aminoalkyl;

—

(CH₂CH₂O)_pCH₃, —

(CH₂CH₂O)_pH, or —

(CH₂CH₂O)_pCOOR⁴ wherein p is an integer from 2 to 8 and R⁴ is H, alkyl, substituted alkyl, aryl, or substituted aryl;

—

(CH₂CH₂O)₈COOH;

—

CH₂CH₂OH;

—

(CH₂CH₂O)₄OH;

—

(CH₂CH₂O)₆OH;

—

(CH₂CH₂O)₈OH;

—

(CH₂CH₂O)₈COOMe;

—

(CH₂CH₂O)₄OMe;

—

(CH₂CH₂O)₆OMe;

—

(CH₂CH₂O)₈OMe;

—

CH₂OCH₃;

—

CH₂OCH₂CH₃;

or —

CH₂OCH₂CH₂OCH₃.

3. The composition of claim 1, further comprising at least one agent selected from the group consisting of an excipient, a carrier, a herbicide, a fungicide, an insecticide, and a fertilizer.

4. The composition of claim 1, wherein at least 10%, at least 25%, at least 60%, or at least 90% of the ribonucleotides independently comprise Formula (I).

5. The composition of claim 1, wherein the MdsRNA comprises a sequence having homology and/or complementarity to an expressed RNA in a target host.

6. The composition of claim 5, wherein the target host is an animal, insect, fungus, plant, protozoan, or weed.

7. The composition of claim 6, wherein the insect, fungus, or weed is selected from the group consisting of Coleopteran, Lepidopteran, Dipteran, Hemipteran, Hymenopteran, Colorado potato beetle, corn root worm, red imported fire ant, Aedes aegypti, diamondback moth, Asian citrus psyllid, Hypocreales, Fusarium gramninearum, Fusarium avenacea, Fusarium culmorum, Fusarium oxysporum, Fusarium sporotrichioides, Palmer Amaranth, Common Lambsquarters, Horseweed, Morning Glory, Waterhemp, Nutsedge, Kochia, Common Ragweed, Giant Ragweed, and Nightshade.

8. The composition of claim 3, wherein the excipient is selected from the group consisting of absorption enhancer, anti-adherent, anti-foaming agent, anti-oxidant, binder, binder, buffering agent, chelating agent, coating agent, color, delivery enhancer, dextran, dextrose, diluent, disintegrant, dispersant, emulsifier, extender, filler, foam control agent, glidant, humectant, lubricant, oil, pigment, polymer, preservative, saline, salt, solvent, sugar, surfactant, suspending agent, sustained release matrix, sweetener, thickening agent, tonicity agent, vehicle, water-repelling agent, and wetting agent.

9. The composition of claim 3, where the MdsRNA is present in an amount of less than 5% by weight, less than about 1% by weight, less than about 0.9% by weight, less than about 0.8% by weight, less than about 0.7% by weight, less than about 0.6% by weight, less than about 0.5% by weight, less than about 0.4% by weight, less than about 0.3% by weight, less than about 0.2% by weight, less than about 0.1% by weight, less than about 0.05% by weight, less than about 0.01% by weight, or less than about 0.001% by weight.

10. The composition of claim 1, wherein the MdsRNA comprises a double strand RNA having at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 base pairs.

11. A composition, comprising a post-transcriptionally modified double strand RNA (MdsRNA) wherein the MdsRNA comprises a double strand RNA having more than 30 nucleotide base pairs wherein at least 5% of the nucleotides independently comprise Formula (I):

12. The composition of claim 11, wherein R¹ is alkyl;

substituted alkyl;

alkenyl;

substituted alkenyl;

alkynyl;

substituted alkynyl;

aryl;

substituted aryl;

C1-C10 alkyl, C1-C10 alkenyl, or C1-C10 alkynyl wherein alkyl and alkenyl can be linear, branched or cyclic;

hydrogen if n=1;

methyl;

ethyl;

propyl;

isopropyl;

butyl;

isobutyl;

tert-butyl;

pentyl;

hexyl;

cyclohexyl;

heptyl;

octyl;

nonyl;

decyl;

vinyl;

allyl;

ethynyl;

benzyl;

cinnamyl;

C6-C14 aryl;

C6-C14 substituted aryl;

heterocyclyl;

C5-C14 heterocyclyl;

phenyl;

mono or disubstituted phenyl wherein the substituents are selected from C1-C10 alkyl, C1-C10 alkenyl, C1-C6 alkoxy, halogen, nitro, methylsulfonyl, and trifluoromethyl;

2-nitrophenyl;

4-nitrophenyl;

2,4-dinitrophenyl;

2-trifluromethylphenyl;

4-triflouromethylphenyl;

styryl;

C8-C16 substituted styryl;

2-aminophenyl;

mono or disubstituted 2-aminophenyl wherein the substituents are selected from C1-C10 alkyl, C1-C10 alkenyl, C1-C6 alkoxy, halogen, nitro, methylsulfonyl, and trifluoromethyl;

N-alkyl-2-aminophenyl or N-aryl-2-aminophenyl wherein alkyl has the formula —

C_mH_2m+1 (wherein m is an integer less than or equal to

     12) and aryl is an aromatic moiety;

2-amino-3-methyl-phenyl;

2-amino-5-chlorophenyl;

2-methyl-5-chlorophenyl;

N-methyla-2-minophenyl;

N-ethyl-2-aminophenyl;

N-propyl-2-aminophenyl;

N-butyl2-aminophenyl;

N-pentyl-2-aminophenyl;

N-methyl-2-amino-4-nitrophenyl;

2-methyl-3-furyl;

2-methylnicotyl or N-trifluoromethyl-2-aminophenyl;

silanyl;

substituted silanyl;

C1-C10 alkylsilanyl;

C3-C12 trialkylsilanyl;

C2-C12 alkoxyalkyl;

C2-C12 alkoxyalkenyl;

C2-C12 alkylthioalkyl;

alkylsulfonyl;

C1-C10 alkylsulfonyl;

C1-C10 haloalkyl;

C1-C10 haloalkenyl or C1-C10 aminoalkyl;

—

(CH₂CH₂O)_pCH₃, —

(CH₂CH₂O)_pH, or —

(CH₂CH₂O)_pCOOR⁴ wherein p is an integer from 2 to 8 and R⁴ is H, alkyl, substituted alkyl, aryl, or substituted aryl;

—

(CH₂CH₂O)₈COOH;

—

CH₂CH₂OH;

—

(CH₂CH₂O)₄OH;

—

(CH₂CH₂O)₆OH;

—

(CH₂CH₂O)₈OH;

—

(CH₂CH₂O)₈COOMe;

—

(CH₂CH₂O)₄OMe;

—

(CH₂CH₂O)₆OMe;

—

(CH₂CH₂O)₈OMe;

—

CH₂OCH₃;

—

CH₂OCH₂CH₃;

or —

CH₂OCH₂CH₂OCH₃.

13. The composition of claim 11, further comprising at least one agent selected from the group consisting of an excipient, a carrier, a herbicide, a fungicide, an insecticide, and a fertilizer

14. The composition of claim 11, wherein at least 10%, at least 25%, at least 60%, or at least 90% of the ribonucleotides independently comprise Formula (I).

15. The composition of claim 11, wherein the MdsRNA comprises a sequence having homology and/or complementarity to an expressed RNA in a target host.

16. The composition of claim 15, wherein the target host is an animal, insect, fungus, plant, protozoan, or weed.

17. The composition of claim 16, wherein the insect, fungus, or weed is selected from the group consisting of Coleopteran, Lepidopteran, Dipteran, Hemipteran, Hymenopteran, Colorado potato beetle, corn root worm, red imported fire ant, Aedes aegypti, diamondback moth, Asian citrus psyllid, Hypocreales, Fusarium gramninearum, Fusarium avenacea, Fusarium culmorum, Fusarium oxysporum, Fusarium sporotrichioides, Palmer Amaranth, Common Lambsquarters, Horseweed, Morning Glory, Waterhemp, Nutsedge, Kochia, Common Ragweed, Giant Ragweed, and Nightshade.

18. The composition of claim 13, wherein the excipient is selected from the group consisting of absorption enhancer, anti-adherent, anti-foaming agent, anti-oxidant, binder, binder, buffering agent, chelating agent, coating agent, color, delivery enhancer, dextran, dextrose, diluent, disintegrant, dispersant, emulsifier, extender, filler, foam control agent, glidant, humectant, lubricant, oil, pigment, polymer, preservative, saline, salt, solvent, sugar, surfactant, suspending agent, sustained release matrix, sweetener, thickening agent, tonicity agent, vehicle, water-repelling agent, and wetting agent.

19. The composition of claim 13, where the MdsRNA is present in an amount of less than 5% by weight, less than about 1% by weight, less than about 0.9% by weight, less than about 0.8% by weight, less than about 0.7% by weight, less than about 0.6% by weight, less than about 0.5% by weight, less than about 0.4% by weight, less than about 0.3% by weight, less than about 0.2% by weight, less than about 0.1% by weight, less than about 0.05% by weight, less than about 0.01% by weight, or less than about 0.001% by weight.

20. The composition of claim 11, wherein the MdsRNA comprises a double strand RNA having at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 base pairs.