The present invention provides methods for identifying druggable targets in assays that feature compositions, cells and/or organisms having structured viral non-coding RNAs (svRNAs) and an RNA interference (RNAi) pathway. Methods for identifying antiviral agents and creating vaccines are also featured. The invention further provides methods for inhibiting RNAi involving svRNAs or inhibitory derivatives thereof. The invention also provides compositions for delivering siRNA and miRNA molecules derived from svRNA loci and methods of use thereof. Therapeutic methods are also featured.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that Adenovirus Virus-Associated (VA) RNA, is processed by the ribonuclease Dicer to generate .about.21-23 nt RNA product. The instant inventors further discovered that infection with Adenovirus-5 (AD-5) can inhibit siRNA activity in mammalian cells. VA RNAs of the Adenoviridae family bear a striking resemblance to pre-miRNAs, which are similarly processed by Dicer into miRNAs. Other virus families encode untranslated RNAs having similar structures. Based on these discoveries, VA RNAs or other virally derived untranslated structural RNAs (referred to herein as structural viral RNAs or svRNAs) are believed to be incorporated into a Dicer (or an orthologue or homologue thereof) or RISC complex to function as substrates and/or inhibitors of the RNAi pathway.

Accordingly, the present invention features svRNAs (or derivatives thereof) for use as mediators of RNAi. In one embodiment, the svRNAs (or derivatives thereof) are activators of RNAi. Also featured are svRNAs (or derivatives thereof) for use as inhibitors of RNAi. Also featured are methods for identifying druggable targets, in particular, antiviral targets, mediated by the svRNAs (or derivatives thereof). Such targets are further useful in drug discovery methodologies. Also featured are expression cassettes and vectors (e.g., virus-derived vectors), the cassettes and/or vectors including VA RNA loci modified to deliver miRNA- and siRNA-like molecules.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the surprising discovery that a short non-coding RNA produced by Adenovirus, Adenovirus Virus-Associated (VA) RNA.sub.I, is processed by the ribonuclease Dicer to generate .about.21-23 nt RNA products. The present invention is based on the further discovery that infection with Adenovirus-5 (AD-5) can inhibit siRNA activity in mammalian cells. This processing of VA RNA.sub.I is similar to the processing events by Dicer of pre-miRNA into miRNAs. Pre-miRNAs are complex, double-stranded precursor RNA molecules characterized by key structural features such as stem loops and bulges (4, 10). VA RNAs of the Adenoviridae family bear a striking resemblance to pre-miRNAs. VA RNAs, which are produced at very high levels in infected cells, are generally believed to promote viral infection by binding and inhibiting the protein kinase PKR, where PKR normally functions to activate the interferon response upon binding dsRNA. Other virus families and viruses, e.g. gamma herpesvirinae, lentivirus and retrovirus, encode untranslated RNAs that have similar structures.

Based on the discoveries set forth herein, VA RNAs encoded by viruses within the Adenoviridae family, or alternatively, short untranslated structural RNAs encoded by other viruses, are proposed to act as precursors for cleavage by Dicer, thereby producing miRNA-like molecules that regulate gene expression. The concept of a virus encoding miRNA or miRNA-like sequences represents a hitherto unidentified mechanism by which viruses may control viral or cellular gene expression to produce an environment conducive to infection. Disruption of this viral function would result in attenuation of viral infection, thus providing novel antiviral strategies. Cellular and/or viral genes whose RNA expression is inhibited by VA RNAs make attractive targets for therapeutic anti-viral strategies as well as novel ways to modulate host homeostasis.

Given the high levels of VA RNA expressed in infected cells, VA RNAs are further proposed to act as inhibitors of RNAi by competing with other substrates for interaction with components of the RNAi pathway, e.g. Dicer, or components of RISC. This prevents processing of other potential RNAi triggers, including host miRNA precursors and viral transcripts. Antagonism of host cell RNAi by VA RNAs may further serve to promote virulence. Disabling the capacity of VA RNAs to antagonize host cell RNAi provides novel approaches for the creation of vaccines or in the design for their use as therapeutic vectors. Additionally, VA RNA loci can be modified to express miRNA- and siRNA-like molecules directed to selected target RNAs, thereby providing a highly efficient siRNA/miRNA transduction system.

Based at least in part on the above findings, the invention features, in a first aspect, methods for identifying genes whose expression is modulated by svRNAs (e.g., VA RNAs). In an exemplary aspect, the genes identified are involved in important cellular processes, for example, in the maintenance of cellular homeostasis or in the modulation of an antiviral response. The genes thus make desirable targets for drug discovery (i.e., druggable targets) or desirable antiviral drug targets, respectively.

Accordingly, the invention provides, in this first aspect, a method for identifying a druggable target, involving: (a) obtaining an assay composition comprising an RNAi pathway molecule and a svRNA; (b) assaying for expression of a candidate RNA; wherein a change in expression of the candidate RNA indicates that a gene or protein corresponding to the RNA is a druggable target. In a preferred embodiment, the assay composition is a cell extract, e.g., a mammalian cell extract.

In a related aspect, the invention provides a method for identifying a druggable target, comprising: (a) obtaining a cell or organism comprising an RNAi pathway and a svRNA; (b) assaying for expression of a candidate RNA; wherein a change in expression of the candidate RNA indicates that a gene or protein corresponding to the RNA is a druggable target. In preferred embodiments, the cell is a eukaryotic cell, e.g., a mammalian cell, a murine cell, an avian cell, a human cell and the like.

In one embodiment of these aspects, the RNA is an mRNA, e.g., an mRNA that encodes a viral protein or a cellular protein. In another embodiment, the RNA is a ncRNA, e.g., a ncRNA that regulates gene expression. Preferably, the druggable target is an antiviral drug target.

In one embodiment of these aspects, the change in expression of the candidate RNA is a decrease in the expression of the candidate RNA. In one embodiment, the methods involve a further step of preselecting the candidate RNA. Preferably, the preselection step involves determining a sufficient degree of sequence identity between the svRNA and the candidate RNA, e.g., wherein the svRNA and the candidate RNA share, for example, at least 60%, 70%, 80%, or 90% sequence identity. In other embodiments, the preselection step involves determining a sufficient degree of sequence identity between the svRNA and the candidate RNA, e.g., wherein the svRNA and the candidate RNA share, for example, at least 30%, 40%, 45%, 50% or 55% sequence identity. In another embodiment, the preselection step comprises selecting the candidate RNA based on its encoding a gene or protein having a desired cellular function, e.g., maintenance of cellular homeostasis or maintenance of differentiation.

Preferably, the svRNA in these related aspects is expressed from a virus, a vector, or a cassette. In a preferred embodiment, the svRNA is derived from a virus capable of infecting mammalian cells. In various embodiments, the svRNA is derived from a virus belonging to a family selected from the group consisting of the Herpesviridae, Retroviridae, Reoviridae, Flaviviridae, Poxyiridae and Picornaviridae families. In various embodiments, the svRNA is derived from a virus selected from the group consisting of EBV, HPV, MHV-68, HCMV, HIV, HCV, Dengue Virus, Foot and Mouth Disease Virus, Poliovirus, Vacciniavirus, Small Pox virus and KSHV. In preferred embodiments, the svRNA is selected from the group consisting of EBER 1, EBER 2, MHV-68 short ncRNAs, CMER, RRE, TAR, POLADS, PAN RNA and IRES.

In exemplary embodiments of the invention, the svRNA is derived from a virus belonging to the adenoviridae family, e.g., adenovirus type 2 or adenovirus type 5 virus. In further exemplary embodiments, the svRNA is a VA RNA, e.g., VA-RNA.sub.I or VA-RNA.sub.II.

The invention further features a druggable target, e.g., an antiviral drug target, identified according to the provided methods of the invention. Such antiviral drug targets are useful in methods for identifying an antiviral agent, e.g., methods that involve assaying a test agent for activity against the antiviral drug target. In preferred embodiments, a method for identifying an antiviral agent involves assaying a test agent for the ability to stimulate expression or activity of the antiviral drug target, or to inhibit an interaction between the antiviral drug target and a corresponding svRNA.

The invention provides, in another aspect, a method for identifying an antiviral agent, involving: (a) contacting a cell with a test agent, said cell comprising an RNAi pathway and a svRNA, wherein said RNAi pathway generates a siRNA or miRNA from said svRNA; (b) detecting an indicator of said siRNA or miRNA; wherein an agent is identified based on its ability to inhibit the generation of said siRNA or miRNA.

In a related aspect, a method is provided for identifying an antiviral agent, involving: (a) contacting an assay composition with a test agent, wherein said assay composition comprises an RNAi pathway molecule and a svRNA, wherein said RNAi pathway molecule generates a siRNA or miRNA from said ribonucleotide; (b) detecting an indicator of said siRNA or miRNA; wherein an agent is identified based on its ability to inhibit the generation of said siRNA or miRNA.

The invention also provides an agent that is identified according to the methods of these aspects, as well as a pharmaceutical composition comprising the agent and a pharmaceutically acceptable carrier. These agents and compositions can be administered in an effective dose to an organism or subject in methods for attenuating and/or treating a viral infection. Preferably, the organism or subject is a eukaryotic organism, e.g., a mammal, e.g., a human.

The invention further features svRNA as inhibitors of the RNAi pathway. As inhibitors of RNAi, svRNAs compete with other substrates of the RNAi machinery to modulate expression of those genes regulated by siRNA or miRNA molecules.

Accordingly, in another aspect, the invention provides a method of inhibiting RNAi in a cell, involving introducing into the cell a svRNA or inhibitory derivative thereof, such that RNAi in the cell is inhibited. In a related aspect, a method is provided for inhibiting the incorporation of a siRNA or miRNA into a cellular Dicer or RISC complex, comprising introducing into the cell an isolated svRNA or inhibitory derivative thereof, such that incorporation of the siRNA or miRNA into the complex is inhibited.

In various embodiments, the cell is a eukaryotic cell, e.g., a mammalian cell, preferably a human cell. In another embodiment, the cell is present in an organism, e.g., present in a human subject.

In one embodiment, the svRNA is a VA RNA. In one embodiment, the svRNA is derived from a virus capable of infecting eukaryotic cells, e.g., mammalian cells. In an exemplary embodiment, the svRNA is derived from a virus belonging to the adenoviridae family, e.g., adenovirus type 2 or adenovirus type 5 virus. Preferably, the svRNA is VA-RNA.sub.I or VA-RNA.sub.II.

Preferably, the svRNA in these related aspects is expressed from a virus, a vector, or a cassette. In various embodiments, the svRNA is derived from a virus belonging to a family selected from the group consisting of the Herpesviridae, Retroviridae, Reoviridae, Flaviviridae, Poxyiridae and Picornaviridae families. In various embodiments, the svRNA is derived from a virus selected from the group consisting of EBV, HPV, MHV-68, HCMV, HIV, HCV, Dengue Virus, Foot and Mouth Disease Virus, Poliovirus, Vacciniavirus, Small Pox virus and KSHV. In preferred embodiments, the svRNA is selected from the group consisting of EBER 1, EBER 2, MHV-68 short ncRNAs, CMER, RRE, TAR, POLADS, PAN RNA and IRES.

In yet another aspect of the invention, a method is provided for identifying an antiviral agent, involving: (a) contacting a cell with a test agent, said cell comprising an RNAi pathway and a svRNA, wherein the ribonucleotide inhibits the RNAi pathway; (b) detecting an indicator of the RNAi pathway; wherein an agent is identified based on its ability to alleviate inhibition of the RNAi pathway.

In a related aspect, the invention provides a method for identifying an antiviral agent, involving: (a) contacting an assay composition with a test agent, wherein said assay composition comprises a RNAi pathway molecule and a svRNA which inhibits the activity of said RNAi pathway molecule; (b) detecting activity of said RNAi pathway molecule; wherein said agent is identified based on its ability to restore activity of said RNAi pathway molecule.

In a third related aspect, the invention provides a method for identifying an antiviral agent, involving: (a) contacting an assay composition with a test agent, wherein said assay composition comprises a svRNA and a RNAi pathway molecule capable of interacting with or altering the svRNA; (b) detecting the ability of the RNAi pathway molecule to interact with or alter the svRNA; wherein said agent is identified based on its ability to modulate the interaction of the svRNA with the RNAi pathway molecule or alteration of the svRNA by the RNAi pathway molecule.

In one embodiment of these aspects, the RNAi pathway molecule is a RISC component. In another embodiment, the RNAi pathway molecule is Dicer, or a homologue thereof.

Agents identified according to these aspects are provided in the present invention, as well as pharmaceutical compositions comprising the agent and a pharmaceutically acceptable carrier.

Loci of svRNA and modified derivatives thereof are useful as delivery vehicles for RNAi agents, e.g., siRNA or miRNA-like molecules. Accordingly, in another aspect, the invention provides a vector for delivering a siRNA or miRNA, comprising a svRNA locus that has been modified to comprise a ribonucleotide sequence that encodes a siRNA or miRNA precursor. In one embodiment of this aspect, the vector comprises two svRNA loci. Preferably, the first svRNA locus is derived from adenovirus VA RNA.sub.I and a second svRNA locus is derived from VARNA.sub.II. In one embodiment, the vector is a plasmid. In another embodiment, the vector is derived from a virus.

In a related aspect, a cassette is provided for expressing a siRNA or miRNA, comprising a svRNA locus that has been modified to comprise a ribonucleotide sequence that encodes a siRNA or miRNA precursor. In one embodiment of this aspect, the svRNA locus is derived from a virus of the Adenoviridae family. In exemplary embodiments, the svRNA locus is derived from adenovirus VA RNA.sub.I or VARNA.sub.II.

In various embodiments, the svRNA is derived from a virus belonging to a family selected from the group consisting of the herpesviridae, retroviridae, flaviviridae, poxviridae and picornaviridae families. In various embodiments, the svRNA is derived from a virus is selected from the group consisting of EBV, HPV, MHV-68, HCMV, HIV, HCV, Dengue Virus, Foot and Mouth Disease Virus, Poliovirus, Vacciniavirus, Small Pox Virus and KSHV. In preferred embodiments, the svRNA is selected from the group consisting of EBER 1, EBER 2, MHV-68 short ncRNAs, CMER, RRE, TAR, POLADs, PAN RNA and IRES.

In some embodiments, the vector or cassette further comprises a polymerase III promoter operably linked to the ribonucleotide sequence. In other embodiments, the vector or cassette further comprises a cryptic promoter endogenous to the svRNA locus operably linked to the ribonucleotide sequence. In yet other embodiments, the sequence of the miRNA or siRNA molecule is sufficiently complementary to a RNA sequence to mediate degradation or to inhibit translation of said RNA sequence.

In another aspect, the invention provides a method for delivering a siRNA or miRNA in a cell at a significantly high level, comprising contacting the cell with the vector or cassette of the present invention under conditions such that the ribonucleotide sequences are expressed.

In yet another aspect, the invention features an adenovirus-derived vector that expresses a siRNA or miRNA from a VA RNA locus. In a related aspect, the invention provides an adenovirus-derived vector that expresses multiple (e.g., two, three, four, five, six, seven, eight or more) siRNA or miRNA. In one embodiment, the invention provides an adenovirus-derived vector that expresses a first siRNA or miRNA from a VA RNA.sub.I locus and a second siRNA or miRNA from a VA RNA.sub.II locus. In other embodiments, the invention provides an adenovirus-derived vector that expresses two, three, four, or more siRNA or miRNAs from the VA RNA.sub.I locus, from the VA RNA.sub.II locus, or from both the VA RNA.sub.I locus and the VA RNA.sub.II locus.

In one embodiment, the vector further comprises nucleotide sequences which encode at least one endogenous cellular protein. The invention further features a vaccine comprising these vectors, wherein at least one siRNA or miRNA targets a viral RNA or a cellular gene required for viral replication.

In yet another aspect, a viral-derived vector is provided that expresses a siRNA or miRNA from a svRNA locus and an exogenous gene from second locus. In a preferred embodiment, the siRNA or miRNA targets a mutant form, e.g., a dominant negative form or a dominant active form of a gene. In another preferred embodiment, the exogenous gene rescues haploinsufficiency.

The invention further provides a composition comprising the vectors of the invention and a pharmaceutically acceptable carrier. Such compositions are useful in methods for targeting degradation of RNA in a subject. Accordingly, the invention provides, in still another aspect, a method for targeting degradation of a RNA in a subject, comprising administering to the subject a composition of the invention, wherein the siRNA or miRNA has a ribonucleotide sequence having sufficient complementarity to the target RNA, such that the targets are degraded. In a related aspect, a method is provided for targeting degradation of multiple RNAs, e.g., a first and second RNA in a subject, comprising administering to the subject a composition of the invention, wherein, for example, a first siRNA or miRNA has a ribonucleotide sequence having sufficient complementarity to the first target RNA and a second siRNA or miRNA has a ribonucleotide sequence having sufficient complementarity to the second target RNA, such that the multiple targets are degraded. In various embodiments, multiple RNAs, e.g., three, four, five, six, seven, eight, nine, ten or more RNAs, are targeted by a composition of the invention. Preferably, in these aspects, at least one siRNA or miRNA has a ribonucleotide sequence sufficiently complementary to a mutant allelic target RNA, such that the mutant allelic target is degraded.

In still another aspect, a method is provided for targeting a RNA for translational inhibition in a subject, involving administering to the subject the composition of the invention, wherein the siRNA or miRNA has a ribonucleotide sequence having sufficient complementarity to the target RNA, such that the targets are translationally inhibited. In a related aspect, the invention provides a method for targeting multiple, e.g., a first and second RNA, for translational inhibition in a subject, comprising administering to the subject the composition of the invention, wherein, for example, a first siRNA or miRNA has a ribonucleotide sequence having sufficient complementarity to the first target RNA and a second siRNA or miRNA has a ribonucleotide sequence having sufficient complementarity to the second target RNA, such that the multiple targets are translationally inhibited. Preferably, in these aspects, at least one siRNA or miRNA has a ribonucleotide sequence sufficiently complementary to a mutant allelic target RNA, such that the mutant allelic target is translationally inhibited.

In yet another aspect, the invention provides a method for creating an attenuated virus, comprising modifying a svRNA locus of a virus, wherein the modification inhibits the ability of the svRNA to function as a substrate or inhibitor of a RNAi pathway, such that an attenuated virus is created. A vaccine produced according to this method is also provided.

Claim 1 of 31 Claims

1. A method for identifying a druggable target, comprising: (a) contacting an isolated assay composition comprising an RNAi pathway molecule and at least one candidate RNA with a VA RNA, wherein the VA RNA is VA RNA.sub.I or VA RNA.sub.II, and wherein the VA RNA has not been modified to include a heterologous sequence, under conditions such that the VA RNA is processed by Dicer to generate a double-stranded RNA agent; and (b) assaying for degradation of the candidate RNA, wherein the double-stranded RNA agent has sufficient sequence complementarity to the candidate RNA to direct RNAi; wherein degradation of the candidate RNA indicates that a gene or protein corresponding to the candidate RNA is a druggable target.

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