WO2009029688A2 - Compositions of asymmetric interfering rna and uses thereof - Google Patents

Compositions of asymmetric interfering rna and uses thereof Download PDF

Info

Publication number
WO2009029688A2
WO2009029688A2 PCT/US2008/074528 US2008074528W WO2009029688A2 WO 2009029688 A2 WO2009029688 A2 WO 2009029688A2 US 2008074528 W US2008074528 W US 2008074528W WO 2009029688 A2 WO2009029688 A2 WO 2009029688A2
Authority
WO
WIPO (PCT)
Prior art keywords
strand
rna molecule
nucleotides
duplex
length
Prior art date
Application number
PCT/US2008/074528
Other languages
French (fr)
Other versions
WO2009029688A3 (en
Inventor
Chiang Jia Li
Xiangao Sun
Harry Rogoff
Youzhi Li
Original Assignee
Boston Biomedical, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Boston Biomedical, Inc. filed Critical Boston Biomedical, Inc.
Priority to DK08828867.5T priority Critical patent/DK2193140T3/en
Priority to EP16187862.4A priority patent/EP3121281B1/en
Priority to ES08828867.5T priority patent/ES2617877T3/en
Priority to EP21158616.9A priority patent/EP3889261A1/en
Priority to EP08828867.5A priority patent/EP2193140B1/en
Priority to CN200880113236.9A priority patent/CN101835789B/en
Priority to CA2735166A priority patent/CA2735166C/en
Priority to JP2010523120A priority patent/JP2010537639A/en
Publication of WO2009029688A2 publication Critical patent/WO2009029688A2/en
Publication of WO2009029688A3 publication Critical patent/WO2009029688A3/en
Priority to HK11102556.9A priority patent/HK1148534A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/53—Methods for regulating/modulating their activity reducing unwanted side-effects
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • RNAi therapeutics There is a need to develop novel approaches to effective RNAi in mammalian cells through a novel design of siRNAs having better efficacy and potency, rapid onset of action, better durability, and a shorter length of the RNA duplex to avoid non-specific interferon like response and to reduce the cost of synthesis for research and pharmaceutical development of RNAi therapeutics.
  • the second strand is substantially complementary to a target mRNA sequence
  • the eukaryotic cell is a mammalian cell or an avian cell.
  • the duplex RNA molecule is an isolated duplex RNA molecule.
  • the 3'-overhang and/or 5'-overhang is stabilized against degradation.
  • the duplex RNA molecule contains at least one modified nucleotide or its analogue.
  • the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base- modified ribonucleotide.
  • the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide.
  • the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom.
  • the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
  • the present invention provides a method of modifying a first duplex RNA molecule with an antisense strand and a sense strand that form a double-stranded region.
  • the method comprises, shortening the length of the sense strand so that the antisense strand has a 3'-overhang from 1-8 nucleotides and a 5'-ovcrhang from 0-8 nucleotides, and forming a second duplex RNA molecule; wherein at least one property of the first duplex RNA molecule is improved.
  • the property is selected from the group consisting of size, efficacy, potency, the speed of onset, durability, synthesis cost, off-target effects, interferon response, and delivery.
  • the present invention also provides a cell.
  • the cell comprises the expression vector.
  • the cell comprises the duplex RNA molecule.
  • the cell is a mammalian cell, avian cell, or bacterial cell.
  • Figure IB shows the structure of a duplex RNA molecule that has both a 3 '-overhang and a 5 '-overhang on the first strand, and a nick in the duplex.
  • Figure 6d shows the schematic of ⁇ -catenin mRNA cleavage site confirmed by sequencing the 5'-RACE-PCR fragment.
  • Figure 6e shows differential RISC loading efficiency of aiRNA and siRNA.
  • aiRNA or siRNA duplexes were transfected into HeIa cells 48 hours after transfection with pCMV-Ago2.
  • Ago2 was immunoprecipitated at the indicated time points following aiRNA or siRNA transfection, and northern blot analysis was performed to determine levels of Ago2/RISC associated small RNAs. Levels of Ago2 (shown below-) were determined by western blot following IP.
  • Figure 14a shows aiRNA duplex is more efficacious than siRNA in targeting ⁇ -catenin in different mammalian cell lines.
  • Figure 14b shows the western blot analysis o ⁇ Nbsl, Survivin, Parpl, p21 from cells transfected with 20 nM of the indicated aiRNA or siRNA for 48 hours.
  • the first strand is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer than the second strand.
  • the duplex RNA molecule comprises a double-stranded region, a blunt end, and a 5 '-overhang (see, e.g., Fig. 2A).
  • the method comprises changing the length of the antisene and/or sense strand so that the duplex RNA molecule is formed having at least one of a 3'- overhang of 1-6 nucleotides and a 5'-overhang of 1-6 nucleotides.
  • aiRNA molecules possess several advantages that should make a wider ranger of methods available for delivery purpose.
  • aiRNAs can be designed to be smaller than their siRNA and miRNA counterparts, therefore, reducing or eliminating any interferon responses.
  • aiRNAs are more potent, faster-onsetting, more efficacious and lasts longer, therefore, less amount/dosage of aiRNAs is required to achieve a therapeutic goal.
  • ds RNA double stranded RNA
  • PLR protein kinase R
  • Modification to the parental 15/21 aiRNA structure was done by altering the sense strand, anti-sense strand, or both (Table 4). Modified aiRNA duplexes were transfected into HeIa cells at 50 nM for 48 hours. Western blots for ⁇ -catenin and actin were used to examine the degree of gene silencing compared to the parental 15/21 aiRNA and to the traditional siRNA structure. aiRNA modifications were also tested which contained dual sense strand overhangs. These oligonucleotides contain a 21 base sense strand paired to differing length anti-sense strands. In addition, we also examined the activity of aiRNA oligonucleotides that have been modified with DNA bases.
  • aiRNA is more efficacious than siRNA in silencing Stat3, ⁇ -catenin, Rskl, p70S6K, Nbsl, mTOR, and EF2, and is as efficacious as siRNA in silencing NQOl, PCNA, Survivin, PTEN, Parpl, and p21. Since the target sequences were chosen based on the optimization for siRNA, it is possible that the efficacy and potency of aiRNA can be further increased by targeting sites that are optimized for aiRNA.
  • RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Pulmonology (AREA)
  • Diabetes (AREA)
  • Dermatology (AREA)
  • Neurology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Transplantation (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Rheumatology (AREA)

Abstract

The present invention provides asymmetrical duplex RNA molecules that are capable of effecting sequence-specific gene silencing. The RNA molecule comprises a first strand and a second strand. The first strand is longer than the second strand. The RNA molecule comprises a double-stranded region formed by the first strand and the second strand, and two ends independently selected from the group consisting of 5 '-overhang, 3 '-overhang, and blunt end. The RNA molecules of the present invention can be used as research tools and/or therapeutics.

Description

TITLE OF THE INVENTION
Compositions of asymmetric interfering RNA and uses thereof
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to and the benefit of U.S. provisional patent applications Serial Nos. 60/968,257 filed on August 27, 2007, 61/029,753 filed on Feb. 19, 2008, and 61/038,954 filed on March 24, 2008, the entire contents of which applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Gene silencing through RNAi (RNA-interference) by use of small or short interfering RNA (siRNA) has emerged as a powerful tool for molecular biology and holds the potential to be used for therapeutic purposes (de Fougerolles et al, 2007; Kim and Rossi, 2007). RNAi can be theoretically employed to knockdown or silence any disease gene with specificity and potency. Possible applications of RNAi for therapeutic purposes are extensive and include genetic, epigenetic, and infectious diseases, provided that a disease-causing gene is identified.
However, other than the prominent delivery issue, the development of RNAi-based drugs faces challenges of limited efficacy of siRNA, non-specific effects of siRNA such as interferon- like responses and sense-strand mediated off-target gene sliencing, and the prohibitive or high cost associated with siRNA synthesis. The gene silencing efficacy by siRNA is limited to about 50% or less for majority of genes in mammalian cells. The manufacture of these molecules is expensive (much more expensive than manufacturing an ti sense deoxynucleotides), inefficient, and requires chemical modification. Finally, the observation that the extracellular administration of synthetic siRNAs can trigger interferon-like responses has added a significant barrier for RNAi-based research and RNAi-based therapeutic development. RNAi can be selectively triggered by synthetic short interfering RNA (siRNAs) or genetic elements encoding short-hairpin RNAs (shRNAs) that are subsequently cleaved into siRNAs by the ribonuclease Ill-like enzyme, Dicer. The biochemical mechanism of gene silencing, not yet fully understood, appears to involve a multi-protein RNA-induced silencing complex (RISC). RISC binds, unwinds, and incorporates the anti-sense siRNA strand, which then recognizes and targets perfectly complementary mRNAs for cleavage thereby reducing gene expression. Potent gene silencing (1-10 days) is attributable to the catalytic properties of the RISC complex. The power of RNAi stems from the exquisite specificity that can be achieved. However, off-target RNAi effects are known to occur. Another major side effect is the activation of the interferon-like response by siRNA, which is mediated via dsRNA-dependent protein kinase (PKR) and Toll-like receptors (TLR). The capability to induce interferon-like response by siRNA is mainly determined by its length, (ibid.)
For gene silencing in mammalian cells, the current art teaches that the structure of siRNA is a symmetric double stranded RNA with a length of 19-21 nucleotides and 3' overhangs on both ends to be effective in mammalian cells and to avoid cellular innate ''anti-viral" responses, (ibid.) It is now well established in the field that this 'Optimal" structure can still trigger interferon responses, and pose significant challenges to the development of RNAi-based research and RNAi-based therapeutics (Sledz et al., 2003).
There is a need to develop novel approaches to effective RNAi in mammalian cells through a novel design of siRNAs having better efficacy and potency, rapid onset of action, better durability, and a shorter length of the RNA duplex to avoid non-specific interferon like response and to reduce the cost of synthesis for research and pharmaceutical development of RNAi therapeutics.
The references cited herein are not admitted to be prior art to the claimed invention.
SUMMARY OF THE INVENTION
The present invention is about a surprising discovery of a novel class of small duplex RNA that can induce potent gene silencing in mammalian cells, which is termed herein asymmetrical interfering RNAs (aiRNA). The hallmark of this novel class of RNAi-inducers is the length asymmetry of the two RNA strands. Such a novel structural design is not only functionally potent in effecting gene silencing, but offers several advantages over the current state-of-art siRNAs. Among the advantages, aiRNA can have RNA duplex structure of much shorter length than the current siRNA, which should reduce the cost of synthesis and abrogate/reduce the length-dependent triggering of nonspecific interferon-like responses. In addition, the asymmetry of the aiRNA structure abrogates/reduces the sense-strand mediated off- target effects. Furthermore, aiRNA is more efficacious, potent, rapid-onset, and durable than siRNA in inducing gene silencing. AiRNA can be used in all areas that current siRNA or shRNA are being applied or contemplated to be used, including biology research, R&D research in biotechnology and pharmaceutical industry, and RNAi-based therapies.
The present invention provides a duplex RNA molecule. The duplex RNA molecule comprises a first strand with a length from 18-23 nucleotides and a second strand with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein the first strand has a 3'- overhang from 1-9 nucleotides, and a 5'-overhang from 0-8 nucleotides, wherein said duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell. In an embodiment, the first strand comprises a sequence being substantially complementary to a target mRNA sequence. In a further embodiment, the first strand comprises a sequence being at least 70 percent complementary to a target mRNA sequence. In another embodiment, the eukaryotic cell is a mammalian cell or an avian cell.
In an embodiment, at least one nucleotide of the sequence of 5' overhang is selected from the group consisting of A, U, and dT.
In an embodiment, the GC content of the double stranded region is 20%-70%.
In an embodiment, the first strand has a length from 19-22 nucleotides.
In an embodiment, the first strand has a length of 21 nucleotides. In a further embodiment, the second strand has a length of 14-16 nucleotides.
In an embodiment, the first strand has a length of 21 nucleotides, and the second strand has a length of 15 nucleotides. In a further embodiment, the first strand has a 3 '-overhang of 2-4 nucleotides. In an even further embodiment, the first strand has a 3 '-overhang of 3 nucleotides.
In an embodiment, the duplex RNA molecule contains at least one modified nucleotide or its analogue. In a further embodiment, the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base- modified ribonucleotide. In an even further embodiment, the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide. In an embodiment, the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom. In another embodiment, the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
In an embodiment, the at least one modified nucleotide or its analogue is an unusual base or a modified base. In another embodiment, the at least one modified nucleotide or its analogue comprises inosine, or a tritylated base.
In a further embodiment, the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.
In an embodiment, the first strand comprises at least one deoxynucleotide. In a further embodiment, the at least one deoxynucleotides are in one or more regions selected from the group consisting of 3 '-overhang, 5 '-overhang, and double-stranded region. In another embodiment, the second strand comprises at least one deoxynucleotide.
The present invention also provides a method of modulating gene expression in a cell or an organism comprising the steps of contacting said cell or organism with the duplex RNA molecule of claim 1 under conditions wherein selective gene silencing can occur, and mediating a selective gene silencing effected by the duplex RNA molecule towards a target gene or nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA. In a further embodiment, said contacting step comprises the step of introducing said duplex RNA molecule into a target cell in culture or in an organism in which the selective gene silencing can occur. In an even further embodiment, the introducing step is selected from the group consisting of transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical and regional administration. In another embodiment, the introducing step comprises using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid.
In an embodiment, the modulating method is used for determining the function or utility of a gene in a cell or an organism.
In an embodiment, the modulating method is used for treating or preventing a disease or an undesirable condition.
In an embodiment, the target gene is associated with a disease, a pathological condition, or an undesirable condition in a mammal. In a further embodiment, the target gene is a gene of a pathogenic microorganism. In an even further embodiment, the target gene is a viral gene. In another embodiment, the target gene is a tumor-associated gene. In yet another embodiment, the target gene is a gene associated with a disease selected from the group consisting of autoimmune disease, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging.
The present invention provides a research reagent. The reagent comprises the duplex RNA molecule.
The present invention further provides a kit. The kit comprises a first RNA strand with a length from 18-23 nucleotides and a second RNA strand with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and capable of forming a duplex RNA molecule with the first strand, wherein the duplex RNA molecule has a 3'-overhang from 1-9 nucleotides, and a 5'-overhang from 0-8 nucleotides, wherein said duplex RNA molecule is capable of effecting sequence-specific gene silencing in a eukaryotic cell.
The present invention also provides a method of preparing the duplex RNA molecule. The method comprises the steps of synthesizing the first strand and the second strand, and combining the synthesized strands under conditions, wherein the duplex RNA molecule is formed, which is capable of effecting sequence-specific gene silencing. In an embodiment, the method of claim 35 further comprises a step of introducing at least one modified nucleotide or its analogue into the duplex RNA molecule during the synthesizing step, after the synthesizing and before the combining step, or after the combining step. In another embodiment, the RNA strands are chemically synthesized, or biologically synthesized. The present invention provides an expression vector. The vector comprises a nucleic acid or nucleic acids encoding the duplex RNA molecule operably linked to at least one expression- control sequence. In an embodiment, the vector comprises a first nucleic acid encoding the first strand operably linked to a first expression-control sequence, and a second nucleic acid encoding the second strand operably linked to a second expression-control sequence. In another embodiment, the vector is a viral, eukaryotic, or bacterial expression vector.
The present invention also provides a cell. In an embodiment, the cell comprises the vector. In another embodiment, the cell comprises the duplex RNA molecule. In a further embodiment, the cell is a mammalian, avian, or bacterial cell.
The present invention provides a duplex RNA molecule. The duplex RNA molecule comprises a first strand and a second strand, wherein the first strand is longer than the second strand, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein said duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell. In an embodiment, the two ends of the duplex RNA molecule are independently selected from the group consisting of 3 '-overhang from 1-10 nucleotides, 5'-overhang from 0-10 nucleotides, and blunt end. In another embodiment, the first strand is substantially complementary to a target mRNA sequence. In an alternative embodiment, the second strand is substantially complementary to a target mRNA sequence, In an embodiment, the eukaryotic cell is a mammalian cell or an avian cell. In another embodiment, the duplex RNA molecule is an isolated duplex RNA molecule.
In an embodiment, the first strand has a 3'-overhang from 1-8 nucleotides and a 5'- overhang from 1-8 nucleotides.
In another embodiment, the first strand has a 3'-overhang from 1-10 nucleotides and a blunt end.
In yet another embodiment, the first strand has a 5'-overhang from 1-10 nucleotides and a blunt end.
In an alternative embodiment, the RNA duplex has two 5'-overhangs from 1-8 nucleotides, or two 3'-overhangs from 1-10 nucleotides.
In an embodiment, the first strand has a length from 12-100 nucleotides, from 12-30 nucleotides from 18-23 nucleotides, from 19-25 nucleotides. In a further embodiment, the first strand has a length of 21 nucleotides.
In another embodiment, the second strand has a length from 5-30 nucleotides, 12-22 nucleotides, 12-17 nucleotides. In a further embodiment, the second strand has a length of 15 nucleotides. In an embodiment, the first strand has a length from 12-30 nucleotides, and the second strand has a length from 10-29 nucleotides, with the provision that when the double stranded region is 27 bp, the both ends of the RNA molecule are independently 3' overhang or 5' overhang. In a further embodiment, the first strand has a length from 18-23 nucleotides, and the second strand has a length from 12-17 nucleotides.
In another embodiment, the first strand has a length from 19-25 nucleotides, and the second strand has a length from 12-17 nucleotides.
In an alternative embodiment, the first strand has a length from 19-25 nucleotides, and the second strand has a length from 18-24 nucleotides, with the provision that when the first strand is 5 ' -UUCGAAGUAUUCCGCGUACGU 5 ' -UCGAAGUAUUCCGCGUACGUG or 5 ' - CGAAGUAUUCCGCGUACGUGA the second strand has a length of at most 17 nucleotides, or contains at least one mismatch with the first strand, or contains at least one modification.
In an embodiment, the first strand has a length of 21 nucleotides and the second strand has a length of 12-17 nucleotides, or 14-16 nucleotides.
In an embodiment, the first strand is from 1-10 nucleotides longer than the second strand.
In an embodiment, the 3 '-overhang has a length from 2-6 nucleotides.
In another embodiment, the 5'-overhang has a length from 0-5 nucleotides.
In an embodiment, the gene silencing comprises one or two, or all of RNA interference, modulation of translation, and DNA epigenetic modulations.
In an embodiment, the duplex RNA molecule further comprises a nick in at least one of said first and second strands.
In another embodiment, the double stranded region comprises a gap of one or more nucleotides.
In an embodiment, at least one nucleotide of the 5' overhang is not complementary to the target mRNA sequence.
In another embodiment, at least one nucleotide of 5' overhang is selected from the group consisting of A, U, and dT.
In an embodiment, the duplex RNA molecule is conjugated to an entity selected from the group consisting of peptide, antibody, polymer, lipid, oligonucleotide, cholesterol, and aptamer.
In an embodiment, the RNA molecule further comprises at least one unmatched or mismatched nucleotide.
In another embodiment, the GC content of the double stranded region is 20-70%.
In an embodiment, the 3'-overhang and/or 5'-overhang is stabilized against degradation. In an embodiment, the duplex RNA molecule contains at least one modified nucleotide or its analogue. In a further embodiment, the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base- modified ribonucleotide. In a further embodiment, the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide. In another embodiment, the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom. In yet another embodiment, the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
In an embodiment, the at least one modified nucleotide or its analogue comprises a non- natural base or a modified base. In another embodiment, the at least one modified nucleotide or its analogue comprises inosine, or a tritylated base.
In a further embodiment, the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR. NR2, or CN, wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.
In an embodiment, the first strand comprises at least one deoxynucleotide. In a further embodiment, the at least one deoxynucleotides are in one or more regions selected from the group consisting of 3 '-overhang, 5'-overhang, and double-stranded region proximal to the 5' end of the first strand. In another embodiment, the second strand comprises at least one deoxynucleotide.
The present invention also provides a method of modulating gene expression in a cell or an organism. The method comprises the steps of: contacting said cell or organism with the duplex RNA molecule of claim 1 under conditions wherein selective gene silencing can occur, and mediating a selective gene silencing effected by the double-stranded RNA towards a target nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA. In a further embodiment, said contacting comprises the step of introducing said duplex RNA molecule into a target cell in culture or in an organism in which the selective gene silencing can occur. In an even further embodiment, the introducing step is selected from the group consisting of transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical and regional administration. In another embodiment, the introducing step comprises using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid. In an embodiment, the modulating method is used for modulating the expression of a gene in a cell or an organism.
In another embodiment, the modulating method is used for treating or preventing a disease or an undesirable condition. In an embodiment, the target gene is a gene associated with human or animal diseases. In a further embodiment, the gene is a gene of a pathogenic microorganism. In an even further embodiment, the target gene is a viral gene. In another embodiment, the gene is a tumor- associated gene.
In yet another embodiment, the target gene is a gene associated with a disease selected from the group consisting of autoimmune disease, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders. respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging.
The modulating method can also be used for studying drug target in vitro or in vivo.
The present invention provides a reagent comprising the duplex RNA molecule.
The present invention further provides a kit. The kit comprises a first RNA strand and a second RNA strand, wherein the first strand is longer than the second strand, wherein the second strand is substantially complementary to the first strand, and capable of forming a duplex RNA molecule with the first strand, wherein said duplex RNA molecule is capable of effecting sequence-specific gene silencing in a eukaryotic cell.
The present invention also provides a method of preparing the duplex RNA molecule of claim 1 comprising the steps of synthesizing the first strand and the second strand, and combining the synthesized strands under conditions, wherein the duplex RNA molecule is formed, which is capable of effecting sequence-specific gene silencing. In an embodiment, the RNA strands are chemically synthesized, or biologically synthesized. In another embodiment, the first strand and the second strand are synthesized separately or simultaneously.
In an embodiment, the method further comprises a step of introducing at least one modified nucleotide or its analogue into the duplex RNA molecule during the synthesizing step, after the synthesizing and before the combining step, or after the combining step.
The present invention further provides a pharmaceutical composition. The pharmaceutical composition comprises as an active agent at least one duplex RNA molecule and one or more carriers selected from the group consisting of a pharmaceutical carrier, a positive- charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a cholesterol, a lipid, and a lipoid.
The present invention also provides a method of treatment. The method comprises administering an effective amount of the pharmaceutical composition to a subject in need. In an embodiment, the pharmaceutical composition is administered via a route selected from the group consisting of iv. sc, inhalation, topical, po, and regional administration. In an embodiment, the first strand comprises a sequence being substantially complimentary to the target mRNA sequence of a gene selected from the group consisting of a developmental gene, an oncogene, a tumor suppresser gene, and an enzyme gene, and a gene for an adhesion molecule, a cyclin kinase inhibitor, a Wnt family member, a Pax family member, a Winged helix family member, a Hox family member, a cytokine/lymphokinc or its receptor, a growth/differentiation factor or its receptor, a neurotransmitter or its receptor, a kinase, a signal transducer, a viral gene, a gene of an infectious disease, ABLI, BCLl, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2,ETS1 , ETSl, ETV6, FGR, FOS, FYN, HCR, FIRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC,MYCL 1, MYCN, NRAS, PIMl , PML, RET, SRC, TALI, TCL3 and YES, APC, BRCAl, BRCA2, MADH4, MCC,NFl , NF2,RB1 , TP53. WTl, an ACP desaturase or hydroxylase, an ADP-glucose pyrophorylasc, an ATPase, an alcohol dehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygcnase, a decarboxylase, a dextrinase, a DNA or RNA polymerase, a galactosidase, a glucanase. a glucose oxidase, a GTPase, a helicase, a hemicellulase, an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase, a lipoxygenase, a lysozyme, a pectinesterase, a peroxidase, a phosphatase, a phospholipase, a phosphorylase, a polygalacturonase, a proteinase or peptideases, a pullanase, a recombinase, a reverse transcriptase, a topoisomerase, a xylanase, k-RAS, β-Catenin, Rskl, PCNA, p70S6K, Survivin, mTOR, PTEN, Parpl , or p21 .
In another embodiment, the duplex RNA molecule is selected from the group consisting of
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Wherein A, U, G, and C are nucleotides, while a, t, g, and c are deoxynucleotides.
The present invention provides a method of modifying a first duplex RNA molecule with an antisense strand and a sense strand that form a double-stranded region. The method comprises, shortening the length of the sense strand so that the antisense strand has a 3'-overhang from 1-8 nucleotides and a 5'-ovcrhang from 0-8 nucleotides, and forming a second duplex RNA molecule; wherein at least one property of the first duplex RNA molecule is improved. In an embodiment, the property is selected from the group consisting of size, efficacy, potency, the speed of onset, durability, synthesis cost, off-target effects, interferon response, and delivery. In another embodiment, the method further comprises combining the antisense strand and the shortened sense strand under conditions, wherein the second duplex RNA molecule is formed. In a further embodiment, the first duplex RNA molecule is a siRNA, or dicer-substrate siRNA, or a siRNA precursor.
The present invention provides an expression vector. The vector comprises a nucleic acid or nucleic acids encoding the duplex RNA molecule of claim 1 operably linked to at least one expression-control sequence. In an embodiment, the expression vector comprises a first nucleic acid encoding the first strand operably linked to a first expression-control sequence, and a second nucleic acid encoding the second strand operably linked to a second expression-control sequence. In another embodiment, the expression vector is a viral, eukaryotic expression vector, or bacterial expression vector.
The present invention also provides a cell. In an embodiment, the cell comprises the expression vector. In another embodiment, the cell comprises the duplex RNA molecule. In yet another embodiment, the cell is a mammalian cell, avian cell, or bacterial cell.
Other features and advantages of the present invention are apparent from the additional descriptions provided herein including the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure IA shows the structure of a duplex RNA molecule that has both a 3 '-overhang and a 5 '-overhang on the first strand.
Figure IB shows the structure of a duplex RNA molecule that has both a 3 '-overhang and a 5 '-overhang on the first strand, and a nick in the duplex.
Figure 1C shows the structure of a duplex RNA molecule that has both a 3 '-overhang and a 5 '-overhang on the first strand, and a gap in the second strand.
Figure 2A shows the structure of a duplex RNA molecule that has a blunt end, and a 5'- overhang on the first strand.
Figure 2B shows the structure of a duplex RNA molecule that has a blunt end, and a 3'- overhang on the first strand.
Figure 2C shows the structure of a duplex RNA molecule that has 3 '-overhangs on both ends of the duplex and that the first strand is longer than the second strand.
Figure 2D shows the structure of a duplex RNA molecule that has 5 '-overhangs on both ends of the duplex and that the first strand is longer than the second strand.
Figure 3 shows the induction of gene silencing of β-catenin by aiRNA (asymmetric interfering RNAs).
Figure 3 A shows the confirmation of the oligos. After annealing, the oligos were confirmed by 20% poly aery lamide gel. Lane 1 , 21nt/21nt; lane 2, 12nt (a)/21nt; lane 3, 12nt (b)/21 nt; lane 4, 13nt/13nt; lane 5, 13nt/21nt; lane 6, 14nt /14nt; lane 7, 14nt(a)/21nt; lane 8, 14nt(b)/21nt; lane 9, 15nt/15nt; lane 10, 15nt/21nl.
Figure 3B shows the effects of the oligos in gene silencing. HeLa cells were plated at 200,000 cells/well into a 6 well culture plate. 24 hours later they were transfected with scramble siRNA (lane 1 ), 21-bp siRNA targeted E2F1 (lane 2, as a control for specificity) or 21-bp siRNA targeted beta-eaten in (lane 3, as a positive control), or the same concentration of aiRNA of different length mix; 12nt(a)/21 nt (lane 4); 12nt (b)/21nt (lane 5); 13nt/21 nt (lane 6); 14nt (a)/21 nt (lane 7); 14nt (b)/21nt (lane 8); 15nt/21 nt (lane 9). Cells were harvested 48 hours after transfection. Expression of β-catenin was determined by Western blot. E2F1 and actin are used as controls.
Figures 4 and 5 show the structure-activity relationship of aiRNA oligos, with or without base substitutions, in mediating gene silencing. HeIa cells were transfected with the indicated aiRNA. Cells were harvested and lysates generated at 48 hours post transfection. Western blots were performed to detect levels of β-calenin and actin. si stands for β-calenin siRNA oligonucleotide. The numerical labeling above each lane corresponds to the aiRNA oligos in Table 3.
Figure 6 shows the analysis of the mechanism of gene silencing triggered by aiRNA.
Figure 6a shows the northern blot analysis of β-catenin mRNA levels in cells transfected with aiRNA or siRNA for the indicated number of days.
Figure 6b shows the schematic of 5'-RACE-PCR for β-catenin showing cleavage of mRNA and expected PCR product.
Figure 6c shows β-catenin cleavage products mediated by aiRNA were amplified by 5!- RACE-PCR from cells transfected with aiRNA for 4 or 8 hours.
Figure 6d shows the schematic of β-catenin mRNA cleavage site confirmed by sequencing the 5'-RACE-PCR fragment.
Figure 6e shows differential RISC loading efficiency of aiRNA and siRNA. aiRNA or siRNA duplexes were transfected into HeIa cells 48 hours after transfection with pCMV-Ago2. Ago2 was immunoprecipitated at the indicated time points following aiRNA or siRNA transfection, and northern blot analysis was performed to determine levels of Ago2/RISC associated small RNAs. Levels of Ago2 (shown below-) were determined by western blot following IP.
Figure 6f shows the effects of knocking down Ago2 or Dicer on gene siliencing activity of aiRNA and siRNA. Cells were transfected with scramble siRNA (siCon), or siRNA targeting Ago2 (siAgo2), or Dicer (siDicer) 24 hours prior to transfection with scramble aiRNA (Con) or aiRNA targeting Stat3 (ai). Cells were harvested and western blot analysis was performed at 48 hours following aiStat3 transfection.
Figure 7 shows the advantages of incorporation of aiRNA into RISC compared to siRNA.
Figure 7A shows that aiRNA enters RISC with better efficiency than siRNA. Cells transfected with Ago2 expression plasmid were transfected with aiRNA or siRNA for the indicated times. Following cell lysis, Ago2 was immunoprecipitaed, RNA extracted from the immunoprecipitate, and separated on a 15% acrylamide gel. Following transfer, the membrane was hybridized to a probe to detect the 21 mer antisense strand of the aiRNA or siRNA. IgG control lane shows lack of signal compared to Ago2 immunoprecipitate.
Figure 7B shows that the sense strand of aiRNA does not stay in RISC. Membrane from (A) was stripped and re-probed to detect the sense strand of the transfected oligo. Cartoons in (A) and (B) illustrate the position of the sense strand (upper strand), the antisense strand (lower strand), or the duplex on the membrane.
Figure 8 shows that the mechanism of RISC loading by aiRNA.
Figure 8 A shows the immuno precipitation analysis of the interaction between different strands of aiRNA or siRNA and Ago2. HeIa S-IO lysate containing overexpressed Ago2 was incubated with the indicated aiRNA or siRNA duplex containing 32P end labeled sense or antisense strands. The (*) marks the location of the label. Following Ago2 immunoprecipitation, the RNA was isolated and separated on a 15% acrylamide gel and exposed to film. The Ago2- associated RNAs are shown in the pellet fraction, while the non-Ago2 bound RNAs remain in the supernatant (Sup).
Figure 8B shows the role of sense strand cleavage in aiRNA activity. Cells were transfected with aiRNA or aiRNA with sense strand 2'-O-methyl at position 8 (predicted Ago2 cleavage site) or position 9 as a control. RNA was collected at 4 hours post transection and qRT-PCR performed to determine relative levels of β-catenin mRNA remaining.
Figure 9 shows the aiRNA and siRNA competition analysis.
Figure 9A illustrates the siRNA and aiRNA duplex containing 32P end labeled antisense strands. The (*) marks the location of the label.
Figure 9B shows that the cold aiRNA does not compete with labeled siRNA for Ago2. HeIa S-IO lysate containing overexpressed Ago2 was incubated with the 32P end labeled siRNA and cold aiRNA or siRNA duplex prior to Ago2 immunoprecipitation. RNA was then isolated and analyzed on 15% acrylamide gel.
Figure 9C shows that the cold siRNA does not compete with labeled aiRNA for Ago2. The same S-IO lysate used in B was incubated with the 32P end labeled aiRNA and cold aiRNA or siRNA duplex prior to Ago2 immunoprecipitation. RNA wras then isolated and analyzed on 15% acrylamide gel.
FigurelO illustrates the model of aiRNA and siRNA showing observed differences in RISC loading and generation of mature RISC.
Figure 11 shows asymmetric RNA duplexes of 14- 15 bp with antisense overhangs (aiRNA) induced potent, efficacious, rapid, and durable gene silencing.
Figure 1 IA shows the Diagram showing sequence and design of siRNA and aiRNA targeting β-catenin. Figure 1 IB shows the induction of gene silencing by aiRNA of various lengths, β-catenin protein levels were analysed by western blot in cells transfectcd with indicated aiRNA for 48 hours.
Figure 11C shows that aiRNA is more potent and efficacious than siRNA in inducing β- catenin protein depletion. HeIa cells were transfected with aiRNA or siRNA targeting β-catenin at the indicated concentrations. At 48 hours post-transfection, cell lysates were made and western blot analysis was done.
Figure 1 ID shows that the aiRNA is more efficacious, rapid, and durable than siRNA in reducingβ-catenin RNA levels. Cells were transfected with 10 nM 15 bp aiRNA or 21-mer siRNA for the indicated number of days before northern blot analysis.
Figure 12 shows that aiRNA mediates rapid and potent silencing.
Figure 12A shows the sequence and structure of aiRNA and siRNA used to target β- catenin.
Figure 12B shows RT-PCR of β-catenin niRNA levels from cells transfected with control aiRNA or aiRNA targeting β-catenin. RNA was collected at the indicated times post transfection.
Figure 12C shows the quantitative real-time RT-PCR of β-catenin mRNA levels in cells transfected with control, aiRNA, or siRNA for the indicated number of hours.
Figure 12D shows the western blot analysis of β-catenin protein levels in cells transfected with control, aiRNA, or siRNA for the indicated times.
Figure 13 shows the comparison of aiRNA with siRNA in gene silencing efficacy and durability against multiple targets. HeIa cells were transfected with scramble siRNA (c), aiRNA (ai), or siRNA (si) targeting (a) β-catenin at 10 nM, (b) Stat3, (c) EF2, or (d) NQOl at 20 nM. RNA and protein was purified at the indicated time points and analyzed for mRNA levels by quantitative real time polymerase chain reaction (qRT-PCR) and protein levels by western blot. qRT-PCR data is normalized to siCon transfected ceils.
Figure 14 shows aiRNA mediated gene silencing is effective against various genes in multiple cell lines.
Figure 14a shows aiRNA duplex is more efficacious than siRNA in targeting β-catenin in different mammalian cell lines. Figure 14b shows the western blot analysis oϊNbsl, Survivin, Parpl, p21 from cells transfected with 20 nM of the indicated aiRNA or siRNA for 48 hours.
Figure 14c shows the western blot analysis of Rskl, PCNA, p70S6K, mTOR, and PTEN from cells transfected with 20 nM of the indicated aiRNA or siRNA for 48 hours.
Figure 14d shows the allele-specific gene silencing of k-Ras by aiRNA. aiRNA targeting wildtype k-Ras was tested for silencing of k-Ras in both k-Ras wildtype (DLDl) and k-Ras mutant (SW480) cell lines by western blot analysis.
Figure 15 show the lack of off-target gene silencing by sense-strand, immunostimulation, and serum stability of aiRNAs.
Figure 15a shows RT-PCR analysis of the expression of interferon inducible genes in PBMC mock treated or incubated with β-catenin siRNA or aiRNA duplex for 16 hours.
Figure 15b shows RT-PCR analysis of the expression of interferon inducible genes in HeIa cells mock transfected or transfected with EF2 or Survivin aiRNA or siRNA for 24 hours.
Figure 15c shows the microarray analysis for changes in the expression of known interferon response related genes. Total RNA isolated from aiRNA and siRNA transfected HeIa cells was analyzed by microarray.
Figure 15d shows that no sense-strand mediated off-target gene silencing is detected for aiRNA. Cells were co-transfected with aiRNA or siRNA and either a plasmid expressing Stat3 (sense RNA) or a plasmid expressing antisense Stat 3 (antisense RNA). Cells were harvested and RNA collected at 24 hours post transfection and relative levels of Stat 3 sense or antisense RNA were determined by quantitative real time PCR or RT-PCR (inserts).
Figure 15e shows the Stability of aiRNA and siRNA duplexes in human serum. aiRNA and siRNA duplexes were incubated in 10% human serum at 370C for the indicated amount of time prior to gel electrophoresis. Duplex remaining (% of control) is indicated.
Figure 15f illustrates the proposed model for gene specific silencing mediated by the aiRNA duplex.
Figure 16 shows the potent Anti-Tumor Activity of aiRNA against β-catenin in SW480 human colon xenografted mouse model. Immunosurpressed mice with established subcutaneous SW480 human colon cancer were given intravenously (iv) with 0.6 nmol PEI-complexed β- catenin siRNAs, PEI-complexed β-catenin aiRNAs or a PEI-complexed unrelated siRNA as negative control daily. Tumor size was evaluated periodically during treatment. Each point represents the mean + SEM of six tumors.
Figure 17 shows the potent Anti-Tumor Activity of aiRNA against β-catenin in IIT29 human colon xenografted mouse model. Immunosurpressed mice with established subcutaneous HT29 human colon cancer were given intravenously (iv) with 0.6 nmol PEI-complexed β- catenin siRNAs, PEI-complexed β-catenin aiRNAs or a PEI-complexcd unrelated siRNA as negative control every other day. Tumor size was evaluated periodically during treatment. Each point represents the mean + SEM of five tumors.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to an asymmetric duplex RNA molecule that is capable of effecting selective gene silencing in a eukaryotic cell. In an embodiment, the duplex RNA molecule comprises a first strand and a second strand. The first strand is longer than the second strand. The second strand is substantially complementary to the first strand, and forms a double- stranded region with the first strand.
In an embodiment, the duplex RNA molecule has a 3'-overhang from 1-8 nucleotides and a 5'-overhang from 1-8 nucleotides, a 3'-overhang from 1-10 nucleotides and a blunt end, or a 5'- overhang from 1-10 nucleotides and a blunt end. In another embodiment, the duplex RNA molecule has two 5'-overhangs from 1 -8 nucleotides or two 3'-overhangs from 1-10 nucleotides. In a further embodiment, the first strand has a 3'-overhang from 1-8 nucleotides and a 5'- overhang from 1-8 nucleotides. In an even further embodiment, the duplex RNA molecule is an isolated duplex RNA molecule.
In an embodiment, the first strand has a 3 '-overhang from 1-10 nucleotides, and a 5'- overhang from 1-10 nucleotides or a 5 '-blunt end. In another embodiment, the first strand has a 3'-overhang from 1-10 nucleotides, and a 5'-overhang from 1-10 nucleotides. In an alternative embodiment, the first strand has a 3'-overhang from 1-10 nucleotides, and a 5'-blunt end.
In an embodiment, the first strand has a length from 5-100 nucleotides, from 12-30 nucleotides, from 15-28 nucleotides, from 18-27 nucleotides, from 19-23 nucleotides from 20-22 nucleotides, or 21 nucleotides.
In another embodiment, the second strand has a length from 3-30 nucleotides, from 12-26 nucleotides, from 13-20 nucleotides, from 14-23 nucleotides, 14 or 15 nucleotides.
In an embodiment, the first strand has a length from 5-100 nucleotides, and the second strand has a length from 3-30 nucleotides; or the first strand has a length from 10-30 nucleotides, and the second strand has a length from 3-29 nucleotides; or the first strand has a length from 12- 30 nucleotides and the second strand has a length from 10-26 nucleotides; or the first strand has a length from 15-28 nucleotides and the second strand has a length from 12-26 nucleotides; or the first strand has a length from 19-27 nucleotides and the second strand has a length from 14-23 nucleotides; or the first strand has a length from 20-22 nucleotides and the second strand has a length from 14-15 nucleotides. In a further embodiment, the first strand has a length of 21 nucleotides and the second strand has a length of 13-20 nucleotides, 14-19 nucleotides, 14-17 nucleotides, 14 or 15 nucleotides.
In an embodiment, the first strand is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer than the second strand.
In an embodiment, the duplex RNA molecule further comprises 1-10 unmatched or mismatched nucleotides. In a further embodiment, the unmatched or mismatched nucleotides are at or near the 3' recessed end. In an alternative embodiment, the unmatched or mismatched nucleotides are at or near the 5' recessed end. In an alternative embodiment, the unmatched or mismatched nucleotides are at the double-stranded region. In another embodiment, the unmatched or mismatched nucleotide sequence has a length from 1-5 nucleotides. In an even further embodiment, the unmatched or mismatched nucleotides form a loop structure.
In an embodiment, the first strand or the second strand contains at least one nick, or formed by two nucleotide fragments.
In an embodiment, the gene silencing is achieved through one or two, or all of RNA interference, modulation of translation, and DNA epigenetic modulations. As used in the specification and claims, the singular form "a", "an", and "the" include plural references unless the context clearly dictate otherwise. For example, the term "a cell" includes a plurality of cells including mixtures thereof.
As used herein, a "double stranded RNA," a "duplex RNA" or a "RNA duplex" refers to an RNA of two strands and with at least one double-stranded region, and includes RNA molecules that have at least one gap, nick, bulge, and/or bubble either within a double-stranded region or between two neighboring double-stranded regions. If one strand has a gap or a single- stranded region of unmatched nucleotides between two double-stranded regions, that strand is considered as having multiple fragments, A double-stranded RNA as used here can have terminal overhangs on either end or both ends. . In some embodiments, the two strands of the duplex RNA can be linked through certain chemical linker.
As used herein, an "antisense strand" refers to an RNA strand that has substantial sequence complementarity against a target messenger RNA. An antisense strand can be part of an siRNA molecule, part of a miRNA/miRNA* duplex, or a single-strand mature miRNA.
The term "isolated" or "purified" as used herein refers to a material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography.
As used herein, "modulating" and its grammatical equivalents refer to either increasing or decreasing (e.g., silencing), in other words, either up-regulating or down-regulating. As used herein, "gene silencing" refers to reduction of gene expression, and may refer to a reduction of gene expression about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted gene.
As used herein, the term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.
Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" as used herein refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. A subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; and improvement in quality of life.
As used herein, the terms "inhibiting", "to inhibit" and their grammatical equivalents, when used in the context of a bioactivity, refer to a down-regulation of the bioactivity, which may reduce or eliminate the targeted function, such as the production of a protein or the phosphorylation of a molecule. In particular embodiments, inhibition may refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90% or 95% of the targeted activity. When used in the context of a disorder or disease, the terms refer to success at preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition or disorder.
As used herein, the term "substantially complementary" refers to complementarity in a base-paired, double-stranded region between two nucleic acids and not any single-stranded region such as a terminal overhang or a gap region between two double-stranded regions. The complementarity does not need to be perfect; there may be any number of base pair mismatches, for example, between the two nucleic acids. However, if the number of mismatches is so great that no hybridization can occur under even the least stringent hybridization conditions, the sequence is not a substantially complementary sequence. When two sequences are referred to as "substantially complementary" herein, it means that the sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship of nucleic acid complementarity and stringency of hybridization sufficient to achieve specificity is well known in the art. Two substantially complementary strands can be, for example, perfectly complementary or can contain from 1 to many mismatches so long as the hybridization conditions are sufficient to allow, for example discrimination between a pairing sequence and a non-pairing sequence. Accordingly, substantially complementary sequences can refer to sequences with base-pair complementarity of 100, 95, 90, 80, 75, 70, 60, 50 percent or less, or any number in between, in a double-stranded region.
As used herein, antagomirs are miRNA inhibitors, and can be used in silencing endogenous miRNAs. As used herein, mimetics or mimics are miRNA agonists, and can be used to replace endogenous miRNAs as functional equivalents and thereby upregulating pathways affected by such endogenous miRNAs.
1. RNA Interference RNA interference (abbreviated as RNAi) is a cellular process for the targeted destruction of single-stranded RNA (ssRNA) induced by double-stranded RNA (dsRNA). The ssRNA is gene transcript such as a messenger RNA (mRNA). RNAi is a form of post-transcriptional gene silencing in which the dsRNA can specifically interfere with the expression of genes with sequences that are complementary to the dsRNA. The antisense RNA strand of the dsRNA targets a complementary gene transcript such as a messenger RNA (mRNA) for cleavage by a ribonuclease.
In RNAi process, long dsRNA is processed by a ribonuclease protein Dicer to short forms called small interfering RNA (siRNA). The siRNA is separated into guide (or antisense) strand and passenger (or sense) strand. The guide strand is integrated into RNA-induced- silencing-complex (RISC), which is a ribonuclease-containing multi-protein complex. The complex then specifically targets complementary gene transcripts for destruction. RNAi has been shown to be a common cellular process in many eukaryotes. RISC, as well as Dicer, is conserved across the eukaryotic domain. RNAi is believed to play a role in the immune response to virus and other foreign genetic material.
Small interfering RNAs (siRNAs) are a class of short double-stranded RNA (dsRNA) molecules that play a variety of roles in biology. Most notably, it is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition, siRNAs also play roles in the processes such as an antiviral mechanism or shaping the chromatin structure of a genome. In an embodiment, siRNA has a short (19-21 nt) double- strand RNA (dsRNA) region with 2-3 nucleotide 3' overhangs with 5 '-phosphate and 3 '-hydroxy 1 termini.
MicroRNAs (miRNAs) are a class of endogenous, single or double-stranded, about 22 nucleotide-long RNA molecules that regulate as much as 30% of mamalian genes (Czech, 2006; Eulalio et al, 2008; Mack, 2007). MiRNA represses protein production by blocking translation or causing transcript degradation. A miRNA may target 250-500 different mRNAs. MiRNA is the product of the Dicer digestion of pre-miRNA, which is the product of primary miRNA (pri- miRNA).
As used herein, antagomirs are miRNA inhibitors, and can be used in the silencing of endogenous miRNAs. As used herein, mimetics are miRNA agonists, and can be used to replace the miRNAs and downregulate mRNAs.
Dicer is a member of RNase III ribonuclease family. Dicer cleaves long double-stranded RNA (dsRNA), pre-microRNA (miRNA). and short hairpin RNA (shRNA) into short double- stranded RNA fragments called small interfering RNA (siRNA) about 20-25 nucleotides long, usually with a two-base overhang on the 3' end. Dicer catalyzes the first step in the RNA interference pathway and initiates formation of the RNA-induced silencing complex (RISC), whose catalytic component argonaute is an endonucleasc capable of degrading messenger RNA (mRNA) whose sequence is complementary to that of the siRNA guide strand.
As used herein, an effective siRNA sequence is a siRNA that is effective in triggering RNAi to degrade the transcripts of a target gene. Not every siRNA complementary to the target gene is effective in triggering RNAi to degrade the transcripts of the gene. Indeed, time- consuming screening is usually necessary to identify an effective siRNA sequence. In an embodiment, the effective siRNA sequence is capable of reducing the expression of the target gene by more than 90%, more than 80%, more than 70%, more than 60%, more than 50%, more than 40%, or more than 30%.
The present invention provides a novel structural scaffold called asymmetric interfering RNA (aiRNA) that can be used to effect siRNA-like results, and also to modulate miRNA pathway activities (described in detail in co-owned PCT and U.S. applications filed on the same day as the present application under the title "Composition of Asymmetric RNA Duplex as Micro RNA Mimetic or Inhibitor" the entire content of which is incorporated herein by reference).
The novel structural design of aiRNA is not only functionally potent in effecting gene regulation, but also offers several advantages over the current state-of-art, RNAi regulators (mainly antisense, siRNA). Among the advantages, aiRNA can have RNA duplex structure of much shorter length than the current siRNA constructs, which should reduce the cost of synthesis and abrogate or reduce length-dependent triggering of nonspecific interferon-like immune responses from host cells. The shorter length of the passenger strand in aiRNA should also eliminate or reduce the passenger strand's unintended incorporation in IHSC, and in turn, reduce off-target effects observed in miRNA-mediated gene silencing. AiRNA can be used in all areas that current miRNA-based technologies are being applied or contemplated to be applied, including biology research, R&D in biotechnology and pharmaceutical industries, and miRNA- based diagnostics and therapies.
2. The aiRNA structural scaffold
Elbashir, et al, tested several asymmetrical duplex RNA molecules as well as symmetrical siRNA molecules (Elbashir et al.. 2001c). The asymmetrical duplex RNA molecules are listed in table 1 together with the corresponding siRNA molecules.
Figure imgf000024_0001
In comparison with corresponding symmetrical siRNA molecules, however, the asymmetrical duplex RNA molecules are incapable of effecting selective gene silencing {ibid).
The present invention is pertinent to asymmetrical duplex RNA molecules that are capable of effecting sequence-specific gene silencing. In an embodiment, a RNA molecule of the present invention comprises a first strand and a second strand, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein the first strand is longer than the second strand; excluding the asymmetrical duplex RNA molecules disclosed in Elbashir (Elbashir et al., 2001 c). specifically the asymmetrical duplex RNA molecules listed in Table 1. The RNA molecule comprises a double- stranded region, and two ends independently selected from the group consisting of a 5'- overhang, a 3 '-overhang, and a blunt end. The RNA strand can have unmatched or imperfectly matched nucleotides.
In an embodiment, the first strand is at least 1 nt longer than the second strand. In a further embodiment, the first strand is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt longer than the second strand. In another embodiment, the first strand is 20-100 nt longer than the second strand. In a further embodiment, the first strand is 2-12 nt longer than the second strand. In an even further embodiment, the first strand is 3-10 nt longer than the second strand.
In an embodiment, the double-stranded region has a length of 3-98 bp. In a further embodiment, the double-stranded region has a length of 5-28 bp. In an even further embodiment, the double-stranded region has a length of 10-19 bp. The length of the double- stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp.
In an embodiment, the double-stranded region of the RNA molecule does not contain any mismatch or bulge. In another embodiment, the double-stranded region of the RNA molecule contains mismatch and / or bulge.
In an embodiment, when the first strand is 5 ' -UUCGAAGUAUUCCGCGUACGU 5 ' -UCGAAGUAUUCCGCGUACGUG or 5 ' - CGAAGUAUUCCGCGUACGUGA , the second strand has a length of at most 17 nucleotides, or contains at least one mismatch with the first strand, or contains at least one modification.
In an alternative embodiment, the first strand is not 5 ' -UUCGAAGUAUUCCGCGUACGU 5 ' -UCGAAGUAUUCCGCGUACGUG or 5 ' - CGAAGUAUUCCGCGUACGUGA .
In an embodiment, the first strand comprises a sequence being substantially complimentary to a target mRNA sequence. In another embodiment, the second strand comprises a sequence being substantially complimentary to a target mRNA sequence.
The present invention is pertinent to asymmetrical double stranded RNA molecules that are capable of effecting gene silencing. In an embodiment, an RNA molecule of the present invention comprises a first strand and a second strand, wherein the second strand is substantially complementary, or partially complementary to the first strand, and the first strand and the second strand form at least one double-stranded region, wherein the first strand is longer than the second strand (length asymmetry). The RNA molecule of the present invention has at least one double- stranded region, and two ends independently selected from the group consisting of a 5'- overhang, a 3 '-overhang, and a blunt end (e.g., see FIGS. 1 A, 2A-2D).
In the field of making small RNA regulators where changes, additions and deletions of a single nucleotide can critically affect the functionality of the molecule (Elbashir et al., 2001c), the aiRNA scaffold provides a structural platform distinct from the classic siRNA structure of 21-nt double-strand RNA which is symmetric in each strand and their respective 3' overhangs. Further, the aiRNA of the present invention provides a much-needed new approach in designing a new class of small molecule regulators that, as shown by data included in the examples below, can overcome obstacles currently encountered in RNAi-based researches and drug development. For example, data from aiRNAs that structurall) mimic siRNAs show that aiRNAs are more efficacious, potent, rapid-onset, durable, and specific than siRNAs in inducing gene silencing.
Any single-stranded region of the RNA molecule of the invention, including any terminal overhangs and gaps in between two double-stranded regions, can be stabilized against degradation, either through chemical modification or secondary structure. The RNA strands can have unmatched or imperfectly matched nucleotides. Each strand may have one or more nicks (a cut in the nucleic acid backbone, e.g., see FIG. IB), gaps (a fragmented strand with one or more missing nucleotides, e.g, see FIG. 1C), and modified nucleotides or nucelotide analogues. Not only can any or all of the nucleotides in the RNA molecule chemically modified, each strand may be conjugated with one or more moieties to enhance its functionality, for example, with moieties such as one or more peptides, antibodies, antibody fragments, aptamers, polymers and so on.
In an embodiment, the first strand is at least 1 nt longer than the second strand. In a further embodiment, the first strand is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt longer than the second strand. In another embodiment, the first strand is 20- 100 nt longer than the second strand. In a further embodiment, the first strand is 2-12 nt longer than the second strand. In an even further embodiment, the first strand is 3-10 nt longer than the second strand.
In an embodiment, the first strand, or the long strand, has a length of 5-100 nt, or preferably 10-30 or 12-30 nt, or more preferably 15-28 nt. In one embodiment, the first strand is 21 nucleotides in length. In an embodiment, the second strand, or the short strand, has a length of 3-30 nt, or preferably 3-29 nt or 10-26 nt, or more preferably 12-26 nt. In an embodiment, the second strand has a length of 15 nucelotides. In an embodiment, the double-stranded region has a length of 3-98 bp. In a further embodiment, the double-stranded region has a length of 5-28 bp. In an even further embodiment, the double-stranded region has a length of 10-19 bp. 1 he length of the double- stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp.
In an embodiment, the double-stranded region of the RNA molecule does not contain any mismatch or bulge, and the two strands are perfectly complementary to each other in the double- stranded region. In another embodiment, the double-stranded region of the RNA molecule contains mismatch and/or bulge.
In an embodiment, the terminal overhang is 1-10 nucleotides. In a further embodiment, the terminal overhang is 1-8 nucleotides. In another embodiment, the terminal overhang is 3 nt.
2.1. The duplex RNA molecule with both a 5 '-overhang and a 3 '-overhang on the first strand
Referring to FIG. IA, in one embodiment of the present invention, the double stranded RNA molecule has both a 5'-overhang and a 3'-overhang on the first strand. The RNA molecule comprises a first strand and a second strand; the first strand and the second strand form at least one double-stranded region with substantially complementary sequences, wherein the first strand is longer than the second strand. On the first strand, flanking the double-stranded region, there is an unmatched overhang on both the 5' and 3" termini.
In an embodiment, the first strand is at least 2 nt longer than the second strand. In a further embodiment, the first strand is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt longer than the second strand. In another embodiment, the first strand is 20-100 nt longer than the second strand. In a further embodiment, the first strand is 2-12 nt longer than the second strand. In an even further embodiment, the first strand is 3-10 nt longer than the second strand.
In an embodiment, the first strand has a length of 5-100 nt. In a further embodiment, the first strand has a length of 5-100 nt, and the second strand has a length from 3-30 nucleotides. In an even further embodiment, the first strand has a length of 5-100 nt, and the second strand has a length from 3-18 nucleotides.
In an embodiment, the first strand has a length from 10-30 nucleotides. In a further embodiment, the first strand has a length from 10-30 nucleotides, and the second strand has a length from 3-28 nucleotides. In an even further embodiment, the first strand has a length from 10-30 nucleotides, and the second strand has a length from 3-19 nucleotides. In an embodiment, the first strand has a length from 12-26 nucleotides. In a further embodiment, the first strand has a length from 12-26 nucleotides, and the second strand has a length from 10-24 nucleotides. In an even further embodiment, the first strand has a length from 12-26 nucleotides, and the second strand has a length from 10-19 nucleotides.
In an embodiment, the first strand has a length of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nt. In another embodiment, the second strand has a length of 3, 4, 5, 6, 7, 8, 9. 10, 11 , 12, 13, 14, 15, 16. 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nt.
In an embodiment, the first strand has a length of 21 nt, and the second strand has a length of 15 nt.
In an embodiment, the 3 '-overhang has a length of 1-10 nt. In a further embodiment, the 3 '-overhang has a length of 1-8 nt. In an even further embodiment, the 3 '-overhang has a length of 2-6 nt. In one embodiment, the 3'-overhang has a length of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.
In an embodiment, the 5 '-overhang has a length of 1-10 nt. In a further embodiment, the 5'-overhang has a length of 1-6 nt. In an even further embodiment, the 5 "-overhang has a length of 2-4 nt. In one embodiment, the 5'-overhang has a length of 1, 2, 3, 4, 5, 6. 7, 8, 9, or 10 nt.
In an embodiment, the length of the 3 '-overhang is equal to that of the 5 '-overhang. In another embodiment, the 3 '-overhang is longer than the 5 '-overhang. In an alternative embodiment, the 3 '-overhang is shorter than the 5 '-overhang.
In an embodiment, the duplex RNA molecule comprises a double-stranded region of substantially complementary sequences of about 15 nt, a 3-nt 3 '-overhang, and a 3-nt 5'- overhang. The first strand is 21 nt and the second strand is 15 nt. In one feature, the double- stranded region of various embodiments consists of perfectly complementary sequences. In an alternative feature, the double strand region includes at least one nick (FIG. IB), gap (FIG. 1C), or mismatch (bulge or loop).
In an embodiment, the double-stranded region has a length of 3-98 bp. In a further embodiment, the double-stranded region has a length of 5-28 bp. In an even further embodiment, the double-stranded region has a length of 10-19 bp. The length of the double-stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 1 1. 12, 13, 14, 15, 18. 1 9, 20, 21 , 22, 23, 24, 25, 26. 27, 28, 29, or 30 bp. There can be more than one double-stranded region. In an embodiment, the first strand is the antisense strand, which is capable of targeting a substantially complementary gene transcript such as a messenger RNA (mRNA) for gene silencing either by cleavage or by translation repression.
The present invention also provides a duplex RNA molecule comprising a first strand with a length from 18-23 nucleotides and a second strand with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and forms a double- stranded region with the first strand, wherein the first strand has a 3 '-overhang from 1-9 nucleotides, and a 5'-overhang from 1-8 nucleotides, wherein said duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell. In an embodiment, the first strand comprises a sequence being substantially complementary to a target mRNA sequence.
In an embodiment, the first strand has a length of 20, 21, or 22 nucleotides. In another embodiment, the second strand has a length of 14, 15, or 16 nucleotides.
I an embodiment, the first strand has a length of 21 nucleotides, and the second strand has a length of 15 nucleotides. In a further embodiment, the first strand has a 3 '-overhang of 1 , 2, 3, 4, 5, or 6 nucleotides. In an even further embodiment, the first strand has a 3'-overhang of 3 nucleotides.
2.2. The duplex RNA molecule with a blunt end and a 5'-overhang or a 3 '-overhang on the first strand
In one embodiment, the duplex RNA molecule comprises a double-stranded region, a blunt end, and a 5'-overhang or a 3 '-overhang (see. e.g.. FIGS. 2A and 2B). The RNA molecule comprises a first strand and a second strand, wherein the first strand and the second strand form a double-stranded region, wherein the first strand is longer than the second strand.
In an embodiment, the double-stranded region has a length of 3-98 bp. In a further embodiment, the double-stranded region has a length of 5-28 bp. In an even further embodiment, the double-stranded region has a length of 10-18 bp. The length of the double-stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp. The double-stranded region can have features similar to those described with regard to other embodiments and are not necessarily repeated here. For example, the double-stranded region can consist of perfectly complementary sequences or include at least one nick, gap, or mismatch (bulge or loop).
In an embodiment, the first strand is at least 1 nt longer than the second strand. In a further embodiment, the first strand is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt longer than the second strand. In another embodiment, the first strand is 20-1 OO nt longer than the second strand. In a further embodiment, the first strand is 2-12 nt longer than the second strand. In an even further embodiment, the first strand is 4-10 nt longer than the second strand.
In an embodiment, the first strand has a length of 5-100 nt. In a further embodiment, the first strand has a length of 5-100 nt, and the second strand has a length from 3-30 nucleotides. In an even further embodiment, the first strand has a length of 10-30 nt, and the second strand has a length from 3-19 nucleotides. In another embodiment, the first strand has a length from 12-26 nucleotides, and the second strand has a length from 10-19 nucleotides.
In an embodiment, the duplex RNA molecule comprises a double-stranded region, a blunt end, and a 3 '-overhang (see, e.g., Fig. 2B).
In an embodiment, the 3 '-overhang has a length of 1-10 nt. In a further embodiment, the 3'-overhang has a length of 1-8 nt. In an even further embodiment, the 3'-overhang has a length of 2-6 nt. In one embodiment, the 3'-overhang has a length of 1, 2, 3, 4, 5, 6. 7, 8, 9, or 10 nt.
In an alternative embodiment, the duplex RNA molecule comprises a double-stranded region, a blunt end, and a 5 '-overhang (see, e.g., Fig. 2A).
In an embodiment, the 5 '-overhang has a length of 1-10 nt. In a further embodiment, the 5 '-overhang has a length of 1-6 nt. In an even further embodiment, the 5 '-overhang has a length of 2-4 nt. In one embodiment, the 5 '-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.
2.3. The duplex RNA molecule with two 5 '-overhangs or two 3 '-overhangs
In one embodiment, the duplex RNA molecule comprises a double-stranded region, and two 3'-overhangs or two 5 '-overhangs (sec, e.g., FIGS. 2C and 2D). The RNA molecule comprises a first strand and a second strand, wherein the first strand and the second strand form a double-stranded region, wherein the first strand is longer than the second strand.
In an embodiment, the double-stranded region has a length of 3-98 bp. In a further embodiment, the double-stranded region has a length of 5-28 bp. In an even further embodiment, the double-stranded region has a length of 10-18 bp. The length of the double-stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27. 28, 29, or 30 bp. In an embodiment, the first strand is at least 1 nt longer than the second strand. In a further embodiment, the first strand is at least 1, 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nt longer than the second strand. In another embodiment, the first strand is 20- 100 nt longer than the second strand. In a further embodiment, the first strand is 2-12 nt longer than the second strand. In an even further embodiment, the first strand is 4-10 nt longer than the second strand.
In an embodiment, the first strand has a length of 5-100 nt. In a further embodiment, the first strand has a length of 5-100 nt, and the second strand has a length from 3-30 nucleotides. In an even further embodiment, the first strand has a length of 10-30 nt, and the second strand has a length from 3-18 nucleotides. In another embodiment, the first strand has a length from 12-26 nucleotides, and the second strand has a length from 10-16 nucleotides.
In an alternative embodiment, the duplex RNA molecule comprises a double-stranded region, and two 3 '-overhangs (see, e.g., FIG. 2C). The double-stranded region shares similar features as described with regard to other embodiments.
In an embodiment, the 3 '-overhang has a length of 1-10 nt. In a further embodiment, the 3 '-overhang has a length of 1-6 nt. In an even further embodiment, the 3 '-overhang has a length of 2-4 nt. In one embodiment, the 3 '-overhang has a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nt.
In an embodiment, the duplex RNA molecule comprises a double-stranded region, and two 5 '-overhangs (see, e.g., FIG. 2D).
In an embodiment, the 5 '-overhang has a length of 1-10 nt. In a further embodiment, the 5'-overhang has a length of 1-6 nt. In an even further embodiment, the 5'-overhang has a length of 2-4 nt. In one embodiment, the 3 '-overhang has a length of 1, 2, 3, 4. 5, 6, 7, 8, 9, or 10 nt.
3. The design of aiRNAs siRNAs and miRNAs are widely used as research tools, and developed as drug candidates, (see, e.g., Dykxhoorn, Novina & Sharp. Nat, Rev. MoI. Cell Biol. 4:457-467 (2003); Kim & Rossi, Nature Rev. Genet. 8:173-184 (2007); de Fougerolles, et al. Nature Rev. Drug Discov. 6:443-453 (2007); Czech, NEJM 354: 1194-1 195 (2006); and Mack, Nature Biotech. 25:631-638 (2007)). The duplex RNA molecules of the present invention, i.e., aiRNAs, can be derived from siRNAs and miRNAs known in the field. The present invention provides a method of converting an siRNA or a miRNA into an aiRNA. The conversion results in a new duplex RNA molecule that has at least one property improved in comparison to the original molecule. The property can be size, efficacy, potency, the speed of onset, durability, synthesis cost, off-target effects, interferon response, or delivery.
In an embodiment, the original molecule is a duplex RNA molecule, such as an siRNA. The duplex RNA molecule comprises an antisense strand (e.g., a guide strand) and a sense strand (e.g. a passenger strand) that form at least one double-stranded region. The method comprises changing the length of one or both strands so that the antisense strand is longer than the sense strand. In an embodiment, sense passenger strand is shortened. In another embodiment, the antisense strand is elongated. In an even further embodiment, the sense strand is shortened and the antisense strand is elongated. The antisense and sense RNA strands, intact or with changed size, can be synthesized, and then combined under conditions, wherein an aiRNA molecule is formed.
In a further embodiment, the method comprises changing the length of the antisene and/or sense strand so that the duplex RNA molecule is formed having at least one of a 3'- overhang of 1-6 nucleotides and a 5'-overhang of 1-6 nucleotides.
Alternatively, the duplex RNA molecules of the present invention can be designed de novo. A duplex RNA molecule of the present invention can be designed taking advantage of the design methods for siRNAs and miRNAs, such as the method of gene walk.
An RNA molecule of the present invention can be designed with bioinformatics approaches, and then tested in vitro and in vivo to determine its modulating efficacy against the target gene and the existence of any off-target effects. Based on these studies, the sequences of the RNA molecules can then be selected and modified to improve modulating efficacy against the target gene, and to minimize off-target effects, (see e.g., Patzel, Drug Discovery Today 12:139-148 (2007)).
3.1. Unmatched or mismatched region in the duplex RNA molecule
The two single strands of the aiRNA duplex can have at least one unmatched or imperfectly matched region containing, e.g., one or more mismatches. In one embodiment, the unmatched or imperfectly matched region is at least one end region of the RNA molecule, including an end region with a blunt end, an end region with a 3 '-recess or a 5' overhang, and an end region with a 5' recess or a 3' overhang. As used herein, the end region is a region of the RNA molecule including one end and the neighboring area.
In an embodiment, the unmatched or imperfectly matched region is in a double-stranded region of the aiRNA molecule. In a further embodiment, the asymmetric RNA duplex has an unmatched bulge or loop structure.
3.2. Sequence motifs in the duplex RNA molecule
In the design of an aiRNA molecule of the invention, the overall GC content may vary. In an embodiment, the GC content of the double-stranded region is 20-70%. In a further embodiment, the GC content of the double-stranded region is less than 50%, or preferably 30- 50%, to make it easier for strand separation as the G-C pairing is stronger than the A-U pairing.
The nucleotide sequence at a terminal overhang, in some embodiments, e.g., the 5' terminal, can be designed independently from any template sequence (e.g., a target mRNA sequence), i.e., does not have to be substantially complementary to a target mRNA (in the case of an siRNA or miRNA mimetic) or a target miRNA (in the case of miRNA inhibitor). In one embodiment, the overhang, e.g., at the 5' or the 3'. of the longer or antisense strand, is an "AA"', "UU" or "dTdT" motif, which have exhibited increased efficacy in comparison to some other motifs. In an embodiment, the 5' overhang of the longer or antisense strand has an "AA" motif. In another embodiment, the 3' overhang of the longer or antisense strand has a "UU" motif.
3.3. Nucleotide substitution
One or more of the nucleotides in the RNA molecule of the invention can be substituted with deoxynucleotides or modified nucleotides or nucleotide analogues. The substitution can take place anywhere in the RNA molecule, e.g., one or both of the overhang regions, and/or a double-stranded region. In some cases, the substitution enhances a physical property of the RNA molecule such as strand affinity, solubility and resistance to RNase degradation or enhanced stability otherwise.
In one embodiment, the modified nucleotide or analogue is a sugar-, backbone-, and/or base- modified ribonucleotide. The backbone-modified ribonucleotide may have a modification in a phosphodiester linkage with another ribonucleotide. In an embodiment, the phosphodiester linkage in the RNA molecule is modified to include at least a nitrogen and/or sulphur heteroatom. In an embodiment, the modified nucleotide or analogue is an unusual base or a modified base. In an embodiment, the modified nucleotide or analogue is inosine, or a tritylated base.
In a further embodiment, the nucleotide analogue is a sugar-modified ribonucleotide in which the 2'-OH group is replaced by a group selected from the group consisting of H, OR, R, halo, SH, SR, NH2, NHR, NR2, and CN, wherein each R is independently selected from the group consisting of C1-C6 alkyl, alkenyl and alkynyl, and halo is selected from the group consisting of F, Cl, Br and I.
In one embodiment, the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
4. The utilities
The present invention also provides a method of modulating gene expression in a cell or an organism (silencing method). The method comprises the steps of contacting said cell or organism with the duplex RNA molecule under conditions wherein selective gene silencing can occur, and mediating a selective gene silencing effected by the said duplex RNA molecule towards a target nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA.
In an embodiment, the contacting step comprises the step of introducing said duplex RNA molecule into a target cell in culture or in an organism in which the selective gene silencing can occur. In a further embodiment, the introducing step comprises transfection, lipofection, infection, electroporation, or other delivery technologies.
In an embodiment, the silencing method is used for determining the function or utility of a gene in a cell or an organism.
The silencing method can be used for modulating the expression of a gene in a cell or an organism. In an embodiment, the gene is associated with a disease, e.g., a human disease or an animal disease, a pathological condition, or an undesirable condition. In a further embodiment, the gene is a gene of a pathogenic microorganism. In an even further embodiment, the gene is a viral gene. In another embodiment, the gene is a tumor-associated gene.
In an alternative embodiment, the gene is a gene associated with autoimmune disease, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermato logical diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging. 4.1. Research tools
The RNA molecules of the present invention can be used to create gene "knockdown" in animal models as opposed to genetically engineered knockout models to discover gene functions. The methods can also be used to silence genes in vitro. For example, aiRNA can be transfected to cells. AiRNA can be used to 1 as a research tool in drug target/pathway identification and validation, and other biomedical research in drug research and development.
4.2 Therapeutic uses
The RNA molecules of the present invention can be used for the treatment and or prevention of various diseases or undesirable conditions, including the diseases summarized (Czech, 2006; de Fougerolles et al., 2007; Dykxhoorn et al., 2003; Kim and Rossi, 2007; Mack, 2007).
In an embodiment, the present invention can be used as a cancer therapy or to prevent cancer. The RNA molecules of the present invention can be used to silence or knock down genes involved with cell proliferation or other cancer phenolypes. Examples of these genes are k-Ras, β-catenin, Nbsl, EF2, Stat 3, PTEN, p70S6K, mTOR, Rskl, PCNA, Parpl, Survivin, NQOl, and p21. Specifically, k-Ras and β-catenin are therapeutic genes of colon cancer. These oncogenes are active and relevant in the majority of clinical cases.
These RNA molecules can also be used to silence or knockdown non-cancer gene targets. The RNA molecules of the invention can also be used to treat or prevent ocular diseases, {e.g., age-related macular degeneration (AMD) and diabetic retinopathy (DR)); infectious diseases (e.g. HIV/AIDS, hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (I IPV), herpes simplex virus (HSV), RCV, cytomegalovirus (CMV), dengue lever, west Nile virus); respiratory diseases {e.g., respirator}' syncytial virus (RSV), asthma, cystic fibrosis); neurological diseases (e.g., Huntingdon's disease (HD), amyotrophic lateral sclerosis (ALS), spinal cord injury, Parkinson's disease, Alzheimer's disease, pain); cardiovascular diseases; metabolic disorders (e.g., diabetes); genetic disorders; and inflammatory conditions (e g., inflammatory bowel disease (IBD). arthritis, rheumatoid disease, autoimmune disorders), dcrmatological diseases.
Various genes can be silenced using the asymmetrical duplex RNA molecule of the present invention. In an embodiment, the first strand comprises a sequence being substantially complimentary to the target mRNA sequence of a gene selected from the group consisting of a developmental gene, an oncogene, a tumor suppresser gene, and an enzyme gene, and a gene for an adhesion molecule, a cyclin kinase inhibitor, a Wnt family member, a Pax family member, a Winged helix family member, a Hox family member, a cytokine/lymphokine or its receptor, a growth/differentiation factor or its receptor, a neurotransmitter or its receptor, ABLl, BCLl, BCL2, BCL6, CBFA2, CBL, CSFlR, ERBA, ERBB, EBRB2,ETS1 , ETSl, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC,M YCLl , MYCN. NRAS, PIMl, PML, RET, SRC, TALI, TCL3 and YES) (e. g., APC, BRCAl , BRCA2, MADH4, MCC,NFl, NF2,RB1, TP53. WTl , an ACP dcsaturasc or hydroxylase, an ADP- glucosc pyrophorylase, an ATPase, an alcohol dehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase, a decarboxylase, a dextrinase, a DNA or RNA polymerase, a galactosidase, a glucanase, a glucose oxidase, a GTPase, a hclicase, a hemicellulase, an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase, a lipoxygenase, a lysozyme, a pectinesterase, a peroxidase, a phosphatase, a phospholipase, a phosphorylase, a polygalacturonase, a proteinase or peptideases, a pullanase, a recombinase, a reverse transcriptase, a topoisomerase, a xylanase, k-RAS, β-Catcnin, Rskl, PCNA, p70S6K, Survivin, mTOR, PTEN, Parpl. or p21.
The present invention provides a method to treat a disease or undesirable condition. The method comprises using the asymmetrical duplex RNA molecule to effect gene silencing of a gene associated with the disease or undesirable condition.
4.3. Converting the RNA molecules (aiRNA) into drugs 4.3.1. Modifications of the RNA molecules
Naked RNA molecules are relatively unstable and can be degraded in vivo relatively quickly. Chemical modifications can be introduced to the ItNA molecules of the present invention to improve their half-life and to further reduce the risk of non-specific effects of gene targeting, without reducing their biological activities.
The modifications of RNA molecules have been investigated to improve the stability of various RNA molecules, including antisense RNA, ribozyme, aplamer, and RNAi (Chiu and Rana, 2003; Czauderna et al., 2003; de Fougerolles et al, 2007; Kim and Rossi, 2007; Mack, 2007; Zhang et al., 2006; and Schmidt Nature Biotech. 25:273-275 (2007))
Any stabilizing modification known to a skill in the art can be used to improve the stability of the RNA molecules of the present invention. Within the RNA molecules of the present invention, chemical modifications can be introduced to the phosphate backbone (e.g., phosphorothioate linkages), the ribose (e.g., locked nucleic acids, 2"-dcoxy-2'-fluorouridine, T- O-mthyl), and/or the base (e.g., 2'-fluoropyrimidines). Several examples of such chemical modifications are summarized in the following.
Chemical modifications at the 2' position of the ribose, such as 2'-O-mthylpurines and 2'-fluoropyrimidines, which increase resistance to endonuclease activity in serum, can be adopted to the stabilize the RNA molecules of the present invention. The position for the introduction of the modification should be carefully selected to avoid significantly reducing the silencing potency of the RNA molecule. For example, the modifications on 5' end of the guide strand can reduce the silencing activity. On the other hand, 2'-O-methyl modifications can be staggered between the two RNA strands at the double-stranded region to improve the stability while reserving the gene silencing potency. The 2'-O-methyl modifications can also eliminate or reduce the interferon induction.
Another stabilizing modification is phosphorothioate (P=S) linkage. The introduction of phosphorothioate (P=S) linkage into the RNA molecules, e.g., at the 3 "-overhang, can provide protection against exonuclease.
The introduction of deoxyribonucleotidcs into the RNA molecules can also reduce the manufacture cost, and increase stability.
In an embodiment, the 3'-overhang, 5'-overhang, or both are stabilized against degradation.
In an embodiment, the RNA molecule contains at least one modified nucleotide or its analogue. In a further embodiment, the modified ribonucleotide is modified at its sugar, backbone, base, or any combination of the three.
In an embodiment, the nucleotide analogue is a sugar-modified ribonucleotide. In a further embodiment, the 2'-OH group of the nucleotide analogue is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently Cl -C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.
In an alternative embodiment, the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
In an embodiment, the duplex RNA molecule contains at least one deoxynucleotide. In a further embodiment, the first strand comprises 1-6 deoxynucleotides. In an even further embodiment, the first strand comprises 1-3 deoxynucleotides. In another embodiment, the 3'- overhang comprises 1-3 deoxynucleotides. In a further embodiment, the 5 '-overhang comprises 1-3 deoxynucleotides. In an alternative embodiment, the second strand comprises 1-5 deoxynucleotides.
In an embodiment, the duplex RNA molecule comprises a 3 '-overhang or 5'-overhang that contains at least one deoxynucleotide. In another embodiment, the RNA the 3 '-overhang and/or 5'-overhang consists of deoxynucleotides.
In an embodiment, the duplex RNA molecule is conjugated to an entity In a further embodiment, the entity is selected from the group consisting of peptide, antibody, polymer, lipid, oligonucleotide, and aptamer.
In another embodiment, the first strand and the second strand arc joined by a chemical linker. 4.4, In vivo delivery of the RNA molecules
One major obstacle for the therapeutic use of RNAi is the deliver}' of siRNA to the target cell (Zamore and Aronin, 2003). Various approaches have been developed for the delivery of RNA molecules, especially siRNA molecules (de Fougerolles et al., 2007; Dykxhoorn et al., 2003; Kim and Rossi, 2007). Any delivery approach known to a skill of the art can be used for the delivery of the RNA molecules of the present invention.
Major issues in deliver}' include instability in serum, non-specific distribution, tissue barriers, and non-specific interferon response (Lu & Woodle, Methods in MoI Biology 437: 93- 107 (2008)). Compared to their siRNA and miRNA counterparts, aiRNA molecules possess several advantages that should make a wider ranger of methods available for delivery purpose. First, aiRNAs can be designed to be smaller than their siRNA and miRNA counterparts, therefore, reducing or eliminating any interferon responses. Second, aiRNAs are more potent, faster-onsetting, more efficacious and lasts longer, therefore, less amount/dosage of aiRNAs is required to achieve a therapeutic goal. Third, aiRNA are double stranded and more stable than single-stranded antisensc oligos and mi RNAs, and they can be further modified chemically to enhance stability. Therefore, the RNA molecules of the invention can be delivered into a subject via a variety of systemic or local delivery routes. In some embodiments, molecules of the invention are delivered through systemic delivery routes include intra-venous (LV.) and intraperitoneal (ip). In other embodiments, molecules of the invention are delivered through local delivery routes, e.g., intra-nasal, intra- vitreous, intra- tracheal, intra-cerebral, intra-muscle, intraarticular, and intra-tumor.
Examples of the delivery technologies include direct injection of naked RNA molecules, conjugation of the RNA molecules to a natural ligand such as cholesterol, or an aptamer, liposome-formulated delivery, and non-covalently binding to antibody-protaminc fusion proteins. Other carrier choices include positive charged carriers (e.g., cationic lipids and polymers) and various protein carriers. In one embodiment, the delivery of the molecules of the invention uses a ligand-targeted delivery system based on the cationic liposome complex or polymer complex systems (Woodle, et al. J Control Release IA: 309-31 1 ; Song, et al. Nat Biotechnol. 23(6): 709-717 (2005); Morrissey et al. Nat Biotechnol 23(8): 1002-1007 (2005)).
In one embodiment, molecules of the invention is formulated with a collagen carrier, e.g., atelocollagen, for in vivo delivery. Atelocollagen has been reported to protect siRNA from being digested by RNase and to enable sustained release (Minakuchi, et al. Nucleic Acids Res. 32: el 09 (2004); Takei et al. Cancer Res. 64: 3365-3370 (2004)). In another embodiment, molecules of the invention are formulated with nanoparticles or form a nanoemulsion. e.g., RGD peptide ligand targeted nanoparticles. It has been shown that different siRNA oligos can be combined in the same RGD ligand targeted nanoparticle to target several genes at the same time (Woodle et al. Materials Today 8 (suppl 1): 34-41 (2005)).
Viral vectors can also be used for the delivery of the RNA molecules of the present invention. In an embodiment, lentiviral vectors are used to deliver the RNA molecule transgenes that integrate into the genome for stable expression, In another embodiment, adenoviral and adeno-associatcd virus (AAV) are used to deliver the RNA molecule transgenes that do not integrate into the genome and have episomal expression.
Moreover, bacteria can be used for the delivery of the RNA molecules of the present invention (Xiang et al., 2006).
5. The Pharmaceutical Compositions and Formulations
The present invention further provided a pharmaceutical composition. The pharmaceutical comprises as an active agent al least one asymmetrical duplex RNA molecule and one or more carriers selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid. In an embodiment, the composition is for diagnostic applications, or for therapeutic applications.
The pharmaceutical compositions and formulations of the present invention can be the same or similar to the pharmaceutical compositions and formulations developed for siRNA, miRNA, and antisense RNA (de Fougerolles et al., 2007; Kim and Rossi, 2007), except for the RNA ingredient. The siRNA, miRNA, and antisense RNA in the pharmaceutical compositions and formulations can be replaced by the duplex RNA molecules of the present information. The pharmaceutical compositions and formulations can also be further modified to accommodate the duplex RNA molecules of the present information.
A "pharmaceutically acceptable salt" or "salt" of the disclosed duplex RNA molecule is a product of the disclosed duplex RNA molecule that contains an ionic bond, and is typically produced by reacting the disclosed duplex RNA molecule with either an acid or a base, suitable for administering to a subject. Pharmaceutically acceptable salt can include, but is not limited to, acid addition salts including hydrochlorides, hydrobromides, phosphates, sulphates, hydrogen sulphates, alkylsulphonates, arylsulphonates, acetates, benzoates. citrates, maleales, fumarates, succinates, lactates, and tartrates; alkali metal cations such as Na, K, Li, alkali earth metal salts such as Mg or Ca, or organic amine salts.
A "pharmaceutical composition" is a formulation containing the disclosed duplex RNA molecules in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or a vial. The quantity of active ingredient (e.g. , a formulation of the disclosed duplex RNA molecule or salts thereof) in a unit dose of composition is an effecthe amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for the topical or transdermal administration of a duplex RNA molecule of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. In one embodiment, the active duplex RNA molecule is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.
The present invention provides a method of treatment comprising administering an effective amount of the pharmaceutical composition to a subject in need, In an embodiment, the pharmaceutical composition is administered via a route selected from the group consisting of iv, sc, topical, po, and ip. In another embodiment, the effective amount is 1 ng to 1 g per day, 100 ng tol g per day, or 1 μg tol mg per day.
The present invention also provides pharmaceutical formulations comprising a duplex RNA molecule of the present invention in combination with at least one pharmaceutically acceptable excipient or carrier. As used herein, "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in "Remington: The Science and Practice of Pharmacy, Twentieth Edition," Lippincott Williams & Wilkins, Philadelphia, PA., which is incorporated herein by reference. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active duplex RNA molecule, use thereof in the compositions is contemplated. Supplementary active duplex RNA molecules can also be incorporated into the compositions.
Methods for formulation are disclosed in PCT International Application PCT/US02/24262 (WO03/01 1224), U.S. Patent Application Publication No. 2003/0091639 and U.S. Patent Application Publication No. 2004/0071775, each of which is incorporated by- reference herein. A duplex RNA molecule of the present invention is administered in a suitable dosage form prepared by combining a therapeutically effective amount (e.g., an efficacious level sufficient to achieve the desired therapeutic effect through inhibition of tumor growth, killing of tumor cells, treatment or prevention of cell proliferative disorders, etc.) of a duplex RNA molecule of the present invention (as an active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the invention). These procedures may involve mixing, granulating, and compressing or dissolving the ingredients as appropriate to attain the desired preparation. In another embodiment, a therapeutically effective amount of a duplex RNA molecule of the present invention is administered in a suitable dosage form without standard pharmaceutical carriers or diluents.
Pharmaceutically acceptable carriers include solid carriers such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary liquid carriers include syrup, peanut oil, olive oil, water and the like. Similarly, the carrier or diluent may include time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, eihylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like. Other fillers, excipients, flavorants, and other additives such as are known in the art may also be included in a pharmaceutical composition according to this invention.
The pharmaceutical compositions containing active duplex RNA molecules of the present invention may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and/or auxiliaries which facilitate processing of the active duplex RNA molecules into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.
A duplex RNA molecule or pharmaceutical composition of the invention can be administered to a subject in many of the well-known methods currently used for chemotherapeutic treatment. For example, for treatment of cancers, a duplex RNA molecule of the invention may be injected directly into tumors, injected into the blood stream or body cavities or taken orally or applied through the skin with patches. For treatment of psoriatic conditions, systemic administration (e.g., oral administration), or topical administration to affected areas of the skin, are preferred routes of administration. The dose chosen should be sufficient to constitute effective treatment but not as high as to cause unacceptable side effects. The state of the disease condition (e.g., cancer, psoriasis, and the like) and the health of the patient should be closely monitored during and for a reasonable period after treatment.
EXAMPLES
Examples are provided below to further illustrate different features of the present invention. The examples also illustrate useful methodology for practicing the invention. These examples do not limit the claimed invention. RNA interference (RNAi) is a catalytic mechanism of gene-specific silencing in eukaryotic organisms with profound implications for biology and medicine (Fire et al., 1998), 12. RNAi is mediated by the RNA-induced silencing complex (RISC) (Hammond et al., 2000; Martinez and Tuschl, 2004; Rana, 2007) upon incorporating with small interfering RNAs (siRNA) of 19-21 base pairs (bp) with 3' overhangs, the smallest RNA duplex known to enter RISC and mediate RNAi (Elbashir et al., 2001a; Elbashir et al., 2001b; Elbashir et al., 2001c; Fire et al., 1998; Zamore et al., 2000). As the natural substrate of the RISC enzyme complex, siRNA can be chemically synthesized or generated through Dicer-catalyzed processing of its various precursors (Donze and Picard, 2002; Hammond et al., 2000; Kim et al., 2005; Paddison et al., 2002). While being used widely for gene silencing, siRNA has limited efficiency in gene silencing with low silencing efficacy for numerous genes in mammalian cells (de Fougerolles et al., 2007; Iorns et al., 2007). Here we investigate structural scaffold requirements for an efficient RNAi mediator in mammalian cells. To our surprise, we found that asymmetric RNA duplexes of 14-15 bp with dual antisense overhangs mediate potent and efficacious gene silencing in mammalian cells. The asymmetric interfering RNA (aiRNA), structurally characterized by duplex RNA of 14-15 bp with 3'and 5' antisense overhangs, was incorporated into RISC with higher efficiency than siRNA. The aiRNA caused sequence-specific cleavage of the mRNA and targeted gene silencing in mammalian cells. When the identical sequence of β-catenin mRNA was targeted, the aiRNA was more efficacious (near 100%), potent (picoM), rapid-onset (less than 24 h) and durable (up to 1 week) than siRNA in mediating gene silencing in vitro. These results suggest aiRNA as the smallest RNA duplex scaffold incorporated into RISC and non- siRNA type of RNAi mediators that silence genes with better efficiency than siRNA in mammalian cells. Therefore, aiRNA may have significant potential for broad RKAi application.
Methods and Materials
Cell culture and Reagents
HeIa, SW480, DLDl, HT29, and H1299 cells were obtained from ATCC, and cultured in
Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Invitrogen). Fresh peripheral blood mononuclear cells (PBMC) were obtained from AllCells LLC and maintained in RPMI-1640 medium containing 10% FBS and pen/strep (Invitrogen). Small RNAs described in this study were synthesized by Dharmacon, Qiagen, or Integrated DNA technologies (Table 2) and annealed following the manufacturer's instructions (Figure 3a). siRNAs targeting human Ago2, and Dicer (Ambion) were used at 100 nM. Transfections of the RNAs were performed using DharmaFECTl (Dharmacon) at the indicated concentrations. Human Argonaute2 (Ago2) expression vector (OriGene) was transfccted using Lipofectamine 2000 (Invitrogen). Serum stability was determined by incubation of aiRNA or siRNA duplex with 10% human serum (Sigma) for the indicated amount of time followed by non-denaturing TBE-acrylamide gel electrophoresis and ethidium bromide staining.
Northern blot analysis.
To determine levels of β-catenin, total RNA was extracted with TRIZOL (Invitrogen) from siRNA or aiRNA transfected HeIa cells at various time points. 20 μg of total cellular RNA was loaded to each lane of a denaturing agarose gel. After electrophoresis, RNA was transferred to Hybond-XL Nylon membrane (Amersham Bioscicnccs), UV crosslinked, and baked at 800C for 30 min. Probes detecting β-catenin and actin mRNA was prepared using Prime-It II Random Primer Labeling Kit (Stratagene) from β-catenin cDNA fragment (1-568 nt) and actin cDNA fragment (1-500 nt). To analyze small RNA RISC loading, siRNA or aiRNA were transfected into HeIa cells 48 hours after transfection with pCMV-Ago2. Cells were lysed at the indicated timepoints and immunoprecipitated with Ago2 antibody. Immunoprecipitates were washed, RNA isolated from the complex by TRIZOL extraction, and loaded on a 15% TBE-Urea PAGE gel (Bio-Rad). Following electrophoreses, RNA was transferred to Hybond-XL Nylon membrane. mirVana miRNA Probe Kit (Ambion) was used to generate 5' 32P labeled RNA probes. Antisense probe (5'-GUAGCUGAUAUUGAUGGACUU-3'). Sense probe (5'- UCCAUCAAU AUCAGC-3')
In vitro Ago2-RISC loading. aaiiRRNNAA oorr ssiiRRNNAA sseennsse and anti-sense strands were 32P end labeled using T4 kinase (Promega). End labeled RNAs were purified by phenol/chloroform/isoamyl alcohol, precipitated with EtOH, and resuspended in water. Labeled RNAs were then annealed to siRNA or aiRNA anti-sense strands as described. For in vitro lysates, HcIa cells were transfected for 24 hours with human Ago2 expression vector, and SlO lysates generated essentially as described (Dignam et al., 1983). 5' sense strand or anti-sense strand labeled duplex aiRNA or siRNA was then added to the Ago2-S10 lysate. Following a 5 min incubation at 37°C, Ago2 was immunoprecipited as described, and Ago2-associated (pellet) and non-Ago2 associated (supernatant) fractions were separated on a 20% TBE-acrylamide gel and gel exposed to film to detect sense strand-Ago2 association.For aiRNA and siRNA competition experiments, up to 100 folds cold aiRNA and siRNA were used to compete with 32P labeled aiRNA or siRNA to load to RISC. Briefly, SlO lysates were generated from HeIa cells transfected with Ago2 expression vector as described. Labeled aiRNA or siRNA was then added to the SlO lysates followed immediately by addition of unlabeled aiRNA or siRNA. Reaction was incubated for 5 min at 37°C and processed as described above.
qRT-PCR.
Cells transfected with the indicated aiRNA or siRNA were harvested at the indicated time points following transfection. RNA was isolated with TRIZOL, and qRT-PCR performed using TaqMan one-step RT-PCR reagents and primer probe sets for the indicated mRNA (Applied Biosystems). Data is presented relative to control transfected cells and each sample is normalized to aclin mRNA levels. For the experiment in Figure 14d, Stat3 constructs were created by cloning Stat3 cDNA (Origene) into either pcDNA3.1+ or pcDNA3.1- at the Hindlll-Xhol sites. Stat3 forward or reverse expression vectors were then co-transfected into HeIa cells with aiStat3 or siStat3 for 24 hours. Cells were then harvested, RNA isolated by TRTZOL, and qRT-PCR performed using TaqMan one-step RT-PCR reagents and primer probe sets for Stat3 or actin (Applied Biosystems). RT-PCR was performed on the same RNA samples using Superscript One-Step RT-PCR kit (Invitrogen) and Stat3 forward (5'- GGATCTAGAATCAGCTACAGCAGC-3') and Stat3 reverse (5'- TCCTCTAGAGGGCAATCTCCATTG-3') primers and actin forward (5'- CCATGGATGATGATATCGCC-3') and actin reverse (5 '-TAGAAGCATTTGCGGTGGAC-3 ') primers. RT-PCR.
Total RNA was prepared using the TRIZOL, and cDNA was synthesized using random primers with Thermoscript RT-PCR System (Invitrogen). PCR was run for 20 cycles using Pfx polymerase. Primers: ACTIN-I, 5' CCATGGATGATGATATCGCC-3'; ACTIN-2, 5'- TAGAAGCATTTGCGGTGGAC- 3'; β-catenin -/, 5'-GACAATGGCTACTCAAGCTG-3'; β- catenin -2, 5'-CAGGTCAGTATCAAACCAGG-3'.
Western blot.
Cells were washed twice with ice-cold phosphate-buffered saline and lysed in lysis buffer
(50 mM HEPES, pH 7.5, 0.5% NonidetP-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 niM sodium ortho vanadate, 1 mM dithiothreitol, 1 mM NaF, 2 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of pepstatin, leupeptin, and aprotinin). 20 μg of soluble protein was separated by SDS-PAGE and transferred to PVDF membranes. Primary Antibodies against β-catenin, Nbsl, Survivin, p21, Rskl, k-Ras, Stat3, PCNA, NQOl. Actin (Santa Cruz), EF2, p70S6K, mTOR, PTEN (Cell Signaling Technology), Ago2 (Wako), Dicer (Novus), and Parpl (EMD Biosciences) were used in this study. The antigen-antibody complexes were visualized by enhanced chemiluminescence (GE Biosciences).
5' -RACE analysis
Total RNA (5 μg) from HeIa cells treated with non-silencing aiRNA or aiRNA was ligated to GeneRacer™ RNA adaptor (Invitrogen, 5"-
CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3') without any prior processing. Ligated RNA was reverse transcribed into cDNA using a random primer. To detect cleavage product, PCR was performed using primers complementary to the RNA adaptor (GeneRacer™ 5' Nested Primer: 5'-GGACACTGACATGGACTGAAGGAGTA-S ') and β- catenin specific primer (GSP: 5'- CGCATGATAGCGTGTCTGGAAGCTT-3'). Amplification fragments were resolved on 1.4% agarose gel and sized using a 1-kb Plus DNA Ladder (Invitrogen). Specific cleavage site was further confirmed by DNA sequencing.
Interferon-response detection.
For the experiment in Figure 15a. PBMC were incubated directly with 100 nM β-catenin siRNA or aiRNA. Total RNA was purified at 16 hours using TRIZOL, and levels of interferon responsive gene expression were determined by RT-PCR as described by the manufacturer (System Biosciences). For the experiment in Figure 15b, HeIa cells were mock transfected or transfected with 100 iiM of the indicated aiRNA or siRNA for 24 hours. Total RNA was purified using TRlZOL and levels of interferon responsive gene expression were determined by RT-PCR. For microarray analysis, HeIa cells were transfected with 100 nM aiRNA or siRNA. Total RNA was purified at 24 hours using TRIZOL, and RNA was used for hybridization to Human Genome Ul 33 Plus 2.0 GeneChip (Affymetrix) according to the manufacturer's protocol (ExpressionAnalysis, Inc.). RNA from DharmaFECT 1 treated cells was used as control. To calculate transcript expression values, Microarray Suite 5.0 was used with quantile normalization, and transcripts with sufficient hybridization signals to be called present (P) were used in this study.
aiRNA and siRNA sequences
Sequence and structure of aiRNA and siRNA duplexes were listed in Table 2. Location of point mutation is framed in the k-Ras aiRNA.
Figure imgf000047_0001
In Life Evaluations
Daily examinations into the health status of each animal were also conducted. Body weights were checked every three days. Food and water was supplied daily according to the animal husbandry procedures of the facility. Treatment producing >20% lethality and or >20% net body weight loss were considered toxic. Results are expressed as mean tumor volume (mm3) ± SE. P Values < 0.05 are considered to be statistically relevant.
Animal Husbandry
Male or female athymic nude mice 4-5 weeks (Charles River Laboratories, Wilmington, MA.), were acclimated to the animal housing facility for at least 1 week before study initiation. All of the experimental procedures utilized were consistent with the guidelines outlined by the American Physiology Society and the Guide for the Care and Use of Laboratory Animals and were also approved by the Institutional Animal Care and Use Committee of Boston Biomedical Inc. The animals were housed in groups of four in wood chip bedded cages in a room having controlled temperature (68°F-72°F), light (12-h light-dark cycle), and humidity (45-55%). The animals were allowed free access to water and food during the experiment.
Example 1. Asymmetric interfering RNA (aiRNA) causes genc-spccific silencing in mammalian cells
The siRNA structural scaffold is considered the essential configuration for incorporating into RISC and mediating RNAi(Elbashir et al, 2001a; Elbashir ct al., 2001b; Elbashir et al., 2001c; Rana, 2007; Zamore et al., 2000). However, very little is known about RNA duplex scaffold requirements for RISC incorporation and gene silencing. To investigate the structural scaffold requirements for an efficient RNAi mediator and RISC substrate, we first determined if RNA duplexes shorter than siRNAs could mediate gene silencing. The length of double stranded (ds) RNA is an important determinant of its propensity in activating protein kinase R (PKR)- mediated non-specific interferon responses, increased synthesis cost, and delivery challenges (Elbashir et al., 2001b; Sledz et al., 2003). We designed a series of short dsRNAs ranging from 12 to 21 bp with 2 nucleotide 3' overhangs or blunt ends targeting different mammalian genes. No gene silencing was detected after the length was reduced below 19 bp (data not shown), which is consistent with previous reports in Drυsophila Melanogaster cell lysate(Elbashir et al., 2001b) and the notion that 19-21bp is the shortest siRNA duplex that mediates RNAi (Elbashir et al., 2001a; Elbashir et al., 2001b; Elbashir et al., 2001c; Rana, 2007; Zamore et al., 2000).
We next tested if RNA duplexes of non-siRNA scaffold with an asymmetric configuration of overhangs can mediate gene silencing. The siRNA duplex contains a symmetrical sense strand and an antisensc strand. While the duplex siRNA structure containing a 3' overhang is required for incorporation into the RISC complex, following Argonaute (Ago) mediated cleavage of the sense strand, the antisense strand directs cleavage of the target mRNA (Hammond et al., 2001 ; Matranga et al., 2005; Tabara et al., 1999). We sought to make asymmetric RNA duplexes of various lengths with overhangs at the 3' and 5' ends of the antisense strand.
Oligos with sequences shown in Table 3 were confirmed by 20% polyacrylamide gel after annealing. As shown in Fig. 3A, each lane was loaded as follows: lane 1« 21nt/21nt; lane 2, 12nt (a)/21nt; lane 3, 12nt (b)/21nt; lane 4, 13nt/13nt; lane 5, 13nt/21nt; lane 6, 14nt /14nt; lane 7, 14nt(a)/21nt; lane 8, 14nt(b)/21 nt: lane 9, 15nt/15nt; lane 10, 15nt/21 nt.
Table 3
Figure imgf000048_0001
Figure imgf000049_0001
HeLa cells were plated at 200,000 cells/well into a 6 well culture plate. As shown in Fig. 3B, 24 hours later they were transfected with scramble siRNA (lane 1), 21 -bp siRNA targeted E2F1 (lane 2, as a control for specificity) or 21-bp siRNA targeted beta-catenin (lane 3, as a positive control), or the same concentration of aiRNA of different length mix: 12nt(a)/21nt (lane 4); 12nt (b)/21nt (lane 5); 13nt/21nt (lane 6); 14nt (a)/21nt (lane 7); 14nt (b)/21nt (lane 8); 15nt/21nt (lane 9). Cells were harvested 48 hours after transfection, Expression of β-catenin was determined by Western blot. E2F1 and actin are used as controls. The results demonstrate that asymmetric interfering RNA (aiRNA) causes gene-specific silencing in mammalian cells.
In order to determine the structural features of aiRNA important in aiRNA function, we generated multiple aiRNA oligonucleotides based on modification of the core 15/21 dual anti- sense overhang structure (Table 4). The aiRNAs, summarized in Table 4, contained modifications including, but not limited to, length of the sense and anti-sense strands, degree of sense and anti- sense overhangs, and RNA-DNA hybrid oligonucleotides.
Modification to the parental 15/21 aiRNA structure was done by altering the sense strand, anti-sense strand, or both (Table 4). Modified aiRNA duplexes were transfected into HeIa cells at 50 nM for 48 hours. Western blots for β-catenin and actin were used to examine the degree of gene silencing compared to the parental 15/21 aiRNA and to the traditional siRNA structure. aiRNA modifications were also tested which contained dual sense strand overhangs. These oligonucleotides contain a 21 base sense strand paired to differing length anti-sense strands. In addition, we also examined the activity of aiRNA oligonucleotides that have been modified with DNA bases. DNA substitutions were done on both the anti-sense and sense strands (Table 3). RNA-DNA hybrid oligonucleotides tested contained 1 or more DNA substitutions in either the sense or anti-sense strand, or contained 21 base anti-sense RNA paired with indicated length of DNA sense strand. The gene silencing results of these various aiRNAs were shown in Figures 4 and 5.
Taken together, these data provide structural clues to aiRNA function.
Regarding the sense strand, our data indicate that the length of 15 bases works well, while lengths between 14 and 19 bases remain functional. The sense strand can match any part of the anti-sense strand, provided that the anti-sense overhang rules are met. Replacement of a single RNA base with DNA at either the 5' or 3' end of the sense strand is tolerated and may even provide increased activity.
With respect to the anti-sense strand length, the length of 21 bases works well, 19-22 bases retains activity, and activity is decreased when the length falls below 19 bases or increases above 22 bases. The 3' end of the anti-sense strand requires an overhang of 1-5 bases with a 2-3 base overhang being preferred, blunt end shows a decrease in activity. Base pairing with the target RNA sequence is preferred, and DNA base replacement up to 3 bases is tolerated without concurrent 5' DNA base replacement. The 5' end of the anti-sense strand prefers a 0-4 base overhang, and does not require an overhang to remain active. The 5' end of the anti-sense strand can tolerate 2 bases not matching the target RNA sequence, and can tolerate DNA base replacement up to 3 bases without concurrent 3' DNA base replacement.
With respect to mismatched or chemically modified bases, we find that both mismatches and one or more chemically modified bases in either the sense or anti-sense strand is tolerated by the aiRNA structure.
Table 4: aiRNA sequences used for figures 4-5
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
In table 4, A, U, G, C represent nucleotides, while a, t, g, c represent deoxynucleotides.
Example 2, Mechanism of gene silencing triggered by aiRNA
To investigated the mechanism of gene knockdown induced by aiRNA, we first determined if the gene silencing by aiRNA occurs at translational or mRNA level. Northern blot analysis of β-catenin in cells transfected with 10 nM of the 15 bp aiRNA showed that the aiRNA reduced mRNA levels by over 95% within 24 hours and the decrease lasted more than 4 days (Fig. 6a), suggesting that aiRNA mediates gene silencing at the mRNA level. The reduction of β-catenin mRNA induced by aiRNA was substantially more rapid, efficacious and durable than by siRNA (Fig. 6a). We further determined if the 15 bp aiRNA catalyzed the site-specific cleavage of β- calenin mRNA. Total RNA isolated from cells transfected with the 15 bp aiRNA was examined by rapid amplification of cDNA ends (5'-RACE) and PCR for the presence of the β-catenin mRNA cleavage fragments (Fig. 6b). We detected β-catenin cleavage fragments at 4 and 8 hours following aiRNA transfection (Fig. 6c). Sequence analysis showed that cleavage was taking place within the aiRNA target sequence between bases 10 and 1 1 relative to the 5' end of the aiRNA antisense strand (Fig. 6d). No such cleavage fragments were observed following transfection with a scrambled aiRNA (Fig. 6c). These results demonstrate that aiRNA induced potent and efficacious gene silencing through sequence-specific cleavage of the target mRNA.
We next determined whether the novel asymmetric scaffold of aiRNA can be incorporated into the RISC. RNAi is catalyzed by RISC enzyme complex with an Argonaute protein (Ago) as the catalytic unit of the complex (Liu et al., 2004; Matranga et al, 2005). To determine if aiRNA is incorporated into the Ago/RISC complex, we immunoprecipitated myc-taggcd human Agol from cells expressing myc-tagged Agol (Siolas et al., 2005) after cells were transfected with aiRNA. Small RNAs associated with the RISC complex were detected by northern blotting of Ago immunoprecipitates. Northern blot analysis revealed that the aiRNA entered the RISC complex with high efficiency (Fig. 6e). These data suggest the asymmetric scaffold of aiRNA can be efficiently incorporated into RISC.
Since aiRNA induced more efficient gene silencing than siRNA, we tested if aiRNA can give rise to RISC complex more efficiently than siRNA. As shown in Fig. 6e, aiRNA- Ago2/RISC complexes formed faster and more efficient than the siRNA-Ago2/RISC complexes, with more aiRNA contained in the RISC complex than the corresponding siRNA (Fig. 6e and Fig. 7A). Of note, siRNA displayed a typical pattern (21) that is consistent with formation of secondary structures by siRNA (Fig. 6e and Fig. 7). In contrast, aiRNA displayed a single band, suggesting that the shorter length of aiRNA may reduce or eliminate the secondary structure formation as occurred with siRNA.
Further, the asymmetric configuration of aiRNA may facilitate the formation of active RISC with antisense strand and reduce the ineffective RISC formed with the sense strand (Ref. 16). Our data proved this is true as shown in Fig. 7B, no sense strand can be detected in the RISC complex. Fig. 8A also demonstrates that while the anti-scnsc strand of the aiRNA strongly associates with Ago 2, the sense-strand does not. In contrast, both the anti-sense and sense strand of the siRNA associate with Ago 2. These data suggest that aiRNA has higher efficiency in forming RISC than siRNA in cells, which may underlie the superior gene silencing efficiency of aiRNA.
In addition, it has been shown that the sense strand of siRNA is required to be cleaved in order to be functional. Therefore, we tested if the same requirement is true for aiRNA. To do that, the nucleotide at position 8 or 9 of the aiRNA sense strand was modified with 2'-O-methyl to make it uncleavable. Our results show that the aiRNAs with the uncleavable sense strand are still functional (Fig. 8B), demonstrating aiRNA is quite different than siRNA in terms of their mechanism. Further we asked if there is any different loading pocket for aiRNA and siRNA. We used cold aiRNA or siRNA to compete with the radioactively labelled siRNA or aiRNA for the RISC complex (Fig. 9). Surprisingly, the results show that cold aiRNA does not compete with the siRNA for RISC complex (Fig. 9B) and cold siRNA does not compete with aiRNA for the RISC complex either (Fig. 9C). These data indicate that aiRNA and siRNA may load to different pockets of RISC complex.
Together, the data above suggest that aiRNA represents the first non-siRNA scaffold that is incorporated into RISC, providing a novel structural scaffold that interacts with RISC. The difference of the RISC loading of aiRNA and siRNA is illustrated in our model shown in Fig. 10. Briefly, because of the asymmetric property, only the anti-sense strand is selected to stay in the RISC complex and results in a 100% efficiency in strand selection. In contrast, siRNA is structurally symmetric. Both anti-sense strand and sense strand of the siRNA has a chance to be selected to stay in the RISC complex and therefore siRNA has an inefficient strand selection and at the same time may cause non-specific gene silencing due to the sense strand RISC complex.
Example 3. aiRNA mediates a more rapid, potent, efficacious, and durable gene silencing than siRNA
To compare aiRNA with siRNA in gene silencing properties, we first determined the optimal aiRNA structure for gene silencing.
The siRNA duplex contains a symmetrical sense strand and an antisense strand. While the duplex siRNA structure containing a 3' overhang is required for incorporation into the RISC complex, following Argonaute (Ago) mediated cleavage of the sense strand, the antisense strand directs cleavage of the target mRNA(Hammond et al., 2001 ; Matranga et al., 2005; Tabara et al., 1999). We sought to make asymmetric RNA duplexes of various lengths with overhangs at the 3' and 5' ends of the antisense strand. We designed one set of such asymmetrical RNA duplexes of 12 tol5 bp with 3' and 5' antisense overhangs to target β-catenin (Fig. 1 IA), an endogenous gene implicated in cancer and stem cells (Clevers, 2006). An optimized siRNA of the standard configuration has been designed to target β-catenin for triggering RNAi (Xiang et al., 2006). AU aiRNAs against β-catenin were designed within the same sequence targeted by the siRNA (Fig. 1 IA). The results showed that the optimal gene silencing achieved was with the 15 bp aiRNA (Fig. 1 IB). Therefore, we used 15 bp aiRNA to be compared with 21-mcr siRNA duplex in the subsequent experiments.
To our surprise, we found that aiRNA induced potent and highly efficacious reduction of β-catenin protein while sparing the non-targeted control genes actin (Fig. 11C). We next examined the onset of gene silencing by aiRNA and siRNA targeting β-catenin. The sequence of the aiRNA and siRNA used is shown in Fig. 1 1 A. As shown in Fig. 12, aiRNA has a more rapid onset (Fig. 12C and D) and also a better efficacy (Fig. 12B and D).
We also compared the gene silencing effects of aiRNA and siRNA on various targets and multiple human cell lines. The aiRNAs were designed to target genes of different functional categories including Stat3 (Fig. 13b), NQOl (Fig. 12d), elongation factor 2 (EF2) (Fig. 13c), Nbsl (Fig. 14b), Survivin (Fig. 14b), Parpl (Fig. 14b), p21 (Fig. 14b), Rskl (Fig. 14c), PCNA (Fig. 14c), p70S6K (Fig. 14c), mTOR (Fig. 14c), and PTEN (Fig. 14c), besides β-calenin (Fig. 13 a) at the same sequences that have been targeted with siRNA with low efficiency (Rogoff et al, 2004). As shown in Fig. 13 and 14, aiRNA is more efficacious than siRNA in silencing Stat3, β-catenin, Rskl, p70S6K, Nbsl, mTOR, and EF2, and is as efficacious as siRNA in silencing NQOl, PCNA, Survivin, PTEN, Parpl, and p21. Since the target sequences were chosen based on the optimization for siRNA, it is possible that the efficacy and potency of aiRNA can be further increased by targeting sites that are optimized for aiRNA. In addition, our data also shows that aiRNA is more efficacious than siRNA against b-catenin in multiple cell lines including HeIa (Fig. 13a), H1299 (Fig. 14a, left panel) and Dldl (Fig. 14a, right panel).
Taken together, these data demonstrate that aiRNA is more efficacious, potent, rapid-onset, and durable than siRNA in mediating gene silencing in mammalian cells.
Example 4. Specificity of gene silencing mediated by aiRNA
We next investigated the specificity of gene silencing mediated by aiRNAs. We first analyzed aiRNAs that target the wildtype k-Rαs allele. DLDl cells contain wild-type k-Rαs while SW480 cells contain mutant k-Rαs that has a single base pair substitution (Fig. 14d). Transfection of DLDl cells with aiRNA targeting wildtype k-Rαs showed effective silencing, but no silencing of mutant k-Rαs was observed in the SW480 cells. These data demonstrate that aiRNA mediates allele specific gene silencing.
The activation of an interferon-like response is a major non-specific mechanism of gene silencing. A primary reason that siRNAs are used for gene silencing is that the dsRNA of shorter than 30 bp has reduced ability to activate the interferon-like response in mammalian cells (Bernstein et al., 2001 ; Martinez and Tuschl, 2004; Sledz et al., 2003). We tested if aiRNA showed any signs of activating the interferon-like response in mammalian cells. RNA collected from PBMC cells transfected with aiRNA against β-cαtenin and HeIa cells transfected with aiRNA against EF2 or Survivin was analyzed by RT-PCR for interferon inducible genes. We found that aiRNA transfection showed no increase by RT-PCR of any of the interferon inducible genes tested, while levels of targeted mRNAs were reduced relative to control transfected cells (Fig. 15a and b). Microarray analysis was also performed to compare the changes in the expression of known interferon response related genes induced by aiRNA and raiRNA. As shown in Fig. 15c, much less changes were observed for aiRNA compared to siRNA.
In addition, as mentioned above, sense strand-RISC complex may cause non-specific gene silencing. To compare aiRNA and siRNA on the non-specific gene silencing mediated by sense- strand-RISC complex, cells were co-transfected with aiRNA or siRNA and either a plasmid expressing Stat3 (sense RNA) or a plasmid expressing antisense Stat3 (antisense RNA). Cells were harvested and RNA collected at 24 hours post transfection and relative levels of Stal3 sense or antisense RNA were determined by quantitative real time PCR or RT-PCR (inserts). The results show that aiRNA has no effect on the antisense Stat3 niRNAs while siRNA does (Fig. 15d). This result demonstrate aiRNA completely abolish the undesired non-specific gene- silencing mediated by the sense strand-RISC complex.
In summary, we have shown that aiRNA is a novel class of gene-silencing inducers, the non-siRNA type and the smallest structural scaffold for RISC substrates and RNAi mediators (Fig. 15f). Our data suggest that aiRNA works through RISC, the cellular RNAi machinery. After incorporation into RISC, aiRNA mediates sequence-specific cleavage of the mRNA between base 10 and 11 relative to the 5' end of the aiRNA antisense strand. The asymmetrical configuration of aiRNA can interact more efficiently with RJSC than siRNA. Consistent with high RISC binding efficiency, aiRNA is more potent, efficacious, rapid-onset, and durable than siRNA in mediating gene-specific silencing against genes tested in our study. While previous studies have proposed a role of Dicer in facilitating efficient RISC formation, our data suggest aiRNA can give rise to active RISC complexes with high efficiency independent of Dicer- mediated processing.
The key feature of this novel RNA duplex scaffold is antisense overhangs at the 3' and 5' ends. The 12-15 bp aiRNA are the shortest RNA duplex known to induce RNAi. While long dsRNAs triggered potent gene silencing in C elegans and Drosophila Melanogaster, gene- specific silencing in mammalian cells was not possible until siRNA duplexes were used. The siRNA scaffold, as defined by Dicer digestion, is characterized by symmetry in strand lengths of 19-21 bp and 3' overhangs (Bernstein ct al., 2001), which has been considered the essential structure for incorporating into RISC to mediate RNAi. Therefore, optimization efforts for RNAi inducers have been focused on siRNA precursors, which are invariably larger than siRNA (Soutschek et al., 2004; Zhang and Farwell, 2007). Our data suggest that siRNA is not the essential scaffold for incorporating into RISC to mediate RNAi. The aiRNAs of different lengths displayed a spectrum of gene silencing efficacy and RISC incorporation efficiency, offering unique opportunity for understanding the mechanism of RISC incorporation and activation. Research is needed to further understand the structure-activity relationship of aiRNAs in RISC incorporation and RNAi induction, which should help establish a rational basis for optimizing aiRNAs with regards to target sequence selection, length, structure, chemical composition and modifications for various RNAi applications.
Example 5. aiRNA is more efficacious than siRNA in vivo
To investigate if aiRNA is efficacious in vivo and to compare it with siRNA, we tested the effects of aiRNA and siRNA in human colon cancer xenograft models.
Human Colon Cancer is the second leading cause of cancer death in the U.S. The Wnt β- catenin signaling pathway is tightly regulated and has important functions in development, tissue homeostasis, and regeneration. Deregulation of Wnt/β-catenin signaling is frequently found in various human cancers. Eighty percent of colorectal cancers alone reveal activation of this pathway by either inactivation of the tumor-suppressor gene adenomatous polyposis coli or mutation of the proto-oncogene β-catenin.
Activation of Wnt β-catenin signaling has been found to be important for both initiation and progression of cancers of different tissues. Therefore, targeted inhibition of Wnt β-catenin signaling is a rational and promising new approach for the therapy of cancers of various origins.
In vitro, by ribozyme-targeting we have demonstrated the redviction of β-catenin expression in human colon cancer SW480 cells and associated induction of cell death, indicating that β-catenin expression is rate-limiting for tumor growth in vitro.
SW480 human colon cancer cells were inoculated subcutaneously into female athymic nude mice (8x106 cells/mouse) and allowed to form palpable tumors. In this study, dosing began when the tumors reached approximately 120 mm3. Animals were treated intravenously (iv) with 0.6 nmol PEI-complexed β-catenin siRNAs, PEl-complexed β-catenin aiRNAs or a PEI- complexed unrelated siRNA as a negative control daily. The animals received a total of 10 doses of siRNA, aiRNA or control. Tumors were measured throughout treatment. As shown in Figure 16, intravenous treatment with siRNA and aiRNA as a monotherapy at 0.6nmol mg/kg significantly inhibited tumor growth. The %T/C value of siRNA was calculated to be 48.8% with a p value of 0.0286. The treatment with the β-catenin -specific aiRNAs, however, resulted in a much more potent reduction in tumor growth. The %T/C value was calculated to be 9.9% with a p value of 0.0024. There was no significant change in body weight due to iv administration of the siRNA, aiRNA or control. These data suggest that the systemic in vivo application of aiRNAs through PEI complexation upon targeting of the β-catenin offers an avenue for the development of highly efficient, specific and safe agents for therapeutic applications for patients with colon cancer.
In addition, we also tested the effects of aiRNA and siRNA in HT29 human colon cancer xenograft model. HT29 human colon cancer cells were inoculated subcutaneously into female athymic nude mice (6x106 cells/mouse) and allowed to form palpable tumors. In this study, dosing began when the tumors reached approximately 200 mm . Animals were treated intravenously (iv) with 0.6 nmol PEI-complexed β-catenin siRNAs, PEI-complexed β-calenin aiRNAs or a PEI-complexed unrelated siRNA as a negative control eveiy other day. The animals received a total of 8 doses of siRNA, aiRNA or control. Tumors were measured throughout treatment. As shown in Figure 17, intravenous treatment with siRNA and aiRNA as a monotherapy at 0.6nmol mg/kg significantly inhibited tumor growth. The %T/C value of siRNA was calculated to be 78% with a p value of 0.21. Again, the treatment with the β-catenin -specific aiRNAs resulted in an even more potent reduction in tumor growth. The %T/C value was calculated to be 41% with a p value of 0.016. There was no significant change in body weight due to iv administration of the siRNA, aiRNA or control. These data second that the systemic in vivo application of aiRNAs through PEI complexation upon targeting of the β- calenin offers an avenue for the development of highly efficient, specific and safe agents for therapeutic applications for patients with colon cancer.
Together, the aiRNA may significantly improve broad RNAi applications. The siRNA- based therapeutics have met with challenges, including limited efficacy, delivery difficulty, interferon- like responses and manufacture cost (de Fougerolles et al., 2007; Iorns et al., 2007; Rana, 2007). The improved efficacy, potency, durability, and smaller size of aiRNAs may help or overcome these challenges since aiRNA is smaller and may need less material for its delivery. Therefore, aiRNA represents new and smallest RNA duplexes that enter RISC and mediates gene silencing of better efficacy, potency, onset of action, and durability than siRNA in mammalian cells, holding significant potential for broad RNAi applications in gene function study and RNAi-based therapies.
Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications may be made without departing from the spirit and scope of the present invention. References
Bernstein, E., Caudy, A.A., Hammond, S. M.. and Hannon, GJ. (2001 ). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363-366. Chiu, Y.L., and Rana, T. M. (2003). siRNA function in RNAi: a chemical modification analysis. RNA (New York, NY 9, 1034-1048.
Clevers, H. (2006). Wnt/beta-catenin signaling in development and disease. Cell 127, 469-480. Czauderna, F., Fechtner, M., Dames, S., Aygun, H., Klippel, A., Pronk, GJ. , Giese, K., and
Kaufmann, J. (2003). Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells. Nucleic acids research 31, 2705-2716. Czech, M. P. (2006). MicroRNAs as therapeutic targets. The New England journal of medicine
354, 1 194-1195. de Fougerolles, A., Vornlocher, H.P., Maraganore, J., and Lieberman, J. (2007). Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 6, 443-
453. Dignam, J.D., Lebovitz, R.M., and Roedcr. R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic acids research 11, 1475-1489. Donze, O., and Picard, D. (2002). RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res JO, e46. Dykxhoorn, D.M., Novina, CD., and Sharp, P.A. (2003). Killing the messenger: short RNAs that silence gene expression. Nat Rev MoI Cell Biol 4, 457-467. Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. (2001a).
Duplexes of 21 -nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494-498. Elbashir, S. M., Lendeckel, W., and Tuschl, T. (2001 b). RNA interference is mediated by 21- and
22-nucleolide RNAs. Genes Dev 15, 188-200. Elbashir, S. M., Martinez, J., Patkaniowska, A., Lendeckel, W., and Tuschl, T. (2001c).
Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. Embo J 20, 6877-6888. Eulalio, A., Huntzinger, E., and Izaurralde, E. (2008). Getting to the root of miRNA-mediated gene silencing. Cell 132, 9-14. Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, CC. (1998). Potent and specific genetic interference by double-stranded RNA in Cacnorhabditis elegans.
Nature 391, 806-811. Hammond, S. M., Bernstein, E., Beach, D.. and Harmon, G.J. (2000). An RNA-dircctcd nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293-296. Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R., and Hannon, G.J. (2001). Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293,
1146-1150. Iorns, E., Lord, C. J., Turner, N., and Ashworth, A. (2007). Utilizing RNA interference to enhance cancer drug discovery. Nature reviews 6, 556-568. Kim, D.H., Behlke, M.A., Rose, S.D., Chang, M.S., Choi, S., and Rossi, JJ. (2005). Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat Biotechnol 23, 222-
226. Kim, D.H., and Rossi, JJ. (2007). Strategies for silencing human disease using RNA interference. Nature reviews 8, 173-184. Liu, J., Carmcll, M.A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, JJ., Hammond, S. M.,
Joshua-Tor, L., and Hannon, GJ. (2004). Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437-1441.
Mack, G. S. (2007). MicroRNA gets down to business. Nature biotechnology 25, 631-638. Martinez, J., and Tuschl, T. (2004). RISC is a 5' phosphomonocstcr-producing RNA endonuclease. Genes Dev 18, 975-980. Matranga, C, Tomari, Y., Shin, C, Bartel, D.P., and Zamore, P. D. (2005). Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes.
Cell 123, 607-620. Paddison, P.J., Caudy, A.A., Bernstein, E., Ilannon. GJ , and Conklin, D, S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16,
948-958.
Patzel, V. (2007). In silico selection of active siRNA. Drug discovery today 12, 139-148. Rana, T.M. (2007). Illuminating the silence: understanding the structure and function of small RNAs. Nat Rev MoI Cell Biol 8, 23-36. Rogoff, H.A., Pickering, M.T., Frame, F. M., Debatis, M. E., Sanchez, Y., Jones, S., and Kowalik,
T. F. (2004). Apoptosis associated with deregulated E2F activity is dependent on E2F1 and Atm/Nbsl/Chk2. MoI Cell Biol 24, 2968-2977. Siolas, D., Lerner, C, Burchard, J., Ge, W., Linsley, P. S., Paddison, P. J., Hannon, GJ., and
Cleary, M.A. (2005). Synthetic shRNAs as potent RNAi triggers. Nat Biotechnol 23.
227-231. Sledz, C.A., Holko, M., de Veer, MJ. , Silverman, R.H., and Williams, B.R. (2003). Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5, 834-839. Soutschek, J., Akinc, A., Bramlage, B., Charisse. K., Constien, R., Donoghue, M., Elbashir, S.,
Geick, A., Hadwiger, P., Harborth, J., et al. (2004). Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432, 173-178. Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., and
Mello, CC. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123-132. Xiang, S., Fruehauf, J., and Li, CJ. (2006). Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nature biotechnology 24, 697-702. Zamore, P. D., and Aronin, N. (2003). siRNAs knock down hepatitis. Nature medicine 9, 266-
267. Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. (2000). RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101,
25-33. Zhang, B., and Farwell, M.A. (2007). microRNAs: a new emerging class of players for disease diagnostics and gene therapy. J Cell MoI Med. Zhang, H.Y., Du, Q., Wahlestedt, C, and Liang, Z. (2006). RNA Interference with chemically modified siRNA. Current topics in medicinal chemistry 6, 893-900.

Claims

WHAT IS CLAIMED IS:
1. A duplex RNA molecule comprising a first strand with a length from 18-23 nucleotides and a second strand with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein the first strand has a 3 '-overhang from 1-9 nucleotides, and a 5'-overhang from 0-8 nucleotides, wherein said duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell.
2. The duplex RNA molecule of claim 1 , wherein the first strand comprises a sequence being at least 70 percent complementary to a target mRNA sequence,
3. The RNA molecule of claim 1 wherein at least one nucleotide of the 5' overhang is selected from the group consisting of A, U, and dT.
4. The RNA molecule of claim 1 wherein the GC content of the double stranded region is 30%-70%.
5. The duplex RNA molecule of claim 1 wherein the first strand has a length from 19-23 nucleotides.
6. The duplex RNA molecule of claim 1 wherein the first strand has a length of 21 nucleotides.
7. The duplex RNA molecule of claim 6 wherein the second strand has a length of 14-16 nucleotides.
8. The duplex RNA molecule of claim 6 wherein the second strand has a length of 15 nucleotides.
9. The duplex RNA molecule of claim 8 wherein the first strand has a 3 '-overhang of 2-4 nucleotides.
10. The duplex RNA molecule of claim 8 wherein the first strand has a 3'-overhang of 3 nucleotides.
11. The duplex RNA molecule of claim 1, which contains at least one modified nucleotide or its analogue.
12. The duplex RNA molecule of claim 11 , wherein the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base- modified ribonucleotide.
13. The duplex RNA molecule of claim 12 wherein the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide.
14. The duplex RNA molecule of claim 13 wherein the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom,
15. The duplex RNA molecule of claim 1 1 , wherein the at least one modified nucleotide or its analogue is an unusual base or a modified base.
16. The duplex RNA molecule of claim 1 1 , wherein the at least one modified nucleotide or its analogue comprises inosine. or a tritylatcd base.
17. The duplex RNA molecule of claim 11 , wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.
18. The duplex RNA molecule of claim 11, wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
19. The duplex RNA molecule of claim 11 wherein the first strand comprises at least one deoxynucleotide.
20. The duplex RNA molecule of claim 19 wherein the at least one deoxynucleotides are in one or more regions selected from the group consisting of 3 '-overhang, 5'-overhang. and double-stranded region.
21. The duplex RNA molecule of claim 11 wherein the second strand comprises at least one deoxynucleotide.
22. The RNA molecule of claim 1 wherein the eukaryotic cell is a mammalian cell or an avian cell.
23. A method of modulating gene expression in a cell or an organism comprising the steps of: contacting said cell or organism with the duplex RNA molecule of claim 1 under conditions wherein selective gene silencing can occur, and mediating a selective gene silencing effected by said duplex RNA molecule towards a target gene nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA.
24. The method of claim 23, wherein said contacting step comprises a step of introducing said duplex RNA molecule into a target cell in culture or in an organism in which the selective gene silencing can occur.
25. The method of claim 24 wherein the introducing step is selected from the group consisting of transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical and regional administration.
26. The method of claim 24 wherein the introducing step comprises using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid.
27. Use of the method of claim 23 for determining the function or utility of a gene in a cell or an organism.
28. Use of the method of claim 23 for treating or preventing a disease or an undesirable condition.
29. The use of claim 23 wherein the target gene is associated with a disease, a pathological condition, or an undesirable condition in a mammal.
30. The use of claim 23, wherein the target gene is a gene of a pathogenic microorganism.
31. The use of claim 23, wherein the target gene is a viral gene.
32. The use of claim 23, wherein the target gene is a tumor-associated gene.
33. The use of claim 23, wherein the target gene is a gene associated with a disease selected from the group consisting of autoimmune disease, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging.
34. A reagent comprising the duplex RNA molecule of claim 1.
35. A kit comprising a first RNA strand with a length from 18-23 nucleotides and a second RNA strand with a length from 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand, and capable of forming a duplex RNA molecule with the first strand, wherein the duplex RNA molecule has a 3'-overhang from 1-9 nucleotides, and a 5'-overhang from 0-8 nucleotides, wherein said duplex RNA molecule is capable of effecting sequence-specific gene silencing in a eukaryotic cell.
36. A method of preparing the duplex RNA molecule of claim 1 comprising the steps of: synthesizing the first strand and the second strand, and combining the synthesized strands under conditions, wherein the duplex RNA molecule is formed, which is capable of effecting sequence-specific gene silencing.
37. The method of claim 36 further comprising a step of introducing at least one modified nucleotide or its analogue into the duplex RNA molecule during the synthesizing step, after the synthesizing and before the combining step, or after the combining step.
38. The method of claim 36, wherein the RNA strands are chemically synthesized, or biologically synthesized.
39. An expression vector comprising a nucleic acid or nucleic acids encoding the duplex RNA molecule of claim 1 operably linked to at least one expression-control sequence.
40. The expression vector of claim 39 comprising a first nucleic acid encoding the first strand operably linked to a first expression-control sequence, and a second nucleic acid encoding the second strand operably linked to a second expression-control sequence.
41. The expression vector of claim 39 is a viral, eukaryotic, or bacterial expression vector.
42. A cell comprising the vector of claim 39.
43. A cell comprising the duplex RNA molecule of claim 1.
44. The cell of claim 43 wherein the cell is a mammalian, avian, or bacterial cell.
45. A duplex RNA molecule comprising a first strand and a second strand, wherein the first strand is longer than the second strand, wherein the second strand is substantially complementary to the first strand, and forms a double-stranded region with the first strand, wherein said duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell.
46. The duplex RNA molecule of claim 45 wherein the two ends of the duplex RNA molecule are independently selected from the group consisting of 3'-overhang from 1-10 nucleotides, 5'-overhang from 0-10 nucleotides, and blunt end.
47. The RNA molecule of claim 45 wherein the first strand is substantially complementary to a target mRNA sequence.
48. The RNA molecule of claim 45 wherein the first strand has a 3'-overhang from 1- 8 nucleotides and a 5'-overhang from 1 -8 nucleotides.
49. The RNA molecule of claim 45 wherein the first strand has a 3'-overhang from 1- 10 nucleotides and a blunt end.
50. The RNA molecule of claim 45 wherein the first strand has a 5'-overhang from 1- 10 nucleotides and a blunt end.
51. The RNA molecule of claim 45 wherein the RNA duplex has two 5'-overhangs from 1-8 nucleotides.
52. The RNA molecule of claim 45 wherein the RNA duplex has two 3'-ovcrhangs from 1-10 nucleotides.
53. The RNA molecule of claim 45 wherein the first strand has a length from 12-100 nucleotides.
54. The RNA molecule of claim 45 wherein the first strand has a length from 12-30 nucleotides.
55. The RNA molecule of claim 45 wherein the first strand has a length from 18-23 nucleotides.
56. The RNA molecule of claim 45 wherein the first strand has a length from 19-25 nucleotides.
57. The RNA molecule of claim 45 wherein the first strand has a length of 21 nucleotides.
58. The RNA molecule of claim 45 wherein the second strand has a length from 5-30 nucleotides.
59. The RNA molecule of claim 45 the second strand has a length from 12-22 nucleotides.
60. The RNA molecule of claim 45 wherein the second strand has a length of 12-17 nucleotides.
61. The RNA molecule of claim 45 wherein the second strand has a length of 15 nucleotides.
62. The RNA molecule of claim 45 wherein the first strand has a length from 12-30 nucleotides, and the second strand has a length from 10-29 nucleotides, with the provision that when the double stranded region is 27 bp, the overhangs at both ends of the RNA molecule are independently 3' overhang or 5' overhang.
63. The RNA molecule of claim 45 wherein the first strand has a length from 18-23 nucleotides, and the second strand has a length from 12-17 nucleotides.
64. The RNA molecule of claim 45 wherein the first strand has a length from 19-25 nucleotides, and the second strand has a length from 12-17 nucleotides.
65. The RNA molecule of claim 45 wherein the first strand has a length from 19-25 nucleotides, and the second strand has a length from 18-24 nucleotides, with the provision that when the first strand is 5 ' -UUCGAAGUAUUCCGCGUACGU 5 ' -UCGAAGUAUUCCGCGUACGUG or
5' -CGAAGUAUUCCGCGUACGUGA the second strand has a length of at most 17 nucleotides, or contains at least one mismatch with the first strand, or contains at least one modification.
66. The RNA molecule of claim 45 wherein the first strand has a length of 21 nucleotides and the second strand has a length of 12-17 nucleotides.
67. The RNA molecule of claim 45 wherein the first strand has a length of 21 nucleotides and the second strand has a length of 14-16 nucleotides.
68. The RNA molecule of claim 45 wherein the first strand is from 1-10 nucleotides longer than the second strand.
69. The RNA molecule of claim 46 wherein the 3'-overhang has a length from 2-6 nucleotides.
70. The RNA molecule of claim 46 wherein the 5'-overhang has a length from 0-5 nucleotides.
71. The duplex RNA molecule of claim 45 wherein the gene silencing comprises one or two, or all of RNA interference, modulation of translation, and DNA epigenetic modulations.
72. The duplex RNA molecule of claim 45 further comprising a nick in at least one of said first and second strands.
73. The duplex RNA molecule of claim 45 wherein the double stranded region comprises a gap of one or more nucleotides.
74. The RNA molecule of claim 47 wherein at least one nucleotide of the 5' overhang is not complementary to the target niRNA sequence.
75. The RNA molecule of claim 45 wherein at least one nucleotide of 5' overhang is selected from the group consisting of A. U, and dT.
76. The duplex RNA molecule of claim 45 being conjugated to an entity selected from the group consisting of peptide, antibody, polymer, lipid, oligonucleotide, cholesterol, and aptamer.
77. The RNA molecule of claim 45 further comprising at least one unmatched or mismatched nucleotide.
78. The RNA molecule of claim 45 wherein the GC content of the double stranded region is 20-70%.
79. The RNA molecule of claim 45, wherein the 3 '-overhang and/or 5 '-overhang is stabilized against degradation.
80. The duplex RNA molecule of claim 45, which contains at least one modified nucleotide or its analogue.
81. The duplex RNA molecule of claim 80, wherein the at least one modified nucleotide or its analogue is sugar-, backbone-, and/or base- modified ribonucleotide.
82. The duplex RKA molecule of claim 81 wherein the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide.
83. The duplex RNA molecule of claim 82 wherein the phosphodiester linkage is modified to include at least one of a nitrogen or sulphur heteroatom.
84. The duplex RNA molecule of claim 80, wherein the at least one modified nucleotide or its analogue comprises a non-natural base or a modified base
85. The duplex RNA molecule of claim 80, wherein the at least one modified nucleotide or its analogue comprises inosine, or a tritylated base.
86. The duplex RNA molecule of claim 80, wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2. NHR, NR2, or CN, wherein each R is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F, Cl, Br or I.
87. The duplex RNA molecule of claim 80, wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group.
88. The duplex RNA molecule of claim 80 wherein the first strand comprises at least one deoxynucleotide.
89. The duplex RNA molecule of claim 88 wherein the at least one deoxynuclcotidcs are in one or more regions selected from the group consisting of 3'-overhang, 5 '-overhang, and double-stranded region proximal to the 5' end of the first strand.
90. The duplex RNA molecule of claim 80 wherein the second strand comprises at least one deoxynucleotide.
91. The duplex RNA molecule of claim 45 wherein the eukaryotic cell is a mammalian cell or an avian cell.
92. The duplex RNA molecule of claim 45 wherein the duplex RNA molecule is an isolated duplex RNA molecule.
93. A method of modulating gene expression in a cell or an organism comprising the steps of: contacting said cell or organism with the duplex RNA molecule of claim 1 under conditions wherein selective gene silencing can occur, and mediating a selective gene silencing effected by the double-stranded RNA towards a target gene or nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA.
94. The method of claim 93, wherein said contacting comprises a step of introducing said duplex RNA molecule into a target cell in culture or in an organism in which the selective gene silencing can occur.
95. The method of claim 94 wherein the introducing step is selected from the group consisting of transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical and regional administration.
96. The method of claim 93 wherein the introducing step comprises using a pharmaceutically acceptable excipient, carrier, or diluent selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a nanoemulsion, a lipid, and a lipoid.
97. Use of the method of claim 93 for modulating the expression of a gene in a cell or an organism.
98. Use of the method of claim 93 for treating or preventing a disease or an undesirable condition.
100. The use of claim 93 wherein the target gene is a gene associated with human or animal diseases.
101. The use of claim 93. wherein the target gene is a gene of a pathogenic microorganism.
102. The use of claim 93, wherein the target gene is a viral gene.
103. The use of claim 93 , wherein the target gene is a tumor-associated gene.
104. The use of claim 93, wherein the target gene is a gene associated with a disease selected from the group consisting of autoimmune disease, inflammatory diseases, degenerative diseases, infectious diseases, proliferative diseases, metabolic diseases, immune-mediated disorders, allergic diseases, dermatological diseases, malignant diseases, gastrointestinal disorders, respiratory disorders, cardiovascular disorders, renal disorders, rheumatoid disorders, neurological disorders, endocrine disorders, and aging.
105. Use of the method of claim 93 for studying drug target in vitro or in vivo.
106. A reagent comprising the duplex RNA molecule of claim 1.
107 A kit comprising a first RNA strand and a second RN A strand, wherein the first strand is longer than the second strand. wherein the second strand is substantially complementary to the first strand, and capable of forming a duplex RNA molecule with the first strand, wherein said duplex RNA molecule is capable of effecting sequence-specific gene silencing in a eukaryotic cell.
108. A method of preparing the duplex RNA molecule of claim 1 comprising the steps of: synthesizing the first strand and the second strand, and combining the synthesized strands under conditions, wherein the duplex RNA molecule is formed, which is capable of effecting sequence-specific gene silencing.
109. The method of claim 108 further comprising a step of introducing at least one modified nucleotide or its analogue into the duplex RNA molecule during the synthesizing step, after the synthesizing and before the combining step, or after the combining step.
110. The method of claim 108. wherein the RNA strands are chemically synthesized, or biologically synthesized.
111. The method of claim 108 wherein the first strand and the second strand are synthesized separately or simultaneously.
1 12. A pharmaceutical composition comprising as an active agent at least one duplex RNA molecule of claim 1 and one or more carriers selected from the group consisting of a pharmaceutical carrier, a positive-charge carrier, a liposome, a protein carrier, a polymer, a nanoparticle, a cholesterol, a lipid, and a lipoid.
113. A method of treatment comprising administering an effective amount of the pharmaceutical composition of claim 1 12 to a subject in need.
1 14. The method of claim 1 13 wherein the pharmaceutical composition is administered via a route selected from the group consisting of iv, po, sc, im, inhalation, topical, and regional administration.
115. The duplex RNA molecule of claim 1, wherein the first strand comprises a sequence being substantially complimentary to the target mRN A sequence of a gene selected from the group consisting of a developmental gene, an oncogene, a tumor suppresser gene, and an enzyme gene, and a gene for an adhesion molecule, a cyclin kinase inhibitor, a Wnt family member, a Pax family member, a Winged helix family member, a IIox family member, a cytokine/lymphokine or its receptor, a growth/differentiation factor or its receptor, a neurotransmitter or its receptor, a kinase, a signal transducer, a viral gene, a gene of an infectious disease, ABLI, BCLl, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2,ETS1, ETSl, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC,MYCLl, MYCN, NRAS, PIMl, PML, RET, SRC, TALI , TCL3 and YES, APC, BRCAL BRCA2, MADH4, MCC,NFl, NF2,RB1, TP53, WTl, an ACP desaturase or hydroxylase, an ADP-glucose pyrophorylase, an ATPase, an alcohol dehydrogenase, an amylase, an amyloglucosidase, a catalase, a cellulase, a cyclooxygenase, a decarboxylase, a dextrinase, a DNA or RNA polymerase, a galactosidase, a glucanase. a glucose oxidase, a GTPase, a helicase, a hemicellulase, an integrase, an invertase, an isomerase, a kinase, a lactase, a lipase, a lipoxygenase, a lysozyme, a pectinesterasc, a peroxidase, a phosphatase, a phospholipase, a phosphorylase, a polygalacturonase, a proteinase or peptideases, a pullanase, a recombinase, a reverse transcriptase, a topoisomerase, a xylanase, k-RAS, β-Catenin, Rskl, PCNA, p70S6K, Survivin, mTOR, PTEN, Parpl, or p21.
116. A du lex RNA molecule selected from the rou consistin of
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Wherein A, U, G, and C are nucleotides, while a, t, g, and c are deoxynucleotides.
117. A method of modifying a first duplex RNA molecule with an antisense strand and a sense strand that form a double- stranded region, the method comprising, shortening the length of the sense strand so that the antisense strand has a 3'-overhang from 1-8 nucleotides and a 5'- overhang from 0-8 nucleotides, and forming a second duplex RNA molecule; wherein at least one property of the first duplex RNA molecule is improved.
118. The method of claim 1 17 further comprising combining the antisense strand and the shortened sense strand under conditions, wherein the second duplex RNA molecule is formed.
1 19. The method of claim 117 wherein the first duplex RNA molecule is a siRNA, or dicer-substrate siRNA, or a siRNA precursor.
120. An expression vector comprising a nucleic acid or nucleic acids encoding the duplex RNA molecule of claim 1 operably linked to at least one expression-control sequence.
121. The expression vector of claim 120 comprising a first nucleic acid encoding the first strand operably linked to a first expression-control sequence, and a second nucleic acid encoding the second strand operably linked to a second expression-control sequence.
122. The expression vector of claim 120 is a viral, eukaryotic expression vector, or bacterial expression vector.
123. A cell comprising the expression vector of claim 120.
124. A cell comprising the duplex RNA molecule of claim 1.
125. The cell of claim 124 is a mammalian cell, avian cell, or bacterial cell.
PCT/US2008/074528 2007-08-27 2008-08-27 Compositions of asymmetric interfering rna and uses thereof WO2009029688A2 (en)

Priority Applications (9)

Application Number Priority Date Filing Date Title
DK08828867.5T DK2193140T3 (en) 2007-08-27 2008-08-27 COMPOSITIONS OF ASYMMETRIC INTERFERRING RNA AND ITS APPLICATIONS
EP16187862.4A EP3121281B1 (en) 2007-08-27 2008-08-27 Compositions of asymmetric interfering rna and uses thereof
ES08828867.5T ES2617877T3 (en) 2007-08-27 2008-08-27 Asymmetric interfering RNA compositions and their use
EP21158616.9A EP3889261A1 (en) 2007-08-27 2008-08-27 Compositions of asymmetric interfering rna and uses thereof
EP08828867.5A EP2193140B1 (en) 2007-08-27 2008-08-27 Compositions of asymmetric interfering rna and uses thereof
CN200880113236.9A CN101835789B (en) 2007-08-27 2008-08-27 Compositions of asymmetric interfering RNA and uses thereof
CA2735166A CA2735166C (en) 2007-08-27 2008-08-27 Compositions of asymmetric interfering rna and uses thereof
JP2010523120A JP2010537639A (en) 2007-08-27 2008-08-27 Asymmetric interfering RNA compositions and uses thereof
HK11102556.9A HK1148534A1 (en) 2007-08-27 2011-03-14 Compositions of asymmetric interfering rna and uses thereof rna

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US96825707P 2007-08-27 2007-08-27
US60/968,257 2007-08-27
US2975308P 2008-02-19 2008-02-19
US61/029,753 2008-02-19
US3895408P 2008-03-24 2008-03-24
US61/038,954 2008-03-24

Publications (2)

Publication Number Publication Date
WO2009029688A2 true WO2009029688A2 (en) 2009-03-05
WO2009029688A3 WO2009029688A3 (en) 2009-07-02

Family

ID=40387778

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2008/074528 WO2009029688A2 (en) 2007-08-27 2008-08-27 Compositions of asymmetric interfering rna and uses thereof
PCT/US2008/074531 WO2009029690A1 (en) 2007-08-27 2008-08-27 Composition of asymmetric rna duplex as microrna mimetic or inhibitor

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US2008/074531 WO2009029690A1 (en) 2007-08-27 2008-08-27 Composition of asymmetric rna duplex as microrna mimetic or inhibitor

Country Status (9)

Country Link
US (6) US20100286378A1 (en)
EP (4) EP3889261A1 (en)
JP (7) JP2010537640A (en)
CN (3) CN101835789B (en)
CA (2) CA2735167A1 (en)
DK (1) DK2193140T3 (en)
ES (2) ES2617877T3 (en)
HK (2) HK1148534A1 (en)
WO (2) WO2009029688A2 (en)

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010039536A2 (en) 2008-09-23 2010-04-08 President And Fellows Of Harvard College Sirt4 and uses thereof
EP2222848A2 (en) * 2007-12-18 2010-09-01 Lee, Dong Ki Novel sirna structure for minimizing off-target effects and relaxing saturation of rnai machinery and the use thereof
CN101979558A (en) * 2010-10-28 2011-02-23 百奥迈科生物技术有限公司 Small interfering RNA (siRNA) molecule of targeted hepatitis B virus (HBV) gene and application thereof
WO2011041582A2 (en) 2009-09-30 2011-04-07 President And Fellows Of Harvard College Methods for modulation of autophagy through the modulation of autophagy-inhibiting gene products
EP2352744A2 (en) * 2008-10-15 2011-08-10 Somagenics, Inc. Short hairpin rnas for inhibition of gene expression
EP2513334A1 (en) * 2009-12-18 2012-10-24 Dicerna Pharmaceuticals, Inc. Dicer substrate agents and methods for the specific inhibition of gene expression
CN103555722A (en) * 2009-04-03 2014-02-05 戴瑟纳制药公司 Methods and compositions for the specific inhibition of kras by asymmetric double-stranded rna
WO2014089121A2 (en) * 2012-12-03 2014-06-12 Thomas Ichim Retrograde delivery of cells and nucleic acids for treatment of cardiovascular diseases
US8871730B2 (en) 2009-07-13 2014-10-28 Somagenics Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression
US9080171B2 (en) 2010-03-24 2015-07-14 RXi Parmaceuticals Corporation Reduced size self-delivering RNAi compounds
WO2015182913A1 (en) * 2014-05-27 2015-12-03 주식회사 제노포커스 Method for overexpression of target protein by gene silencing
EP2953646A4 (en) * 2013-02-05 2017-02-15 1Globe Health Institute LLC Biodegradable and clinically-compatible nanop articles as drug delivery carriers
US9637742B2 (en) 2010-10-22 2017-05-02 Sungkyunkwan University Foundation For Corporate Collaboration Nucleic acid molecules inducing RNA interference, and uses thereof
WO2017100127A1 (en) 2015-12-06 2017-06-15 Boston Biomedical, Inc. ASYMMETRIC INTERFERING RNAs, AND COMPOSITIONS, USE, OR PREPARATION THEREOF
US9745574B2 (en) 2009-02-04 2017-08-29 Rxi Pharmaceuticals Corporation RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
US9938530B2 (en) 2008-09-22 2018-04-10 Rxi Pharmaceuticals Corporation RNA interference in skin indications
US9963702B2 (en) 2010-03-24 2018-05-08 Rxi Pharmaceuticals Corporation RNA interference in dermal and fibrotic indications
CN108064155A (en) * 2014-12-26 2018-05-22 日东电工株式会社 For the RNA agent of P21 Gene regulations
WO2018098352A2 (en) 2016-11-22 2018-05-31 Jun Oishi Targeting kras induced immune checkpoint expression
US10125362B2 (en) 2012-05-22 2018-11-13 Olix Pharmaceuticals, Inc. RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor
US10167471B2 (en) 2009-01-05 2019-01-01 Rxi Pharmaceuticals Corporation Inhibition of PCSK9 through RNAI
US10184124B2 (en) 2010-03-24 2019-01-22 Phio Pharmaceuticals Corp. RNA interference in ocular indications
WO2019074071A1 (en) 2017-10-11 2019-04-18 日東電工株式会社 Regulation of nucleic acid molecule expression
US10266821B2 (en) 2007-08-27 2019-04-23 1Globe Health Institute Llc Compositions of asymmetric interfering RNA and uses thereof
US10358647B2 (en) 2015-12-13 2019-07-23 Nitto Denko Corporation siRNA structures for high activity and reduced off target
US10392619B2 (en) 2009-10-12 2019-08-27 Larry J. Smith Methods and compositions for modulating gene expression using oligonucleotide based drugs administered in vivo or in vitro
US10519449B2 (en) 2016-02-02 2019-12-31 Olix Pharmaceuticals, Inc. Treatment of angiogenesis-associated diseases using RNA complexes that target ANGPT2 and PDGFB
US10590423B2 (en) 2015-11-16 2020-03-17 Olix Pharmaceuticals, Inc. Treatment of age-related macular degeneration using RNA complexes that target MyD88 or TLR3
US10808247B2 (en) 2015-07-06 2020-10-20 Phio Pharmaceuticals Corp. Methods for treating neurological disorders using a synergistic small molecule and nucleic acids therapeutic approach
US10829761B2 (en) 2016-04-11 2020-11-10 Olix Pharmaceuticals, Inc. Treatment of idiopathic pulmonary fibrosis using RNA complexes that target connective tissue growth factor
US10900039B2 (en) 2014-09-05 2021-01-26 Phio Pharmaceuticals Corp. Methods for treating aging and skin disorders using nucleic acids targeting Tyr or MMP1
US10934550B2 (en) 2013-12-02 2021-03-02 Phio Pharmaceuticals Corp. Immunotherapy of cancer
US10947541B2 (en) 2016-02-02 2021-03-16 Olix Pharmaceuticals, Inc. Treatment of atopic dermatitis and asthma using RNA complexes that target IL4Rα, TRPA1, or F2RL1
US11001845B2 (en) 2015-07-06 2021-05-11 Phio Pharmaceuticals Corp. Nucleic acid molecules targeting superoxide dismutase 1 (SOD1)
US11021707B2 (en) 2015-10-19 2021-06-01 Phio Pharmaceuticals Corp. Reduced size self-delivering nucleic acid compounds targeting long non-coding RNA
US11040057B2 (en) 2016-06-29 2021-06-22 Olix Pharmaceuticals, Inc. Pharmaceutical compositions and methods for potentiating gene silencing
US11045488B2 (en) 2014-12-26 2021-06-29 Nitto Denko Corporation RNA interference agents for GST-π gene modulation
US11279934B2 (en) 2014-04-28 2022-03-22 Phio Pharmaceuticals Corp. Methods for treating cancer using nucleic acids targeting MDM2 or MYCN
WO2022175749A1 (en) 2021-02-18 2022-08-25 Oneglobe Holdings Limited Compositions for conjugating oligonucleotides and carbohydrates
WO2022256351A1 (en) 2021-05-29 2022-12-08 1Globe Health Institute Llc Asymmetric short duplex dna as a novel gene silencing technology and use thereof
US11591600B2 (en) 2017-02-10 2023-02-28 OliX Pharmaceuticals. Inc. Long double-stranded RNA for RNA interference
WO2024040531A1 (en) 2022-08-25 2024-02-29 1Globe Health Institute, Nanjing Novel compositions for conjugating oligonucleotides and carbohydrates

Families Citing this family (93)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0720219A2 (en) * 2006-12-08 2013-12-24 Univ Iowa State Res Found Inc PLANT GENES INVOLVED IN ABSORPTION AND NITRATE METABOLISM
US8097712B2 (en) 2007-11-07 2012-01-17 Beelogics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
WO2010141933A1 (en) * 2009-06-05 2010-12-09 Dicerna Pharmaceuticals, Inc. Specific inhibition of gene expression by nucleic acid containing a dicer substrate
US8796238B2 (en) 2009-09-17 2014-08-05 Sigma-Aldrich Co. Llc Short RNA mimetics
US8962584B2 (en) 2009-10-14 2015-02-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Compositions for controlling Varroa mites in bees
KR101237036B1 (en) * 2009-11-04 2013-02-25 성균관대학교산학협력단 Novel siRNA Structure for Minimizing Off-target Effects by Antisense Strand and the Use Thereof
WO2011072390A1 (en) * 2009-12-16 2011-06-23 The University Of Western Ontario Compositions and methods related to mirna in diabetic conditions
WO2011112570A1 (en) 2010-03-08 2011-09-15 Monsanto Technology Llc Polynucleotide molecules for gene regulation in plants
EP3502254A1 (en) 2010-04-23 2019-06-26 Cold Spring Harbor Laboratory Novel structurally designed shrnas
GB201006841D0 (en) * 2010-04-26 2010-06-09 Thomsen Lars Method, device and system for targetted cell lysis
WO2012140234A1 (en) 2011-04-13 2012-10-18 Vib Vzw Modulation of mirna in diseases with aberrant angiogenesis
WO2012149646A1 (en) * 2011-05-05 2012-11-08 Sunnybrook Research Institute Mirna inhibitors and their uses
BR112014002737B1 (en) 2011-08-04 2022-08-02 Yeda Research And Development Co. Ltd. MIR-135
US10760086B2 (en) 2011-09-13 2020-09-01 Monsanto Technology Llc Methods and compositions for weed control
US10829828B2 (en) 2011-09-13 2020-11-10 Monsanto Technology Llc Methods and compositions for weed control
UA116089C2 (en) 2011-09-13 2018-02-12 Монсанто Текнолоджи Ллс Methods and compositios for weed control
US10806146B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US10184151B2 (en) * 2011-10-11 2019-01-22 The Brigham And Women's Hospital, Inc. Micrornas in neurodegenerative disorders
US9593331B2 (en) 2011-11-02 2017-03-14 Osaka City University Double-stranded nucleic acid molecule for gene expression control
US20130273655A1 (en) * 2012-03-12 2013-10-17 Shannon Eaker ENGRAFTMENT POTENTIAL OF EMBRYONIC STEM CELLS AND PLURIPOTENT STEM CELLS USING MICRO RNAs
MX360866B (en) 2012-05-24 2018-11-09 A B Seeds Ltd Compositions and methods for silencing gene expression.
WO2013180038A1 (en) 2012-05-26 2013-12-05 株式会社ボナック Single-stranded nucleic acid molecule for regulating expression of gene having delivering function
ES2534210T3 (en) * 2012-08-29 2015-04-20 Chanel Parfums Beauté Micro-RNA inhibitors for use to prevent and / or reduce skin aging
US9096853B2 (en) * 2012-09-24 2015-08-04 U.S. Department Of Veterans Affairs Modified siRNA molecules incorporating 5-fluoro-2′-deoxyuridine residues to enhance cytotoxicity
US9694050B2 (en) 2012-10-21 2017-07-04 University Of Rochester THY1 (CD90) as a novel therapy to control adipose tissue accumulation
WO2014164797A2 (en) 2013-03-13 2014-10-09 Monsanto Technology Llc Methods and compositions for weed control
UY35379A (en) 2013-03-13 2014-09-30 Monsanto Technology Llc ? METHODS AND COMPOSITIONS FOR WEED CONTROL ?.
US10568328B2 (en) 2013-03-15 2020-02-25 Monsanto Technology Llc Methods and compositions for weed control
WO2014169078A2 (en) 2013-04-09 2014-10-16 Boston Biomedical, Inc. Methods for treating cancer
MX359191B (en) 2013-07-19 2018-09-18 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa.
UA120426C2 (en) 2013-11-04 2019-12-10 Монсанто Текнолоджі Елелсі Compositions and methods for controlling arthropod parasite and pest infestations
UA119253C2 (en) 2013-12-10 2019-05-27 Біолоджикс, Інк. Compositions and methods for virus control in varroa mite and bees
JP6486836B2 (en) 2013-12-26 2019-03-20 学校法人東京医科大学 Artificial mimic miRNA for gene expression control and use thereof
AU2014370829B2 (en) * 2013-12-27 2021-03-11 Bonac Corporation Artificial match-type miRNA for controlling gene expression and use therefor
EP3116303B1 (en) 2014-01-15 2020-07-22 Monsanto Technology LLC Methods and compositions for weed control using epsps polynucleotides
CN106715695B (en) 2014-02-05 2020-07-31 耶达研究及发展有限公司 micro-RNAs for therapy and diagnosis and compositions comprising same
TW201620525A (en) * 2014-03-14 2016-06-16 波士頓生醫公司 Asymmetric interfering RNA compositions that silence K-RAS and methods of uses thereof
WO2015153339A2 (en) 2014-04-01 2015-10-08 Monsanto Technology Llc Compositions and methods for controlling insect pests
EP3145550B1 (en) * 2014-05-19 2019-03-27 BioNTech AG Particles comprising protamine and rna in combination with endosome destabilizing agents
CA2953347A1 (en) * 2014-06-23 2015-12-30 Monsanto Technology Llc Compositions and methods for regulating gene expression via rna interference
EP3161138A4 (en) 2014-06-25 2017-12-06 Monsanto Technology LLC Methods and compositions for delivering nucleic acids to plant cells and regulating gene expression
CN114009454A (en) 2014-07-29 2022-02-08 孟山都技术公司 Compositions and methods for controlling insect pests
RU2749882C2 (en) 2014-11-14 2021-06-18 Вояджер Терапьютикс, Инк. Modulating polynucleotides
EP3218484A4 (en) 2014-11-14 2018-05-30 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (als)
JP6637652B2 (en) * 2014-11-21 2020-01-29 株式会社バイオシンクタンク Method for selecting antitumor agent and siRNA contained in the antitumor agent
SG11201705223XA (en) 2014-12-27 2017-07-28 Bonac Corp NATURALLY OCCURING miRNA FOR CONTROLLING GENE EXPRESSION, AND USE OF SAME
PL3256589T3 (en) 2015-01-22 2022-02-21 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
CN104673796A (en) * 2015-02-09 2015-06-03 暨南大学 Small-interference RNA of target HSV-1 virus UL18 gene and application of small-interference RNA
JP2018509912A (en) * 2015-03-16 2018-04-12 ミラゲン セラピューティクス， インコーポレイテッド MiRNA mimics and their use in treating sensory conditions
CN108064289A (en) 2015-03-27 2018-05-22 株式会社博纳克 Single stranded nucleic acid molecule with delivery functions and gene expression regulation ability
AU2016270870A1 (en) 2015-06-02 2018-01-04 Monsanto Technology Llc Compositions and methods for delivery of a polynucleotide into a plant
AU2016270913A1 (en) 2015-06-03 2018-01-04 Monsanto Technology Llc Methods and compositions for introducing nucleic acids into plants
EA201792623A1 (en) 2015-06-03 2018-04-30 Бостон Биомедикал, Инк. COMPOSITIONS CONTAINING CANCER STEM BONE INHIBITOR AND IMMUNOTHERAPEUTIC AGENT TO BE USED IN CANCER TREATMENT
RU2718534C2 (en) 2015-06-05 2020-04-08 Мираджен Терапьютикс, Инк. Mir-155 inhibitors for treatment of cutaneous t-cell lymphoma (ctcl)
EP3334499A4 (en) 2015-08-14 2019-04-17 University of Massachusetts Bioactive conjugates for oligonucleotide delivery
GB201516685D0 (en) * 2015-09-21 2015-11-04 Varghese Oommen P And Oommen Oommen P Nucleic acid molecules with enhanced activity
CA3011894A1 (en) 2016-01-31 2017-08-03 University Of Massachusetts Branched oligonucleotides
EP3414329A1 (en) * 2016-02-12 2018-12-19 The U.S.A. as represented by the Secretary, Department of Health and Human Services Rna/dna hybrid nanoparticles modified with single stranded rna toeholds and uses thereof
MA45328A (en) 2016-04-01 2019-02-06 Avidity Biosciences Llc NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF
RU2758488C2 (en) 2016-05-18 2021-10-28 Вояджер Терапьютикс, Инк. Modulating polynucleotides
JP7220080B2 (en) 2016-05-18 2023-02-09 ボイジャー セラピューティクス インコーポレイテッド Compositions and methods for treating Huntington's disease
CA3029596A1 (en) 2016-06-28 2018-01-04 Boston Biomedical, Inc. Methods for treating cancer
CN107630015B (en) * 2016-07-19 2021-03-09 上海市东方医院 Stable DNA-RNA double-stranded structure
CA3033368A1 (en) 2016-08-12 2018-02-15 University Of Massachusetts Conjugated oligonucleotides
JP6842779B2 (en) * 2016-10-31 2021-03-17 国立大学法人岐阜大学 Double-stranded nucleic acid molecules and their uses
US11299469B2 (en) 2016-11-29 2022-04-12 Sumitomo Dainippon Pharma Oncology, Inc. Naphthofuran derivatives, preparation, and methods of use thereof
JP6962600B2 (en) * 2017-01-31 2021-11-05 キュリジン カンパニー，リミテッド Nucleic acid that simultaneously suppresses the expression of the MTOR gene and the STAT3 gene
WO2018160887A1 (en) * 2017-03-01 2018-09-07 Boston Biomedical, Inc. Pdl1-specific and beta-catenin-specific asymmetric interfering rna compositions, uses or preparation thereof
WO2018204803A1 (en) 2017-05-05 2018-11-08 Voyager Therapeutics, Inc. Compositions and methods of treating huntington's disease
WO2018204786A1 (en) 2017-05-05 2018-11-08 Voyager Therapeutics, Inc. Compositions and methods of treating amyotrophic lateral sclerosis (als)
CA3062656A1 (en) 2017-05-17 2018-11-22 Boston Biomedical, Inc. Methods for treating cancer
EP3642341A4 (en) 2017-06-23 2021-06-16 University Of Massachusetts Two-tailed self-delivering sirna and related methods
US20200115710A1 (en) * 2017-06-30 2020-04-16 National University Corporation Tokyo Medical And Dental University HETERO DOUBLE-STRANDED antimiR
US12054711B2 (en) 2017-07-14 2024-08-06 City Of Hope Meta-stable oligonucleotides junctions for delivery of therapeutics
EP4124658A3 (en) 2017-10-16 2023-04-19 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (als)
EP3697908A1 (en) 2017-10-16 2020-08-26 Voyager Therapeutics, Inc. Treatment of amyotrophic lateral sclerosis (als)
WO2019103581A1 (en) * 2017-11-27 2019-05-31 (주)프로스테믹스 Double stranded oligonucleotide and method for manufacturing same
AU2018378812A1 (en) 2017-12-06 2020-07-09 Avidity Biosciences, Inc. Compositions and methods of treating muscle atrophy and myotonic dystrophy
WO2019195765A1 (en) 2018-04-06 2019-10-10 University Of Massachusetts Mlk-regulated micrornas in angiogenesis and tumor development
WO2020027641A1 (en) * 2018-08-03 2020-02-06 주식회사 레모넥스 Pharmaceutical composition for preventing or treating atopic diseases
JP2021533762A (en) 2018-08-10 2021-12-09 ユニバーシティ・オブ・マサチューセッツUniversity Of Massachusetts Modified oligonucleotides that target SNPs
KR102321425B1 (en) * 2019-01-18 2021-11-04 올릭스 주식회사 Asymmetric siRNA Inhibiting Expression of NRL
MX2021013418A (en) * 2019-05-03 2021-12-10 Dicerna Pharmaceuticals Inc Double-stranded nucleic acid inhibitor molecules with shortened sense strands.
EP4010476A4 (en) 2019-08-09 2023-12-27 University Of Massachusetts Chemically modified oligonucleotides targeting snps
CA3172111A1 (en) 2020-03-19 2021-09-23 Barbora MALECOVA Compositions and methods of treating facioscapulohumeral muscular dystrophy
WO2022240824A1 (en) 2021-05-13 2022-11-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Compositions and methods for treating sickle cell diseases
AU2022286925A1 (en) 2021-05-29 2023-12-21 1Globe Health Institute Llc Short duplex dna as a novel gene silencing technology and use thereof
AR126207A1 (en) 2021-06-23 2023-09-27 Univ Massachusetts ANTI-FLT1 OLIGONUCLEOTIDE COMPOUNDS OPTIMIZED FOR THE TREATMENT OF PRE-ECLAMPIA AND OTHER ANGIOGENIC DISORDERS
CN118202046A (en) * 2021-07-22 2024-06-14 中天(上海)生物科技有限公司 Modified small interfering RNA molecules with reduced off-target effects
AU2022345098A1 (en) 2021-09-16 2024-04-04 Avidity Biosciences, Inc. Compositions and methods of treating facioscapulohumeral muscular dystrophy
TW202333750A (en) * 2021-11-08 2023-09-01 美商黛瑟納製藥公司 Rnai oligonucleotide conjugates
WO2023207615A1 (en) * 2022-04-26 2023-11-02 南京明德新药研发有限公司 Class of double-stranded rnai compounds containing overhang consisting of natural nucleotides
WO2024124216A1 (en) * 2022-12-08 2024-06-13 1Globe Health Institute Llc Short duplex rna with interspersed deoxyribonucleotides as a gene silencing technology and use thereof

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ308053B6 (en) 2000-12-01 2019-11-27 Max Planck Gesellschaft Isolated double-stranded RNA molecule, process for producing it and its use
US20070042983A1 (en) * 2001-05-18 2007-02-22 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using short interfering nucleic acid (siNA)
US20060211642A1 (en) * 2001-05-18 2006-09-21 Sirna Therapeutics, Inc. RNA inteference mediated inhibition of hepatitis C virus (HVC) gene expression using short interfering nucleic acid (siNA)
US6962944B2 (en) 2001-07-31 2005-11-08 Arqule, Inc. Pharmaceutical compositions containing beta-lapachone, or derivatives or analogs thereof, and methods of using same
US7074824B2 (en) 2001-07-31 2006-07-11 Arqule, Inc. Pharmaceutical compositions containing beta-lapachone, or derivatives or analogs thereof, and methods of using same
DK2284266T3 (en) * 2002-11-14 2014-01-13 Thermo Fisher Scient Biosciences Inc SIRNA MOLECULE MOD TP53
US20040248299A1 (en) * 2002-12-27 2004-12-09 Sumedha Jayasena RNA interference
US20050266552A1 (en) 2003-12-05 2005-12-01 Doench John G Reagents and methods for identification of RNAi pathway genes and chemical modulators of RNAi
WO2005079533A2 (en) * 2004-02-17 2005-09-01 University Of Massachusetts Methods and compositions for mediating gene silencing
WO2005086896A2 (en) * 2004-03-10 2005-09-22 Ulrich Thomann Delivery vectors for short interfering rna, micro-rna and antisense rna
CA2559955C (en) * 2004-03-15 2016-02-16 City Of Hope Methods and compositions for the specific inhibition of gene expression by double-stranded rna
US20060142228A1 (en) 2004-12-23 2006-06-29 Ambion, Inc. Methods and compositions concerning siRNA's as mediators of RNA interference
JP2010537640A (en) 2007-08-27 2010-12-09 ボストン バイオメディカル， インコーポレイテッド Compositions of asymmetric RNA duplexes as microRNA mimetics or inhibitors
KR100949791B1 (en) * 2007-12-18 2010-03-30 이동기 Novel siRNA Structure for Minimizing Off-target Effects and Relaxing Saturation of RNAi Machinery and the Use Thereof
TW201620525A (en) 2014-03-14 2016-06-16 波士頓生醫公司 Asymmetric interfering RNA compositions that silence K-RAS and methods of uses thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None
See also references of EP2193140A4

Cited By (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10266821B2 (en) 2007-08-27 2019-04-23 1Globe Health Institute Llc Compositions of asymmetric interfering RNA and uses thereof
EP2222848A4 (en) * 2007-12-18 2011-06-22 Lee Dong Ki Novel sirna structure for minimizing off-target effects and relaxing saturation of rnai machinery and the use thereof
EP2222848A2 (en) * 2007-12-18 2010-09-01 Lee, Dong Ki Novel sirna structure for minimizing off-target effects and relaxing saturation of rnai machinery and the use thereof
JP2011505868A (en) * 2007-12-18 2011-03-03 ドンギ イ Novel siRNA structure that suppresses off-target effect as much as possible and does not saturate RNAi mechanism and use thereof
US10774330B2 (en) 2008-09-22 2020-09-15 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAI compounds
US10876119B2 (en) 2008-09-22 2020-12-29 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAI compounds
US11396654B2 (en) 2008-09-22 2022-07-26 Phio Pharmaceuticals Corp. Neutral nanotransporters
US9938530B2 (en) 2008-09-22 2018-04-10 Rxi Pharmaceuticals Corporation RNA interference in skin indications
US10815485B2 (en) 2008-09-22 2020-10-27 Phio Pharmaceuticals Corp. RNA interference in skin indications
US10041073B2 (en) 2008-09-22 2018-08-07 Rxi Pharmaceuticals Corporation Reduced size self-delivering RNAi compounds
US10138485B2 (en) 2008-09-22 2018-11-27 Rxi Pharmaceuticals Corporation Neutral nanotransporters
WO2010039536A2 (en) 2008-09-23 2010-04-08 President And Fellows Of Harvard College Sirt4 and uses thereof
EP2352744A2 (en) * 2008-10-15 2011-08-10 Somagenics, Inc. Short hairpin rnas for inhibition of gene expression
EP2352744A4 (en) * 2008-10-15 2013-04-10 Somagenics Inc Short hairpin rnas for inhibition of gene expression
US8779115B2 (en) 2008-10-15 2014-07-15 Somagenics Inc. Short hairpin RNAs for inhibition of gene expression
US10167471B2 (en) 2009-01-05 2019-01-01 Rxi Pharmaceuticals Corporation Inhibition of PCSK9 through RNAI
US9745574B2 (en) 2009-02-04 2017-08-29 Rxi Pharmaceuticals Corporation RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
US11667915B2 (en) 2009-02-04 2023-06-06 Phio Pharmaceuticals Corp. RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
US10479992B2 (en) 2009-02-04 2019-11-19 Phio Pharmaceuticals Corp. RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality
CN103555722A (en) * 2009-04-03 2014-02-05 戴瑟纳制药公司 Methods and compositions for the specific inhibition of kras by asymmetric double-stranded rna
CN103555722B (en) * 2009-04-03 2016-08-31 戴瑟纳制药公司 Asymmetric double-stranded RNA is utilized specifically to suppress the method and composition of KRAS
US10870850B2 (en) 2009-07-13 2020-12-22 Somagenics, Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression
US9816091B2 (en) 2009-07-13 2017-11-14 Somagenics, Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression
US8871730B2 (en) 2009-07-13 2014-10-28 Somagenics Inc. Chemical modification of short small hairpin RNAs for inhibition of gene expression
WO2011041582A2 (en) 2009-09-30 2011-04-07 President And Fellows Of Harvard College Methods for modulation of autophagy through the modulation of autophagy-inhibiting gene products
WO2011041584A2 (en) 2009-09-30 2011-04-07 President And Fellows Of Harvard College Methods for modulation of autophagy through the modulation of autophagy-enhancing gene products
US11359201B2 (en) 2009-10-12 2022-06-14 Larry J. Smith Methods and compositions for modulating gene expression using oligonucleotide based drugs administered in vivo or in vitro
US10392619B2 (en) 2009-10-12 2019-08-27 Larry J. Smith Methods and compositions for modulating gene expression using oligonucleotide based drugs administered in vivo or in vitro
EP2513334A1 (en) * 2009-12-18 2012-10-24 Dicerna Pharmaceuticals, Inc. Dicer substrate agents and methods for the specific inhibition of gene expression
JP2013514089A (en) * 2009-12-18 2013-04-25 ダイセルナ ファーマシューティカルズ， インコーポレイテッド Dicer substrate agents and methods for specific inhibition of gene expression
EP2513334A4 (en) * 2009-12-18 2014-01-08 Dicerna Pharmaceuticals Inc Dicer substrate agents and methods for the specific inhibition of gene expression
US10240149B2 (en) 2010-03-24 2019-03-26 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAi compounds
US11118178B2 (en) 2010-03-24 2021-09-14 Phio Pharmaceuticals Corp. Reduced size self-delivering RNAI compounds
US10913948B2 (en) 2010-03-24 2021-02-09 Phio Pharmaceuticals Corp. RNA interference in dermal and fibrotic indications
US10184124B2 (en) 2010-03-24 2019-01-22 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US10662430B2 (en) 2010-03-24 2020-05-26 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US11584933B2 (en) 2010-03-24 2023-02-21 Phio Pharmaceuticals Corp. RNA interference in ocular indications
US9080171B2 (en) 2010-03-24 2015-07-14 RXi Parmaceuticals Corporation Reduced size self-delivering RNAi compounds
US9963702B2 (en) 2010-03-24 2018-05-08 Rxi Pharmaceuticals Corporation RNA interference in dermal and fibrotic indications
US10214744B2 (en) 2010-10-22 2019-02-26 Sungkyunkwan University Foundation For Corporate Collaboration Nucleic acid molecules inducing RNA interference, and uses thereof
US9637742B2 (en) 2010-10-22 2017-05-02 Sungkyunkwan University Foundation For Corporate Collaboration Nucleic acid molecules inducing RNA interference, and uses thereof
US10829760B2 (en) 2010-10-22 2020-11-10 Olix Pharmaceuticals, Inc. Nucleic acid molecules inducing RNA interference, and uses thereof
CN101979558A (en) * 2010-10-28 2011-02-23 百奥迈科生物技术有限公司 Small interfering RNA (siRNA) molecule of targeted hepatitis B virus (HBV) gene and application thereof
US10125362B2 (en) 2012-05-22 2018-11-13 Olix Pharmaceuticals, Inc. RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor
US10883105B2 (en) 2012-05-22 2021-01-05 Olix Pharmaceuticals, Inc. RNA-interference-inducing nucleic acid molecule able to penetrate into cells, and use therefor
WO2014089121A3 (en) * 2012-12-03 2014-07-31 Thomas Ichim Retrograde delivery of cells and nucleic acids for treatment of cardiovascular diseases
WO2014089121A2 (en) * 2012-12-03 2014-06-12 Thomas Ichim Retrograde delivery of cells and nucleic acids for treatment of cardiovascular diseases
EP3845248A1 (en) 2013-02-05 2021-07-07 1Globe Health Institute LLC Biodegradable and clinically-compatible nanoparticles as drug delivery carriers
US11001840B2 (en) 2013-02-05 2021-05-11 1Globe Health Institute Llc Biodegradable and clinically-compatible nanoparticles as drug delivery carriers
EP2953646A4 (en) * 2013-02-05 2017-02-15 1Globe Health Institute LLC Biodegradable and clinically-compatible nanop articles as drug delivery carriers
US10934550B2 (en) 2013-12-02 2021-03-02 Phio Pharmaceuticals Corp. Immunotherapy of cancer
US11279934B2 (en) 2014-04-28 2022-03-22 Phio Pharmaceuticals Corp. Methods for treating cancer using nucleic acids targeting MDM2 or MYCN
WO2015182913A1 (en) * 2014-05-27 2015-12-03 주식회사 제노포커스 Method for overexpression of target protein by gene silencing
US11926828B2 (en) 2014-09-05 2024-03-12 Phio Pharmaceuticals Corp. Methods for treating aging and skin disorders using nucleic acids targeting TYR or MMP1
US10900039B2 (en) 2014-09-05 2021-01-26 Phio Pharmaceuticals Corp. Methods for treating aging and skin disorders using nucleic acids targeting Tyr or MMP1
US10405749B2 (en) 2014-12-26 2019-09-10 Nitto Denko Corporation RNA agents for P21 gene modulation
EP3236975A4 (en) * 2014-12-26 2018-09-12 Nitto Denko Corporation Rna agents for p21 gene modulation
USRE49431E1 (en) 2014-12-26 2023-02-28 Nitto Denko Corporation RNA interference agents for GST-PI gene modulation
CN108064155B (en) * 2014-12-26 2021-06-08 日东电工株式会社 RNA agents for P21 gene regulation
CN108064155A (en) * 2014-12-26 2018-05-22 日东电工株式会社 For the RNA agent of P21 Gene regulations
US11045488B2 (en) 2014-12-26 2021-06-29 Nitto Denko Corporation RNA interference agents for GST-π gene modulation
US10808247B2 (en) 2015-07-06 2020-10-20 Phio Pharmaceuticals Corp. Methods for treating neurological disorders using a synergistic small molecule and nucleic acids therapeutic approach
US11001845B2 (en) 2015-07-06 2021-05-11 Phio Pharmaceuticals Corp. Nucleic acid molecules targeting superoxide dismutase 1 (SOD1)
US11021707B2 (en) 2015-10-19 2021-06-01 Phio Pharmaceuticals Corp. Reduced size self-delivering nucleic acid compounds targeting long non-coding RNA
US10590423B2 (en) 2015-11-16 2020-03-17 Olix Pharmaceuticals, Inc. Treatment of age-related macular degeneration using RNA complexes that target MyD88 or TLR3
WO2017100127A1 (en) 2015-12-06 2017-06-15 Boston Biomedical, Inc. ASYMMETRIC INTERFERING RNAs, AND COMPOSITIONS, USE, OR PREPARATION THEREOF
US10358647B2 (en) 2015-12-13 2019-07-23 Nitto Denko Corporation siRNA structures for high activity and reduced off target
US11926831B2 (en) 2015-12-13 2024-03-12 Nitto Denko Corporation SiRNA structures for high activity and reduced off target
US11390871B2 (en) 2015-12-13 2022-07-19 Nitto Denko Corporation SiRNA structures for high activity and reduced off target
US10519449B2 (en) 2016-02-02 2019-12-31 Olix Pharmaceuticals, Inc. Treatment of angiogenesis-associated diseases using RNA complexes that target ANGPT2 and PDGFB
US10947541B2 (en) 2016-02-02 2021-03-16 Olix Pharmaceuticals, Inc. Treatment of atopic dermatitis and asthma using RNA complexes that target IL4Rα, TRPA1, or F2RL1
US10829761B2 (en) 2016-04-11 2020-11-10 Olix Pharmaceuticals, Inc. Treatment of idiopathic pulmonary fibrosis using RNA complexes that target connective tissue growth factor
US11040057B2 (en) 2016-06-29 2021-06-22 Olix Pharmaceuticals, Inc. Pharmaceutical compositions and methods for potentiating gene silencing
WO2018098352A2 (en) 2016-11-22 2018-05-31 Jun Oishi Targeting kras induced immune checkpoint expression
US11591600B2 (en) 2017-02-10 2023-02-28 OliX Pharmaceuticals. Inc. Long double-stranded RNA for RNA interference
WO2019074071A1 (en) 2017-10-11 2019-04-18 日東電工株式会社 Regulation of nucleic acid molecule expression
US11298371B2 (en) 2017-10-11 2022-04-12 Nitto Denko Corporation Regulation of nucleic acid molecule expression
WO2022175749A1 (en) 2021-02-18 2022-08-25 Oneglobe Holdings Limited Compositions for conjugating oligonucleotides and carbohydrates
WO2022256351A1 (en) 2021-05-29 2022-12-08 1Globe Health Institute Llc Asymmetric short duplex dna as a novel gene silencing technology and use thereof
WO2024040531A1 (en) 2022-08-25 2024-02-29 1Globe Health Institute, Nanjing Novel compositions for conjugating oligonucleotides and carbohydrates

Also Published As

Publication number Publication date
CN105018492A (en) 2015-11-04
US20100286378A1 (en) 2010-11-11
HK1217349A1 (en) 2017-01-06
WO2009029690A1 (en) 2009-03-05
EP3121281A1 (en) 2017-01-25
JP2016171815A (en) 2016-09-29
CA2735167A1 (en) 2009-03-05
JP2010537640A (en) 2010-12-09
US10927376B2 (en) 2021-02-23
US11015195B2 (en) 2021-05-25
CN101835789A (en) 2010-09-15
JP6236498B2 (en) 2017-11-22
JP2017127333A (en) 2017-07-27
US10266821B2 (en) 2019-04-23
JP5986596B2 (en) 2016-09-06
US9328345B2 (en) 2016-05-03
EP2193140A4 (en) 2011-07-13
JP2010537639A (en) 2010-12-09
CA2735166C (en) 2020-12-01
EP2201022A4 (en) 2012-01-04
EP2193140B1 (en) 2016-11-02
EP2201022A1 (en) 2010-06-30
US20200063128A1 (en) 2020-02-27
HK1148534A1 (en) 2011-09-09
CA2735166A1 (en) 2009-03-05
ES2617877T3 (en) 2017-06-20
EP3121281B1 (en) 2021-03-31
CN105018492B (en) 2018-08-24
EP3889261A1 (en) 2021-10-06
WO2009029688A3 (en) 2009-07-02
CN101842381A (en) 2010-09-22
ES2873350T3 (en) 2021-11-03
EP2193140A2 (en) 2010-06-09
US20200071696A1 (en) 2020-03-05
JP6453275B2 (en) 2019-01-16
US20160304868A1 (en) 2016-10-20
JP2014097072A (en) 2014-05-29
JP2016168050A (en) 2016-09-23
JP2015130885A (en) 2015-07-23
DK2193140T3 (en) 2017-01-23
US20090208564A1 (en) 2009-08-20
CN101835789B (en) 2015-06-24
US20210301292A1 (en) 2021-09-30

Similar Documents

Publication Publication Date Title
US10927376B2 (en) Compositions of asymmetric interfering RNA and uses thereof
US11359198B2 (en) Extended dicer substrate agents and methods for the specific inhibition of gene expression
JP2014097072A5 (en)
CA2784252C (en) Dicer substrate agents and methods for the specific inhibition of gene expression
US11597929B2 (en) Extended dicer substrate agents and methods for the specific inhibition of gene expression

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200880113236.9

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08828867

Country of ref document: EP

Kind code of ref document: A2

ENP Entry into the national phase

Ref document number: 2010523120

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

REEP Request for entry into the european phase

Ref document number: 2008828867

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2008828867

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2735166

Country of ref document: CA