HK1217349B - Compositions of asymmetric interfering rna and uses thereof - Google Patents

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 therapeutic agents.

Description

Compositions for asymmetrically interfering RNA and uses thereof

The application date is 2008, 8 and 27, the date of the PCT application entering the country is 2010, 4 and 26, and the application numbers are as follows: 200880113236.9 entitled "composition of asymmetric interfering RNA and uses thereof".

Technical Field

The invention relates to a composition of asymmetric interfering RNA and application thereof.

Background

Gene silencing via RNAi (RNA interference) through the use of small or short interfering RNAs (siRNAs) has emerged as a powerful molecular biological tool and has potential for therapeutic applications (de Fougerolles et al, 2007; Kim and Rossi, 2007).

It is theorized that RNAi can be used to specifically and efficiently knock down (knock down) or silence any disease gene. The range of possible applications of RNAi for therapeutic purposes is broad, including genetic, epigenetic and infectious diseases, provided that the causative gene is identified.

However, in addition to the outstanding delivery problems, the development of RNAi-based drugs faces the following problems: limited efficacy of siRNA, non-specific effects of siRNA such as interferon-like response and sense strand mediated off-target gene silencing, and prohibitively high or high costs associated with siRNA synthesis. The gene silencing efficacy of siRNA is limited to 50% or less for most genes in mammalian cells. These molecules are expensive to manufacture (much more expensive than antisense deoxynucleotides), inefficient, and require chemical modification. Finally, it was observed that extracellular administration of synthetic siRNAs may trigger interferon-like responses, which adds a significant obstacle to RNAi-based research and RNAi-based therapeutic development.

RNAi can be triggered by synthetic short interfering RNAs (siRNAs), or genetic elements encoding short hairpin RNAs (shRNAs) that can be subsequently cleaved into siRNAs by the ribonuclease III-like enzyme Dicer. The biochemical mechanisms of gene silencing are not fully understood and appear to involve the multiprotein RNA-induced silencing complex (RISC). RISC binds, unfolds, and incorporates antisense siRNA strands, which then recognize and target the cleavage of fully complementary mRNA, thereby reducing gene expression. Effective gene silencing (1-10 days) can be attributed to the catalytic properties of the RISC complex. The strength 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 an interferon-like response activated by siRNA, which is mediated by dsRNA-dependent Protein Kinases (PKRs) and Toll-like receptors (TLRs). The ability of siRNA to induce an interferon-like response is largely determined by its length. (same as above).

For gene silencing in mammalian cells, the prior art teaches that the structure of the siRNA can be a symmetric double stranded RNA 19-21 nucleotides in length and having a 3' overhang at 2 ends to be effective in mammalian cells and to avoid the cellular innate "anti-viral" response. (same as above). It is now well established in the art that this "optimal" structure still triggers an interferon response, constituting a significant obstacle to RNAi-based research and RNAi-based therapeutic development (Sledz et al, 2003).

There is a need to develop new methods for achieving effective RNAi in mammalian cells by newly designing siRNAs with better efficacy and potency, fast onset of action, better persistence, and RNA duplexes with shorter length to avoid non-specific interferon-like responses and reduce the synthetic costs for research and drug development of RNAi therapeutics.

Citation of a reference herein shall not be construed as an admission that it is prior art to the claimed invention.

Disclosure of Invention

The present invention relates to the surprising discovery of a new class of small duplex RNA, which can induce efficient gene silencing in mammalian cells, referred to herein as asymmetric interfering RNA (airna). The hallmark of this new class of RNAi inducers is the asymmetry in the length of the 2 RNA strands. This new structural design is not only functionally effective in achieving gene silencing, but also provides several advantages over prior art siRNAs. Among these advantages, aiRNA can have a much shorter RNA duplex structure than existing siRNA lengths, which will reduce synthesis costs and eliminate/reduce the triggering of length-dependent non-specific interferon-like responses. In addition, asymmetry in aiRNA structure also eliminates/reduces sense strand mediated off-target effects. In addition, aiRNA is more effective, potent, rapid onset and persistent in inducing gene silencing than siRNA. aiRNA can be used in all areas where siRNA or shRNA are currently being applied or considered for use, including biological research, R & D research in biotechnology and pharmaceutical industries, and RNAi-based therapies.

The present invention provides duplex RNA molecules. The duplex RNA molecule comprises a first strand having a length of 18-23 nucleotides and a second strand having a length of 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 a3 '-overhang of 1-9 nucleotides and a 5' -overhang of 0-8 nucleotides, wherein the duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell. In one embodiment, the first strand comprises a sequence that is substantially complementary to a target mRNA sequence. In yet another embodiment, the first strand comprises a sequence that is at least 70% complementary to the target mRNA sequence. In another embodiment, the eukaryotic cell is a mammalian cell or an avian cell.

In one embodiment, at least one nucleotide of the sequence of the 5' overhang is selected from A, U and dT.

In one embodiment, the GC content of the double-stranded region is 20% to 70%.

In one embodiment, the first strand has a length of 19-22 nucleotides.

In one embodiment, the first strand has a length of 21 nucleotides. In yet another embodiment, the second strand has a length of 14-16 nucleotides.

In one 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 a further embodiment, the first strand has a 3' -overhang of 3 nucleotides.

In one embodiment, the duplex RNA molecule comprises at least one modified nucleotide or an analogue thereof. In a further embodiment, the at least one modified nucleotide or analog thereof is a 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 one embodiment, the phosphodiester linkage is modified to include at least one of a nitrogen or sulfur heteroatom. In another embodiment, the nucleotide analog is a backbone-modified ribonucleotide that comprises a phosphorothioate group.

In one embodiment, the at least one modified nucleotide or analog thereof is a rare base or a modified base. In another embodiment, the at least one modified nucleotide or analog thereof comprises an inosine or a tritylated base.

In yet another embodiment, the nucleotide analog is a sugar-modified ribonucleotide wherein the 2' -OH group is replaced by a group selected from the group consisting of H, OR, R, halogen, SH, SR, NH₂、NHR、NR₂Or CN, wherein each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halogen is F, Cl, Br, or I.

In one embodiment, the first strand comprises at least one deoxynucleotide. In a further embodiment, the at least one deoxynucleotide is in one or more regions selected from the group consisting of a3 '-overhang, a 5' -overhang, and a 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 organism comprising the steps of: contacting said cell or organism with the duplex RNA molecule of claim 1 under conditions whereby selective gene silencing can occur, and mediating selective gene silencing by said duplex RNA molecule against a target gene or nucleic acid having a sequence portion substantially corresponding to said duplex RNA. In a further embodiment, the contacting step comprises the steps of: introducing the duplex RNA molecule into a target cell in culture or in an organism in which selective gene silencing can occur. In a still further embodiment, the introducing step is selected from transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical and regional administration. In another embodiment, the introducing step comprises the use of 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 (nanoemulsion), a lipid, and a lipoid (lipoid).

In one embodiment, the modulation method is used to determine the function or use of a gene in a cell or organism.

In one embodiment, the modulation method is used to treat or prevent a disease or an undesired condition.

In one embodiment, the target gene is associated with a disease, pathological condition, or undesired condition in a mammal. In a further embodiment, the target gene is a gene of a pathogenic microorganism. In a further embodiment, the target gene is a viral gene. In another embodiment, the target gene is a tumor-associated gene. In another embodiment, the target gene is a gene associated with a disease selected from the group consisting of: autoimmune diseases, 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 invention provides research reagents. The reagent comprises a duplex RNA molecule.

The invention further provides a kit. The kit comprises a first RNA strand having a length of 18-23 nucleotides and a second RNA strand having a length of 12-17 nucleotides, wherein the second strand is substantially complementary to the first strand and is capable of forming a duplex RNA molecule with the first strand, wherein the duplex RNA molecule has a3 '-overhang of 1-9 nucleotides and a 5' -overhang of 0-8 nucleotides, wherein the duplex RNA molecule is capable of effecting sequence-specific gene silencing in a eukaryotic cell.

The invention also provides methods of making duplex RNA molecules. The method comprises the following steps: the first and second strands are synthesized, and the synthesized strands are combined under conditions to form a duplex RNA molecule capable of effecting sequence-specific gene silencing. In one embodiment, the method of claim 35 further comprises the step of introducing at least one modified nucleotide or analog thereof 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 strand is chemically synthesized or biosynthesized.

The present invention provides expression vectors. The vector includes one or more nucleic acids encoding a duplex RNA molecule operably linked to at least one expression control sequence. In one embodiment, the vector comprises a first nucleic acid encoding a first strand operably linked to a first expression control sequence, and a second nucleic acid encoding a second strand operably linked to a second expression control sequence. In another embodiment, the vector is a viral, eukaryotic or bacterial expression vector.

The invention also provides cells. In one embodiment, the cell comprises a vector. In another embodiment, the cell comprises a duplex RNA molecule. In a further embodiment, the cell is a mammalian, avian or bacterial cell.

The present invention provides duplex RNA molecules. 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 the duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell. In one embodiment, the 2 ends of the duplex RNA molecule are independently selected from the group consisting of a 1-10 nucleotide 3 '-overhang, a 0-10 nucleotide 5' -overhang, and a blunt end. In one embodiment, the first strand is substantially complementary to the target mRNA sequence. In an alternative embodiment, the second strand is substantially complementary to the target mRNA sequence. In one 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 one embodiment, the first strand has a3 '-overhang of 1-8 nucleotides, and a 5' -overhang of 1-8 nucleotides.

In another embodiment, the first strand has a 3' -overhang of 1-10 nucleotides and a blunt end.

In another embodiment, the first strand has a 5' -overhang of 1-10 nucleotides and a blunt end.

In an alternative embodiment, the RNA duplex has two 5 '-overhangs of 1-8 nucleotides, or two 3' -overhangs of 1-10 nucleotides.

In one embodiment, the first strand has a length of 12-100 nucleotides, 12-30 nucleotides, 18-23 nucleotides, 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 of 5-30 nucleotides, 12-22 nucleotides, 12-17 nucleotides. In a further embodiment, the second strand has a length of 15 nucleotides.

In one embodiment, the first strand has a length of 12-30 nucleotides and the second strand has a length of 10-29 nucleotides, with the proviso that when the double stranded region is 27bp, the 2 ends of the RNA molecule are independently a3 'overhang or a 5' overhang. In a further embodiment, the first strand has a length of 18-23 nucleotides and the second strand has a length of 12-17 nucleotides.

In another embodiment, the first strand has a length of 19-25 nucleotides and the second strand has a length of 12-17 nucleotides.

In an alternative embodiment, the first strand has a length of 19-25 nucleotides and the second strand has a length of 18-24 nucleotides,

provided that when the first chain is

5’-UUCGAAGUAUUCCGCGUACGU(SEQ ID NO:1)

5' -UCGAAGUAUUCCGCGUACGUG (SEQ ID NO:2) or

5' -CGAAGUAUUCCGCGUACGUGA (SEQ ID NO:3)

The second strand has a length of at most 17 nucleotides, or comprises at least one mismatch with the first strand, or comprises at least one modification.

In one 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 one embodiment, the first strand is 1-10 nucleotides longer than the second strand.

In one embodiment, the 3' -overhang has a length of 2-6 nucleotides.

In another embodiment, the 5' -overhang has a length of 0-5 nucleotides.

In one embodiment, gene silencing includes one or both or all of RNA interference, translational regulation, and DNA epigenetic regulation.

In one 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 one 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 the 5' overhang is selected from A, U and dT.

In one embodiment, the duplex RNA molecule is conjugated to an entity selected from the group consisting of a peptide, an antibody, a polymer, a lipid, an oligonucleotide, cholesterol, and an aptamer (aptamer).

In one embodiment, the RNA molecule further comprises at least one unpaired or mismatched nucleotide.

In another embodiment, the GC content of the double-stranded region is 20-70%.

In one embodiment, the 3 '-overhang and/or the 5' -overhang are stabilized against degradation.

In one embodiment, the duplex RNA molecule comprises at least one modified nucleotide or an analogue thereof. In a further embodiment, the at least one modified nucleotide or analog thereof is a 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 sulfur heteroatom. In another embodiment, the nucleotide analog is a backbone-modified ribonucleotide that comprises a phosphorothioate group.

In one embodiment, the at least one modified nucleotide or analog thereof comprises a non-natural base or a modified base. In another embodiment, the at least one modified nucleotide or analog thereof comprises an inosine or a tritylated base.

In yet another embodiment, the nucleotide analog is a sugar-modified ribonucleotide wherein the 2' -OH group is replaced by a group selected from H, OR, R, halogen, SH, SR, NH2, NHR, NR2, OR CN, wherein each R is independently C1-C6 alkyl, alkenyl, OR alkynyl and the halogen is F, Cl, Br, OR I.

In one embodiment, the first strand comprises at least one deoxynucleotide. In a further embodiment, the at least one deoxynucleotide is in one or more regions selected from the group consisting of a 3' -overhang, a 5' -overhang, and a double-stranded region near the 5' -end of the first strand. In another embodiment, the second strand comprises at least one deoxynucleotide.

The invention also provides methods of modulating gene expression in a cell or organism. The method comprises the following steps: contacting the cell or organism with the duplex RNA molecule of claim 1 under conditions whereby selective gene silencing can occur, and effecting selective gene silencing by the double-stranded RNA molecule against a target nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA. In a further embodiment, the contacting comprises the steps of: introducing the duplex RNA molecule into a target cell in culture or in an organism in which selective gene silencing can occur. In a still further embodiment, the introducing step is selected from transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical and regional administration. In another embodiment, the introducing step comprises the use of 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 one embodiment, the method of modulation is for modulating expression of a gene in a cell or organism.

In another embodiment, the modulation method is used to treat or prevent a disease or an undesired condition.

In one embodiment, the target gene is a gene associated with a human or animal disease. In a further embodiment, the target gene is a gene of a pathogenic microorganism. In a further embodiment, the target gene is a viral gene. In another embodiment, the target gene is a tumor-associated gene.

In another embodiment, the target gene is a gene associated with a disease selected from the group consisting of: autoimmune diseases, 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 modulation methods may also be used to study drug targets in vitro or in vivo.

The invention provides reagents comprising duplex RNA molecules.

The invention also 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 is capable of forming a duplex RNA molecule with the first strand, wherein the 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 a first strand and a second strand, and combining the synthesized strands under conditions to form a duplex RNA molecule capable of effecting sequence-specific gene silencing. In one embodiment, the RNA strand is chemically synthesized or biosynthesized. In another embodiment, the first strand and the second strand are synthesized separately or simultaneously.

In one embodiment, the method further comprises the step of introducing at least one modified nucleotide or analog thereof into the duplex RNA molecule during the synthesizing step, after the synthesizing and before the combining step, or after the combining step.

The invention further provides pharmaceutical compositions. The pharmaceutical composition comprises at least one duplex RNA molecule as an active agent and one or more carriers selected from the group consisting of pharmaceutical carriers, positive charge carriers, liposomes, protein carriers, polymers, nanoparticles, cholesterol, lipids and lipids.

The invention also provides methods of treatment. The method comprises administering to a subject in need thereof an effective amount of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is administered via a route selected from iv, sc, inhalation, topical, po and regional administration.

In one embodiment, the first strand comprises a sequence substantially complementary to a target mRNA sequence of a gene selected from the group consisting of developmental genes, oncogenes, tumor suppressor genes and enzyme genes, as well as adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines or their receptors, growth/differentiation factors or their receptors, neurotransmitters or their receptors, kinases, signal transduction proteins, viral genes, infectious disease genes, ABLI, BCLl, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM 5, MLL, MDMA B, MYC, MYCL1, CN, NRAS, FAs 1, FAS, PAP family members, enzymes, PHR, ATP-A-PAP-A, ATP-G, ATP-DNA polymerase, ATP-DNA-lyase, xylanase.

In another embodiment, the duplex RNA molecule is selected from

Wherein A, U, G and C are nucleotides and a, t, g and C are deoxynucleotides.

The present invention provides methods of modifying a first duplex RNA molecule having an antisense strand and a sense strand that form a double-stranded region. The method comprises the following steps: shortening the length of the sense strand such that the antisense strand has a3 '-overhang of 1-8 nucleotides and a 5' -overhang of 0-8 nucleotides, and forming a second duplex RNA molecule; wherein at least one property of the first duplex RNA molecule is improved. In one embodiment, the property is selected from the group consisting of size, efficacy, potency, speed of onset, persistence, cost of synthesis, 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 to form a second duplex RNA molecule. In a further embodiment, the first duplex RNA molecule is an siRNA, or a dicer-substrate siRNA, or a precursor of an siRNA.

The present invention provides expression vectors. The vector comprises one or more nucleic acids encoding the duplex RNA molecule of claim 1 operably linked to at least one expression control sequence. In one embodiment, the expression vector comprises a first nucleic acid encoding a first strand operably linked to a first expression control sequence, and a second nucleic acid encoding a second strand operably linked to a second expression control sequence. In another embodiment, the expression vector is a viral, eukaryotic or bacterial expression vector.

The invention also provides cells. In one embodiment, the cell comprises an expression vector. In another embodiment, the cell comprises a duplex RNA molecule. In another embodiment, the cell is a mammalian cell, an avian cell, or a bacterial cell.

Other features and advantages of the invention will be apparent from the other descriptions provided herein, including the different embodiments. The examples provided illustrate different components and methods useful in practicing the present invention. The embodiments do not limit the claimed invention. Based on the present disclosure, one of ordinary skill in the art can identify and employ other components and methods useful for practicing the present invention.

Drawings

FIG. 1A shows the structure of a duplex RNA molecule with a3 '-overhang and a 5' -overhang on the first strand.

FIG. 1B shows the structure of a duplex RNA molecule with 3 '-and 5' -overhangs on the first strand and a nick in the duplex.

FIG. 1C shows the structure of a duplex RNA molecule with a3 '-overhang and a 5' -overhang on the first strand and a nick in the second strand.

FIG. 2A shows the structure of a duplex RNA molecule with one blunt end and a 5' -overhang on the first strand.

FIG. 2B shows the structure of a duplex RNA molecule with one blunt end and a 3' -overhang on the first strand.

Figure 2C shows the structure of a duplex RNA molecule with 3' -overhangs on both 2 ends of the duplex and a first strand longer than a second strand.

Figure 2D shows the structure of a duplex RNA molecule with 5' -overhangs on both 2 ends of the duplex and a first strand longer than a second strand.

FIG. 3 shows gene silencing of β -catenin induced by airRNA (asymmetric interfering RNAs).

FIG. 3A shows the validation of the oligomers. After annealing, the oligomer was confirmed by 20% polyacrylamide gel. Lane 1, 21nt/21 nt; lane 2, 12nt (a)/21 nt; lane 3, 12nt (b)/21 nt; lane 4, 13nt/13 nt; lane 5, 13nt/21 nt; lanes 6, 14nt/14 nt; lane 7, 14nt (a)/21 nt; lane 8, 14nt (b)/21 nt; lanes 9, 15nt/15 nt; lanes 10, 15nt/21 nt.

HeLa cells were seeded into 6-well plates at 200,000 cells/well 24 hours later with scramble siRNA (lane 1), 21-bp siRNA targeting E2F1 (lane 2, as a specific control) or 21-bp siRNA targeting β -catenin (lane 3, as a positive control), or a mixture of different lengths of airRNA at the same concentration 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), transfected, cells were harvested 48 hours after transfection. expression of the β -catenin is determined by western blot E2F1and actin are used as controls.

FIGS. 4 and 5 show the structure-activity relationship of the aiRNA oligos with or without base substitutions in mediating gene silencing Hela cells were transfected with the indicated aiRNA, cells were harvested 48 hours post transfection and lysates were generated, Western blots were performed to detect the levels of β -catenin and actin Si represents the β -catenin siRNA oligonucleotide.

Fig. 6 shows an analysis of the mechanism of aiRNA-triggered gene silencing.

FIG. 6a shows a northern blot analysis of β -catenin mRNA levels in cells transfected with airRNA or siRNA for the indicated days.

FIG. 6b shows a schematic representation of 5' -RACE-PCR of β -catenin showing mRNA cleavage and expected PCR products.

FIG. 6c shows the β -catenin cleavage product mediated by airRNA amplified by 5' -RACE-PCR from cells transfected with airRNA for 4 or 8 hours.

FIG. 6d shows a schematic representation of the β -catenin mRNA cleavage site verified by sequencing the 5' -RACE-PCR fragment.

FIG. 6e shows the differential RISC loading efficiency of airRNA and siRNA. AIRNA or siRNA duplexes were transfected into Hela cells 48 hours after transfection with pCMV-Ago 2. Ago2 was immunoprecipitated at the indicated time points after aiRNA or siRNA transfection and northern blot analysis was performed to determine the level of small RNAs bound to Ago 2/RISC. The level of Ago2 was determined by western blotting after IP (shown below).

FIG. 6f shows the effect of knocking down Ago2 or Dicer on the gene silencing activity of airRNA and siRNA. After transfecting cells with scramblesiRNA (siCon), or siRNAs targeting Ago2(siAgo2) or dicer (siDicer) for 24 hours, they were transfected with scramble airRNA (Con) or airRNA (ai) targeting Stat 3. Cells were harvested 48 hours after aitat 3 transfection and western blot analysis was performed.

FIG. 7 shows the advantage of airRNA incorporation into RISC compared to siRNA.

FIG. 7A shows that airRNA enters RISC more efficiently than siRNA. Cells transfected with Ago2 expression plasmid were transfected with aiRNA or siRNA for the indicated times. After cell lysis, Ago2 was immunoprecipitated, RNA was extracted from the immunoprecipitates, and separated on a 15% acrylamide gel. After transfer, the membrane is hybridized to a probe to detect the 21mer antisense strand of aiRNA or siRNA. The IgG control lane showed a lack of signal compared to Ago2 immunoprecipitates.

FIG. 7B shows that the sense strand of airRNA is not retained in RISC. Stripping off the probe from the membrane of (A) and probing again with the probe to detect the sense strand of the transfected oligo. (A) The cartoon figures in (a) and (B) illustrate the position of the sense strand (top strand), antisense strand (bottom strand), or duplex on the membrane.

FIG. 8 shows the RISC loading mechanism by airRNA.

Fig. 8A shows an immunoprecipitation analysis of the interaction between different strands of aiRNA or siRNA and Ago 2. Hela S-10 lysate comprising overexpressed Ago2 and methods of producing same³²The indicated aiRNA or siRNA duplexes of the P-terminally labeled sense or antisense strand were incubated together. The position of the marker is indicated by the asterisk. Following Ago2 immunoprecipitation, RNA was isolated and separated on a 15% acrylamide gel and the film was exposed. RNAs bound to Ago2 were shown in the pellet fraction, while RNA not bound to Ago2 remained in the supernatant (Sup).

Cells were transfected with aiRNA, or with aiRNA with the sense strand having a 2' -O-methyl group at position 8 (predicted Ago2 cleavage site) or position 9 (as control). RNA was collected 4 hours post transfection and qRT-PCR was performed to determine the relative level of remaining β -catenin mRNA.

FIG. 9 shows the airRNA and siRNA competition assay.

FIG. 9A is an example of a system including³²siRNA and aiRNA duplexes of the P-terminally labeled antisense strand. The (. + -.) number indicates the position of the marker.

Fig. 9B shows that cold aiRNA does not compete with labeled siRNA for Ago 2. Hela S-10 lysate comprising overexpressed Ago2 and³²p-end labeled siRNA and cold aiRNA or siRNA duplex were incubated together before immunoprecipitation of Ago 2. The RNA was subsequently isolated and analyzed on a 15% acrylamide gel.

Fig. 9C shows that cold siRNA does not compete with labeled aiRNA for Ago 2. The same S-10 lysate as used in B was mixed with³²P-end labeled aiRNA was incubated with cold aiRNA or siRNA duplexes prior to immunoprecipitation of Ago 2. The RNA was subsequently isolated and analyzed on a 15% acrylamide gel.

FIG. 10 illustrates models of airRNA and siRNA, showing the differences observed in RISC loading and mature RISC production.

FIG. 11 shows that 14-15bp asymmetric RNA duplexes (airRNAs) with antisense overhangs induce potent, efficient, rapid and durable gene silencing.

Fig. 11A is a schematic diagram showing the sequence and design of siRNA and aiRNA targeting β -catenin.

FIG. 11B shows gene silencing induced by various lengths of airRNA in cells transfected with the indicated airRNA for 48 hours, the protein level of β -catenin was analyzed by Western blotting.

Fig. 11C shows that aiRNA is more potent and effective than siRNA in inducing protein depletion of β -catenin Hela cells were transfected with either aiRNA or siRNA targeting β -catenin at the indicated concentrations, 48 hours post transfection, cell lysates were prepared and subjected to western blot analysis.

FIG. 11D shows that airRNA is more potent, rapid and durable than siRNA in reducing the RNA level of β -catenin cells were subjected to northern blot analysis after 10nM transfection with 15bp airRNA or 21-mer siRNA for the indicated days.

Fig. 12 shows that aiRNA mediates rapid and robust silencing.

Fig. 12A shows the sequences and structures of aiRNA and siRNA for targeting β -catenin.

Fig. 12B shows RT-pcr performed on levels of β -catenin mRNA from cells transfected with control aiRNA or with aiRNA targeting β -catenin.

FIG. 12C shows quantitative real-time RT-PCR for β -catenin mRNA levels in cells transfected with control, airRNA or siRNA for the indicated hours.

FIG. 12D shows Western blot analysis of protein levels of β -catenin in cells transfected with control, airRNA or siRNA for the indicated times.

Fig. 13 shows a comparison of aiRNA to siRNA in terms of efficacy and persistence of gene silencing against multiple targets Hela cells were transfected with scarmbled siRNA (c), aiRNA (ai) targeted to (a) β -catenin (at 10nM), (b) Stat3, (c) EF2, or (d) NQO1 (at 20nM) or siRNA (si) — RNA and protein were purified at the indicated time points and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) for mRNA levels and by western blot for protein levels.

FIG. 14 shows that airRNA mediated gene silencing was effective for various genes in various cell lines.

Fig. 14a shows that the aiRNA duplex targets β -catenin more efficiently than siRNA in different mammalian cell lines.

FIG. 14b shows Western blot analysis of Nbs1, survivin, Parp1, p21 from cells transfected with either airRNA or siRNA indicated at 20nM for 48 hours.

FIG. 14c shows Western blot analysis of Rsk1, PCNA, p70S6K, mTOR, and PTEN from cells transfected with either airRNA or siRNA indicated at 20nM for 48 hours.

FIG. 14d shows allele-specific gene silencing of k-Ras by airRNA. Silencing of k-Ras by aiRNA targeting wild-type k-Ras was tested in k-Ras wild-type (DLD1) and k-Ras mutant (SW480) cell lines by western blot analysis.

Fig. 15 shows the lack of off-target gene silencing of aiRNAs by sense strand, as well as the immunostimulatory effect and serum stability of aiRNAs.

FIG. 15a shows RT-PCR analysis of interferon inducible gene expression in PBMCs mock (mock) treated or incubated with β -catenin siRNA or airRNA duplexes for 16 hours.

FIG. 15b shows RT-PCR analysis of the expression of interferon inducible genes in Hela cells mock transfected or transfected with EF2 or survivin airRNA or siRNA for 24 hours.

Figure 15c shows microarray analysis of changes in expression of genes associated with known interferon responses. Total RNA isolated from ai RNA and siRNA transfected Hela cells was analyzed by microarray.

FIG. 15d shows that sense strand mediated off-target gene silencing was not detected for airRNA. 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 24 hours post transfection and RNA was collected and the relative levels of the Stat3 sense or antisense RNA (insert) were determined by quantitative real-time PCR or RT-PCR.

Fig. 15e shows the stability of the aiRNA and siRNA duplexes in human serum. The aiRNA and siRNA duplexes were incubated in 10% human serum at 37 ℃ for the indicated times, followed by gel electrophoresis. Remaining duplexes are shown (% control).

Fig. 15f illustrates the proposed model for aiRNA duplex-mediated gene-specific silencing.

Figure 16 shows potent antitumor activity of aiRNA against β -catenin in a SW480 human colon xenograft mouse model immunosuppressive mice with established subcutaneous SW480 human colon cancer were administered 0.6nmol PEI-complexed β -catenin siRNAs, PEI-complexed β -catenin aiRNAs, or PEI-complexed irrelevant sirna as negative controls intravenously every day (iv) tumor size was assessed periodically during treatment.

Figure 17 shows potent antitumor activity of aiRNA against β -catenin in HT29 human colon xenograft mouse model immunosuppressed mice with established subcutaneous HT29 human colon cancer were administered intravenously every other day (iv) with 0.6nmol PEI-complexed β -catenin siRNAs, PEI-complexed β -catenin aiRNAs, or PEI-complexed irrelevant sirna as a negative control.

Detailed Description

The present invention relates to asymmetric duplex RNA molecules capable of effecting selective gene silencing in eukaryotic cells. In one embodiment, the duplex RNA molecule comprises a first strand and a second strand. The first chain is longer than the second chain. The second strand is substantially complementary to the first strand and forms a duplex region with the first strand.

In one embodiment, the duplex RNA molecule has a3 '-overhang of 1-8 nucleotides and a 5' -overhang of 1-8 nucleotides, a3 '-overhang and a blunt end of 1-10 nucleotides, or a 5' -overhang and a blunt end of 1-10 nucleotides. In another embodiment, the duplex RNA molecule has two 1-8 nucleotide 5 '-overhangs or two 1-10 nucleotide 3' -overhangs. In a further embodiment, the first strand has a3 '-overhang of 1-8 nucleotides and a 5' -overhang of 1-8 nucleotides. In a further embodiment, the duplex RNA molecule is an isolated duplex RNA molecule.

In one embodiment, the first strand has a 3' -overhang of 1-10 nucleotides, and a 5' -overhang or 5' -blunt end of 1-10 nucleotides. In another embodiment, the first strand has a3 '-overhang of 1-10 nucleotides and a 5' -overhang of 1-10 nucleotides. In an alternative embodiment, the first strand has a3 '-overhang of 1-10 nucleotides and a 5' -blunt end.

In one embodiment, the first strand has a length of 5-100 nucleotides, 12-30 nucleotides, 15-28 nucleotides, 18-27 nucleotides, 19-23 nucleotides, 20-22 nucleotides, or 21 nucleotides.

In another embodiment, the second strand has a length of 3-30 nucleotides, 12-26 nucleotides, 13-20 nucleotides, 14-23 nucleotides, 14 or 15 nucleotides.

In one embodiment, the first strand has a length of 5-100 nucleotides and the second strand has a length of 3-30 nucleotides; or the first strand has a length of 10-30 nucleotides and the second strand has a length of 3-29 nucleotides; or the first strand has a length of 12-30 nucleotides and the second strand has a length of 10-26 nucleotides; or the first strand has a length of 15-28 nucleotides and the second strand has a length of 12-26 nucleotides; or the first strand has a length of 19-27 nucleotides and the second strand has a length of 14-23 nucleotides; or the first strand has a length of 20-22 nucleotides and the second strand has a length of 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 one 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 one embodiment, the duplex RNA molecule further comprises 1-10 unpaired or mismatched nucleotides. In a further embodiment, the unpaired or mismatched nucleotide is at or near the 3' notch end. In an alternative embodiment, the unpaired or mismatched nucleotide is at or near the end of the 5' notch. In an alternative embodiment, the unpaired or mismatched nucleotides are at the double-stranded region. In another embodiment, the unpaired or mismatched nucleotide sequence has a length of 1-5 nucleotides. In a further embodiment, unpaired or mismatched nucleotides form a loop structure.

In one embodiment, the first strand or the second strand comprises at least one nick or is formed from a2 nucleotide fragment.

In one embodiment, gene silencing is achieved by one or both or all of RNA interference, translational regulation, and DNA epigenetic regulation.

In the specification and claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

As used herein, "double-stranded RNA," "duplex RNA," or "RNA duplex" refers to RNA having 2 strands and having at least one duplex region, including RNA molecules having at least one nick, protrusion, and/or bubble within a duplex region or between 2 adjacent duplex regions. A strand is considered to have multiple fragments if it has gaps between 2 double-stranded regions or unpaired nucleotide regions that are single-stranded. As used herein, a double-stranded RNA can have an end overhang on either end or 2 ends. In certain embodiments, the 2 strands of the duplex RNA may be joined by a chemical linker.

As used herein, "antisense strand" refers to an RNA strand having substantial sequence complementarity with a target messenger RNA. The antisense strand may be a portion of an siRNA molecule, a portion of a miRNA/miRNA duplex, or a single-stranded mature miRNA.

As used herein, the term "isolated" or "purified" means that a substance is substantially or essentially free of components with which it normally accompanies in its natural state. Purity and homogeneity are generally determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

As used herein, "modulate" and grammatical equivalents thereof refers to increasing or decreasing (e.g., silencing), in other words, up-regulating or down-regulating. As used herein, "gene silencing" refers to a reduction in gene expression, and may refer to about a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% reduction in gene expression of a target gene.

As used herein, the term "subject" refers to any animal (e.g., mammal) that will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, and the like. In general, the terms "subject" and "patient" are used interchangeably herein with respect to a human subject.

As used herein, terms such as "treating" or "alleviating" refer both to 1) a therapeutic measure that cures, alleviates, reduces the symptoms of, and/or interrupts the progression of a diagnosed condition or disorder, and to 2) a preventative or prophylactic measure that prevents or slows the development of the targeted condition or disorder. Thus, those in need of treatment include those already having the disorder; those susceptible to the disorder; and those in which the disorder is to be prevented. A subject is successfully "treated" by the methods of the invention if the subject exhibits one or more of the following conditions: a reduction in the number of cancer cells or complete cancer cell-free; a reduction in tumor size; inhibition or absence of infiltration of cancer cells into peripheral organs, including spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; reduction of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; and improvement of quality of life.

As used herein, the term "inhibit" and its grammatical equivalents, when used in the context of biological activity, refers to a down-regulation of biological activity that can reduce or eliminate a targeted function, such as production of a protein or 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 term refers to the success of preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition, or disorder.

The term "substantially complementary" as used herein refers to complementarity in the base-pairing double-stranded region between 2 nucleic acids, but not in any single-stranded region, such as an end overhang or a gap region between the 2 double-stranded regions. The complementarity need not be complete; for example, there may be any number of base pair mismatches between 2 nucleic acids. However, if the number of mismatches is so large that hybridization does not occur even under the lowest stringency hybridization conditions, then the sequence is not a substantially complementary sequence. When 2 sequences are referred to herein as "substantially complementary," it is meant that the two sequences are sufficiently complementary to each other to allow hybridization to occur under the selected reaction conditions. The relationship between nucleic acid complementarity and hybridization stringency sufficient for specificity is well known in the art. The 2 substantially complementary strands may, for example, be perfectly complementary, or may contain 1to more mismatches, provided that the hybridization conditions are sufficient to allow, for example, discrimination between the paired and unpaired sequences. Thus, a substantially complementary sequence may refer to a sequence having 100, 95, 90, 80, 75, 70, 60, 50% or less, or any percentage between base pair complementarity in the double-stranded region.

As used herein, antagomirs are miRNA inhibitors that can be used to silence endogenous mirnas. As used herein, a mimetics or mimics is a miRNA agonist that can be used to replace an endogenous miRNA as a functional equivalent and thereby up-regulate a pathway affected by this endogenous miRNA.

RNA interference

RNA interference (abbreviated RNAi) is a cellular process induced by double-stranded RNA (dsrna) for targeted destruction of single-stranded RNA (ssrna). The ssRNA may be a gene transcript such as messenger RNA (mRNA). RNAi is a form of post-transcriptional gene silencing in which dsRNA can specifically interfere with the expression of genes having sequences complementary to dsRNA. The antisense RNA strand of the dsRNA targets a complementary gene transcript, such as messenger RNA (mrna), to effect cleavage by a ribonuclease.

During RNAi, long dsRNA is processed by the ribonuclease protein Dicer into a short form called small interfering rna (sirna). The siRNA is separated into a guide (or antisense) strand and a passenger (or sense) strand. The guide strand is integrated into the RNA-induced silencing complex (RISC), which is a multiprotein complex containing ribonucleases. The complex is then specifically targeted to the complementary gene transcript for disruption.

RNAi has been demonstrated to be a common cellular process in many eukaryotes. RISC and Dicer are conserved in the eukaryotic world. RNAi is thought to play a role in immune responses against viruses and other foreign genetic material.

Small interfering rna (sirna) is a class of short double-stranded rna (dsrna) molecules that exert a variety of biological effects. Most notably, it is involved in the RNA interference (RNAi) pathway, where sirnas interfere with the expression of specific genes. In addition, sirnas may also play a role in processes such as antiviral mechanisms or chromatin structure formation of the genome. In one embodiment, the siRNA has a short (19-21nt) double stranded RNA (dsRNA) region and a 2-3 nucleotide 3' overhang and 5' -phosphate and 3' -hydroxyl termini.

Micrornas (mirnas) are a class of endogenous, single-or double-stranded, RNA molecules of about 22 nucleotides in length that regulate up to 30% of mammalian genes (Czech, 2006; Eulalio et al, 2008; Mack, 2007). mirnas repress protein production by blocking translation or causing transcript degradation. mirnas can target 250-500 different mrnas. mirnas are Dicer digestions of miRNA precursors, which are products of primary mirnas (pri-mirnas).

As used herein, antagomirs are miRNA inhibitors that can be used to silence endogenous mirnas. As used herein, mimetics are miRNA agonists that can be used to replace mirnas and down-regulate mrnas.

Dicer is a member of the rnase III ribonuclease family. Dicer cleaves long double-stranded RNA (dsrna), microrna (mirna) precursors, and short hairpin RNA (shrna) into short double-stranded RNA fragments of about 20-25 nucleotides in length, typically with a2 base overhang at the 3' end, called small interfering RNAs (sirnas). Dicer catalyzes the first step in the RNA interference pathway and initiates the formation of an RNA-induced silencing complex (RISC), the catalytic component of which, argonaute is an endonuclease able to degrade messenger RNA (mrna), the sequence of which is complementary to that of the siRNA guide strand.

As used herein, an effective siRNA sequence is an siRNA that is effective in triggering RNAi to degrade the transcript of a target gene. Not every siRNA complementary to the target gene can effectively trigger RNAi to degrade the transcript of the gene. In fact, time consuming screening is often required to identify effective siRNA sequences. In one embodiment, the effective siRNA sequence is capable of reducing target gene expression by greater than 90%, greater than 80%, greater than 70%, greater than 60%, greater than 50%, greater than 40%, or greater than 30%.

The present invention provides a new structural scaffold, termed asymmetric interfering RNA (airna), which can be used to achieve siRNA-like results, and can also be used to modulate miRNA pathway activity (described in detail in commonly owned PCT and us applications entitled "Composition of asymmetric RNA Duplex as MicroRNA or inhibition", filed on the same day as the present application, the entire contents of which are incorporated herein by reference).

The new structural design of aiRNA is not only functionally effective in achieving gene regulation, but also offers several advantages over prior art RNAi modulators (mainly antisense, siRNA). Among these advantages, aiRNA can have a much shorter length RNA duplex structure than existing siRNA constructs, which will reduce synthesis costs and eliminate or reduce length-dependent triggering of non-specific interferon-like immune responses from host cells. The shorter length of the passenger strand in aiRNA will also eliminate or reduce unintended incorporation of the passenger strand in RISC, which in turn can reduce off-target effects observed in miRNA-mediated gene silencing. aiRNA can be used in all areas where miRNA-based technologies are currently in use or are considered for use, including biological research, R & D research in biotechnology and pharmaceutical industries, and miRNA-based diagnostics and therapeutics.

Ai RNA structural scaffold

Elbashir et al tested several asymmetric duplex RNA molecules as well as symmetric siRNA molecules (Elbashir et al, 2001c). The asymmetric duplex RNA molecules and the corresponding siRNA molecules are listed in table 1.

TABLE 1

However, these asymmetric duplex RNA molecules failed to achieve selective gene silencing compared to the corresponding symmetric siRNA molecules (supra).

The present invention relates to asymmetric duplex RNA molecules capable of effecting sequence-specific gene silencing. In one embodiment, the RNA molecule of the 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; asymmetric duplex RNA molecules disclosed in Elbashir (Elbashir et al, 2001c), in particular those listed in table 1, were excluded. The RNA molecule comprises a double-stranded region, and 2 ends independently selected from a 5 '-overhang, a 3' -overhang, and a blunt end. The RNA strand may have unpaired or incompletely paired nucleotides.

In one embodiment, the first strand is at least 1nt longer than the second strand. In a further embodiment, the first strand is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nt longer than the second strand. In another embodiment, the first chain is 20 to 100nt longer than the second chain. In a further embodiment, the first strand is 2 to 12nt longer than the second strand. In a further embodiment, the first strand is 3-10nt longer than the second strand.

In one 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 a further embodiment, the double-stranded region has a length of 10-19 bp. The double-stranded region may 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 30bp in length.

In one embodiment, the double-stranded region of the RNA molecule does not comprise any mismatches or bulges. In another embodiment, the double-stranded region of the RNA molecule comprises mismatches and/or bulges.

In one embodiment, when the first strand is

5’-UUCGAAGUAUUCCGCGUACGU(SEQ ID NO:1)

5' -UCGAAGUAUUCCGCGUACGUG (SEQ ID NO:2) or

5' -CGAAGUAUUCCGCGUACGUGA (SEQ ID NO:3),

the second strand has a length of at most 17 nucleotides, or comprises at least one mismatch with the first strand, or comprises at least one modification.

In an alternative embodiment, the first strand is not

5’-UUCGAAGUAUUCCGCGUACGU(SEQ ID NO:1)

5' -UCGAAGUAUUCCGCGUACGUG (SEQ ID NO:2) or

5’-CGAAGUAUUCCGCGUACGUGA(SEQ ID NO:3)。

In one embodiment, the first strand comprises a sequence that is substantially complementary to a target mRNA sequence. In another embodiment, the second strand comprises a sequence that is substantially complementary to the target mRNA sequence.

The present invention relates to asymmetric double-stranded RNA molecules capable of effecting gene silencing. In one embodiment, the RNA molecule of the 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 (asymmetric in length). The RNA molecules of the invention have at least one double-stranded region, and 2 ends independently selected from a 5 '-overhang, a 3' -overhang, and a blunt end (e.g., see fig. 1A, 2A-2D).

In the field of small RNA modulator preparation where single nucleotide changes, additions and deletions can critically affect the functionality of the molecule (Elbashir et al, 2001c), the aiRNA scaffold provides a different structural platform than classical siRNA structures with 21-nt double stranded RNA, which are symmetrical on each strand and their corresponding 3' overhangs. Furthermore, aiRNA of the present invention provides a highly desirable new approach to designing novel small molecule modulators that can overcome the obstacles currently encountered in RNAi-based research and drug development, as shown by the data in the examples below. For example, data obtained from aiRNA structurally mimicking siRNA show that aiRNA is more effective, potent, rapid onset, persistent and specific in inducing gene silencing than siRNA.

Any single-stranded region of the RNA molecules of the invention, including any terminal overhangs and gaps between 2 double-stranded regions, can be stabilized against degradation by chemical modification or secondary structure. The RNA strand may have unpaired or incompletely paired nucleotides. Each strand may have one or more nicks (nicks in the nucleic acid backbone, see, e.g., fig. 1B), nicks (broken strands with one or more deleted nucleotides, see, e.g., fig. 1C), and modified nucleotides or nucleotide analogs. Not only can any or all of the nucleotides in an RNA molecule be chemically modified, but each chain can be conjugated to one or more moieties to enhance its functionality, such as one or more peptides, antibodies, antibody fragments, aptamers, polymers, and the like.

In one embodiment, the first strand is at least 1nt longer than the second strand. In a further embodiment, the first strand is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20nt longer than the second strand. In another embodiment, the first chain is 20 to 100nt longer than the second chain. In a further embodiment, the first strand is 2 to 12nt longer than the second strand. In a further embodiment, the first strand is 3-10nt longer than the second strand.

In one embodiment, the first or long strand has a length of 5 to 100nt, or preferably 10 to 30 or 12 to 30nt, or more preferably 15 to 28 nt. In one embodiment, the first strand is 21 nucleotides in length. In one embodiment, the second or short chain has a length of 3 to 30nt, or preferably 3 to 29nt or 10 to 26nt, or more preferably 12 to 26 nt. In one embodiment, the second strand has a length of 15 nucleotides.

In one 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 a further embodiment, the double-stranded region has a length of 10-19 bp. The double-stranded region may be 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, 28, 29 or 30bp in length.

In one embodiment, the double-stranded region of the RNA molecule does not comprise any mismatches or bulges, and the 2 strands are fully complementary to each other in the double-stranded region. In another embodiment, the double-stranded region of the RNA molecule comprises mismatches and/or bulges.

In one 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. Duplex RNA molecules having a 5 '-overhang and a 3' -overhang on the first strand

Referring to FIG. 1A, in one embodiment of the invention, a double stranded RNA molecule has 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 having substantially complementary sequences, wherein the first strand is longer than the second strand. On the first strand, flanking the double-stranded region, unpaired overhangs are present on both the 5 'and 3' ends.

In one embodiment, the first strand is at least 2nt 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 20nt longer than the second strand. In another embodiment, the first chain is 20 to 100nt longer than the second chain. In a further embodiment, the first strand is 2 to 12nt longer than the second strand. In a further embodiment, the first strand is 3-10nt longer than the second strand.

In one embodiment, the first strand has a length of 5-100 nt. In a further embodiment, the first strand has a length of 5-100nt and the second strand has a length of 3-30 nucleotides. In a further embodiment, the first strand has a length of 5-100nt and the second strand has a length of 3-18 nucleotides.

In one embodiment, the first strand has a length of 10-30 nucleotides. In a further embodiment, the first strand has a length of 10-30 nucleotides and the second strand has a length of 3-28 nucleotides. In a further embodiment, the first strand has a length of 10-30 nucleotides and the second strand has a length of 3-19 nucleotides.

In one embodiment, the first strand has a length of 12-26 nucleotides. In a further embodiment, the first strand has a length of 12-26 nucleotides and the second strand has a length of 10-24 nucleotides. In a further embodiment, the first strand has a length of 12-26 nucleotides and the second strand has a length of 10-19 nucleotides.

In one 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 one embodiment, the first strand has a length of 21nt and the second strand has a length of 15 nt.

In one 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 a 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 one 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 a 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 one embodiment, the length of the 3 '-overhang is equal to the length 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 one embodiment, the duplex RNA molecule includes a double-stranded region of substantially complementary sequence of about 15nt, a 3-nt3 '-overhang, and a 3-nt 5' -overhang. The first strand is 21nt and the second strand is 15 nt. In one feature, the double-stranded regions of various embodiments consist of sequences that are fully complementary. In an alternative feature, the double-stranded region comprises at least one nick (FIG. 1B), nick (FIG. 1C) or mismatch (bulge or loop).

In one 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 a further embodiment, the double-stranded region has a length of 10-19 bp. The double-stranded region may 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 30bp in length. More than one double stranded region may be present.

In one embodiment, the first strand is an antisense strand capable of targeting a substantially complementary gene transcript, such as messenger rna (mrna), gene silencing by cleavage or by translational repression.

The present invention also provides a duplex RNA molecule comprising a first strand having a length of 18-23 nucleotides and a second strand having a length of 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 a3 '-overhang of 1-9 nucleotides and a 5' -overhang of 1-8 nucleotides, wherein the duplex RNA molecule is capable of effecting selective gene silencing in a eukaryotic cell. In one embodiment, the first strand comprises a sequence that is substantially complementary to a target mRNA sequence.

In one 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.

In one 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 a further embodiment, the first strand has a 3' -overhang of 3 nucleotides.

2.2. Duplex RNA molecules with a blunt end and a 5 '-overhang or 3' -overhang on the first strand Seed of Japanese apricot

In one embodiment, the duplex RNA molecule comprises a double-stranded region, a blunt end, and a 5 '-overhang or 3' -overhang (see, e.g., fig. 2A and 2B). The RNA molecule includes 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 one 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 a further embodiment, the double-stranded region has a length of 10-18 bp. The double-stranded region may 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 30bp in length. The double-stranded region may have similar properties to those described with respect to other embodiments and need not be repeated here. For example, the double-stranded region may consist of a completely complementary sequence, or include at least one nick, gap, or mismatch (bulge or loop).

In one embodiment, the first strand is at least 1nt 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 20nt longer than the second strand. In another embodiment, the first chain is 20 to 100nt longer than the second chain. In a further embodiment, the first strand is 2 to 12nt longer than the second strand. In a further embodiment, the first chain is 4-10nt longer than the second chain.

In one embodiment, the first strand has a length of 5-100 nt. In a further embodiment, the first strand has a length of 5-100nt and the second strand has a length of 3-30 nucleotides. In a further embodiment, the first strand has a length of 10-30nt and the second strand has a length of 3-19 nucleotides. In another embodiment, the first strand has a length of 12-26 nucleotides and the second strand has a length of 10-19 nucleotides.

In one embodiment, the duplex RNA molecule comprises a double-stranded region, a blunt end, and a 3' -overhang (see, e.g., fig. 2B).

In one 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 a 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 one 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 a 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. Duplex RNA molecules 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 (see, e.g., fig. 2C and 2D). The RNA molecule includes 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 one 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 a further embodiment, the double-stranded region has a length of 10-18 bp. The double-stranded region may 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 30bp in length.

In one embodiment, the first strand is at least 1nt 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 20nt longer than the second strand. In another embodiment, the first chain is 20 to 100nt longer than the second chain. In a further embodiment, the first strand is 2 to 12nt longer than the second strand. In a further embodiment, the first chain is 4-10nt longer than the second chain.

In one embodiment, the first strand has a length of 5-100 nt. In a further embodiment, the first strand has a length of 5-100nt and the second strand has a length of 3-30 nucleotides. In a further embodiment, the first strand has a length of 10-30nt and the second strand has a length of 3-18 nucleotides. In another embodiment, the first strand has a length of 12-26 nucleotides and the second strand has a length of 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 has similar properties as described for the other embodiments.

In one 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 a 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 one embodiment, the duplex RNA molecule comprises a double-stranded region and two 5' -overhangs (see, e.g., fig. 2D).

In one 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 a 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.

Design of airRNA

sirnas and mirnas have been widely used as research tools and developed as drug candidates. (see, for example, Dykxhoorn, Novina & Sharp. Nat. Rev. mol. cell biol.4: 457-. The duplex RNA molecules of the present invention, i.e., aiRNA, may be derived from siRNA and miRNA known in the art.

The present invention provides methods for converting siRNA or miRNA into aiRNA. This transformation results in a new duplex RNA molecule with at least one improved property compared to the original molecule. The property may be size, efficacy, potency, speed of onset, persistence, cost of synthesis, off-target effects, interferon response or delivery.

In one embodiment, the original molecule is a duplex RNA molecule, such as an siRNA. The duplex RNA molecule includes an antisense strand (e.g., guide strand) and a sense strand (e.g., passenger strand) that form at least one double-stranded region. The method includes altering the length of one or both strands such that the antisense strand is longer than the sense strand. In one embodiment, the sense passenger chain is shortened. In another embodiment, the antisense strand is extended. In a further embodiment, the sense strand is shortened and the antisense strand is extended. The antisense and sense RNA strands, which are invariant or of varying size, can be synthesized and subsequently combined under conditions to form an aiRNA molecule.

In yet another embodiment, the method comprises altering the length of the antisense and/or sense strand such that a duplex RNA molecule is formed having at least one of a 1-6 nucleotide 3 '-overhang and a 1-6 nucleotide 5' -overhang.

Alternatively, the duplex RNA molecules of the invention can be designed de novo. The duplex RNA molecules of the invention can be designed using methods for designing siRNA and miRNA, such as gene walking.

The RNA molecules of the invention can be designed using bioinformatic methods and subsequently tested in vitro and in vivo to determine their regulatory efficacy on target genes and the presence of any off-target effects. Based on these studies, the sequence of the RNA molecule can then be selected and modified to improve the regulatory efficacy on the target gene and minimize off-target effects. (see, e.g., Patzel, Drug Discovery Today12:139-148 (2007)).

3.1. Unpaired or mismatched regions in duplex RNA molecules

The 2 single strands of the aiRNA duplex may have at least one unpaired or incompletely paired (containing, for example, one or more mismatches) region. In one embodiment, the unpaired or incompletely paired region is at least one terminal region of the RNA molecule, including a terminal region with a blunt end, a terminal region with a3 '-notch or 5' overhang, and a terminal region with a 5 'notch or 3' overhang. As used herein, a terminal region is a region of an RNA molecule that includes one terminal and adjacent regions.

In one embodiment, the unpaired or incompletely paired region is in the double-stranded region of the aiRNA molecule. In a further embodiment, the asymmetric RNA duplex has an unpaired bulge or loop structure.

3.2. Sequence motifs in duplex RNA molecules

In the design of the aiRNA molecules of the invention, the overall GC content may vary. In one 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 strand separation easier because G-C pairings are stronger than A-U pairings.

In certain embodiments, the nucleotide sequence of the terminal overhang, e.g., the 5' end, may be designed independently of any template sequence (e.g., a target mRNA sequence), i.e., not necessarily substantially complementary to a target mRNA (in the case of siRNA or miRNA mimics) or a target miRNA (in the case of miRNA inhibitors). In one embodiment, the overhang of the longer or antisense strand, e.g., at 5 'or 3', is an "AA", "UU" or "dTdT" motif that has shown increased efficacy compared to certain other motifs. In one 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 nucleotides in the RNA molecules of the invention may be replaced with deoxynucleotides or modified nucleotides or nucleotide analogs. Substitutions can occur anywhere in the RNA molecule, for example, in one or both overhang regions, and/or in the double-stranded region. In certain instances, the substitutions enhance physical properties of the RNA molecule, such as strand affinity, solubility, and resistance to degradation by rnases or otherwise enhanced stability.

In one embodiment, the modified nucleotide or analog 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 one embodiment, the phosphodiester bond in the RNA molecule is modified to include at least a nitrogen and/or sulfur heteroatom. In one embodiment, the modified nucleotide or analog is a rare base or a modified base. In one embodiment, the modified nucleotide or analog is an inosine or tritylated base.

In a further stepIn an embodiment, the nucleotide analog is a sugar-modified ribonucleotide wherein the 2' -OH group is replaced by a group selected from the group consisting of H, OR, R, halogen, SH, SR, NH₂、NHR、NR₂And CN, wherein each R is independently selected from the group consisting of C1-C6 alkyl, alkenyl, and alkynyl, and halogen is selected from the group consisting of F, Cl, Br, and I.

In one embodiment, the nucleotide analog is a backbone-modified ribonucleotide that comprises a phosphorothioate group.

4. Use of

The invention also provides methods of modulating gene expression in a cell or organism (silencing methods). The method comprises the following steps: contacting the cell or organism with a duplex RNA molecule under conditions in which selective gene silencing can occur, and effecting selective gene silencing mediated by the duplex RNA molecule against a target nucleic acid having a sequence portion substantially corresponding to the double-stranded RNA.

In one embodiment, the contacting step comprises the step of introducing the duplex RNA molecule into a target cell in culture or in an organism in which selective gene silencing can occur. In a still further embodiment, the introducing step comprises transfection, lipofection, infection, electroporation, or other delivery techniques.

In one embodiment, the silencing method is used to determine the function or use of a gene in a cell or organism.

The silencing method may be used to regulate expression of a gene in a cell or organism. In one embodiment, the gene is associated with a disease, such as 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 a 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 a disease selected from the group consisting of: autoimmune diseases, 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.

4.1. Research tool

The RNA molecules of the invention can be used to cause gene "knockdown" in animal models to discover gene function, relative to genetically engineered knock-out models. The method can also be used to silence genes in vitro. For example, aiRNA may be transfected into cells. aiRNA can be used as a research tool in drug research and development for identification and validation of drug targets/pathways, as well as other biomedical research.

4.2 therapeutic uses

The RNA molecules of the invention may be used to treat and/or prevent a variety of diseases or undesirable conditions, including the diseases outlined (Czech, 2006; de Fougerolles et al, 2007; Dykxhoorn et al, 2003; Kim and Rossi, 2007; Mack, 2007).

The RNA molecules of the invention can be used to silence or knock down genes involved in cell proliferation or other cancer phenotypes examples of these genes are k-Ras, β -catenin, Nbs1, EF2, Stat3, PTEN, p70S6K, mTOR, Rsk1, PCNA, Parp1, survivin, NQO 1and p 21. in particular, k-Ras and β -catenin are therapeutic genes for colon cancer.

These RNA molecules can also be used to silence or knock down non-oncogene targets. The RNA molecules of the invention may also be used to treat or prevent ocular diseases such as age-related macular degeneration (AMD) and Diabetic Retinopathy (DR); infectious diseases (e.g., HIV/AIDS, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Human Papilloma Virus (HPV), Herpes Simplex Virus (HSV), RCV, Cytomegalovirus (CMV), dengue fever, west nile virus); respiratory diseases (e.g., Respiratory Syncytial Virus (RSV), asthma, cystic fibrosis); neurological diseases (e.g., Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), spinal injury, parkinson's disease, alzheimer's disease, pain); cardiovascular diseases; metabolic disorders (e.g., diabetes); a genetic disorder; and inflammatory conditions (e.g., Inflammatory Bowel Disease (IBD), arthritis, rheumatoid disease, autoimmune disorders), dermatological diseases.

In one embodiment, the first strand comprises a sequence that is substantially complementary to a target mRNA sequence of a gene selected from the group consisting of developmental genes, oncogenes, tumor suppressor genes and enzyme genes, as well as adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines or receptors thereof, growth/differentiation factors or receptors thereof, neurotransmitters or receptors thereof, ABLI, BCLl, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, MYR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, C, CL1, CN, PIM, 1, PMAS, DNAS, JUN, KRAS, JUN, KR, KRAS, LAK, LYN, MLL, MYB 2, MYB, C, CN, DNAS, DNA transferase, DNA lyase, DNA transferase, xylanase.

The present invention provides methods of treating diseases or undesirable conditions. The method includes the use of asymmetric duplex RNA molecules to effect gene silencing of a gene associated with a disease or an undesirable condition.

4.3. Conversion of RNA molecules (airRNA) into drugs

4.3.1. modification of RNA molecules

Naked RNA molecules are relatively unstable and can be degraded relatively rapidly in vivo. Chemical modifications can be introduced into the RNA molecules of the present invention to improve their half-life and further reduce the risk of non-specific gene targeting effects without reducing their biological activity.

To improve the stability of various RNA molecules, including antisense RNA, ribozymes, aptamers, and RNAi, modifications to RNA molecules have been investigated (Chiu and Rana, 2003; Czauderna et al, 2003; de Fougeroles et al, 2007; Kim and Rossi, 2007; Mack, 2007; Zhang et al, 2006; and Schmidt, Nature Biotech.25:273-275 (2007)).

Any stabilization modification known to those skilled in the art may be used to improve the stability of the RNA molecules of the invention. Within the RNA molecules of the invention, chemical modifications can be introduced into the phosphate backbone (e.g., phosphorothioate linkages), ribose (e.g., locked nucleic acids, 2 '-deoxy-2' -fluorouridine, 2 '-O-methyl), and/or base (e.g., 2' -fluoropyrimidine). Several examples of such chemical modifications are outlined below.

Chemical modifications at the 2' position of the ribose sugar, such as 2' -O-methylpurine and 2' -fluoropyrimidine (which can increase resistance against endonuclease activity in serum), can be employed to stabilize the RNA molecules of the invention. The location at which the modification is introduced should be carefully selected to avoid significantly reducing the silencing efficacy of the RNA molecule. For example, this modification at the 5' end of the guide strand may reduce silencing activity. On the other hand, 2' -O-methyl modifications can be staggered between 2 RNA strands in the double-stranded region to improve stability while preserving gene silencing efficacy. 2' -O-methyl modification can also eliminate or reduce interferon induction.

Another stabilizing modification is phosphorothioate (P ═ S) linkage. The introduction of phosphorothioate (P ═ S) linkages into RNA molecules, for example at the 3' -overhang, can provide protection against exonucleases.

The incorporation of deoxyribonucleotides into RNA molecules can also reduce manufacturing costs and increase stability.

In one embodiment, the 3 '-overhang, the 5' -overhang, or both are stabilized against degradation.

In one embodiment, the RNA molecule comprises at least one modified nucleotide or an analogue thereof. In a further embodiment, the modified ribonucleotide is modified on its sugar, backbone, base or any combination of the three.

In one embodiment, the nucleotide analog is a sugar-modified ribonucleotide. In a further embodiment, the 2' -OH group of the nucleotide analog is replaced by a group selected from H, OR, R, halogen, SH, SR, NH2, NHR, NR2, OR CN, wherein each R is independently C1-C6 alkyl, alkenyl, OR alkynyl, and halogen is F, Cl, Br, OR I.

In an alternative embodiment, the nucleotide analog is a backbone-modified ribonucleotide that comprises a phosphorothioate group.

In one embodiment, the duplex RNA molecule comprises at least one deoxynucleotide. In a further embodiment, the first strand comprises 1-6 deoxynucleotides. In a further embodiment, the first strand comprises 1-3 deoxynucleotides. In another embodiment, the 3' -overhang includes 1-3 deoxynucleotides. In a further embodiment, the 5' -overhang includes 1-3 deoxynucleotides. In an alternative embodiment, the second strand comprises 1-5 deoxynucleotides.

In one embodiment, the duplex RNA molecule comprises a3 '-overhang or a 5' -overhang comprising at least one deoxynucleotide. In another embodiment, the RNA 3 '-overhang and/or 5' -overhang consists of deoxynucleotides.

In one embodiment, the duplex RNA molecule is conjugated to an entity. In a further embodiment, the entity is selected from the group consisting of a peptide, an antibody, a polymer, a lipid, an oligonucleotide, and an aptamer.

In another embodiment, the first strand and the second strand are linked by a chemical linker.

In vivo delivery of RNA molecules

One major obstacle to the therapeutic application of RNAi is the delivery of siRNA to target cells (Zamore and Aronin, 2003). Various methods have been developed for the delivery of RNA molecules, particularly siRNA molecules (de Fougerolles et al, 2007; Dykxhoorn et al, 2003; Kim and Rossi, 2007). Any delivery method known to those skilled in the art may be used to deliver the RNA molecules of the present invention.

Major problems in delivery include instability in serum, nonspecific distribution, tissue barrier, and nonspecific interferon response (Lu & Woodle, Methods in Mol Biology 437: 93-107 (2008)). aiRNA molecules have several advantages compared to their siRNA and miRNA counterparts, which enable a wider range of methods for delivery purposes. First, aiRNA can be designed to be smaller than its siRNA and miRNA counterparts, thereby reducing or eliminating any interferon response. Second, aiRNA is more potent, starts more rapidly, is more effective, and lasts longer, thus requiring smaller amounts/doses of aiRNA to achieve therapeutic targets. Third, aiRNA is double-stranded and more stable than single-stranded antisense oligomers and mirnas, and they can be further chemically modified to enhance stability. Thus, the RNA molecules of the invention can be delivered into a subject via a variety of systemic or local delivery routes. In certain embodiments, the molecules of the invention are delivered by systemic delivery routes, including intravenous (i.v.) and intraperitoneal (ip). In other embodiments, the molecules of the invention are delivered by local delivery routes, such as intranasal, intravitreal, intratracheal, intracerebral, intramuscular, intraarticular, and intratumoral.

Examples of delivery techniques include direct injection of naked RNA molecules, conjugation of RNA molecules to natural ligands such as cholesterol or aptamers, delivery of liposomal formulations, and non-covalent binding to antibody-protamine fusion proteins. Other carrier options include positively 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 a cationic liposome complex or polymer complex system (Woodle et al J Control Release 74: 309-.

In one embodiment, the molecules of the invention are formulated with a collagen carrier, such as atelocollagen, for in vivo delivery. Atelocollagen has been reported to protect siRNA from RNase degradation and to enable sustained release (Minakuchi et al Nucleic Acids Res.32: e109 (2004); Takei et al Cancer Res.64: 3365-. In another embodiment, the molecules of the present invention are formulated with or form a nanoemulsion, such as an RGD peptide ligand targeted nanoparticle. It has been demonstrated that different siRNA oligomers can be combined in the same RGD ligand targeting nanoparticle to target several genes simultaneously (Woodle et al Materials Today 8 (supl 1): 34-41 (2005)).

Viral vectors may also be used to deliver the RNA molecules of the invention. In one embodiment, lentiviral vectors are used to deliver an RNA molecule transgene that can be integrated into the genome for stable expression. In another embodiment, adenoviruses and adeno-associated viruses (AAV) are used to deliver RNA molecule transgenes that do not integrate into the genome and have episomal expression.

Furthermore, bacteria can be used to deliver the RNA molecules of the invention (Xiang et al, 2006).

5. Drug groupCompounds and formulations

The invention further provides pharmaceutical compositions. The medicament comprises at least one asymmetric duplex RNA molecule as an active agent and one or more carriers selected from the group consisting of pharmaceutical carriers, positive charge carriers, liposomes, protein carriers, polymers, nanoparticles, nanoemulsions, lipids and lipids. In one embodiment, the composition is for diagnostic applications or for therapeutic applications.

The pharmaceutical compositions and formulations of the invention, apart from the RNA component, may be the same or similar to those developed for siRNA, miRNA and antisense RNA (de Fougeroles et al, 2007; Kim and Rossi, 2007). The siRNA, miRNA and antisense RNA in these pharmaceutical compositions and formulations may be replaced by the duplex RNA molecules of the invention. The pharmaceutical compositions and formulations may be further modified to accommodate the duplex RNA molecules of the present invention.

A "pharmaceutically acceptable salt" or "salt" of the disclosed duplex RNA molecules is a product of the disclosed duplex RNA molecules, which comprises an ionic bond, and is generally produced by reacting the disclosed duplex RNA molecules with an acid or a base, and is suitable for administration to a subject. Pharmaceutically acceptable salts may include, but are not limited to, acid addition salts including hydrochloride, hydrobromide, phosphate, sulfate, bisulfate, alkylsulfonate, arylsulfonate, acetate, benzoate, citrate, maleate, fumarate, succinate, lactate, and tartrate; alkali metal cations, such as Na, K, Li, alkaline earth metal salts, such as Mg or Ca, or organic amine salts.

A "pharmaceutical composition" is a formulation comprising the disclosed duplex RNA molecules in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form can be in 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 amount of active ingredient (e.g., a disclosed preparation of a duplex RNA molecule or salt thereof) in a unit dosage composition is an effective amount and can vary depending on the particular treatment involved. Those skilled in the art will appreciate that, depending on the age and condition of the patient, routine variations in dosage are sometimes necessary. The dosage will also depend on the route of administration. Various routes are contemplated, including oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, intranasal, and the like. Dosage forms for topical or transdermal administration of the duplex RNA molecules of the 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 any preservatives, buffers, or propellants that are required.

The present invention provides a method of treatment comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition. In one embodiment, the pharmaceutical composition is administered via a route selected from iv, sc, topical, po and ip. In another embodiment, an effective amount is 1ng to 1 g/day, 100ng to 1 g/day, or 1 μ g to 1 mg/day.

The invention also provides pharmaceutical formulations comprising a duplex RNA molecule of the 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, 20 th 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 solution, 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. Unless any convenient medium or agent is incompatible with the active duplex RNA molecule, its use in compositions is contemplated. Supplemental active duplex RNA molecules can also be incorporated into the compositions.

Methods for formulation are disclosed in PCT International application PCT/US02/24262(WO03/011224), U.S. patent application publication No. 2003/0091639, and U.S. patent application publication No. 2004/0071775, each of which is incorporated herein by reference.

The duplex RNA molecules of the invention may be administered in a suitable dosage form prepared by: a therapeutically effective amount (e.g., an effective level sufficient to achieve a desired therapeutic effect by inhibiting tumor growth, killing tumor cells, treating or preventing cell proliferative disorders, etc.) of a duplex RNA molecule of the invention (as an active ingredient) is admixed with standard pharmaceutical carriers or diluents according to conventional procedures (i.e., by producing a pharmaceutical composition of the invention). These processes may include mixing, granulating and tableting or dissolving the ingredients as appropriate to obtain the desired formulation. In another embodiment, a therapeutically effective amount of a duplex RNA molecule of the 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 a time delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate, or the like. Other fillers, excipients, flavoring agents and other additives, such as those known in the art, may also be included in the pharmaceutical compositions of the present invention.

Pharmaceutical compositions comprising the active duplex RNA molecules of the invention can be manufactured in a generally known manner, for example by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions may be formulated in 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 which can be used pharmaceutically. Of course, the appropriate formulation depends on the route of administration chosen.

The duplex RNA molecules or pharmaceutical compositions of the invention can be administered to a subject by a number of well-known methods currently used for chemotherapy. For example, for the treatment of cancer, the duplex RNA molecules of the invention can be injected directly into a tumor, injected into the bloodstream or into a body cavity or orally or applied through the skin with a patch. For the treatment of psoriasis, systemic administration (e.g., oral administration) or topical administration to the affected skin area is the preferred route of administration. The dosage selected should be sufficient to constitute an effective treatment, but not so high as to cause unacceptable side effects. The status of the disease (e.g., cancer, psoriasis, etc.) and the health of the patient should be closely monitored during and for a reasonable period of time after treatment.

Examples

Examples are provided below to further illustrate various features of the present invention. These examples also illustrate methods useful for practicing the invention. These examples do not limit the claimed invention.

RNA interference (RNAi) is a catalytic gene-specific silencing mechanism in eukaryotes 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) after incorporation of 19-21 base pairs (bp) of small interfering RNA (siRNA) with 3 'overhangs, which is the smallest RNA duplex known to enter RISC and mediate this 3' overhang (Elbashir et al, 2001 a; Elbashir et al, 2001 b; Elbashir et al, 2001 c; Fire et al, 1998; Zamore Z ore RISC et al, 2000.) as a natural substrate for the enzyme complex, siRNA can be chemically synthesized or generated by the surprising processing of its various precursors via Dicer catalysis (Donze and Picard, 2002; Hammond et al, 2000; Kim et al, 2005; Paison et al, 2002; Paison et al, 35dsna et al, the efficiency of siRNA is more efficient than the RNA-specific silencing duplex found in mammalian RNAi when these RNA-mediated by siRNA are not more efficient than the antisense RNA-mediated by siRNA-RNA-mediated gene-mediated duplexes (siRNA), thus the efficient silencing mechanism in mammalian-mediated by siRNA-RNA-mediated silencing-RNA-mediated by siRNA-RNA-mediated in-RNA-mediated silencing-RNA-mediated by siRNA-RNA-antisense-RNA-mediated targeting RNA-antisense-RNA-mediated targeting-RNA-antisense-mediated silencing duplexes-mediated silencing-RNA-mediated, which-mediated silencing-antisense-mediated silencing-mediated by-mediated silencing-antisense.

Method and material

Cell culture and reagents

Hela, SW480, DLD1, 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. mu.g/ml streptomycin and 2mM 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). The small RNAs described in this study were synthesized by Dharmacon, Qiagen or Integrated DNA technologies (table 2) and annealed according to the manufacturer's instructions (figure 3 a). siRNAs targeting human Ago2 and Dicer (Ambion) were used at 100 nM. Transfection of RNA was performed using DharmaFECT1(Dharmacon) at the indicated concentrations. Human Argonaute2(Ago2) expression vector (OriGene) was transfected using Lipofectamine 2000 (Invitrogen). Serum stability was determined by incubating the aiRNA or siRNA duplexes with 10% human serum (Sigma) for the indicated amount of time, followed by non-denaturing TBE-acryloyl gel electrophoresis and ethidium bromide staining.

Northern blot analysis

To determine the levels of β -catenin, total RNA was extracted from siRNA or airRNA transfected Hela cells at different time points with TRIZOL (Invitrogen). 20 μ g of total cellular RNA was loaded into each lane of a denaturing agarose gel after electrophoresis, the RNA was transferred to Hybond-XL Nylon membrane (Amersham Biosciences), UV cross-linked, and baked at 80 ℃ for 30 minutes.probes for detecting β -catenin and actin mRNA were prepared using the Prime-It II Random Primer laboratory Kit (Stratagene), from the β -catenin cDNA fragment (1-568) and the actin cDNA fragment (1-500). for analysis of small RNA RISC loading, siRNA or airRNA was transfected into the α cells 48 hours after transfection with pCMV-Ago 2. cells were lysed at the time points shown and immunoprecipitated with Ago2 antibody, the immunoprecipitates were washed, the immunoprecipitates were extracted from TRIZOL and the miRNA was loaded into 15% miRNA-BioZO membrane (BioJE-PAGE) for generation after electrophoresis of the KirZOL.³²P-labeled RNA probes. Antisense probe (5'-GUAGCUGAUAUUGAUGGACUU-3' (SEQ ID NO: 71)). Sense Probe (5'-UCCAUCAAU AUCAGC-3' (SEQ ID NO:72))

In vitro Ago2-RISC loading

airRNA or siRNA sense and antisense strands were performed using T4 kinase (Promega)³²The P-terminal is labeled. The end-labeled RNA was purified by phenol/chloroform/isoamyl alcohol, precipitated with EtOH, and resuspended in water. The labeled RNA is then annealed to the siRNA or aiRNA antisense strand. For in vitro lysates, Hela cells were transfected with the human Ago2 expression vector for 24 hours and S10 lysates were generated essentially as described (Dignam et al, 1983). The 5' sense or antisense strand labeled duplex aiRNA or siRNA is then added to the Ago2-S10 lysate. After incubation at 37 ℃ for 5 min, immunoprecipitated Ago2 as described, separated on a 20% TBE-acrylamide gelAgo 2-bound (pellet) and Ago 2-unbound (supernatant) fractions, the film was exposed with a gel to detect sense strand-Ago 2 binding. For the airRNA and siRNA competition experiments, up to 100-fold cold airRNA and siRNA were used in combination with³²P-labeled aiRNA or siRNA competes for loading into RISC. Briefly, S10 lysates were generated from Hela cells transfected with the Ago2 expression vector. The labeled aiRNA or siRNA was then added to the S10 lysate, followed by the addition of unlabeled aiRNA or siRNA. The reaction was incubated at 37 ℃ for 5 minutes and processed as described above.

qRT-PCR

Cells transfected with the indicated aiRNA or siRNA were harvested at the indicated time points after transfection. RNA was isolated with TRIZOL and qRT-PCR was performed using TaqMan one-step RT-PCR reagents and primer probe sets for the indicated mRNA (Applied Biosystems). Data are presented relative to control transfected cells and each sample is normalized to actin mRNA levels. For the experiment in FIG. 14d, the plasmid was prepared by cloning Stat3 cDNA (origin) into pcDNA3.1⁺Or pcDNA3.1^-The Stat3 construct was made at the HindIII-Xho1 site. HeLa cells were then co-transfected with Stat3 forward or reverse expression vectors with either AIStat3 or siStat3 for 24 hours. Cells were then harvested, RNA isolated by TRIZOL, and qRT-PCR was 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 the Superscript One-Step RT-PCR kit (Invitrogen), as well as the Stat3 forward (5'-GGATCTAGAATCAGCTACAGCAGC-3' (SEQ ID NO:73)) and Stat3 reverse (5'-TCCTCTAGAGGGCAATCTCCATTG-3' (SEQ ID NO:74)) primers, and the actin forward (5'-CCATGGATGATGATATCGCC-3' (SEQ ID NO:75)) and actin reverse (5'-TAGAAGCATTTGCGGTGGAC-3' (SEQ ID NO:76)) primers.

RT-PCR

Total RNA was prepared using TRIZOL and cDNA was synthesized using a random primer using a Thermoscript RT-PCR System (Invitrogen). PCR was run for 20 cycles using Pfx polymerase primers actin-1, 5 'CCATGGATGATGATATCGCC-3' (SEQ ID NO:75), actin-2, 5'-TAGAAGCATTTGCGGTGGAC-3' (SEQ ID NO:76), β -catenin-1, 5'-GACAATGGCTACTCAAGCTG-3' (SEQ ID NO:77), β -catenin-2, 5'-CAGGTCAGTATCAAACCAGG-3' (SEQ ID NO: 78).

Western blot

Cells were washed 2 times with ice-cold phosphate buffered saline and lysed in lysis buffer (50mM HEPES, pH 7.5, 0.5% Nonidet P-40, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1mM sodium orthovanadate, 1mM dithiothreitol, 1mM NaF, 2mM phenylmethylsulfonyl fluoride, and 10. mu.g/ml each of pepstatin, leupeptin, and aprotinin.) 20. mu.g of soluble protein was isolated by SDS-PAGE and transferred to PVDF membrane. in this study, anti- β -catenin, Nbs1, survivin, P21, Rsk1, k-Ras, Stat3, PCNA, NQO1, actin (Santa Cruz), EF 7, P70S6 GE K, mTOR, PTEN (Cell Signag linology), Ago2(Wako), Novus), and Pare P (EMies) antibody developed by chemiluminescence through Biosciences-antibody (Biosciences).

5' -RACE analysis

Total RNA (5. mu.g) from Hela cells treated with non-silenced airRNA or airRNA without any prior processing with GeneRacer^TMRNA adaptors (Invitrogen, 5'-CGACUGGAGCACGAG GACAC UGACAUGGACU GAAGGAGUAGAAA-3' (SEQ ID NO:79)) were ligated. The ligated RNA was reverse transcribed into cDNA using random primers. To detect cleavage products, primers complementary to the RNA adapters (GeneRacer) were used^TM5' Nested Primer: 5'-GGACACTGACATGGACTGAAGGAGTA-3' (SEQ ID NO:80)) and β -catenin specific Primer (GSP: 5'-CGCATGATAGCGTGTCTGGAAGCTT-3' (SEQ ID NO:81)) PCR was performed the amplified fragments were separated on a 1.4% agarose gel and sized using 1-kb Plus DNA Ladder (Invitrogen).

Interferon response detection

For the experiment in FIG. 15a, PBMC were incubated directly with 100nM β -catenin siRNA or airRNA, total RNA was purified at 16 hours using TRIZOL, and the level of gene expression in response to interferon was determined by RT-PCR as described by the manufacturer (System Biosciences). for the experiment in FIG. 15b, Hela cells were either mock transfected or transfected with 100nM of airRNA or siRNA for 24 hours. total RNA was purified using TRIZOL, and the level of gene expression in response to interferon was determined by RT-PCR. for Microarray analysis, Hela cells were transfected with 100nM airRNA or siRNA, total RNA was purified at 24 hours using TRIZOL, and RNA from Hamameter U133 us 2.0Gen chip hybridization (Affymetrix) FECT1 treated cells was used as a control for calculating transcript expression values, using a Microarray with a hybridization signal of 0.5. for the study, and a hybridization signal was sufficient for the study in the present of the sample (Plus) in the study.

aiRNA and siRNA sequences

The sequences and structures of the aiRNA and siRNA duplexes are listed in table 2. The position of the point mutation is boxed in the k-Ras airRNA.

TABLE 2

In Life evaluation

Daily checks of the health status of each animal were also performed. Body weight was checked every 3 days. Food and water were supplied daily according to the animal feeding operations in the laboratory. Generating>20% lethality and/or>Treatments with 20% net weight loss were considered toxic. Results are expressed as mean tumor volume (mm)³) Plus or minus SE. P value<0.05 was considered statistically relevant.

Animal management:

male or female athymic nude mice (Charles River Laboratories, Wilmington, MA.) of 4-5 weeks of age were acclimatized to animal housing facilities for at least one week prior to study initiation. All experimental methods utilized are in accordance with guidelines given by the American Society of Physiology (American Physiology Society) and guidelines for the Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals), and are also approved by the Institutional Animal Care and Use Committee of Boston Biomedical Inc. Animals 4 were housed in groups of wood chip bedding cages and placed in a room with controlled temperature (68-72F), light (12 hours light-dark cycle) and humidity (45-55%). During the experiment the animals had free access to water and food.

Example 1 asymmetric interfering RNA (airRNA) causes Gene-specific silencing in mammalian cells

siRNA structural scaffolds are thought to be an essential configuration for incorporation into RISC and mediation of RNAi (Elbashir et al, 2001 a; Elbashir et al, 2001 b; Elbashir et al, 2001 c; Rana, 2007; Zamore et al, 2000). However, little is known about the requirements of RNA duplex scaffolds for RISC incorporation and gene silencing. To investigate the structural scaffold required for efficient RNAi mediators and RISC substrates, we first determined whether RNA duplexes shorter than siRNA are capable of mediating gene silencing. The length of double-stranded (ds) RNA is an important factor in determining its propensity to activate protein kinase R- (PKR) -mediated non-specific interferon responses, increased synthetic costs and delivery problems (Elbashir et al, 2001 b; Sledz et al, 2003). We designed a series of 12 to 21bp short dsrnas targeting different mammalian genes with 2 nucleotide 3' overhangs or blunt ends. No gene silencing was detected after the length was reduced to less than 19bp (data not shown), consistent with previous reports in Drosophila Melanogaster cell lysates (Elbashir et al, 2001b), and with the notion that 19-21bp is the shortest siRNA duplex mediating RNAi (Elbashir et al, 2001 a; Elbashir et al, 2001 b; Elbashir et al, 2001 c; Rana, 2007; Zamore et al, 2000).

Next we tested whether non-siRNA scaffold RNA duplexes with overhangs of asymmetric configuration could mediate gene silencing. The siRNA duplex comprises a symmetrical sense strand and an antisense strand. Although duplex siRNA structures containing 3' overhangs are required for incorporation into the RISC complex, cleavage of the target mRNA is guided by the antisense strand following Argonaute (ago) mediated sense strand cleavage (Hammond et al, 2001; Matranga et al, 2005; Tabara et al, 1999). We sought to prepare asymmetric RNA duplexes of various lengths with overhangs at the 3 'and 5' ends of the antisense strand.

The oligomers with the sequences shown in table 3 were verified by 20% polyacrylamide gel after annealing. As shown in fig. 3A, each lane was loaded as follows: lane 1, 21nt/21 nt; lane 2, 12nt (a)/21 nt; lane 3, 12nt (b)/21 nt; lane 4, 13nt/13 nt; lane 5, 13nt/21 nt; lanes 6, 14nt/14 nt; lane 7, 14nt (a)/21 nt; lane 8, 14nt (b)/21 nt; lanes 9, 15nt/15 nt; lanes 10, 15nt/21 nt.

TABLE 3

HeLa cells were seeded at 200,000 cells/well in 6-well plates as shown in FIG. 3B, 24 hours later they were transfected with scarmbled siRNA (lane 1), 21-bp siRNA targeting E2F1 (lane 2, as a specific control) or 21-bp siRNA targeting β -catenin (lane 3, as a positive control), or a mixture of different lengths of aiRNA of 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), at the same concentration, cells were harvested 48 hours after transfection, expression of β -catenin was determined by Western blotting E2F1and used as a control for actin results (asymmetric interfering RNA) (AIRNA) caused gene specific silencing in mammalian cells.

To determine the structural features of aiRNA that are important in aiRNA function, we generated a number of aiRNA oligonucleotides based on modifications to the double antisense overhang structure of core 15/21 (table 4). The aiRNA summarized in table 4 contains modifications including, but not limited to, the length of the sense and antisense strands, the extent of the sense and antisense overhangs, and RNA-DNA hybrid oligonucleotides.

The parental 15/21aiRNA structure was modified by altering the sense strand, antisense strand, or both (table 4). Hela cells were transfected with the modified aiRNA duplex at 50nM for 48 hours using western blotting of β -catenin and actin to examine the degree of gene silencing compared to the parental 15/21aiRNA and conventional siRNA structures.

These data collectively provide structural clues about aiRNA function.

In the case of the sense strand, our data indicate that a length of 15 bases works well, while lengths of 14 to 19 bases remain functional. The sense strand may match any portion of the antisense strand, provided that the antisense overhang rules are satisfied. DNA substitutions of a single RNA base at the 5 'or 3' end of the sense strand are tolerated and may even provide increased activity.

In terms of antisense strand length, a length of 21 bases works well, 19-22 bases retain activity, and activity decreases when the length decreases below 19 bases or increases beyond 22 bases. The 3' end of the antisense strand requires a 1-5 base overhang, with 2-3 base overhangs being preferred and blunt ends showing a reduction in activity. Base pairing with the target RNA sequence is preferred, and DNA base substitutions of no more than 3 bases are tolerated without simultaneous 5' DNA base substitutions. The 5' end of the antisense strand preferably has a 0-4 base overhang, and no overhang is required to maintain activity. The 5 'end of the antisense strand can tolerate 2 base mismatches in the target RNA sequence and can tolerate DNA base substitutions of up to 3 bases without simultaneous 3' DNA base substitutions.

In the case of mismatched or chemically modified bases, we have found that aiRNA structures are tolerant to both mismatches and one or more chemically modified bases in the sense or antisense strand.

Table 4: airRNA sequences for FIGS. 4-5

In Table 4, A, U, G and C represent nucleotides, and a, t, g and C represent deoxynucleotides.

Example 2 mechanism of Gene silencing triggered by airRNA

To investigate the mechanism of gene knock-down induced by aiRNA, we first determined whether gene silencing by aiRNA occurred at the translation or mRNA level, northern blot analysis of β -catenin performed in cells transfected with 10nM 15bp aiRNA, showing that aiRNA reduced mRNA levels by more than 95% within 24 hours and that the reduction lasted for more than 4 days (fig. 6a), suggesting that aiRNA mediated gene silencing at the mRNA level β -catenin mRNA reduction induced by aiRNA was substantially faster, more efficient and durable compared to siRNA (fig. 6a), we further determined whether 15bp aiRNA catalyzes site-specific cleavage of β -catenin mRNA, by rapid amplification of cDNA ends (5 '-RACE) and PCR, for the presence of β -catenin mRNA cleavage fragments, examined total RNA isolated from cells transfected with 15bp aiRNA (fig. 6b), detected β -catenin cleavage fragments at 4 and 8 hours after transfection of the mRNA (fig. 6c), analysis showed that such efficient cleavage of mRNA fragments within target strand was observed by aiRNA cleavage at the target strand after transfection with aiRNA transfection (fig. 6c) and strong induction of mRNA cleavage by aimb 5' 11.

Next we determined whether the new aiRNA asymmetric scaffold could be incorporated into RISC. RNAi is catalyzed by RISC enzyme complex with the Argonaute protein (Ago) as the catalytic unit of the complex (Liu et al, 2004; Matranga et al, 2005). To determine whether aiRNA was incorporated into the Ago/RISC complex, myc tag-human Ago1 was immunoprecipitated from cells expressing myc tag-Ago 1 after transfection of the cells with aiRNA (sioles et al, 2005). Small RNAs that bind to the RISC complex are detected by northern blotting of the Ago immunoprecipitate. Northern blot analysis revealed that aiRNA entered the RISC complex with high efficiency (fig. 6 e). These data indicate that the asymmetric scaffold of aiRNA can be incorporated efficiently into RISC.

Since aiRNA induces more efficient gene silencing than siRNA, we tested whether aiRNA is able to produce RISC complex more efficiently than siRNA. As shown in fig. 6e, the aiRNA-Ago2/RISC complex formed faster and more efficiently than the siRNA-Ago2/RISC complex, where more aiRNA was contained in the RISC complex than the corresponding siRNA (fig. 6e and fig. 7A). Notably, sirnas showed typical patterns (21), which are consistent with the formation of secondary structures by sirnas (fig. 6e and fig. 7). Conversely, aiRNA exhibits a single band, suggesting that shorter aiRNA lengths may reduce or eliminate secondary structure formation that accompanies siRNA.

In addition, the asymmetric configuration of aiRNA can promote the formation of active RISC with the antisense strand, reducing inefficient RISC formation by the sense strand (ref 16). Our data demonstrates that this is true (as shown in fig. 7B), the sense strand cannot be detected in the RISC complex. Fig. 8A also demonstrates that although the antisense strand of aiRNA binds strongly to Ago2, the sense strand does not bind. In contrast, both the antisense and sense strands of the siRNA bind to Ago 2. These data suggest that aiRNA has a higher RISC forming efficiency in cells than siRNA, which may explain the superior gene silencing efficiency of aiRNA.

Furthermore, it was demonstrated that the sense strand of the siRNA needs to be cleaved in order to be functional. Therefore, we tested whether the same requirements exist for aiRNA. For this, the nucleotide at position 8 or 9 of the sense strand of aiRNA was modified with 2' -O-methyl so that it could not be cleaved. The results show that the aiRNA with the sense strand that was not cleaved is still functional (fig. 8B), suggesting that aiRNA is very different from siRNA in its mechanism.

Further, we investigated whether there were any different loading pockets for aiRNA and siRNA. Cold airRNA or siRNA was used to compete with radiolabeled siRNA or airRNA for the RISC complex (FIG. 9). Surprisingly, the results show that cold aiRNA does not compete with siRNA for the RISC complex (fig. 9B), and that cold siRNA does not compete with aiRNA for the RISC complex either (fig. 9C). These data indicate that the aiRNA and siRNA may be loaded into different pockets of the RISC complex.

The above data collectively indicate that aiRNA is the first non-siRNA scaffold that can be incorporated into RISC, thus providing a new structural scaffold for interaction with RISC. The RISC loading differences of aiRNA and siRNA are illustrated in the model shown in fig. 10. In short, because of the asymmetric nature, only the antisense strand is selected to remain in the RISC complex, thus resulting in a strand selection efficiency of 100%. In contrast, siRNA is symmetrical in structure. Both the antisense and sense strands of the siRNA have the opportunity to be selected for retention in the RISC complex, thus siRNA has inefficient strand selection and at the same time can cause non-specific gene silencing due to the sense strand RISC complex.

Example 3 aiRNA mediated Gene silencing more rapidly, potently, effectively and persistently than siRNA

To compare the gene silencing properties of aiRNA with siRNA, we first determined the optimal aiRNA structure for gene silencing.

Although duplex siRNA structures containing 3' overhangs are required for incorporation into RISC complexes, cleavage of target mRNA is guided by antisense strands following argonaute (ago) mediated sense strand cleavage (Hammond et al, 2001; Matranga et al, 2005; Tabara et al, 1999). we sought to prepare asymmetric RNA duplexes of various lengths with overhangs at the 3' and 5' ends of the antisense strand a 12 to 15bp set of asymmetric RNA duplexes with 3' and 5' antisense overhangs was designed to target β -catenin (fig. 11A), β -catenin is an endogenous gene involved in cancer and stem cells (clers, 2006). optimized sirnas with standard configurations have been designed to target β -catenin for triggering RNAi (Xiang et al, 2006). all the aai for β -catenin designed within the same sequence targeted by siRNA (fig. 11A) are compared to the optimal RNA duplexes using RNA duplexes for silencing (fig. 11A) and fig. 15bp RNA silencing results are shown in subsequent experiments using RNA-mer fig. 11B.

Surprisingly, aiRNA was found to induce a potent and efficient reduction of β -catenin protein while sparing the non-target control gene actin (fig. 11C).

Next, we examined the onset of gene silencing mediated by airRNA and siRNA targeting β -catenin the sequences of airRNA and siRNA used are shown in FIG. 11A. as shown in FIG. 12, airRNA has a faster onset (FIGS. 12C and D) and better efficacy (FIGS. 12B and D).

aiRNA was designed to target genes of different functional types, including Stat3 (fig. 13b), NQO1 (fig. 12d), elongation factor 2(EF2) (fig. 13c), Nbs1 (fig. 14b), survivin (fig. 14b), Parp1 (fig. 14b), p21 (fig. 14b), Rsk1 (fig. 14c), PCNA (fig. 14c), p70S6K (fig. 14c), mTOR (fig. 14c) and PTEN (fig. 14c) and p 48-catenin (fig. 13a), as shown in fig. 13 and 14, aiRNA is more effective in silencing Stat3, β -catenin, Rsk 2, p70S 6S K, nb 1, nb 2 and siRNA for various targets and various human cell lines, and further, as shown by the efficacy of siRNA against the siRNA sequence of siRNA, siRNA map 11a, siRNA map 11a 3614 b, irq 9, irna is more effective in silencing against the right siRNA, siRNA 14a, siRNA map 11a, and optionally more effective against the target proteins, as shown in the right siRNA sequence of siRNA, map, siRNA, map 11a, siRNA, map, and map.

Taken together, these data demonstrate that aiRNA can mediate gene silencing in mammalian cells more efficiently, potently, rapidly, and persistently than siRNA.

Example 4 specificity of Gene silencing mediated by airRNA

Next, we investigated the specificity of gene silencing mediated by aiRNA. First, airRNA targeting the wild-type k-Ras allele was analyzed. DLD1 cells contained wild-type k-Ras, while SW480 cells contained mutant k-Ras with single base pair substitutions (FIG. 14 d). Transfection of DLD1 cells with airRNA targeting wild-type k-Ras showed potent silencing, but no silencing of mutant k-Ras was observed in SW480 cells. These data demonstrate that aiRNA mediates allele-specific gene silencing.

Activation of interferon-like responses is the major non-specific mechanism of gene silencing A major reason for siRNA use for gene silencing is that dsRNA shorter than 30bp has a reduced ability to activate interferon-like responses in mammalian cells (Bernstein et al, 2001; Martinez and Tuschl, 2004; Sledz et al, 2003). We examined whether the aiRNA shows any indication of activation of interferon-like responses in mammalian cells, RNA was collected from PBMC cells transfected with anti- β -catenin aiRNA and Hela cells transfected with anti-EF 2 or survivin aiRNA, interferon-inducible genes were analyzed by RT-PCR, aiRNA transfection was found to show no increase in any interferon-inducible genes tested by RT-PCR, while levels of target mRNA were reduced relative to control transfected cells (FIGS. 15a and b). microarray analysis was also performed to compare the changes in expression of known interferon-response-related genes induced by aiRNA and siRNA, as shown in FIG. 15c, a much less change in ai RNA compared to siRNA.

In addition, as described above, the sense strand-RISC complex can cause non-specific gene silencing. To compare non-specific gene silencing mediated by the sense strand-RISC complex for aiRNA and siRNA, 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 24 hours post transfection and RNA was collected and the relative levels of the Stat3 sense or antisense RNA (insert) were determined by quantitative real-time PCR or RT-PCR. The results showed that aiRNA had no effect on antisense Stat3 mRNA, while siRNA had an effect (fig. 15 d). This result indicates that aiRNA completely eliminates the undesired non-specific gene silencing mediated by the sense strand-RISC complex.

In summary, aiRNA has been shown to be a new class of gene silencing inducers, a minimal RISC substrate scaffold of the non-siRNA type, and RNAi mediators (fig. 15 f). Our data suggest that aiRNA works through RISC, the cellular RNAi machinery. Upon incorporation into RISC, the aiRNA mediates sequence-specific cleavage of mRNA between bases 10 and 11 relative to the 5' end of the aiRNA antisense strand. The asymmetric configuration of aiRNA can interact with RISC more efficiently than siRNA. Consistent with high RISC binding efficiency, aiRNA is more potent, efficient, rapid onset and durable than siRNA in mediating gene-specific silencing against the genes tested in this study. Although previous studies have suggested a role for Dicer in promoting efficient RISC formation, our data suggest that aiRNA can lead to an active RISC complex with high efficiency independent of Dicer-mediated processing.

The key feature of this novel RNA duplex scaffold is the antisense overhang at the 3 'and 5' ends. 12-15bp airRNA is the shortest RNA duplex known to induce RNAi. Although long dsrnas trigger potent gene silencing in caenorhabditis elegans (c. elegans) and drosophila melanogaster, gene-specific silencing in mammalian cells is not possible prior to the use of siRNA duplexes. siRNA scaffolds defined by Dicer digestion are characterized by 19-21bp strand length and symmetry at the 3' overhang (Bernstein et al, 2001), which has been considered to be a necessary structure for incorporation into RISC to mediate RNAi. Therefore, efforts to optimize the inducers of RNAi have focused on siRNA precursors, which are always larger than siRNAs (Soutschek et al, 2004; Zhang and Farwell, 2007). Our data suggest that siRNA is not an essential scaffold for incorporation into RISC to mediate RNAi. aiRNA of different lengths shows a range of gene silencing efficacy and RISC incorporation efficiency, providing a unique opportunity to understand RISC incorporation and activation mechanisms. Studies are needed to further understand the structure-activity relationship of aiRNA in RISC incorporation and RNAi induction, which will help establish the theoretical basis for optimizing aiRNA for various RNAi applications through target sequence selection, length, structure, chemical composition and modification.

Example 5 airRNA is more effective than siRNA in vivo

To investigate whether aiRNA was effective in vivo and compared to siRNA, the role of aiRNA and siRNA in a human colon cancer xenograft model was tested.

The Wnt β -catenin signaling pathway is tightly regulated and has important functions in development, tissue homeostasis, and regeneration.dysregulation of Wnt/β -catenin signaling occurs frequently in various human cancers 80% of colorectal cancers alone reveal activation of this pathway by inactivation of the tumor suppressor adenomatous polyposis coli or mutation of the proto-oncogene β -catenin.

Thus, targeted inhibition of Wnt/β -catenin signaling is a rational and promising new approach for the treatment of cancers of various origins.

In vitro, by ribozyme targeting, a reduction in β -catenin expression and a related reduction in cell death induction in human colon cancer SW480 cells has been demonstrated, suggesting that β -catenin expression is rate-limiting for tumor growth in vitro.

SW480 human colon cancer cells were subcutaneously inoculated into female athymic nude mice (8X 10)⁶Cells/mouse) and allowed to form palpable tumors. In this study, when tumors reached about 120mm³The animals received a total of 10 doses of siRNA, aiRNA or control during treatment the tumors were measured as shown in figure 16, intravenous treatment with 0.6nmol mg/kg using siRNA and aiRNA as monotherapy significantly inhibited tumor growth,% T/C value of siRNA was calculated to be 48.8%, p value was 0.0286 whereas treatment with β -catenin-specific aiRNA resulted in a much more potent reduction in tumor growth,% T/C value was calculated to be 9.9%, p value was 0.0024.

In addition, we also tested the effects of aiRNA and siRNA in HT29 human colon cancer xenograft model. HT29 human colon cancer cells were subcutaneously inoculated into female athymic nude mice (6X 10)⁶Cells/mouse) and allowed to form palpable tumors. In this study, when tumors reached about 200mm³Animals were dosed every other day with 0.6nmol PEI-complexed β -catenin siRNA, PEI-The animals received 8 total doses of siRNA, aiRNA or control the tumors were measured in the treatments as shown in figure 17 intravenous treatment with siRNA and aiRNA as monotherapy at 0.6nmol mg/kg significantly inhibited tumor growth,% T/C value of siRNA calculated as 78%, p value 0.21 again treatment with β -catenin-specific aiRNA resulted in an even more robust reduction of tumor growth,% T/C value calculated as 41%, p value 0.016 again, there was no significant change in body weight due to iv administration of siRNA, aiRNA or control these data again demonstrate that targeting β -catenin by PEI complexing systemic in vivo provides a way to develop a highly effective, specific and safe agent for therapeutic application to colon cancer patients.

In summary, aiRNA can significantly improve a wide range of RNAi applications. siRNA-based therapies have encountered challenges including limited efficacy, delivery difficulties, interferon-like responses, and manufacturing costs (de Fougerolles et al, 2007; Iorns et al, 2007; Rana, 2007). The improved efficacy, durability, and smaller size of aiRNA may help or overcome these challenges because aiRNA is smaller and may require less material for its delivery. Thus, aiRNA represents a new minimal RNA duplex into RISC that mediates gene silencing in mammalian cells more efficiently, potently, faster onset and more persistently than siRNA, with significant potential for gene function studies and for broad RNAi-based applications in RNAi-based therapies.

Other embodiments are within the following claims. While several embodiments have been shown and described, various modifications can be made without departing from the spirit and scope of the invention.

Reference to the literature

Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J, (2001) Role for antisense nucleic acid in the initiation step of RNA interference. Nature 409, 363-.

Chiu, y.l. and Rana, t.m. (2003). RNA (New York, NY 9, 1034-1048).

Clevers，H.(2006).Wnt/beta-catenin signaling in development anddisease.Cell127，469-480.

Czaudernan, F., Fechtner, M., Dames, S., Aygun, H., Klippel, A., Pronk, G.J., Giese, K., and Kaufmann, J. (2003). Structural variations and statistical modifications of synthetic siRNA in mammalian cells, nucleic acids research31, 2705-.

Czech，M.P.(2006).MicroRNA as therapeutic targets.The New Englandjournal of medicine 354，1194-1195.

de fougeroles, a., Vornlocher, h.p., Maraganore, j. and Lieberman, J. (2007). a progressive report on siRNA-based therapeutics. NatRev Drug Discov 6, 443-.

Dignam, J.D., Lebovitz, R.M., and Roeder, R.G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated nucleic acids research 11, 1475-1489.

Donze, O. and Picard, D. (2002). RNA interference in a mammalian cell using siRNA synthesized with T7RNA polymerase, nucleic Acids Res 30, e46.

Dykxhoorn, d.m., Novina, c.d., and Sharp, p.a. (2003). short RNA that gene expression. nat Rev Mol Cell Biol 4, 457-.

Elbashir, s.m., Harborth, j., Lendeckel, w., Yalcin, a., Weber, k. and Tuschl, t. (2001 a.) duplex of 21-nucleotide RNA media RNA interference in simplified mammalian cells, nature 411, 494-498.

Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001 b.) RNA interference amplified by 21-and 22-nucleotide RNA genes Dev 15, 188-.

Elbashir, s.m., Martinez, j., Patkaniowska, a., leneckel, w, and Tuschl, t. (2001 c.) Functional anatomi of siRNA for mediating effective RNAi.

Cell 132, 9-14, cell to the root of mirna-mediated gene cloning, Eulalio, a., huntsinger, e., and izuralded, E. (2008).

Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C, (1998) content and specific genetic interaction by double-stranded RNA in Caenorhabditis elegans 391, 806-.

Hammond, s.m., Bernstein, e., Beach, d., and Hannon, G.J, (2000) An RNA-directed nucleic acids post-transcriptional gene sizing in drosophilalels 404, 293-.

Hammond, s.m., Boettcher, s., Caudy, a.a., Kobayashi, r., and Hannon, G.J, (2001) Argonaute2, a link beta gene and biochemical analytes of rnai. science293, 1146-.

Iorns, E., Lord, C.J., Turner, N., and Ashworth, A. (2007) Utilizing RNAInterferce to enhance register device driver discovery Nature reviews 6, 556-.

Kim, d.h., Behlke, m.a., Rose, s.d., Chang, m.s., Choi, s.and Rossi, J.J (2005). Synthetic dsRNA Dicer substrates RNAi force and efficacy. natbiotechnol23, 222-.

Kim, D.H. and Rossi, J.J. (2007). Strategies for slimming human disease RNA interference Nature reviews 8, 173- "184".

Liu, j., Carmell, m.a., Rivas, f.v., Marsden, c.g., Thomson, j.m., Song, j.j., Hammond, s.m., Joshua-Tor, l., and Hannon, G.J. (2004) Argonaute2is the cataliting of mammalian rnai.science 305, 1437-.

Mack，G.S.(2007).MicroRNA gets down to business.Nature biotechnology25，631-638.

Martinez, J. and Tuschl, T. (2004). RISC is a 5' phosphoester-producing RNA endonuclease Dev 18, 975-.

Matrana, C., Tomari, Y., Shin, C., Bartel, D.P., and Zamore, P.D, (2005.) Passenger-strand close tolerances evaluation of siRNA intro Ago 2-accompanying RNAi enzyme complexes cell 123, 607-.

Paddison, P.J., Caudy, A.A., Bernstein, E., Hannon, G.J., and Conklin, D.S. (2002). Short hairpin RNA (shRNA) index sequence-specific cloning in a cellular cells. genes Dev 16, 948-.

Patzel，V.(2007).In silico selection of active siRNA.Drug discoverytoday 12，139-148.

Rana，T.M.(2007).Illuminating the silence：understanding the structureand function of small RNA.Nat Rev Mol Cell Biol 8，23-36.

Rogoff, H.A., Pickering, M.T., Frame, F.M., Debatis, M.E., Sanchez, Y., Jones, S, and Kowaiik, T.F, (2004). Apoptosis associated with a truncated E2F activity dependent on E2F1and Atm/Nbs1/Chk2.mol Cell Biol 24, 2968-.

Solas, d., Lerner, c., Burchard, j., Ge, w., Linsley, p.s., Paddison, p.j., Hannon, g.j., and clean, M.A, (2005).

Sledz, c.a., Holko, m., de Veer, m.j., Silverman, r.h., and Williams, b.r. (2003).

Soutschek, j., akin, a., Bramlage, b., Charisse, k., continen, r., Donoghue, m., Elbashir, s., Geick, a., Hadwiger, p., Harborth, j. et al (2004) the therapeutic administration of an end genes by system administration of modifidsirna.nature 432, 173- "178.

Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C.C, (1999) The rde-1gene, RNA interference, and transsoxilnening in C.elegans.cell 99, 123-.

Xiao ang, S., Fruehauf, J., and Li, C.J. (2006). Short hairpin RNA-expression bacteria elici RNA interference in mammals. Nature biotechnology 24, 697-.

Zamore, P.D. and Arnin, N. (2003). siRNA knock down hepatics. Naturemedine 9, 266-.

Zamore, p.d., Tuschl, t., Sharp, p.a. and Bartel, d.p. (2000). double-stranded RNA directions the ATP-dependent cleavage of mRNA at 21to 23nucleotideintervals. Cell101, 25-33.

Zhang, b. and Farwell, M.A. (2007). a new embedding class of plasma diagnostics and gene therapy.

Zhang, h.y., Du, q., Wahlestedt, c, and Liang, z. (2006) RNA Interference with chemical modified sirna current topocs in medical chemistry 6, 893-.

Claims (37)

1. An asymmetrically interfering RNA duplex molecule comprising

-an antisense strand consisting of 19, 20, 21 or 22 nucleotides, wherein the antisense strand comprises a3 'overhang of 1-6 nucleotides and a 5' overhang of 0-5 nucleotides,

-a sense strand consisting of 14, 15, 16 or 17 nucleotides,

wherein the sense strand is substantially complementary to the antisense strand, wherein the antisense strand is at least 70% complementary to a target mRNA sequence, and the sense strand and the antisense strand form a double-stranded region of 14, 15, 16, or 17 base pairs.

2. The asymmetric interfering RNA duplex molecule of claim 1 wherein the sense strand is 15 nucleotides.

3. The asymmetric interfering RNA duplex molecule of claim 1 wherein the 3' overhang has a length of 2, 3, 4, 5 or 6 nucleotides.

4. The asymmetric interfering RNA duplex molecule of claim 1 wherein the antisense strand comprises a 5' blunt end.

5. The asymmetric interfering RNA duplex molecule of claim 1 wherein at least one nucleotide of the 5' overhang is not complementary to a target mRNA sequence.

6. The asymmetric interfering RNA duplex molecule of claim 1 wherein at least one nucleotide of the 5' overhang is selected from A, U or dT.

7. The asymmetric interfering RNA duplex molecule of claim 1 wherein the double-stranded region comprises at least one nick, nick or mismatch.

8. The asymmetric interfering RNA duplex molecule of claim 1 conjugated to an entity selected from the group consisting of a peptide, an antibody, a polymer, a lipid, an oligonucleotide, cholesterol, and an aptamer.

9. The asymmetric interfering RNA duplex molecule of claim 1 wherein the 3 'overhang and/or 5' overhang is stabilized against degradation.

10. The asymmetrically interfering RNA duplex molecule of claim 1, comprising at least one deoxynucleotide, or comprising at least one modified nucleotide or an analog thereof.

11. The asymmetric interfering RNA duplex molecule of claim 10 wherein the at least one modified nucleotide or its analogue is a sugar, backbone and/or base modified ribonucleotide.

12. The asymmetric interfering RNA duplex molecule of claim 11 wherein the backbone-modified ribonucleotide has a modification in a phosphodiester linkage with another ribonucleotide.

13. The asymmetric interfering RNA duplex molecule of claim 12 wherein the phosphodiester linkage is modified to include at least one of a nitrogen or sulfur heteroatom.

14. The asymmetric interfering RNA duplex molecule of claim 10 wherein the at least one modified nucleotide or its analogue is a rare base or a modified base.

15. The asymmetrically-interfering RNA duplex molecule of claim 10, wherein said nucleotide analog is a sugar-modified ribonucleotide wherein the 2' -OH group is selected from the group consisting of H, OR, R, halogen, SH, SR, NH₂、NHR、NR₂Or CN, wherein each R is independently C1-C6 alkyl, alkenyl, or alkynyl, and halogen is F, Cl, Br, or I.

16. The asymmetrically-interfering RNA duplex molecule of claim 10, wherein said nucleotide analog is a backbone-modified ribonucleotide comprising a phosphorothioate group.

17. The asymmetric interfering RNA duplex molecule according to claim 1 wherein the antisense strand comprises a sequence that is substantially complementary to a target mRNA sequence of a gene selected from the group consisting of developmental genes, oncogenes, tumor suppressor genes and enzyme genes, and adhesion molecules, cyclin kinase inhibitors, Wnt family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines or receptors thereof, growth/differentiation factors or receptors thereof, neurotransmitters or receptors thereof, kinases, signal transduction proteins, genes of infectious diseases, ABLI, BCLl, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETS1, MYS 1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYL, MYS 46B, MYC, CN, NRAS 1, PAS, PAP, DNAS, PAP, DNAS, DNA lyase, cellulase, DNA transferase, cellulase, DNA transferase, xylanase.

18. An expression vector comprising one or more nucleic acids encoding the asymmetric interfering RNA duplex RNA molecule of claim 1 operably linked to at least one expression control sequence.

19. The expression vector of claim 18, 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.

20. The expression vector of claim 18, which is a viral, eukaryotic or bacterial expression vector.

21. Use of an expression vector according to any one of claims 18 to 20 in the manufacture of a medicament for modulating gene expression in a cell or organism.

22. A cell comprising the vector of claim 18.

23. A cell comprising the asymmetric interfering RNA duplex RNA molecule of claim 1.

24. The cell of claim 23, wherein the cell is a mammalian, avian, or bacterial cell.

25. Use of a cell according to any one of claims 22 to 24 in the manufacture of a medicament for modulating gene expression in a cell or organism.

26. Use of the asymmetric interfering RNA duplex molecule of claim 1 in the preparation of a medicament for modulating gene expression in a cell or organism, wherein the medicament, when administered, comprises the steps of: contacting said cell or organism with an asymmetric interfering RNA duplex molecule according to claim 1 under conditions wherein selective gene silencing can occur, and

mediates selective gene silencing effected by the RNA duplex molecule against a target gene or nucleic acid having a sequence portion substantially corresponding to the RNA duplex.

27. The use of claim 26, wherein said contacting step comprises the step of introducing said RNA duplex molecule into a target cell in culture or in an organism where selective gene silencing can occur.

28. The use of claim 27, wherein said introducing step is selected from the group consisting of transfection, lipofection, electroporation, infection, injection, oral administration, inhalation, topical and regional administration.

29. The use of claim 27, wherein said introducing step comprises the use of 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.

30. The use of claim 26, wherein the target gene is a gene associated with a human or animal disease.

31. The use of claim 26, wherein the target gene is a gene of a pathogenic microorganism.

32. The use of claim 26, wherein the target gene is a viral gene.

33. The use of claim 26, wherein the target gene is a tumor-associated gene.

34. The use of claim 26, wherein the target gene is a gene associated with a disease selected from the group consisting of: autoimmune diseases, 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.

35. A pharmaceutical composition comprising at least one asymmetric interfering RNA duplex molecule of claim 1 as an active agent and one or more carriers selected from the group consisting of pharmaceutical carriers, positive charge carriers, liposomes, protein carriers, polymers, nanoparticles, cholesterol, lipids and lipoids.

36. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition is administered via a route selected from the group consisting of iv, po, sc, im, inhalation, topical, and regional administration.

37. A duplex RNA molecule selected from

Wherein A, U, G and C are nucleotides and a, t, g and C are deoxynucleotides.