WO2008073957A2

WO2008073957A2 - Compounds and methods for modulating the silencing of a polynucleotide of interest

Info

Publication number: WO2008073957A2
Application number: PCT/US2007/087172
Authority: WO
Inventors: Peng Jin
Original assignee: Emory University
Priority date: 2006-12-12
Filing date: 2007-12-12
Publication date: 2008-06-19
Also published as: WO2008073957A3; WO2008073957A8

Abstract

Methods and compositions comprising chemical compounds that modulate the silencing of a polynucleotide of interest in a cell are provided. Such chemical compounds when used in combination with an appropriate silencing element can be used to modulate (e.g., increase) the level of the polynucleotide targeted by the silencing element. Methods of using such compositions both in therapies involving RNAi -mediated suppression of gene expression, as well as, in vitro methods that allow for the targeted modulation of expression of a polynucleotide of interest are provided. Pharmaceutical or cosmetic compositions comprising such compounds and silencing elements also are disclosed. Methods for screening a compound of interest for the ability to modulate the activity of a heterologous silencing element also are provided.

Description

COMPOUNDS AND METHODS FOR MODULATING THE SILENCING OF A POLYNUCLEOTIDE OF INTEREST

FIELD OF THE INVENTION

The presently disclosed subject matter generally relates to methods and compositions for modulating the silencing of a polynucleotide of interest in a cell. More particularly, the presently disclosed subject matter relates to the use of small molecules, which when used in combination with silencing elements modulate expression of a polynucleotide of interest.

BACKGROUND

Gene regulation by RNA interference (RNAi) has been recognized as one of the major regulatory pathways in eukaryotic cells. See Plasterk (2006) Cell 124 (5): 877-81. Long double-stranded RNAs (dsRNAs) can be used to suppress or silence the expression of target genes in a variety of organisms and cell types. Upon introduction into an organism or a cell, the long dsRNAs enter a natural cellular pathway, referred to as the RNA interference (RNAi) pathway, wherein the dsRNA is degraded by the cytoplasmic nuclease Dicer. Dicer cleaves the long dsRNA into 20 to 25 base pair (bp) small interfering RNAs (siRNAs), which then unwind and assemble into RNA-induced silencing complexes (RISCs). The antisense siRNA strand then guides the RISC to complementary RNA molecules, wherein the RISC cleaves the target mRNA, leading to specific gene silencing. If the complementary RNA is not perfect, the RISC may only bind to the mRNA, which also blocks translation, thereby inhibiting expression.

Two types of small regulatory RNAs that regulate gene expression have been identified: small interference RNAs (siRNAs) and micro RNAs (miRNAs).

Referring now to Figure 1, in animals, siRNAs direct target messenger RNA (mRNA) cleavage, i.e., mRNA degradation, whereas miRNAs block target mRNA translation, i.e., translation suppression. Thus, in animals, RNAi operates through two post- transcriptional mechanisms: targeted mRNA degradation (siRNA) and the suppression of translation (miRNA). In animals, an RNAi pathway in which silencing occurs by a process involving cleavage of a target transcript mediated by a short RNA that binds to a target transcript to form a duplex structure is referred to as an siRNA RNAi pathway. An RNAi pathway in which silencing occurs by a process involving translational suppression mediated by a short RNA that binds to a target transcript to form a duplex structure is referred to as an miRNA translational suppression pathway. Recent data suggest that siRNAs and miRNAs incorporate into similar protein complexes and that a critical determinant of mRNA degradation versus translation regulation is the degree of sequence complementary between the small RNAs and their mRNA target.

RNAi is a powerful method for the study of gene function in animals and plants. RNAi by small dsRNAs is highly specific because only mRNAs with sequences complementary to the interfering RNA are degraded or blocked. RNAi can be induced by endogenous dsRNA, as well as by exogenous siRNA, for example, after transfection of synthetic siRNA molecules. This feature allows the function of a gene by the selective abrogation of its transcript (i.e., gene knock-down) to be studied through RNAi. Because RNAi technology involves natural cellular mechanisms, RNAi technology can be more efficient than the artificial antisense RNA approach that has failed in numerous experimental settings. Further, because most mammalian cells initiate a potent antiviral response upon introduction of dsRNAs longer than about 30 bp, siRNAs ranging from about 20 bp to about 25 bp typically are used to induce RNAi in mammalian systems without eliciting an antiviral response.

Although RNAi is a relatively new technique, its potential therapeutic applications are significant and far-reaching. The RNAi mechanism has been co- opted by researchers and has achieved broad utility in gene-function analysis, drug- target discovery and validation, and therapeutic development. See Dykxhoorn and Lieberman (2005) Annu Rev Med 56: 401-23; Dykxhoorn and Liebermann (2006) Annu Rev Biomed Eng 8: 377-402. More particularly, RNAi represents a promising therapeutic approach for diseases that result from aberrant protein synthesis. For example, RNAi has been used as a therapy for treating genetic disorders and viral infections. Because RNAi can be used to target virtually any protein, RNAi -based therapies can be developed for almost any therapeutic area. In this way, inhibiting or eliminating a target mRNA would result in a significant decrease in the expression level of a specific protein, thereby serving as a powerful therapeutic tool. Therapeutics based on RNAi potentially have significant advantages over current disease treatments, including, but not limited to, broad applicability, high therapeutic specificity, and target RNA destruction resulting in a decrease or termination of disease progression. Due to its importance in endogenous gene regulation and its use as a research tool, the RNAi mechanism has been extensively studied for the past several years. Although the major components of the endogenous RNAi machinery have been identified, little is known about the regulation of the RNAi pathway itself. Thus, the mechanisms involved in RNAi, remain poorly understood. For example, many of the molecular components that mediate RNAi remain unidentified. In addition, synthetic siRNAs are easily degraded by RNase before entering the target cell and before exerting their silencing functionalities. Thus, the design and delivery of interfering RNAs to efficiently knock down endogenous genes have been a challenge in the art. To fully exploit the potential of RNAi there is a need in the art for reagents and methods that can be used to identify molecules that can influence the efficacy of

RNAi. Methods known in the art involve stabilizing synthetic siRNAs against RNase degradation to achieve higher efficacy extracellularly. The effects of such methods, however, are limited. Therefore, there is a need in the art for molecules that are capable of modulating the suppressive effect of interfering RNA on gene expression, that is molecules that can regulate, control, or modulate, e.g., enhance or inhibit RNAi pathways, and for tools that allow identification of such molecules. The presently disclosed subject matter addresses, in whole or in part, these and other needs in the art.

SUMMARY

In some embodiments, the presently disclosed subject matter provides a method for modulating the silencing of a target polynucleotide by RNAi inside a cell.

The method comprises administering to a cell a reagent comprising a heterologous silencing element, which decreases the level of a target polynucleotide when inside the cell, and an effective amount of at least one modulator. In some embodiments, the presently disclosed modulator is an RNAi enhancer, which increases the silencing element's ability to decrease the level of a target polynucleotide when inside the cell.

In some embodiments, the presently disclosed RNAi enhancer comprises at least one compound of Formula (aa) as disclosed herein. In some embodiments, the presently disclosed subject matter provides a method for decreasing the level of a target polynucleotide in a cell, the method comprising administering to the cell an effective amount of a compound of Formula (aa), or a derivative or analog thereof, wherein the cell further comprises at least one heterologous silencing element.

In some embodiments, the presently disclosed subject matter provides a pharmaceutical or cosmetic composition comprising a polynucleotide comprising a silencing element, at least one modulating compound, and a pharmaceutically or cosmetically acceptable carrier. More particularly, in some embodiments, the presently disclosed subject matter provides a pharmaceutical or cosmetic composition comprising one or more polynucleotide comprising a silencing element, which, when administered to the cell, decreases the level of a target polynucleotide; a compound of Formula (aa) as disclosed herein; and a pharmaceutically or cosmetically acceptable carrier. In some embodiments, the presently disclosed subject matter provides a method for treating a disease state or unwanted condition, the method comprising administering to a subject in need of treatment thereof an effective amount of a polynucleotide comprising a silencing element, which, when administered to said subject, decreases the level of the target polynucleotide and an effective amount of a compound of Formula (aa), as disclosed herein, and pharmaceutically or cosmetically acceptable salts thereof.

In some embodiments, the presently disclosed subject matter provides a method for screening a compound of interest for the ability to modulate the activity of a heterologous silencing element, the method comprising: (a) providing a host cell that stably expresses a reporter gene, wherein said host cell further comprises at least one heterologous silencing element capable of inhibiting the expression of the reporter gene; (b) administering to the cell a compound of interest; and (c) measuring the expression of the reporter gene.

The presently disclosed modulating agents represent a novel way to improve the efficiency of RNA interference and gene knock-down. This finding has clinical applications as it improves the efficiency of gene knock-down for RNAi -mediated therapeutic intervention.

Accordingly, it is an object of the presently disclosed subject matter to provide compounds and methods for modulating RNA interference and gene knock-down. It is another object of the presently disclosed subject matter to provide compounds and methods for modulating, i.e., enhancing or inhibiting, RNA interference and gene knock-down intracellularly. It is another object of the presently disclosed subject matter to use the presently disclosed compounds of Formula (aa) to enhance RNA interference and gene knock-down. It is another object of the presently disclosed subject matter to provide a pharmaceutical or cosmetic composition comprising at least one compound of Formula (aa), a pharmaceutically or cosmetically acceptable carrier, and, at least one polynucleotide comprising a silencing element, which, when administered to the subject, decreases the level of a target polynucleotide. It is another object of the presently disclosed subject matter to provide a method for treating a disease state or unwanted condition.

It is another object of the presently disclosed subject matter to provide a method for screening a compound of interest for the ability to modulate the activity of a heterologous silencing element. Certain objects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other objects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will now be made to the accompanying Drawings, which are not necessarily drawn to scale, and wherein:

Figure 1 (prior art) shows a schematic representation of the common biological pathway of gene knock-down mechanisms involving siRNA-mediated mRNA degradation and miRNA-mediated translation suppression.

Figure 2 shows a schematic representation of the development of the presently disclosed reporter system for the chemical screening for compounds that modulate siRNA-mediated mRNA degradation and gene knock-down. Figure 3 shows the identification of an inhibitor of siRNA-mediated mRNA degradation and gene knock-down.

Figure 4 shows enhancement of mRNA-mediated mRNA degradation and gene knock-down by a presently disclosed quinolone compound, e.g., enoxacin. Figure 5 shows enhancement of mRNA-mediated mRNA degradation and gene knock-down by presently disclosed quinolone compounds, e.g., enoxacin, ciproflaxin, and ofloxacin.

Figure 6 shows a schematic representation of a microRNA (miRNA) sensor in mammalian cells. (A) Design of a siRNA duplex against miR-30a precursor (siRNA- p) and a control siRNA duplex (siRNA-c). The miR-30a precursor is provided as SEQ ID NO : 1. The siRNA target region is represented by nucleotides 21 -40 of SEQ ID NO: 1. The siRNA-p is provided as SEQ ID NO:2 and the siRNA-c is provided as SEQ ID NO:3. The double stranded siRNA is shown on the bottom and predicted pairing between one siRNA strand (blue) and the precursor target (green).

Figure 7 shows the relative miRNA-mediated suppression exhibited by a presently disclosed RNAi inhibitor (RNAi-I) as compared to "no drug" and enoxacin.

Figure 8 shows the suppression of the translation of Lin28 by the expression of miR-125a, wherein the addition of RNAi-E further enhances the suppression. Figures 9a-9e show the identification of a small molecule enhancing RNA interference through a chemical screen.

Figure 9a illustrates an embodiment of the presently disclosed chemical screening method as shown in Figure 2, wherein human 293 cells stably expressing EGFP (293 -EGFP) were infected with lentivirus producing shRNA-EGFP and the resulting 293-EGFP-siRNA cells with the reduced expression of GFP were isolated.

The 293-EGFP-siRNA cells additionally transfected with 2-0-methyl RNA against the GFP siRNA are shown on the right. The 293-EGFP-siRNA cells were used for chemical screen, which led to the identification of RNAi-E, the structure of which is shown on the right. Figure 9b illustrates an embodiment of the presently disclosed chemical screening method wherein RNAi-E enhances shRN A-EGFP -mediated gene silencing. Without RNAi-E (top image of Fig. 9b) 293-EGFP-siRNA cells express moderate levels of GFP. In the presence of RNAi-E (bottom image of Fig. 9b), the level of GFP is greatly reduced. This effect also can be observed in western blots using anti- EGFP antibody (bottom panel of Fig. 9b) with GAPDH as a loading control. In the absence of shGFP, RNAi-E has no effect on GFP expression.

Figure 9c illustrates an embodiment of the presently disclosed chemical screening method wherein RNAi-E enhances shGAPDH-mediated gene silencing. Relative GAPDH mRNA levels in cells transfected with control vector, shRNA against GAPDH, and shRNA plus RNAi-E, respectively, are determined by quantitative RT-PCR.

Figure 9d illustrates an embodiment of the presently disclosed chemical screening method wherein RNAi-E reduces the dosage of siRNA duplex required for gene silencing. NIH 3T3 cells were transfected with different amounts of siRNA duplexes against mouse GAPDH in the absence or presence of RNAi-E. Relative GAPDH mRNA levels in the cells transfected with control and different doses of siRNA are determined by quantitative RT-PCR.

Figure 9e illustrates an embodiment of the presently disclosed chemical screening method wherein RNAi-E enhances miR-125a-mediated translational suppression. Luciferase constructs containing (Lin-28) or lacking (Lin-28-A) miR- 125 a target sequences were transfected into cells stably expressing human miR-125a in the absence or presence of RNAi-E. Normalized relative luciferase activities as shown in Figure 8 also are shown here. Figures 10a and 10b show the determination of the structural components of

RNAi-E derivatives and analogs that influence RNAi-E activity.

Figure 10a shows the chemical structures of RNAi-E derivatives and analogs.

Figure 10b shows the relative RNAi-E activities of RNAi-E derivatives and analogs. The RNAi-E activity was determined using 293-EGFP-siRNA cells and fluorescent quantification on an Analyst HT plate reader. After subtraction of background fluorescence, the reduction of GFP fluorescence by RNAi-E was set as 100%. Relative fluorescence intensity reductions measured from the cells treated with the other compounds were normalized to the fluorescence reduction by RNAi-E.

Figures 1 Ia-I Ie show embodiments of the presently disclosed subject matter wherein RNAi-E promotes microRNA processing by facilitating the interaction between TRBP and RNAs.

Figure 11a shows Northern blots that demonstrate that RNAi-E enhances the level of mature siRNAs and miR-125a in cells transfected with shRNA against GFP, Luciferase, and miR-125a, respectively. 5S was used as loading control. Figure l ib shows embodiments of the presently disclosed subject matter wherein quantitative RT-PCR was used to measure the levels of endogenous pri-, pre- and mature forms of miR-125x, miR-124a and miR-199b in the control and RNAi-E treated HEK-293 cells. The probe for each pre-miRNA also recognizes the corresponding pri-miRNA, and therefore measures both forms. The relative expression levels as determined by DACt analyses are shown.

Figure l ie shows the miRNA profiles in control and RNAi-E treated HEK- 293 cells examined by MiRNA TagMan® assay. The miRNAs with consistent and significant changes from two independent experiments are shown. All miRNA profiles except for Let-7i and MiR- 128b (bottom two of Fig. 1 Ic) exhibit an increase in the mature miRNA level in the presence of RNAi-E. Let-7i and MiR- 128b exhibit a decrease. The results for two replicate experiments are shown.

Figure 1 Id shows in vitro processing of radioactively-labeled let- 7 precursor by Dicer or Dicer with TRBP. Arrows indicate the precursor and processed miRNAs.

Figure l ie shows an RNA gel-shift assay with recombinant TRBP protein and let- 7 precursor RNA in the presence or absence of RNAi-E or an RNAi-E derivative or analog. The TRBP-precursor RNA complex and precursor RNA alone are indicated. Figures 12a and 12b demonstrate that RNAi-E enhances RNA interference in vivo.

Figure 12a shows the lethality of wildtype N2 worms fed with different dsRNAs in the absence or presence of RNAi-E. P<0.001 RNAi-E treated versus untreated in mom-5 and let-2 RNAi experiments. *P<0.001 treated compared to untreated with RNAi-E.

Figure 12b shows an embodiment of the presently disclosed subject matter wherein the ears of GFP transgenic mice (ACTB-EGFP) were injected with shRNA- EGFP expressing lentivirus, Lv-siGFP, with or without RNAi-E treatment. The relative GFP mRNA levels in the control ears, injected with Lv-siGFP only, injected with both Lv-siGFP and RNAi-E, and injected with RNAi-E alone, are shown.

*P<0.001 when Lv-siGFP compared to control and Lv-siGFP+RNAi-E compared to Lv-siGFP alone.

Figure 13 shows that RNAi-E can enhance the duration of the siRNA effect.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Drawings, in which some, but not all embodiments of the presently disclosed subject matter are shown. Indeed, the presently disclosed subject matter can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Throughout the specification and claims, a given chemical formula or name shall encompass all optical and stereoisomers, as well as racemic mixtures where such isomers and mixtures exist.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Throughout this specification and the claims, the words "comprise," "comprises" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise.

I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms "a", "an", and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a cell" includes a plurality of such cells, unless the context clearly is to the contrary (e.g., a plurality of cells), and so forth.

As used herein, the term "about," when referring to a value is meant to encompass variations of in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ± 1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions. The term "complementary" is used herein in accordance with its art-accepted meaning to refer to the capacity for precise pairing via hydrogen bonds (e.g., Watson- Crick base pairing or Hoogsteen base pairing) between two nucleosides, nucleotides or nucleic acids, and the like. For example, if a nucleotide at a certain position of a first nucleic acid is capable of stably hydrogen bonding with a nucleotide located opposite to that nucleotide in a second nucleic acid, when the nucleic acids are aligned in opposite 5' to 3' orientation (i.e., in anti-parallel orientation), then the nucleic acids are considered to be complementary at that position (where position may be defined relative to either end of either nucleic acid, generally with respect to a 5' end). The nucleotides located opposite one another can be referred to as a "base pair." A complementary base pair contains two complementary nucleotides, e.g., A and U, A and T, G and C, and the like, whereas a noncomplementary base pair contains two noncomplementary nucleotides (also referred to as a mismatch). Two polynucleotides are said to be complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that hydrogen bond with each other, i.e., a sufficient number of base pairs are complementary.

The compositions disclosed herein, such as the presently disclosed compounds of Formula (aa) and the one or more polynucleotide comprising a silencing element are used in combination. The term "combination" is used in its broadest sense and means that a cell or a subject is administered at least two agents, more particularly a chemical compound disclosed herein and a silencing element of interest.

The timing of administration of the chemical compound and the silencing element can be varied so long as the beneficial effects of the combination of these agents are achieved (i.e., modulating the silencing of a target polynucleotide of interest in a cell or in a subject). The phrase "in combination with" refers to the administration of a chemical compound with a silencing element either simultaneously, sequentially, or a combination thereof. Therefore, a cell or a subject administered a combination of the invention can receive a chemical compound and the silencing element at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the cell or the subject. When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the chemical compound and the silencing element are administered simultaneously, they can be administered to the cell or administered to the subject as separate pharmaceutical or cosmetic compositions, each comprising either a chemical compound or a silencing element, or they can contact the cell as a single composition or be administered to a subject as a single pharmaceutical or cosmetic composition comprising both agents.

When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

An "effective amount" of an active agent refers to the amount of the active agent sufficient to elicit a desired biological response. As will be appreciated by one of ordinary skill in the art, the absolute amount of a particular agent that is effective can vary depending on such factors as the desired biological endpoint, the agent to be delivered, the target cell or tissue, and the like. One of ordinary skill in the art will further understand that an effective amount can be administered in a single dose, or can be achieved by administration of multiple doses. An entity such as a gene or an expression product thereof, is considered

"endogenous" to a cell if it is naturally present within the cell in the absence of modification of the cell, or an ancestor of the cell, by the hand of man. It will be appreciated that the amount of an endogenous RNA (and thus of a protein encoded by the RNA) present within a cell can be increased above its naturally occurring level by introducing a template for transcription of the RNA, operably linked to appropriate regulatory elements, into the cell. As applied to genes, RNAs, proteins, and the like, the term endogenous is generally understood to refer to genes, RNAs, proteins, and the like, as they naturally exist within a cell, unless otherwise indicated.

As used herein, the term "intracellular" or "intracellularly" has its ordinary meaning as understood in the art. In general, the space inside of a cell, which is encircled by a membrane, is defined as "intracellular" space. Similarly, as used herein, the term "extracellular" or "extracellualary" has its ordinary meaning as understood in the art. In general, the space outside of the cell membrane is defined as "extracellular" space.

As used herein, the term "gene" has its meaning as understood in the art. In general, a gene is taken to include gene regulatory sequences (e.g., promoters, enhancers, and the like) and/or intron sequences, in addition to coding sequences (open reading frames). It will further be appreciated that definitions of "gene" include references to nucleic acids that do not encode proteins but rather encode functional

RNA molecules, or precursors thereof, such as microRNA or siRNA precursors, tRNAs, and the like.

The term "gene knock-down" generally refers to the use of a reagent to decrease the level of a polynucleotide of interest. As discussed in further detail elsewhere herein, polynucleotides comprising RNAi silencing elements can be used in such knock-down methods. For example, one reagent-based gene knock-down method employs siRNA or miRNAs as silencing elements. Gene knock-down by RNAi is a research tool that can be used for the analysis of gene function and for target identification and target validation. A "gene product" or "expression product" is, in general, an RNA transcribed from the gene (e.g., either pre- or post-processing) or a polypeptide encoded by an RNA transcribed from the gene (e.g., either pre- or post-modification).

The term "hybridize" as used herein refers to the interaction between two complementary nucleic acid sequences in which the two sequences remain associated with one another under appropriate conditions.

As used herein, the term "isolated" means separated from at least some of the components with which it is usually associated in nature; prepared or purified by a process that involves the hand of man; not occurring in nature; and/or not present as an integral part of an organism. The terms "nucleic acid," "polynucleotide," or "oligonucleotide" generally are used herein in their art-accepted manners to refer to a polymer of nucleotides. As used herein, an oligonucleotide is typically less than 100 nucleotides in length. Naturally occurring nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The polynucleotide or oligonucleotide may include natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine), or synthetic nucleosides, such as, nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5-propynyluridine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, and 2-thiocytidine), and/or nucleosides comprising chemically or biologically modified bases, (e.g., methylated bases), intercalated bases, and/or modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose). The phosphate groups in a polynucleotide or oligonucleotide are typically considered to form the internucleoside backbone of the polymer. In naturally occurring nucleic acids (e.g., DNA or RNA), the backbone linkage is via a 3' to 5' phosphodiester bond. Polynucleotides and oligonucletides containing modified backbones or non-naturally occurring internucleoside linkages, however, also can be used in the presently disclosed subject matter. Such modified backbones include backbones that have a phosphorus atom in the backbone and others that do not have a phosphorus atom in the backbone. Examples of modified linkages include, but are not limited to, phosphorothioate and 5'-N-phosphoramidite linkages. Polynucleotides and oligonucleotides need not be uniformly modified along the entire length of the molecule. For example, different nucleotide modifications, different backbone structures, and the like, may exist at various positions in the polynucleotide or oligonucleotide. Any of the polynucleotides described herein may utilize these modifications.

As used herein, the term "small molecule" refers to organic compounds, whether naturally-occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds. The term "subject" refers to an organism to which the presently disclosed compounds and/or pharmaceutical or cosmetic compositions can be administered. In specific embodiments, a subject is a mammal. In other embodiments, a subject is a primate, a human, a domestic animal or an agricultural animal. A cell can also be employed in the methods and compositions of the invention. Any cell can be used; however, in specific embodiments, the cell is from a mammal, a primate, a human, a domestic animal or an agricultural animal. Such host cells include cultured cells (in vitro), explants and primary cultures (in vitro and ex vivo) and cells in vivo. As used herein, the term "treating" generally can include reversing, alleviating, inhibiting the progression of, preventing or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed active agents can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition. The presently disclosed active agents also can be used for cosmetic applications, as well.

As used herein, the term "modulator" means a reagent that can influence, either enhance or inhibit, the activity of another reagent or element, e.g., a silencing element, when administered in combination a cell.

II. Modulating Levels of Target Polynucleotides

As disclosed hereinabove, RNA interference (RNAi) is a well-conserved mechanism that uses small noncoding RNAs to silence gene expression post- transcriptionally. See Hannon (2002) Nature 418(6894): 244-51; Bartel (2004) Cell 116(2): 281-97. The endogenous small RNAs can shape diverse cellular pathways, including chromosome architecture, development, growth control, apoptosis, and stem cell maintenance. See Zamore and Haley (2005) Science 309(5740): 1519-24. Because small double-stranded RNAs, called small interfering RNAs (siRNAs), induce sequence-specific mRNA degradation when introduced into cells, RNAi has become an attractive tool for functional genomics and a potential class of molecules for human therapeutics. See Dykxhoorn and Lieberman (2006) Cell 126(2): 231-5. Although major components in the RNAi pathway have been identified, regulatory mechanisms for this pathway remain largely unknown.

The presently disclosed subject matter demonstrates that the RNAi pathway can be modulated intracellularly by small molecules. More particularly, the presently disclosed subject matter provides a novel cell-based assay to monitor the activity of the RNAi pathway and identify the RNAi pathway modulators. As disclosed herein, this cell-based assay can be used to screen a chemical library of diversified compounds to identify one or more small molecules that enhance siRNA-mediated mRNA degradation. One such small molecule, enoxacin, a second generation quinolone, is referred to herein as "RNAi-E."

In some embodiments, the presently disclosed subject matter demonstrates that in cells, RNAi-E enhances the efficiency of target gene knock-down mediated by either short hairpin RNA or siRNA duplexes. As provided in more detail herein below, the increased knock-down efficiency in the presence of RNAi-E reduces the required dosage of siRNA duplex by, in some embodiments, at least five fold. By preparing and testing derivatives and analogs of RNAi-E, the presently disclosed subject matter describes structural components of RNAi-E that influence the level of its RNAi-enhancing activity.

Further, the presently disclosed subject matter discloses that, in relation to the role of RNAi-E in the RNAi pathway, RNAi-E promotes the precursor processing/ loading step in the RNAi pathway by facilitating the interaction between the human immunodeficiency virus trans-activating response RNA-binding protein (TRBP) and RNAs. In some embodiments, the RNAi-enhancing effects of RNAi-E improve gene knock-down efficiency in vivo in Caenorhabditis elegans (C. elegans) and mouse models. Accordingly, the identification of RNAi-E and other RNAi-enhancing molecules provides greater insight into the modulation of the RNAi pathway and facilitates the development of siRNAs as therapeutic agents. Thus, in some embodiments, the presently disclosed methods and compositions provide small molecules which modulate RNA interference in a cell and when administered in combination with at least one silencing element are employed to reduce the level of expression of a target polynucleotide of interest. As used herein, a "target sequence" comprises any sequence that one desires to decrease the level of expression. By "reduces" or "reducing" the expression level of a polynucleotide or a polypeptide encoded thereby is intended to mean, the polynucleotide or polypeptide level of the target sequence is statistically lower than the polynucleotide level or polypeptide level of the same target sequence in an appropriate control which is not exposed to the silencing element. In particular embodiments, reducing the polynucleotide level and/or the polypeptide level of the target sequence according to the presently disclosed subject matter results in less than 95%, less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the polynucleotide level, or the level of the polypeptide encoded thereby, of the same target sequence in an appropriate control. Methods to assay for the level of the RNA transcript, the level of the encoded polypeptide, or the activity of the polynucleotide or polypeptide are discussed elsewhere herein.

In other embodiments, the chemical compound decreases the activity of a silencing element. By "decrease" the activity of a silencing element is intended that the ability of the silencing element to decrease expression of a target polynucleotide is decreased by any statistically significant amount including a decrease of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

A. Silencing Elements

As used herein, the term "silencing element" refers to a polynucleotide comprising or encoding an interfering RNA that is capable of reducing or eliminating the level of expression of a target polynucleotide or the polypeptide encoded thereby. The term "interfering RNA" or "RNAi" refers to any RNA molecule which can enter an RNAi pathway and thereby reduce the level of a target polynucleotide of interest or reduce the level of expression of a polynucleotide of interest. RNAi is distinct from so-called "antisense" mechanisms that typically involve inhibition of a target transcript by a single-stranded oligonucleotide through RNase H mediated pathway. See Crooke (ed.) (2001) "Antisense Drug Technology: Principles, Strategies, and Applications," 1st ed., (Marcel Dekker; ISBN: 0824705661).

Thus, a silencing element can comprise the interfering RNA, a precursor to the interfering RNA, a template for the transcription of an interfering RNA or a template for the transcription of a precursor interfering RNA, wherein the precursor is processed within the cell to produce an interfering RNA. Thus, for example, a dsRNA silencing element includes a dsRNA molecule, a transcript or polyribonucleotide capable of forming a dsRNA, more than one transcript or polyribonucleotide capable of forming a dsRNA, a DNA encoding dsRNA molecule, or a DNA encoding one strand of a dsRNA molecule. When the silencing element comprises a DNA molecule encoding an interfering RNA, it is recognized that the DNA can be transiently expressed in a cell or stably incorporated into the genome of the cell. Such methods are discussed in further detail elsewhere herein.

The silencing element can reduce or eliminate the expression level of a target sequence by influencing the level of the target RNA transcript, by influencing translation and thereby affecting the level of the encoded polypeptide, or by influencing expression at the pre-transcriptional level (i.e., via the modulation of chromatin structure, methylation pattern, etc., to alter gene expression). See, e.g., Verdel et al. (2004) Science 303:672-676; Pal-Bhadra et al. (2004) Science 303:669- 672; Allshire (2002) Science 297:1818-1819; Volpe et al (2002) Science 297:1833- 1837; Jenuwein (2002) Science 297:2215-2218; and Hall et al. (2002) Science

297:2232-2237. Methods to assay for functional interfering RNA that are capable of reducing or eliminating the level of a sequence of interest are disclosed elsewhere herein.

Any region of the target polynucleotide can be used to design a domain of the silencing element that shares sufficient sequence identity to allow for the silencing element to decrease the level of the target polynucleotide. For instance, the silencing element can be designed to share sequence identity to the 5' untranslated region of the target polynucleotide(s), the 3' untranslated region of the target polynucleotide(s), exonic regions of the target polynucleotide(s), intronic regions of the target polynucleotide(s), and any combination thereof.

The ability of a silencing element to reduce the level of the target polynucleotide may be assessed directly by measuring the amount of the target transcript using, for example, Northern blots, nuclease protection assays, reverse transcription (RT)-PCR, real-time RT-PCR, microarray analysis, and the like. Alternatively, the ability of the silencing element to reduce the level of the target polynucleotide may be measured directly using a variety of affinity-based approaches (e.g., using a ligand or antibody that specifically binds to the target polypeptide) including, but not limited to, Western blots, immunoassays, ELISA, flow cytometry, protein microarrays, and the like. In still other methods, the ability of the silencing element to reduce the level of the target polynucleotide can be assessed indirectly, e.g., by measuring a functional activity of the polypeptide encoded by the transcript or by measuring a signal produced by the polypeptide encoded by the transcript.

Representative types of silencing elements are discussed in further detail below.

i. Double Stranded RNA Silencing Elements

In one embodiment, the silencing element comprises or encodes a double stranded RNA molecule. As used herein, a "double stranded RNA" or "dsRNA" refers to a polyribonucleotide structure formed either by a single self-complementary RNA molecule or a polyribonucleotide structure formed by the expression of least two distinct RNA strands. Accordingly, as used herein, the term "dsRNA" is meant to encompass other terms used to describe nucleic acid molecules that are capable of mediating RNA interference or gene silencing, including, for example, small RNA (sRNA), short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-

RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA), and others. See, e.g., Meister and Tuschl (2004) Nature 431 :343-349 and Bonetta et al. (2004) Nature Methods 1 :79-86.

In specific embodiments, at least one strand of the duplex or double-stranded region of the dsRNA shares sufficient sequence identity or sequence complementarity to the target polynucleotide to allow for the dsRNA to reduce the level of expression of the target sequence. As used herein, the strand that is complementary to the target polynucleotide is the "antisense strand," and the strand homologous to the target polynucleotide is the "sense strand." In one embodiment, the dsRNA comprises a hairpin RNA. A hairpin RNA comprises an RNA molecule that is capable of folding back onto itself to form a double stranded structure. Multiple structures can be employed as hairpin elements. For example, the hairpin RNA molecule that hybridizes with itself to form a hairpin structure can comprises a single-stranded loop region and a base-paired stem. The base-paired stem region can comprise a sense sequence corresponding to all or part of the target polynucleotide and further comprises an antisense sequence that is fully or partially complementary to the sense sequence. Thus, the base-paired stem region of the silencing element can determine the specificity of the silencing. See, e.g., Chuang and Meyerowitz (2000) Proc. Natl. Acad. ScL USA 97:4985-4990, herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga et al. (2003) MoI. Biol. Rep. 30:135-140, herein incorporated by reference.

H. siRNA Silencing Elements A "short interfering RNA" or "siRNA" comprises an RNA duplex (double- stranded region) and can further comprises one or two single-stranded overhangs, e.g., 3 ' or 5' overhangs. The duplex can be approximately 19 base pairs (bp) long, although lengths between 17 and 29 nucleotides, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 nucleotides, can be used. An siRNA can be formed from two RNA molecules that hybridize together or can alternatively be generated from a single RNA molecule that includes a self-hybridizing portion. The duplex portion of an siRNA can include one or more bulges containing one or more unpaired and/or mismatched nucleotides in one or both strands of the duplex or can contain one or more noncomplementary nucleotide pairs. One strand of an siRNA (referred to herein as the antisense strand) includes a portion that hybridizes with a target transcript. In certain embodiments, one strand of the siRNA (the antisense strand) is precisely complementary with a region of the target transcript over at least about 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides or more meaning that the siRNA antisense strand hybridizes to the target transcript without a single mismatch

(i.e., without a single noncomplementary base pair) over that length. In other embodiments, one or more mismatches between the siRNA antisense strand and the targeted portion of the target transcript can exist. In embodiments in which perfect complementarity is not achieved, any mismatches between the siRNA antisense strand and the target transcript can be located at or near 3' end of the siRNA antisense strand. For example, in certain embodiments, nucleotides 1-9, 2-9, 2-10, and/or 1-10 of the antisense strand are perfectly complementary to the target.

Considerations for design of effective siRNA molecules are discussed in McManus et al. (2002) Nature Reviews Genetics 3: 737-747 and in Dykxhoorn et al. (2003) Nature Reviews Molecular Cell Biology 4: 457-467. Such considerations include the base composition of the siRNA, the position of the portion of the target transcript that is complementary to the antisense strand of the siRNA relative to the 5' and 3' ends of the transcript, and the like. A variety of computer programs also are available to assist with selection of siRNA sequences, e.g., from Ambion (web site having URL www.ambion.com), at web site having URL www.sinc.sunysb.edu/Stu/shilin/rnai.html. Additional design considerations that also can be employed are described in Semizarov et al. Proc. Natl. Acad. Sci. 100: 6347- 6352.

Hi. Short Hairpin RNA Silencing Elements

The term "short hairpin RNA" or "shRNA" refers to an RNA molecule comprising at least two complementary portions hybridized or capable of hybridizing to form a double-stranded (duplex) structure sufficiently long to mediate RNAi (generally between approximately 17 and 29 nucleotides in length, including 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 nucleotides in length, and in some embodiments, typically at least 19 base pairs in length), and at least one single- stranded portion, typically between approximately 1 and 20 or 1 to 10 nucleotides in length that forms a loop connecting the two nucleotides that form the base pair at one end of the duplex portion. The duplex portion can, but does not require, one or more bulges consisting of one or more unpaired nucleotides. In specific embodiments, the shRNAs comprise a 3' overhang. Thus, shRNAs are precursors of siRNAs and are, in general, similarly capable of inhibiting expression of a target transcript.

In particular, RNA molecules having a hairpin (stem-loop) structure can be processed intracellularly by Dicer to yield an siRNA structure referred to as short hairpin RNAs (shRNAs), which contain two complementary regions that hybridize to one another (self-hybridize) to form a double-stranded (duplex) region referred to as a stem, a single-stranded loop connecting the nucleotides that form the base pair at one end of the duplex, and optionally an overhang, e.g., a 3' overhang. The stem can comprise about 19, 20, or 21 bp long, though shorter and longer stems (e.g., up to about 29 nt) also can be used. The loop can comprise about 1-20, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nt, about 4-10, or about 6-9 nt. The overhang, if present, can comprise approximately 1-20 nt or approximately 2-10 nt. The loop can be located at either the 5 ' or 3' end of the region that is complementary to the target transcript whose inhibition is desired (i.e., the antisense portion of the shRNA).

Although shRNAs contain a single RNA molecule that self-hybridizes, it will be appreciated that the resulting duplex structure can be considered to comprise sense and antisense strands or portions relative to the target mRNA and can thus be considered to be double-stranded. It will therefore be convenient herein to refer to sense and antisense strands, or sense and antisense portions, of an shRNA, where the antisense strand or portion is that segment of the molecule that forms or is capable of forming a duplex with and is complementary to the targeted portion of the target polynucleotide, and the sense strand or portion is that segment of the molecule that forms or is capable of forming a duplex with the antisense strand or portion and is substantially identical in sequence to the targeted portion of the target transcript. In general, considerations for selection of the sequence of the antisense strand of an shRNA molecule are similar to those for selection of the sequence of the antisense strand of an siRNA molecule that targets the same transcript. iv. MicroRNA Silencing Elements

In one embodiment, the silencing element comprises an miRNA. "MicroRNAs" or "miRNAs" are regulatory agents comprising about 19 ribonucleotides which are highly efficient at inhibiting the expression of target polynucleotides. See, e.g., Saetrom et al. (2006) Oligonucleotides 16:115-144, Wang et al. (2006) MoI. Cell 22:553-60, Davis et al. (2006) Nucleic Acid Research 34:2294- 304, Pasquinelli (2006) Dev. Cell 10:419-24, all of which are herein incorporated by reference. For miRNA interference, the silencing element can be designed to express a dsRNA molecule that forms a hairpin structure containing a 19-nucleotide sequence that is complementary to the target polynucleotide of interest. The miRNA can be synthetically made, or transcribed as a longer RNA which is subsequently cleaved to produce the active miRNA. Specifically, the miRNA can comprise 19 nucleotides of the sequence having homology to a target polynucleotide in sense orientation and 19 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence.

Referring now to Figure 1, a schematic diagram of the miRNA suppression pathway is shown. It is recognized that various forms of an miRNA can be transcribed including, for example, the primary transcript (termed the "pri-miRNA") which is processed through various nucleolytic steps to a shorter precursor miRNA

(termed the "pre -miRNA"); the pre -miRNA; or the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) and miRNA*. The pre-miRNA is a substrate for a form of dicer that removes the miRNA/miRNA* duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex. It has been demonstrated that miRNAs can be transgenically expressed and be effective through expression of a precursor form, rather than the entire primary form (McManus et al. (2002) RNA 8:842-50). In specific embodiments, 2-8 nucleotides of the miRNA are perfectly complementary to the target. A large number of endogenous human miRNAs have been identified. For structures of a number of endogenous miRNA precursors from various organisms, see Lagos-Quintana et al. (2003) RNA 9(2):175-9; see also Bartel (2004) Cell 116:281-297.

A miRNA or miRNA precursor can share at least about 80%, 85%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity with the target transcript for a stretch of at least about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In specific embodiments, the region of precise sequence complementarity is interrupted by a bulge. See Ruvkun (2001) Science 294: 797-799, Zeng et al. (2002) Molecular Cell 9:1-20, and Mourelatos et al. (2002) Genes Dev 16:720-728.

B. Preparing Silencing Elements

Those of ordinary skill in the art will readily appreciate that a silencing element can be prepared according to any available technique including, but not limited to, chemical synthesis, enzymatic or chemical cleavage in vivo or in vitro, template transcription in vivo or in vitro, or combinations of the foregoing.

i. Recombinant Expression Vectors and Host Cells

As discussed above, the silencing elements employed in the methods and compositions of the invention can comprise a silencing element. In specific embodiments, the silencing element comprises a DNA molecule which when transcribed produces an interfering RNA or a precursor thereof. In such embodiments, the DNA molecule encoding the silencing element is found in an expression cassette. The expression cassette comprises one or more regulatory sequences, selected on the basis of the cells to be used for expression, operably linked to a polynucleotide encoding the silencing element. "Operably linked" is intended to mean that the nucleotide sequence of interest (i.e., a DNA encoding a silencing element) is linked to the regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a cell when the expression cassette or vector is introduced into a cell). "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, e.g., Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, California). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression cassette can depend on such factors as the choice of the host cell to be transformed, the level of expression of the silencing element desired, and the like. Such expression cassettes typically include one or more appropriately positioned sites for restriction enzymes, to facilitate introduction of the nucleic acid into a vector.

It will further be appreciated that appropriate promoter and/or regulatory elements can readily be selected to allow expression of the relevant transcription units/silencing elements in the cell of interest. In certain embodiments, the promoter utilized to direct intracellular expression of a silencing element is a promoter for RNA polymerase III (Pol III). References discussing various Pol III promoters, include, for example, Yu et al. (2002) Proc. Natl. Acad. ScL 99(9), 6047-6052; Sui et al. (2002) Proc. Natl. Acad. ScL 99(8), 5515-5520; Paddison et al. (2002) Genes and Dev. 16,

948-958; Brummelkamp et al. (2002) Science 296, 550-553; Miyagashi (2002) Biotech. 20, 497-500; Paul et al. (2002) Nat. Biotech. 20, 505-508; Tuschl et al. (2002) Nat. Biotech. 20, 446-448. According to other embodiments, a promoter for RNA polymerase I, e.g., a tRNA promoter, can be used. See McCown et al. (2003) Virology 313(2):514-24; Kawasaki (2003) Nucleic Acids Res. 31 (2):700-7.

The regulatory sequences can also be provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus, and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells, see Chapters 16 and 17 of Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory

Press, Plainview, New York). See Goeddel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, California).

In vitro transcription may be performed using a variety of available systems including the T7, SP6, and T3 promoter/polymerase systems (e.g., those available commercially from Promega, Clontech, New England Biolabs, and the like). Vectors including the T7, SP6, or T3 promoter are well known in the art and can readily be modified to direct transcription of silencing elements. When silencing elements are synthesized in vitro the strands may be allowed to hybridize before introducing into a cell or before administration to a subject. As noted above, silencing elements can be delivered or introduced into a cell as a single RNA molecule including self- complementary portions (e.g., an shRNA that can be processed intracellularly to yield an siRNA), or as two strands hybridized to one another. In other embodiments, the silencing elements employed are transcribed in vivo. As discussed elsewhere herein, regardless of if the silencing element is transcribed in vivo or in vitro, in either scenario, a primary transcript can be produced which is then be processed (e.g., by one or more cellular enzymes) to generate the interfering RNA that accomplishes gene inhibition.

Such expression cassettes can be contained in a vector which allow for the introduction of the expression cassette into a cell. In specific embodiments, the vector allows for autonomous replication of the expression cassette in a cell or may be integrated into the genome of a cell. Such vectors are replicated along with the host genome (e.g., nonepisomal mammalian vectors). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno- associated viruses).

Accordingly, the interfering RNA may be generated by transcription from a promoter, either in vitro or in vivo. For instance, a construct may be provided containing two separate transcribable regions, each of which generates a 21 nt transcript containing a 19 nt region complementary with the other. Alternatively, a single construct may be utilized that contains opposing promoters and terminators positioned so that two different transcripts, each of which is at least partly complementary to the other, are generated. Alternatively, an RNA-inducing agent may be generated as a single transcript, for example by transcription of a single transcription unit encoding self complementary regions. A template is employed that includes first and second complementary regions, and optionally includes a loop region connecting the portions. Such a template may be utilized for in vitro transcription or in vivo transcription, with appropriate selection of promoter and, optionally, other regulatory elements, e.g., a terminator.

H. Administering a Silencing Element to a Cell

"Administering" a silencing element to a cell comprises any method that allows for the introduction of the polynucleotide into the cell including any conventional transformation or transfection techniques. Exemplary art-recognized techniques for introducing foreign polynucleotides into a host cell, including calcium phosphate or calcium chloride co -precipitation, DEAE-dextran-mediated transfection, lipofection, particle gun, or electroporation and viral vectors. Suitable methods for transforming or transfecting host cells can be found in U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York) and other standard molecular biology laboratory manuals. One of skill will recognize that depending on the method by which a polynucleotide is introduced into a cell, the silencing element can be stably incorporated into the genome of the cell, replicated on an autonomous vector or plasmid, or present transiently in the cell.

Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of viral vector procedures, see Anderson (1992) Science 256:808-813; Haddada et al. (1995) Current Topics in Microbiology and Immunology Doerfler and Bohm (eds); and Yu et al. (1994) Gene Therapy 1 :13-26.

Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of silencing elements could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Viral vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

In specific embodiments, the silencing element administered to a cell is heterologous to the cell. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a heterologous silencing element is from a species different from the species of the host cell, or, if from the same/analogous species, the silencing element is substantially modified from its native form in composition, length, and genomic locus. The silencing element could be a portion of the original form. III. Methods for Screening Compounds that Modulate the Activity of Silencing Elements

Methods and compositions are provided that increase or decrease the efficacy of RNAi in a cell or an organism, e.g., to control the extent to which a silencing element, such as an siRNA, hairpin RNA, shRNA, miRNA, and the like, present in the cell or organism is able to inhibit expression of a target polynucleotide. In specific embodiments, methods and compositions are provided that enhance the activity of a silencing element and thereby enhance the ability of a silencing element to reduce the level of a target polynucleotide.

In principle, interfering RNA libraries can be used to perform effective genome-scale functional genetic screens in mammalian cells or whole animals to identify molecules that modulate the activity of silencing elements. Accordingly, in some embodiments, the presently disclosed subject matter provides a cell-based reporter system to identify chemical compounds that could modulate the activity of an interfering RNA.

In this system, a host cell expressing a heterologous reporter gene is used to screen compounds of interest for their ability to modulate (e.g., enhance or inhibit) the effects of a heterologous silencing element by introducing a heterologous silencing element directed against the reporter gene expressed by the host cell and exposing the cell to the compound of interest. A compound that leads to an increase in expression of the reporter gene in this system is considered an inhibitor of RNAi, whereas a compound that leads to a decrease in expression of the reporter gene in this system is considered an enhancer of RNAi activity. In this context, an "increase" or a "decrease" in the expression of the reporter gene in the presence of the reporter gene- specific RNAi is relative to the expression of the reporter gene in the presence of the reporter gene-specific RNAi without exposure to the compound of interest.

The reporter gene used in the methods of the presently disclosed subject matter is one whose level of expression can be monitored. In specific embodiments, the level of expression of the reporter gene is visible to the aided or unaided eye. By

"aided" is intended the use of a device to facilitate visualization (i.e., light of any visible wavelength, a microscope or other magnifying instrument, computer hardware or software, or other device capable of detecting, quantitating and/or displaying the visible medium corresponding to the expression level of the reporter gene, such as a fluorometer, luminometer or densitometer). In some embodiments, expression is correlated directly or relatively (e.g., precisely or semi-precisely) with the visible medium (e.g., the amount of light, color, density, and the like, resulting from expression of the reporter gene). The visible medium can be measured manually or automatically by, for example, counting the number of cells in a designated field of view that are colored, fluorescent, luminescent, pixilated, or otherwise visible to the aided or unaided eye, or by quantitating the amount of visible fluorescence, luminescence, color, density, and the like, in all or part of a designated field of view using an instrument or device for performing such a function. Examples of such reporter genes include, but are not limited to, those that encode green fluorescent protein (GFP), luciferase, or beta-galactosidase. The reporter gene can be introduced into the host cell using methods routine to those of skill in the art. In some embodiments, the DNA encoding the reporter gene is found in an expression cassette as described elsewhere herein. Such expression cassettes can be contained in a vector which allows for the introduction of the expression cassette comprising the reporter gene into a host cell. In specific embodiments, a viral vector, particularly a lentiviral vector, is used to introduce the reporter gene into a host cell. A host cell used in the presently disclosed methods allows for the introduction and expression of heterologous nucleic acids and can be maintained in a liquid, solid or semi-solid cell culture media. Any appropriate host cell can be used.

In one embodiment, the human 293 cell line stably expressing the reporter gene GFP is used. One of ordinary skill in the art to which the presently disclosed subject matter pertains, would recognize upon review of the presently disclosed subject matter that any cell line, from insect to human cells, could be used in the presently disclosed screening methods. Exemplary cell lines include, but are not limited to, S2 cells, NKH3T3, NS20Y, HeLa, and HepG2 cells.

To screen for novel compounds that modulate the activity of interfering RNA, cells should be selected that express a moderate level of the reporter gene (i.e., GFP) in the presence of the interfering RNA. In specific embodiments, the moderate level of expression comprises a level of expression that allows one to detect a statistically significant increase or decrease in the level of the expression of the reporter gene. By moderate level is intended between 10% and 70%, between 15% and 60%, between 20% and 50%, or between 25% and 45% of the level of expression of the reporter gene in the absence of interfering RNA. Further, an appropriate clone is selected after the addition of siRNAs.

Thus, in some embodiments, the presently disclosed subject matter provides a method for screening a compound of interest for the ability to modulate the activity of a heterologous silencing element, the method comprising: (a) providing a host cell that stably expresses a reporter gene, wherein said host cell further comprises at least one heterologous silencing element capable of inhibiting the expression of the reporter gene; (b) administering to the cell a compound of interest; and (c) measuring the expression of the reporter gene. In some embodiments, the silencing element comprises an siRNA, an miRNA, a double stranded RNA, or a hairpin RNA. In some embodiments, the reporter gene encodes green fluorescent protein.

Figure 2 discloses one embodiment of the cell-based reporter system of the invention. In this example, a human 293 -cell line stably expressing a reporter gene, GFP, was used. This cell line was further transfected with a construct that expresses siRNA hairpin against GFP, which can decrease the level of GFP expression.

Individual clones were isolated with greatly (although not completely) reduced GFP expression.

Referring now to Figure 3, to verify that the decrease of GFP expression in the presently disclosed reporter system is due to GFP siRNA, 2-O-methyl RNA, which has been shown to block the siRNA effect previously, was transfected against GFP siRNA into those cells. As shown in Figure 3, second panel, the GFP expression increased.

Using the above -identified clones, a screen was performed using The Spectrum Collection (MicroSource Discovery Systems, Inc., Gaylordsville, Connecticut, United States of America), which contains 2000 biologically active and structurally diverse compounds from libraries of known drugs, experimental bioactives, and pure natural products. For example, in one embodiment, a pilot screen of this library identified one inhibitor and one enhancer of siRNA-mediated mRNA degradation. Referring once again to Figure 3, the compound "trimethobenzamide" was found to inhibit siRNA-mediated mRNA degradation and gene knock-down

(Figure 3, third panel). Referring now to Figure 4, the compound "enoxacin" was found to enhance siRNA-mediated mRNA degradation and gene knock-down.

Enoxacin is a quinolone-type antibiotic that has been approved for clinical use by the FDA for the treatment of certain infections caused by bacteria, such as gonorrhea and urinary tract infections. Referring now to Figure 5, other quinolones, e.g., ciprofloxacin and ofloxacin, also enhance siRNA-mediated mRNA degradation. Such enhancers can be directly applied to RNAi technology to increase the efficiency of knocking down the endogenous genes and can be directly applied to different experimental systems to improve the efficiency of decreasing the level of a polynucleotide of interest.

An embodiment of the presently disclosed reporter system for monitoring RNAi activity is now described in more detail, with reference to Figures 9a-9e. Referring now to Figure 9a, in one embodiment of the presently disclosed system, an HEK 293 -derived stable cell line expressing a GFP reporter gene (293 -EGFP) is infected with a lentivirus expressing a short-hairpin RNA (shRNA) that resembles endogenous miRNA precursors and is processed by the endogenous miRNA machinery into siRNAs that specifically target GFP. See Tiscornia et al. (2003) Proc Natl Acad Sci USA 100 (4): 1844-8. Referring once again to Figure 9a, the transduction can lead to reduced levels of GFP in 293 -EGFP cells. As disclosed in

Figure 3 and as also described hereinabove, to verify that the reduction of GFP expression is due to siRNAs against GFP, the clones can be transfected with 2-0- methyl-RNAs, which can block the activity of the lentivirus-encoded GFP siRNA, and lead to increased GFP expression. Further, to reduce variation between experiments, individual cell clones with moderate reductions of GFP expression can be isolated and used to screen for both inhibitors and enhancers of the RNAi effects.

Using this system, a collection of 2,000 FDA-approved compounds and natural products were screened and a small molecule that could enhance siRNA- mediated mRNA degradation was identified. Referring once again to Figure 9a, and as also described hereinabove, this small molecule was identified as enoxacin and was termed RNAi-E (RNAi-Enhancer). Referring now to Figure 9b, RNAi-E could enhance siGFP-mediated gene knock-down in the presently disclosed cell-based reporter system in a dose-dependent manner, with a median effective concentration (EC₅o) of -40 μM, while it had no effect on the cells expressing GFP only. Further, RNAi-E is nontoxic, even at a high concentration of 150 μM, which is higher than the concentration required for maximal enhancement of RNAi. This observation makes RNAi-E a useful tool for studying the modulation of the RNAi pathway. Referring now to Figure 9c, using cells stably expressing a shRNA against GAPDH, a similar enhancement of shGAPDH-mediated gene knock-down was observed, indicating that the effect of RNAi-E on RNAi is universal.

Two approaches exist for harnessing the RNAi machinery to induce specific suppression of gene expression in cells: shRNAs and siRNA duplexes. Thus, the presently disclosed subject matter also addresses whether RNAi-E has an effect on an siRNA duplex. See Pasquinelli (2006) Dev Cell 10(4): 419-24. Referring now to Figure Id, RNAi-E also can increase the knock-down efficiency of an siRNA duplex against GAPDH. Further, by comparing the gene knock-down efficiency among different concentrations of siRNA duplex used for transfection, RNAi-E also significantly reduced the required siRNA dosage to achieve similar knock-down efficiency. Referring once again to Figure Id, 1-nM GAPDH siRNA duplex in the presence of RNAi-E achieved more GAPDH silencing than can be achieved by 5-nM siRNA duplex alone. Further, referring now to Figure 13, the effect of RNAi-E in a time course of siRNA duplex function in a cell culture also was examined, wherein the prolonged duration of siRNA-mediated gene knock-down in the presence of

RNAi-E was observed. Without wishing to be bound to any one particular theory, these data suggest that the small molecule RNAi-E enhances RNA interference, reduces the dosage required to achieve gene knock-down, and prolongs the siRNA effect in mammalian cells. Further, because the endogenous RNAi machinery also is used in miRNA- mediated translational suppression, the effect of RNAi-E on this process also was examined. See Pasquinelli (2006) Dev Cell 10(4): 419-24. An embodiment of the presently disclosed reporter system, in which miR-125a suppresses the translation of a Luciferase reporter containing the 3'-UTR of the Lin-28 gene was used. See Wu and Belasco (2005) MoI Cell Biol 25(21): 9198-208. A stable cell line expressing human miR-125a was generated and the reporter construct was transfected with or without miR-125a target sequences. Referring now to Figure 9e, the addition of RNAi-E enhances miR-125a-mediated translational suppression, whereas RNAi-E has no effect on the reporter construct without miR-125a target sequences. Thus, in addition to enhancing siRNA-mediated mRNA degradation, RNAi-E also enhances miRNA- mediated translational suppression. IV. Compounds that Modulate the Activity of a Silencing Element

In some embodiments, the presently disclosed subject matter provides a method for modulating the level of a target polynucleotide in a cell, the method comprising administering to the cell an effective amount of at least one RNAi modulating compound, wherein said cell further comprises at least one heterologous silencing element. In some embodiments, the RNAi modulating compound comprises at least one RNAi enhancing compound. In some embodiments, the RNAi modulating compound comprises at least one RNAi inhibitory compound.

A. Enhancers of RNAi

In some embodiments, the presently disclosed modulator is an RNAi enhancer, which increases the silencing element's ability to decrease the level of a target polynucleotide when inside a cell. One small molecule that has been found to enhance the RNAi pathway is enoxacin, which is referred to herein as RNAi-E. RNAi-E belongs to a family of synthetic antibacterial compounds based on a fluoroquinolone skeleton. See Bhanot et al. (2001) CurrPharm Des 7(5): 311-35. Fluoroquinolones have a broad antimicrobial spectrum and are very successful in treating a variety of bacterial infections. See Hopper and Wolfson, Quinolone antimicrobial agents, 3rd ed., (Washington, D. C, 2003; Vol. ASM Press). As a family, fluoroquinolones target bacterial type II topoisomerases, such as DNA gyrase in gram-negative bacteria and DNA topoisomerase IV in gram-positive bacteria. See Mitscher (2005) Chem Rev 105(2): 559-92. These agents do not inhibit eukaryotic topoisomerase II. Fluoroquinolones work by forming a ternary complex with gyrase (topoisomerase IV) and DNA. This event interferes with DNA transcription, replication, repair, and ultimately kills the bacterial cells.

The presently disclosed subject matter demonstrates that in addition to RNAi- E, other small molecules of the quinolone family also exhibit RNA-i enhancing activity. Referring now to Figure 10a, the RNAi-enhancing capabilities of commercially available quinolones (e.g., RNAi-E-Vl-6) and synthetically modified molecules (e.g., RNAi-E-V7-9) were investigated using an embodiment of the presently disclosed RNAi reporter system. Referring now to Figure 10b, two of those compounds have RNAi-E activity (e.g., RNAi-E-Vl and RNAi-E- V3); but are less effective than RNAi-E. Referring once again to Figure 10b, other quinolones tested (e.g., RNAi-E- V2, RNAi-E-V4, RNAi-E-V5 and RNAi-E-V6) had less RNAi-E activity. Addition of sterically blocking groups to the C-3 carboxylate (e.g., RNAi-E- V7 and RNAi -E -V9) and the piperazine terminal nitrogen atom of RNAi-E (e.g., RNAi-E-V8) reduced the RNAi-E activity. Substitutions at N-I, C-6, and C-7 positions of RNAi-E also interfered with its RNAi enhancing activity. Again, without wishing to be bound to any one particular theory, the sensitivity of RNAi-E activity to chemical substitution suggests it forms a specific complex distinct from the known targets of quinolones. Thus, it appears that a specific interaction of the small molecule with the nucleic acids and protein(s) involved in the RNAi pathway is required for the observed RNAi-E activity.

In some embodiments, the presently disclosed subject matter provides a method for decreasing the level of a target polynucleotide in a cell, the method comprising administering to the cell an effective amount of a compound of Formula (aa), or a derivative or analog thereof, wherein said cell further comprises at least one heterologous silencing element:

wherein:

X₈ is CH or N;

X9 is C or N, under the proviso that when X9 is N, R₁₆ is absent, and, when X9 is C, R₁₆ is halo; or

R_1S and R₁₆ together form a portion of a 4- to 6-member heterocyclic ring structure, wherein the 4-to 6-member heterocyclic ring structure comprises atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur, and combinations thereof;

R₁₄ is selected from the group consisting of H, alkyl, substituted alkyl, alkylamino, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl; R_1S is selected from the group consisting of heterocycloalkyl and substituted heterocycloalkyl;

R₁₇ is selected from the group consisting of H, halo, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, and substituted heteroaryl;

R_1S is selected from the group consisting of H, alkyl, substituted alkyl, amino, substituted amino, alkoxyl, hydroxyl, and halo;

Ri9 is selected from the group consisting of H, alkyl, and substituted alkyl; and a pharmaceutically or cosmetically acceptable salt thereof.

In some embodiments, the compound of Formula (aa) is selected from the group consisting of:

and RNAl-EΛ/9

In some embodiments, the silencing element comprises an siRNA, an miRNA, a double stranded RNA, or a hairpin RNA. In some embodiments, the cell is in a subject. In some embodiments, the presently disclosed subject matter provides a method for decreasing the level of a target polynucleotide in a cell, the method comprising administering to said cell a combination of an effective concentration of a polynucleotide comprising a heterologous silencing element and an effective amount of a compound of Formula (aa), derivatives and analogs thereof, as defined hereinabove.

In some embodiments, the polynucleotide comprising the heterologous silencing element comprises an expression cassette encoding a siRNA, a miRNA, a dsRNA, or a hairpin RNA. In some embodiments, the polynucleotide is in a viral vector. In some embodiments, the polynucleotide comprises a siRNA, a miRNA, a dsRNA, or a hairpin RNA.

In some embodiments, the compound of Formula (aa) and the heterologous silencing element are administered to the cell simultaneously or sequentially. In some embodiments, the cell is in a subject. In some embodiments, the cell is from a mammal.

In some embodiments, the presently disclosed subject matter provides a pharmaceutical or cosmetic composition comprising at least one of a compound of Formula (aa) and a pharmaceutically or cosmetically acceptable carrier and one or more polynucleotides comprising a silencing element. In some embodiments, the silencing element comprises a siRNA, a miRNA, a double stranded RNA, or a hairpin

RNA.

B. Definition of Chemical Terms

While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

When the term "independently selected" is used, the substituents being referred to (e.g., R groups, such as groups R₁, R₂, and the like, or groups X₁ and X₂), can be identical or different. For example, both R₁ and R₂ can be substituted alkyls, or R₁ can be hydrogen and R₂ can be a substituted alkyl, and the like.

A named "R" or "X" group will generally have the structure that is recognized in the art as corresponding to a group having that name, unless specified otherwise herein. For the purposes of illustration, certain representative "R" and "X" groups as set forth above are defined below. These definitions are intended to supplement and illustrate, not preclude, the definitions that would be apparent to one of ordinary skill in the art upon review of the present disclosure.

As used herein the term "alkyl" refers to C_1-2O inclusive, linear (i.e., "straight- chain"), branched, or cyclic, saturated or at least partially and in some cases fully unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. "Lower alkyl" refers to an alkyl group having 1 to about 8 carbon atoms

(i.e., a C₁-S alkyl), e.g., 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about 10 to about 20 carbon atoms, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In certain embodiments, "alkyl" refers, in particular, to C_1-S straight-chain alkyls. In other embodiments, "alkyl" refers, in particular, to C_1-S branched-chain alkyls.

Alkyl groups can optionally be substituted (a "substituted alkyl") with one or more alkyl group substituents, which can be the same or different. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, halo, arylamino, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl, oxo, and cycloalkyl. There can be optionally inserted along the alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, lower alkyl (also referred to herein as "alkylaminoalkyl"), or aryl.

Thus, as used herein, the term "substituted alkyl" includes alkyl groups, as defined herein, in which one or more atoms or functional groups of the alkyl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto.

"Cyclic" and "cycloalkyl" refer to a non-aromatic mono- or multicyclic ring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms. The cycloalkyl group can be optionally partially unsaturated. The cycloalkyl group also can be optionally substituted with an alkyl group substituent as defined herein, oxo, and/or alkylene. There can be optionally inserted along the cyclic alkyl chain one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent is hydrogen, alkyl, substituted alkyl, aryl, or substituted aryl, thus providing a heterocyclic group. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl, and cycloheptyl. Multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

The term "cycloalkylalkyl," as used herein, refers to a cycloalkyl group as defined hereinabove, which is attached to the parent molecular moiety through an alkyl group, also as defined above. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl. The terms "cycloheteroalkyl" or "heterocycloalkyl" refer to a non-aromatic ring system, such as a 3- to 7-member substituted or unsubstituted cycloalkyl ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of N, O, and S, and optionally can include one or more double bonds. The cycloheteroalkyl ring can be optionally fused to or otherwise attached to other cycloheteroalkyl rings and/or non-aromatic hydrocarbon rings. Representative cycloheteroalkyl ring systems include, but are not limited to pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperidyl, piperazinyl, indolinyl, quinuclidinyl, morpholinyl, thiomorpholinyl, thiadiazinanyl, tetrahydrofuranyl, and the like. The term "alkenyl" as used herein refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon double bond. Examples of "alkenyl" include vinyl, allyl, 2-methyl-3-heptene, and the like.

The term "cycloalkenyl" as used herein refers to a cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadiene, cyclohexenyl,

1,3-cyclohexadiene, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

The term "alkynyl" as used herein refers to a straight or branched hydrocarbon of a designed number of carbon atoms containing at least one carbon-carbon triple bond. Examples of "alkynyl" include propargyl, propyne, and 3-hexyne. "Alkylene" refers to a straight or branched bivalent aliphatic hydrocarbon group having from 1 to about 20 carbon atoms, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. The alkylene group can be straight, branched or cyclic. The alkylene group also can be optionally unsaturated and/or substituted with one or more "alkyl group substituents." There can be optionally inserted along the alkylene group one or more oxygen, sulfur or substituted or unsubstituted nitrogen atoms (also referred to herein as "alkylaminoalkyl"), wherein the nitrogen substituent is alkyl as previously described. Exemplary alkylene groups include methylene (-CH₂-); ethylene (-CH₂-CH₂-); propylene (-(CH₂)₃-); cyclohexylene (-C₆H₁₀-); -CH=CH-CH=CH-; -CH=CH-CH₂-; -(CH₂)_q-N(R)-

(CH₂)_r-, wherein each of q and r is independently an integer from 0 to about 20, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and R is hydrogen or lower alkyl; methylenedioxyl (-0-CH₂-O-); and ethylenedioxyl (-O-(CH₂)₂-O-). An alkylene group can have about 2 to about 3 carbon atoms and can further have 6-20 carbons.

The term "aryl" is used herein to refer to an aromatic substituent that can be a single aromatic ring, or multiple aromatic rings that are fused together, linked covalently, or linked to a common group, such as, but not limited to, a methylene or ethylene moiety. The common linking group also can be a carbonyl, as in benzophenone, or oxygen, as in diphenylether, or nitrogen, as in diphenylamine. The term "aryl" specifically encompasses heterocyclic aromatic compounds. The aromatic ring(s) can comprise phenyl, naphthyl, biphenyl, diphenylether, diphenylamine and benzophenone, among others. In particular embodiments, the term "aryl" means a cyclic aromatic comprising about 5 to about 10 carbon atoms, e.g., 5, 6, 7, 8, 9, or 10 carbon atoms, and including 5- and 6-membered hydrocarbon and heterocyclic aromatic rings.

The aryl group can be optionally substituted (a "substituted aryl") with one or more aryl group substituents, which can be the same or different, wherein "aryl group substituent" includes alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, aryloxyl, aralkyloxyl, carboxyl, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene, and -NR'R", wherein R and R" can each be independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, and aralkyl. Thus, as used herein, the term "substituted aryl" includes aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl group are replaced with another atom or functional group, including for example, alkyl, substituted alkyl, halogen, aryl, substituted aryl, alkoxyl, hydroxyl, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto. Specific examples of aryl groups include, but are not limited to, cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, benzimidazole, isothiazole, isoxazole, pyrazole, pyrazine, triazine, pyrimidine, quinoline, isoquinoline, indole, carbazole, and the like. The term "heteroaryl" refers to an aromatic ring system, such as, but not limited to a 5- or 6-member ring system, including one or more heteroatoms, which can be the same or different, and are selected from the group consisting of N, O, and S. The heteroaryl ring can be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocycloalkyl rings. Representative heteroaryl ring systems include, but are not limited to, pyridyl, pyrimidyl, pyrrolyl, pyrazolyl, azolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, imidazolyl, furanyl, thienyl, quinolinyl, isoquinolinyl, indolinyl, indolyl, benzothienyl, benzothiazolyl, enzofuranyl, benzimidazolyl, benzisoxazolyl, benzopyrazolyl, triazolyl, tetrazolyl, and the like. A structure represented generally by the formula, wherein the ring structure can be aromatic or non-aromatic:

as used herein refers to a ring structure, for example, but not limited to a 3 -carbon, a 4-carbon, a 5-carbon, a 6-carbon, and the like, aliphatic and/or aromatic cyclic compound, including a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure as defined herein, comprising a substituent R group, wherein the R group can be present or absent, and when present, one or more R groups can each be substituted on one or more available carbon atoms of the ring structure. The presence or absence of the R group and number of R groups is determined by the value of the integer n. Each R group, if more than one, is substituted on an available carbon of the ring structure rather than on another R group. For example, the structure above where n is 0 to 2 would comprise compound groups including, but not limited to:

and the like.

A dashed line representing a bond in a cyclic ring structure indicates that the bond can be either present or absent in the ring. That is, a dashed line representing a bond in a cyclic ring structure indicates that the ring structure is selected from the group consisting of a saturated ring structure, a partially saturated ring structure, and an unsaturated ring structure.

When a named atom of an aromatic ring or a heterocyclic aromatic ring is defined as being "absent," the named atom is replaced by a direct bond. As used herein, the term "acyl" refers to an organic acid group wherein the -

OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RCO-, wherein R is an alkyl or an aryl group as defined herein). As such, the term "acyl" specifically includes arylacyl groups, such as an acetylfuran and a phenacyl group. Specific examples of acyl groups include acetyl and benzoyl. "Alkoxyl" refers to an alkyl-O- group wherein alkyl is as previously described. The term "alkoxyl" as used herein can refer to C_1-2O inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, t-butoxyl, and pentoxyl.

The term "alkoxyalkyl" as used herein refers to an alkyl-O-alkyl ether, for example, a methoxyethyl or an ethoxymethyl group.

"Aryloxyl" refers to an aryl-O- group wherein the aryl group is as previously described, including a substituted aryl. The term "aryloxyl" as used herein can refer to phenyloxyl or hexyloxyl, and alkyl, substituted alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl. The term "alkyl-thio-alkyl" as used herein refers to an alkyl-S-alkyl thioether, for example, a methylthiomethyl or a methylthioethyl group.

"Aralkyl" refers to an aryl-alkyl-group wherein aryl and alkyl are as previously described, and included substituted aryl and substituted alkyl. Exemplary aralkyl groups include benzyl, phenylethyl, and naphthylmethyl. "Aralkyloxyl" refers to an aralkyl-O- group wherein the aralkyl group is as previously described. An exemplary aralkyloxyl group is benzyloxyl.

"Alkoxycarbonyl" refers to an alkyl-O-CO- group. Exemplary alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, butyloxycarbonyl, and t-butyloxycarbonyl. "Aryloxycarbonyl" refers to an aryl-O-CO- group.

Exemplary aryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl. "Aralkoxycarbonyl" refers to an aralkyl-O-CO- group. An exemplary aralkoxycarbonyl group is benzyloxycarbonyl.

"Carbamoyl" refers to an H₂N-CO- group. "Alkylcarbamoyl" refers to a R'RN-CO- group wherein one of R and R' is hydrogen and the other of R and R' is alkyl and/or substituted alkyl as previously described. "Dialkylcarbamoyl" refers to a R'RN-CO- group wherein each of R and R is independently alkyl and/or substituted alkyl as previously described.

"Acyloxyl" refers to an acyl-O- group wherein acyl is as previously described. The term "amino" refers to the -NH₂ group and also refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals. For example, the terms "acylamino" and "alkylamino" refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively. The term "alkylamino" refers to an -NHR group wherein R is an alkyl group and/or a substituted alkyl group as previously described. Exemplary alkylamino groups include methylamino, ethylamino, and the like.

"Dialkylamino" refers to an -NRR' group wherein each of R and R' is independently an alkyl group and/or a substituted alkyl group as previously described. Exemplary dialkylamino groups include ethylmethylamino, dimethylamino, and diethylamino.

"Acylamino" refers to an acyl-NH- group wherein acyl is as previously described. "Aroylamino" refers to an aroyl-NH- group wherein aroyl is as previously described. The term "carbonyl" refers to the -(C=O)- group.

The term "carboxyl" refers to the -COOH group.

The terms "halo", "halide", or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups. The term "hydroxyl" refers to the -OH group.

The term "hydroxyalkyl" refers to an alkyl group substituted with an -OH group.

The term "mercapto" refers to the -SH group. The term "oxo" refers to a compound described previously herein wherein a carbon atom is replaced by an oxygen atom.

The term "nitro" refers to the -NO₂ group.

The term "thio" refers to a compound described previously herein wherein a carbon or oxygen atom is replaced by a sulfur atom. The term "sulfate" refers to the -SO₄ group.

As used herein, an "analog" refers to a chemical compound in which one or more individual atoms or functional groups of a parent compound have been replaced, either with a different atom or with a different functional group. For example, thiophene is an analog of furan, in which the oxygen atom of the five-membered ring is replaced by a sulfur atom.

As used herein, a "derivative" refers to a chemical compound which is derived from or obtained from a parent compound and contains essential elements of the parent compound but typically has one or more different functional groups. Such functional groups can be added to a parent compound, for example, to improve the molecule's solubility, absorption, biological half life, and the like, or to decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. An example of a derivative is an ester or amide of a parent compound having a carboxylic acid functional group.

C. Pharmaceutically or Cosmetically Acceptable Salts

Additionally, the active compounds as described herein can be administered as a pharmaceutically or cosmetically acceptable salt. The phrases "pharmaceutically acceptable salt(s)" or "cosmetically acceptable salt(s)," as used herein, means those salts of the presently disclosed compounds that are safe and effective for use in a subject and that possess the desired biological activity. Pharmaceutically or cosmetically acceptable salts include salts of acidic or basic groups present in compounds of the invention. Pharmaceutically or cosmetically acceptable acid addition salts include, but are not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, borate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate, pamoate (i.e., l,r-methylene-bis-(2-hydroxy-3- naphthoate)), mesylate salts. Certain of the presently disclosed compounds can form pharmaceutically or cosmetically acceptable salts with various amino acids. Suitable base salts include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, and diethanolamine salts. For a review on pharmaceutically acceptable salts see Berge et al., 66 J. Pharm. Sci. 1-19 (1977), which is incorporated herein by reference. The salts of the compounds described herein can be prepared, for example, by reacting the appropriate equivalent of the compound with the desired acid or base in solution. After the reaction is complete, the salts are crystallized from solution by the addition of an appropriate amount of solvent in which the salt is insoluble.

D. Pharmaceutical or Cosmetic Formulations or Compositions

RNAi can be used in mammalian cells grown in culture and in mammalian organisms, e.g., for functional studies of genes. In addition, animal studies have indicated that RNAi -inducing agents are likely to have therapeutic applications. Thus, compounds that inhibit or activate RNAi are useful in mammalian tissue culture systems, in animal studies, and for therapeutic purposes. The presently disclosed subject matter therefore provides pharmaceutical or cosmetic compositions comprising one or more silencing elements targeted to one or more specific genes and at least one enhancing compound, including, but not limited to, at least one presently disclosed compound of Formula (aa), as described hereinabove.

The presently disclosed compositions (e.g., compounds that activate or inhibit an RNAi pathway) can be formulated for delivery, i.e., administering to the subject, by any available route including, but not limited, to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, opthalmic, transdermal (topical), transmucosal, rectal, and vaginal routes. Preferred routes of delivery include parenteral, transmucosal, nasal, bronchial, vaginal, and oral. The presently disclosed pharmaceutical or cosmetic compositions also can include an RNAi-inducing agent in combination with a pharmaceutically or cosmetically acceptable carrier. As used herein the terms "pharmaceutically acceptable carrier" or "cosmetically acceptable carriers" include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical or cosmetic administration. Supplementary active compounds also can be incorporated into the compositions. As one of ordinary skill in the art would appreciate, a presently disclosed pharmaceutical or cosmetic composition is formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral (e.g., intravenous), intramuscular, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical or cosmetic compositions suitable for injectable use typically include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition should be sterile and should be fluid to the extent that easy syringability exists. Preferred pharmaceutical or cosmetic compositions are stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as manitol or sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Preferably solutions for injection are free of endotoxin. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral formulations generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral formulations also can be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically or cosmetically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient, such as starch or lactose, a disintegrating agent, such as alginic acid, Primogel, or corn starch; a lubricant, such as magnesium stearate or Sterotes; a glidant, such as colloidal silicon dioxide; a sweetening agent, such as sucrose or saccharin; or a flavoring agent, such as peppermint, methyl salicylate, or orange flavoring. Formulations for oral delivery can advantageously incorporate agents to improve stability within the gastrointestinal tract and/or to enhance absorption. For administration by inhalation, the presently disclosed formulations and compositions are preferably delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Liquid aerosols, dry powders, and the like, also can be used. Systemic administration of the presently disclosed compositions also can be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the composition. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. The compounds also can be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

The presently disclosed compositions also can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials also can be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) also can be used as pharmaceutically or cosmetically acceptable carriers. Such suspensions can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811, which is incorporated herein by reference in its entirety. It is advantageous to formulate oral or parenteral formulations in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical or cosmetic carrier.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical or cosmetic procedures in cell cultures or experimental animals, e.g., for determining the LD_5O (the dose lethal to 50% of the population) and the ED_5O (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit high therapeutic indices are preferred. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the presently disclosed methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

A therapeutically effective amount of a pharmaceutical or cosmetic composition typically ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The pharmaceutical or cosmetic composition can be administered at various intervals and over different periods of time as required, e.g., multiple times per day, daily, every other day, once a week for between about 1 to 10 weeks, between 2 to 8 weeks, between about 3 to 7 weeks, about 4, 5, or 6 weeks, and the like. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, disorder, or unwanted condition, previous treatments, the general health and/or age of the subject, and other diseases or unwanted conditions present. Generally, treatment of a subject can include a single treatment or, in many cases, can include a series of treatments. Further, treatment of a subject can include a single cosmetic application or, in some embodiments, can include a series of cosmetic applications. Exemplary doses include milligram or microgram amounts of the inventive compound per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.) For local administration (e.g., intranasal), smaller doses can be used. It is furthermore understood that appropriate doses of a compound depend upon its potency and can optionally be tailored to the particular recipient, for example, through administration of increasing doses until a preselected desired response is achieved. It is understood that the specific dose level for any particular animal subject can depend on a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated. The presently disclosed compositions can be used for the treatment of nonhuman animals including, but not limited to, horses, swine, and birds.

Accordingly, doses and methods of administration can be selected in accordance with known principles of veterinary pharmacology and medicine. Guidance for appropriate doses and methods of administration can be found, for example, in Adams, R. (ed.), Veterinary Pharmacology and Therapeutics, 8th edition, Iowa State University Press; ISBN: 0813817439; 2001.

The presently disclosed pharmaceutical or cosmetic compositions can be included in a container, pack, or dispenser together with instructions for administration.

Further, one of ordinary skill in the art upon review of the presently disclosed subject matter would appreciate that the presently disclosed compounds, including pharmaceutically or cosmetically acceptable salts and pharmaceutical or cosmetic compositions thereof, can be administered directly to a cell, a cell culture, a cell culture medium, a tissue, a tissue culture, a tissue culture medium, and the like. When referring to an RNA:adjuvant or suppressor, such as a compound of Formula (aa), the term "administering," and derivations thereof, comprises any method that allows for the compound to contact a cell. The presently disclosed compounds, or pharmaceutically or cosmetically acceptable salts or pharmaceutical or cosmetic compositions thereof, can be administered to (or contacted with) a cell or a tissue in vitro or ex vivo. The presently disclosed compounds, or pharmaceutically or cosmetically acceptable salts or pharmaceutical or cosmetic compositions thereof, also can be administered to (or contacted with) a cell or a tissue in vivo by administration to an individual subject, e.g., a patient, for example, by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial administration) or topical application, as described elsewhere herein.

E. Methods for Treating a Disease State or Unwanted Condition In some embodiments, the presently disclosed subject matter provides a method for treating a disease state or unwanted condition. RNAi can be used as a therapeutic approach for treating diseases that result from aberrant protein synthesis, for example diseases arising from genetic disorders or viral infections. In such embodiments, inhibiting or eliminating a target mRNA would result in a significant decrease in the expression level of a specific protein. Because RNAi can be used to target virtually any protein, RNAi-based therapies can be developed for almost any therapeutic area. Such approaches therefore have broad applicability, high therapeutic specificity, and target RNA destruction resulting in a decrease or termination of disease progression.

Accordingly, in some embodiments, the presently disclosed subject matter provides a method for treating a disease state or unwanted condition, the method comprising administering to a subject in need of treatment thereof an effective amount of a polynucleotide comprising a silencing element, which, when administered to said subject, decreases the level of the target polynucleotide and an effective amount of a compound of Formula (aa, as disclosed herein, and pharmaceutically or cosmetically acceptable salts thereof. In some embodiments, the disease state comprises a viral infection. In some embodiments, the disease state comprises a genetic disorder.

V. Biological Targets of RNAi-E and a Mechanism by Which RNAi-E Modulates the RNAi Pathway

Referring now to Figures 1 Ia-I Ie, to investigate the biological target(s) of RNAi-E and the mechanism by which RNAi-E modulates the RNAi pathway, the expression of mature small RNAs in cells expressing different shRNAs was examined. Referring now to Figure 11a, although different promoters were used in these constructs, consistent increases in the expression of mature small RNAs in cells treated with RNAi-E were observed. Further, referring once again to Figure 11a, RNAi-E also increased the level of mature form of miR-125a in cells expressing the primary transcript of miR-125a (Pri-miR125a), as disclosed in Figure 9e.

Referring now to Figure 1 Ib, RNAi-E promoted the processing of miR-125a, as measured by quantitative RT-PCR; the addition of RNAi-E resulted in increases in the mature form of miR-125a and corresponding decreases in the level of the pri- miRNA alone and of the total level of pri-miRNA and pre-miRNA together. The shGFP used in an embodiment of the presently disclosed reporter system mimics miRNA precursors and is processed by Dicer, but not by Drosha, which processes pri- miRNAs. Further, RNAi-E exerted effects on siRNA duplex, which does not require Drosha. Once again, without wishing to be bound to any one particular theory, these data suggest that RNAi-E can function at the level of Dicer-mediated precursor processing and loading into RISCs. See Tiscornia et al. (2003) Proc Natl Acad Sci USA 100(4): 1844-8. Based on this observation, the processing of selective endogenous miRNAs, which exist in both precursor and mature forms in cells, should be enhanced by RNAi-E, as well.

Referring now to Figure 1 Ic, to test this hypothesis, microRNA TaqMan® assays (Applied Biosystems, Foster City, California, United States of America) were used to monitor the profiles (-180 miRNAs) of cells in the presence or absence of RNAi-E. The majority of microRNAs (13/15) that were altered by RNAi-E had increased expression of the mature form, whereas only two had decreased levels of the mature miRNAs, which could reflect the inter-regulation among microRNAs. Referring once again to Figure l ib, consistent with miR-125a, decreased levels of the pri- and pre- forms of the miRNAs, whose mature forms increased in the presence of RNAi-E, also were found. These data demonstrate that RNAi-E promotes the processing of miRNAs from the precursors to the mature forms.

Dicer and TRBP play critical roles in the processing and loading of miRNAs/siRNAs to RISC. See Bernstein et al. (2001) Nature 409(6818): 363-6; Chendrimada et al. (2005) Nature 436(7051); 740-4; Jiang et al. (2005) Genes Dev 19(14): 1674-9; and Forstemann et al. (2005) PLoS Biol 3(7): e236. Whether RNAi- E could be involved in the processing mediated by these two proteins also was examined by using an established in vitro processing assay. Referring now to Figure 1 Id, RNAi-E had no effect on the processing of let-7 miRNA precursor by Dicer alone. The addition of RNAi-E, however, could enhance the processing of let-7 precursor by Dicer and TRBP together. This enhancement was not observed with the addition of RNAi-E- V5, which has less RNAi-E activity (see Figure 10). This result suggests that RNAi-E could target TRBP.

To demonstrate the direct interaction between TRBP and RNAi-E, biotinylated RNAi-E was synthesized and a protein capture assay using streptavidin beads and recombinant TRBP was performed. A direct interaction between TRBP and RNAi-E was not observed (data not shown). Because TRBP binds to miRNA precursors and facilitates the processing and loading of miRNAs, an RNA gel-shift assay was used to examine the effect of RNAi-E on the interaction between TRBP and the let- 7 precursor. Referring now to Figure l ie, RNAi-E, but not RNAi -E -V5, was found to enhance the interaction between TRBP and the let-7 precursor. These results suggest that RNAi-E promotes the processing and loading of miRNAs onto RISC by facilitating the interaction between TRBP and RNAs in mammalian cells.

To determine whether RNAi-E has a similar effect in vivo, two different animal models were tested: C. elegans and mice. It has been established that in C. elegans double-stranded RNAs (dsRNAs) initiate the RNAi pathway and silence expression of a homologous target gene efficiently. To examine the effect of RNAi- E, dsRNAs were used against two C. elegans embryonic lethal genes, mom-5 and let- 2. Mutations in either gene are lethal at early developmental stage. In these experiments, wildtype N2 worms were fed with dsRNAs against either gene and worms with significant knock-down could not survive to adulthood. See Thorpe et al.

(1997) Cell 90(4): 695-705; Sibley et al. (1993) J Cell Biol 123(1): 255-64; and Rocheleau et al. (1997) Cell 90(4): 707-16.

Referring now to Figure 12a, whereas feeding non-specific control dsRNAs had no effect on the viability of wildtype worms, mom-5 or let-2 dsRNAs alone caused -30% lethality. Referring once again to Figure 12a, the addition of RNAi-E enhanced the lethality due to mom-5 or let-2 dsRNAs, whereas RNAi-E alone had no effect on the viability of wildtype worms. In addition to demonstrating an effect of RNAi-E in vivo, these data indicate that, although RNAi-E was initially identified in mammalian cells, it also enhances RNA interference in C elegans. This observation suggests that the interaction between RNAi-E and its target protein might be universal.

Whether RNAi-E has an effect in a more complex in vivo model, e.g., a GFP transgenic mouse, also was examined. It has been demonstrated that in these mice a lentivirus expressing shGFP (Lv-siGFP) can knock down GFP expression when the virus is injected into the mice early in development. See Tiscornia et al. (2003) Proc Natl Acad Sci USA 100(4): 1844-8; Okabe et al (1997) FEBS Lett 407(3): 313-9. Due to the stability of the GFP protein, however, in adult mice, injections of Lv- siGFP do not alter GFP level significantly (data not shown). The effect of RNAi-E through measurements of GFP mRNA following selective injections of Lv-siGFP in young pups was therefore monitored. Mouse ears were chosen for the injections because they are easily accessible and the virus could be delivered to the full ear and enough RNA could be obtained from a single ear for quantitative RT-PCR analysis. Three groups of injections were performed, Lv-siGFP alone, Lv-siGFP with RNAi-E, and RNAi-E alone. Multiple rounds of injections on 10-day old mice were performed with the following results. Lv-siGFP alone reduced the GFP mRNA level to 80% of control tissues (20% gene knockdown). The addition of RNAi-E enhanced the knockdown efficiency to 60% (40% GFP mRNA level remained), whereas RNAi- E alone had no effect on GFP expression, which is consistent with previous cell culture data. These results demonstrate that RNAi-E enhances siRNA-mediated mRNA degradation in vivo.

Small noncoding RNAs play important roles in animal development, evolution and human disease. See Plasterk (2006) Cell 124(5): 877-81. Although the understanding of the mechanism of RNAi has matured to the atomic resolution with the identification of key proteins involved in RNAi pathway, the modulation of the

RNAi pathway in normal and disease states remains poorly understood due to the overlapping, redundant, and compensatory features of the biological pathways regulated by small RNAs. See Pasquinelli (2006) Dev Cell 10(4): 419-24. A chemical biology approach, using small molecules to perturb protein networks of biological systems, is a potential route to elucidating the modulation of the RNAi pathway. See Spring (2005) Chem Soc Rev 34(6): 472-82. The presently disclosed subject matter identified classes of small molecules that enhance RNA interference and promote the biogenesis of miRNA by facilitating the interaction between TRBP and RNAs. These results demonstrate that small molecules can be used to dissect the RNAi pathway.

Other than its important natural role in gene regulation, RNA interference is also a revolutionary tool for biomedical research, and has the potential for treating a variety of illnesses by knocking down expression of disease-related genes. The presently disclosed subject matter demonstrates that the inclusion of clinically proven RNAi-E in RNAi applications can significantly increase the knock-down efficiency, reduce the required dosage of siRNA duplex, and prolong the duration of siRNA effects. Thus, the identification of RNAi-E could facilitate the development of siRNAs for their broad use in functional genomics as well as their therapeutic use.

REFERENCES

The references listed below, as well as all references cited in the specification, are incorporated herein by reference to the extent that they supplement, explain, provide a background for or teach methodology, techniques and/or compositions employed herein.

Hannon, G. J., RNA interference. Nature 2002, 418, (6894), 244-51. Bartel, D. P., MicroRNAs: genomics, biogenesis, mechanism, and function. Ce// 2004, 116, (2), 281-97. Zamore, P. D.; Haley, B., Ribo-gnome: the big world of small RNAs. Science

2005, 309, (5740), 1519-24.

Dykxhoorn, D. M.; Lieberman, J., Knocking down disease with siRNAs. Cell

2006, 126,(2), 231-5.

Plasterk, R. H., Micro RNAs in animal development. Cell 2006, 124, (5), 877- 81.

Dykxhoorn, D. M.; Lieberman, J., The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu Rev Med 2005, 56, 401-23.

Dykxhoorn, D. M.; Lieberman, J., RUNNING INTERFERENCE: Prospects and Obstacles to Using Small Interfering RNAs as Small Molecule Drugs. Annu Rev Biomed Eng 2006, 8, 377-402.

Tiscornia, G.; Singer, O.; Ikawa, M.; Verma, 1. M., A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA. Proc Natl Acad Sci USA 2003, 100, (4), 1844-8.

Pasquinelli, A. E., Demystifying small RNA pathways. Dev Cell 2006, 10, (4), 419-24.

Wu, L.; Belasco, J. G., Micro-RNA regulation of the mammalian lin-28 gene during neuronal differentiation of embryonal carcinoma cells. MoI Cell Biol 2005, 25, (21), 9198-208. Bhanot, S. K.; Singh, M.; Chatterjee, N. R., The chemical and biological aspects of fluoroquinolones: reality and dreams. CurrPharm Des 2001, 7, (5), 311-35.

Hopper, D. C; Wolfson, J. S., Quinolone antimicrobial agents. Third ed.; Washington, DC, 2003; Vol. ASM Press. Mitscher, L. A., Bacterial topoisomerase inhibitors: quinolone and pyridone antibacterial agents. Chem Rev 2005, 105, (2), 559-92.

Bernstein, E.; Caudy, A. A.; Hammond, S. M.; Hannon, G. J., Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001, 409, (6818), 363-6. Chendrimada, T. P.; Gregory, R. L; Kumaraswamy, E.; Norman, J.; Coach,

N.; Nishikura, K.; Shiekhattar, R., TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 2005, 436, (7051), 740-4.

Jiang, F.; Ye, X.; Liu, X.; Fincher, L.; McKearin, D.; Liu, Q., Dicer-1 and R3131-L catalyze microRNA maturation in Drosophila. Genes Dev 2005, 19, (14), 1674-9.

Forstemann, K.; Tomari, Y.; Du, T.; Vagin, V. V.; Denli, A. M.; Bratu, D. P.; Klattenhoff, C; Theurkauf, W. E.; Zamore, P. D., Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA- binding domain protein. PLoS Biol 2005, 3, (7), e236. Thorpe, C. J.; Schlesinger, A.; Carter, J. C; Bowerman, B., Writ signaling polarizes an early C. elegans blastomere to distinguish endoderm from mesoderm. Cell 1997, 90, (4), 695-705.

Sibley, M. H.; Johnson, J. J.; Mello, C. C; Kramer, J. M., Genetic identification, sequence, and alternative splicing of the Caenorhabditis elegans alpha 2(IV) collagen gene. J Cell Biol 1993, 123, (1), 255-64.

Rocheleau, C. E.; Downs, W. D.; Lin, R.; Wittmann, C; Bei, Y.; Cha, Y. H.; AIi, M.; Priess, J. R.; Mello, C. C, Wnt signaling and an APC-related gene specify endoderm in early C. elegans embryos. Cell 1997, 90, (4), 707-16.

Okabe, M.; Ikawa, M.; Kominami, K.; Nakanishi, T.; Nishimune, Y., 'Green mice' as a source of ubiquitous green cells. FEBS Lett 1997, 407, (3), 313-9.

Spring, D. R., Chemical genetics to chemical genomics: small molecules offer big insights. Chem Soc Rev 2005, 34, (6), 472-82. Grimm, D.; Streetz, K. L.; Jopling, C. L.; Storm, T. A.; Pandey, K.; Davis, C. R.; Marion, P.; Salazar, F.; Kay, M. A., Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006, 441, (7092), 537-41.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Example 1 To identify the chemical compounds that modulate the siRNA and miRNA pathway, a screening strategy was used. A human 293 -cell line stably expressing a reporter gene, GFP, was used. This cell line was further transfected with a construct that expresses siRNA hairpin against GFP, which could knock down the expression of GFP. Individual clones were isolated with greatly reduced GFP expression. To verify that the decrease of GFP expression is due to GFP siRNA, 2-0-methyl RNA was transfected against GFP siRNA into those cells, which has been shown to block the siRNA effect previously, and found that GFP expression increased (Figure 3, second panel). Using these clones, a screen was performed using The Spectrum Collection, which contains 2000 biologically active and structurally diverse compounds from libraries of known drugs, experimental bioactives, and pure natural products. The compound trimethobenzamide was found to inhibit siRNA-mediated mRNA degradation and gene knock-down (Figure 3, third panel). The compound enoxacin was found to enhance siRNA-mediated mRNA degradation and gene knockdown (Figure 4).

Example 2

Human 293 cells stably expressing a reporter gene, GFP, were further transfected with a construct that expresses an siRNA hairpin against GFP, which results in the knock-down of the expression of GFP. Transfected cells containing the siRNA suppressed GPF expression were split and then treated with an effective amount of enoxacin (Figure 4, second panel) and were grown for a period of 24-48 hours. Untreated control cells are shown in Figure 4, first panel.

Example 3

Figure 5 shows enhancement of mRNA-mediated mRNA degradation and gene knock-down by presently disclosed quinolone compounds, e.g., enoxacin, ciproflaxin, and ofloxacin. In this example, a luciferase reporter construct GL-3 was transfected into the cells expressing shRNA against Luciferase mRNA. Addition of the disclosed compounds increased the knock-down efficiency of shRNA against luciferase mRNA. More particularly, Figure 5 shows the relative luciferase activity of GL-3 luciferase only; GL-3 luciferase and one of enoxacin, ciprofloxacin, and ofloxacin; GL-3 luciferase and short hairpin RNA specific for luciferase gene (shLuc) only; and GL-3 luciferase, shLuc, and one of enoxacin, ciprofloxacin, and ofloxacin.

Example 4

Figure 6 shows a schematic representation of a microRNA (miRNA) sensor in mammalian cells. More particularly, Figure 6 shows reporter construct and selective translational suppression of reporter containing miR-30a-3p target sites. In Figure 6, top left panel, the arrowheads in Luc-T30 indicate artificial target sequence against miR-30a-3p. Luc-AT30 has an artificial sequence complementary to that in Luc-T30. Figure 6, bottom left panel, shows a dual luciferase assay of HepG2 cells. Relative luciferase activity was calculated by dividing the reporter (Firefly) luciferase activity with co-transfected Renilla luciferase activity. Luciferase activity was decreased by trans fecting Luc-T30 in HepG2 (light gray bars on left) without changes in the amount of its mRNA (dark gray bars on right). Error bars represent the standard deviation (SD) of three triplicate experiments. Figure 6a shows the design of an siRNA duplex against miR-30a precursor (siRNA-p) and a control siRNA duplex (siRNA-c). Figure 6b shows that transfecting siRNA-p reversed the translational suppression of Luc-T30 (fourth bar from left) while siRNA-c had no effect (third bar from left). Figure 6c shows that the recovery is siRNA-p concentration-dependent. Error bars represent the standard deviation (SD) from three triplicate experiments. **P = 0.004; *****P = 0.00001. Example 5

Figure 7 shows the relative miRNA-mediated suppression exhibited by a presently disclosed RNAi inhibitor (RNAi-I) as compared to "no drug" and enoxacin. The reporter system described in Figure 6 was used and the addition of RNAi-I released the miRNA-mediated translational suppression. This reporter contains the target sequence of miR-30. In the miR-30 system, the endogenously expressed miR- 30 was used and, in this cell line, miR-30 has been processed completely. Thus, the addition of enoxacin had no effect. These results suggest that Enoxacin works upstream of the RISC complex.

Example 6

Referring now to Figure 8, the 3'-UTR of Lin28 mRNA, a target mRNA of miR-125a, was inserted into the 3' end of luciferase reporter gene. Lin28-Del is the construct without the target sequence of miR-125a. Lin28 or Lin28-Del was trans fected into cells expressing miR-125a. Although RNAi-E had no effect on

Lin28-Del, the translation of Lin28 was suppressed by the expression of miR-125a. The addition of RNAi-E further enhanced this suppression.

Example 7 DNA Constructs, siRNAs, Cell Lines and Transfections

Lv-siGFP, Luc-Lin28 and Luc-Lin28-Δ plasmids were described previously. See Tiscornia et al. (2003) Proc Natl Acad Sci USA 100(4): 1844-8; Wu and Belasco (2005) MoI Cell Biol 25(21): 9198-208. Short-hairpin vectors, shLuciferase and shGAPDH, were obtained from Open Biosystems (Huntsville, Alabama, United States of America). ShMir-125a was constructed by inserting a 250-bp fragment containing the mature form of human mir-125a and the flanking 115-bp on either side downstream of the U6 promoter of the pSM2 vector (Open Biosystems). 2-O-methyl EGFP oligos were synthesized by IDT. SiGAPDH and siActin were from Ambion (Austin, Texas, United States of America), and siSTABLE-v2 against mouse GAPDH mRNA was from Dharmacon (Lafayette, Colorado, United States of America). In addition, siGAPDH, siGFP, mir-125a and mir-30a duplexes were prepared using Silencer siRNA construction kit (Ambion). All cell lines including HEK 293, HeLa, NIH3T3 and LinX were cultured in DMEM medium with 10% FBS and Penicillin/Streptomycin. Plasmids, 2-O-methy-oligos siRNA and microRNA duplex were transfected into cells by Lipofectamine 2000 (Invitrogen, Carlsbad, California, United States of America). Stable cell lines were generated through selection using appropriate antibiotics.

Example 8

Small-Molecule Screen, Microscopy and Fluorescent Plate Reading Based on the level of GFP fluorescence intensity, individual clones of 293- EGFP-siGFP were isolated. Several clones were used for the chemical screen. Cells were plated into 24 well plates. 2000 individual drugs from The Spectrum Collection (10 mM in DMSO, MicroSource Discovery Systems Inc.) were added into individual well at a final concentration of 50 μM in 24 hrs. GFP fluorescence of each well was then visually inspected in 48 hours using inverted fluorescence microscope. Compounds that obviously changed EGFP fluorescence were identified for follow-up study. Images were taken using a LSM 510 confocal microscope (Carl Zeiss Microimaging, Inc., Thornwood, New York, United States of America). To quantify

RNAi-E activity, EGFP fluorescence intensity was measured in 48 hrs on an Analyst HT plate reader (Molecular Devices, Sunnyvale, California, United States of America). An excitation filter at 485 nm and an emission filter at 520 nm were used with a dichroic mirror of 505 nm.

Example 9

Quantitative RT-PCRs

Total RNAs from cultured cells were isolated with Trizol (Invitrogen). Total RNAs from mouse ears were isolated with RiboPure kit (Ambion). RNA samples were reverse-transcribed into cDNA with Superscript First-strand Synthesis System

(Invitrogen). Real-time PCR was performed with gene specific primers and Power SYBR Green master mix (Applied Biosystems) using a 7500 Fast Real-time PCR system (Applied Biosystems). Relative quantities of mature miRNAs were determined using TaqMan® microRNA Assays (Applied Biosystems). Relative quantities of RNAs were determined using the ΔΔCt method as provided by the manufacturer. Example 10 Western Blot

Protein samples were separated on SDS-PAGE gels and then transferred to PVDF membranes (Millipore). Membranes were processed following ECL western blotting protocol (Amersham, Buckinghamshire, United Kingdom). Anti-GFP (Santa Cruz Biotechnology, Santa Cruz, California, United States of America) and Anti- GAPDH (Ambion) were used as primary antibodies at the concentrations recommended by manufactures. HRP-labeled secondary antibodies were obtained from Sigma. For loading controls, membranes were stripped and re-probed with the antibody to GAPDH.

Example 11 Luciferase Assay HEK-293 cells stably expressing shMir-125a or vector alone were seeded at

0.6x10⁵ cells/well in a 12-well plate in antibiotic-free media the day before transfection. Transfections were performed with 0.5 μg of reporter plasmid and 0.05 μg of pPvL-TK plasmid (Promega Corporation, Madison, Wisconsin, United States of America) per well, and each sample was transfected in triplicate. Transfections were done in a final volume of 600 μL. In twelve hours, a media change was done ±

RNAi-E. Luciferase assays were performed in an additional 48 hrs, using the Dual- Luciferase Reporter Assay System (Promega) and the Luminometer TD-20/20 (Turner Designs, Sunnyvale, California, United States of America).

Example 12

Northern Blot of Small RNAs

RNAs were isolated with Trizol (Invitrogen), and then separated on 15% TBE urea gel, transferred and UV cross-linked to Nylon membrane (Osmonics Inc., Minnetonka, Minnesota, United States of America). ³²p-UTP labeled probes were prepared with Ambion mirVana miRNA probe construction kit. Membranes were prehybridized at 65 ⁰C for 1 hr and hybridized for 12-16 hours at room temperature. Membranes were then washed 3 times at RT and 2 times at 42 ⁰C. Membranes were exposed and scanned with a Typhoon 9200 phosphorimager (Amersham Biosciences, Piscataway, New Jersey, United States of America). Example 13

Purification of Recombinant TRBP

His6-tagged TRBP was expressed in Escherichia coli BL21 (DE3) cells and purified with Ni-NTA Fast Start Kit (Qiagen, Valencia, California, United States of

America). The purity of the protein was analyzed by SDS-PAGE and its size was compared with a His-tagged standard (Invitrogen). Protein concentration was determined by Bradford assay.

Example 14

In vitro miRNA precursor processing assay and TRBP binding assay Let- 7 precursor RNA oligos were described previously and synthesized by Dharmacon. See Forstemann et al. (2005) PLoS Biol 3(7): e236. The let- 7 precursor RNA oligos were radioactively labeled at the 5 ' end with ³²P-γ-ATP using T4 polynucleotide kinase (New England Biolabs, Ipswich, Massachusetts, United States of America). The labeled let-7 precursor was allowed to refold by heating at 95 ⁰C for 2 minutes followed by an incubation at 37 ⁰C for 1 hr. For the processing assay, the labeled let- 7 precursor was incubated with Dicer, or Dicer with TRBP, in the buffer as described previously for 1 hr. See Forstemann et al. (2005) PLoS Biol 3(7): e236. The reaction was terminated and subjected to Phenol/chloroform extraction and ethanol precipitation. Then the samples were separated on a 15% TBE urea gel, transferred and UV cross-linked to Nylon membrane, which was then exposed for phosphorimager scanning.

TRBP binding reaction was carried out in 1 X buffer (20 mM Tris-HCl, pH 8.0, 15 mM NaCl, 2.5 MM MgCl₂) with 100 ng recombinant TRBP, 50 ng ³²P-labeled pre-let-7 in a total volume of 10 μL with or without drug at 30 ⁰C for 1 hr. The reaction mix was then separated on a 10% native gel for 3 hrs at 4 ⁰C. The gel was exposed to a Phosphorimager film and examined with a Typhoon 9200 phosphorimager (Molecular Dynamics, Sunnyvale, California, United States of America). Example 15

C elegans Culture and RNAi Feeding

Wild-type N2 worms were cultivated on NGM agar plates seeded with OP50 E. coli. Worms were well fed on an OP50 food source for a minimum of two generations and never allowed to starve before and during RNAi treatment. E. Coli strains expressing dsRNAs corresponding to let-2 or mom-5 were obtained from GeneService Ltd, United Kingdom. As a control, E. coli was transformed with the RNAi vector L4440. The RNAi constructs were fed to C. elegans on RNAi plates with or without RNAi-E. Starting on the 5th day after L4 hermaphrodites were transferred to the RNAi plates, progenies on the plates were counted for two days.

Example 16 Mice and Lenti viral Injection GFP transgenic line, C57BL16-Tg(ACTB EGFP)lOsb/J, was obtained from

The Jackson Laboratory (Bar Harbor, Maine, United States of America). The lentivirus Lv-siGFP was produced as described previously. See Tiscornia et al. (2003) Proc Natl Acad Sci USA 100(4): 1844-8. Individual GFP transgenic mice (10- day old) were injected with the lentivirus, Lv-siGFP, into one ear (to demonstrate the effect of Lv-siGFP only) or both ears (for injection of RNAi-E or control later).

RNAi-E or control solution was injected once a day for three days into one side of the ears respectively two days after Lv-siGFP injection. As negative control, a group of mice were also injected with RNAi-E or control solution into one side of ears respectively. Mice were then sacrificed and ears were removed and used for RNA isolation and quantitative RT-PCR analysis of GFP mRNA. For each condition, at least 6 mice were injected in three different groups.

Example 17 Chemical Synthesis

Representative chemical syntheses of the presently disclosed derivatives and analogs of RNAi-E are shown in Scheme 1.

Scheme 1

Claims

THAT WHICH IS CLAIMED:

1. A method for decreasing the level of a target polynucleotide in a cell, the method comprising administering to the cell an effective amount of a compound of Formula (aa), or a derivative or analog thereof, wherein said cell further comprises at least one heterologous silencing element:

wherein:

X₈ is CH or N;

X₉ is C or N, under the proviso that when X₉ is N, R₁₆ is absent, and, when X₉ is C, R₁₆ is halo; or

Ri5 and R₁₆ together form a portion of a 4- to 6-member heterocyclic ring structure, wherein the 4-to 6-member heterocyclic ring structure comprises atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur, and combinations thereof;

R₁₄ is selected from the group consisting of H, alkyl, substituted alkyl, alkylamino, cycloalkyl, substituted cycloalkyl, aryl, and substituted aryl;

R₁₅ is selected from the group consisting of heterocycloalkyl and substituted heterocycloalkyl;

R₁S is selected from the group consisting of H, alkyl, substituted alkyl, amino, substituted amino, alkoxyl, hydroxyl, and halo;

R_1P is selected from the group consisting of H, alkyl, and substituted alkyl; and a pharmaceutically or cosmetically acceptable salt thereof.

2. The method of claim 1, wherein the compound of Formula (aa) is selected from the group consisting of:

and RNAl-EΛ/9

3. The method of any of claims 1 -2, wherein said silencing element comprises an siRNA, an miRNA, a double stranded RNA, or a hairpin RNA.

4. The method of any one of claims 1-3, wherein said cell is in a subject.

5. A method for decreasing the level of a target polynucleotide in a cell, the method comprising administering to said cell a combination of an effective concentration of a polynucleotide comprising a heterologous silencing element and an effective amount of a compound of Formula (aa), derivatives and analogs thereof:

wherein:

X₈ is CH or N;

R_1S is selected from the group consisting of heterocycloalkyl and substituted heterocycloalkyl;

R₁₈ is selected from the group consisting of H, alkyl, substituted alkyl, amino, substituted amino, alkoxyl, hydroxyl, and halo;

Ri₉ is selected from the group consisting of H, alkyl, and substituted alkyl; and a pharmaceutically or cosmetically acceptable salt thereof.

6. The method of claim 5, wherein the compound of Formula (aa) is selected from the group consisting of:

nd

7. The method of claim 5, wherein said polynucleotide comprising the heterologous silencing element comprises an expression cassette encoding a siRNA, a miRNA, a dsRNA, or a hairpin RNA.

8. The method of claim 7, wherein said polynucleotide is in a viral vector.

9. The method of claim 5, wherein said polynucleotide comprises a siRNA, a miRNA, a dsRNA, or a hairpin RNA.

10. The method of claim 5, wherein said compound of Formula (a)-(z) or Formula (aa) and said heterologous silencing element are administered to the cell simultaneously or sequentially.

11. The method of any one of claims 5-10, wherein the cell is in a subject.

12. The method of any one of claims 1-11, wherein said cell is from a mammal.

13. A pharmaceutical or cosmetic composition comprising at least one of a compound of Formula (aa) and a pharmaceutically or cosmetically acceptable carrier and one or more polynucleotides comprising a silencing element.

14. The pharmaceutical or cosmetic composition of claim 13, wherein said silencing element comprises a siRNA, a miRNA, a double stranded RNA, or a hairpin RNA.

15. A method for treating a disease state or unwanted condition, the method comprising administering to a subject in need of treatment thereof an effective amount of a polynucleotide comprising a silencing element, which, when administered to said subject, decreases the level of the target polynucleotide and an effective amount of a compound of Formula (aa):

wherein:

X₈ is CH or N;

X9 is C or N, under the proviso that when X9 is N, R₁₆ is absent, and, when X9 is C, R₁₆ is halo; or R_1S and R₁₆ together form a portion of a 4- to 6-member heterocyclic ring structure, wherein the 4-to 6-member heterocyclic ring structure comprises atoms selected from the group consisting of carbon, nitrogen, oxygen, sulfur, and combinations thereof;

Ri5 is selected from the group consisting of heterocycloalkyl and substituted heterocycloalkyl;

16. The method of claim 15, wherein the compound of Formula (aa) is selected from the group consisting of:

RNAι-EΛ/7

and RNAl-EΛ/9

17. The method of any of claims 15-16, wherein said disease state comprises a viral infection.

18. The method of any of claims 15-16, wherein said disease state comprises a genetic disorder.