WO2005012483A2

WO2005012483A2 - Vpr selective rnai agents and methods for using the same

Info

Publication number: WO2005012483A2
Application number: PCT/US2004/023230
Authority: WO
Inventors: Yun Li
Original assignee: International Therapeutics, Inc.
Priority date: 2003-08-01
Filing date: 2004-07-15
Publication date: 2005-02-10
Also published as: WO2005012483A3

Abstract

RNAi agents selective for vpr (a gene that encodes the viral protein R (Vpr) that plays a role in viral, e.g., HIV-1, infection and replication) and methods for using the same are provided. In the subject methods, an effective amount of a vpr selective RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is introduced into a cell, resulting in the inhibition of vpr expression. Also provided are vpr selective RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including therapeutic applications, such as the treatment of hosts suffering from HIV infection and related conditions.

Description

VPR SELECTIVE RNAI AGENTS AND METHODS FOR USING THE SAME CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority (pursuant to 35 U.S.C. § 119 (e)) to the filing date of United States Provisional Patent Application Serial No. 60/491 ,910 filed on August 1 , 2003; the disclosure of which is herein incorporated by reference.

INTRODUCTION Background of the Invention The human immunodeficiency virus type-1 (HIV-1) is the causative agent for the acquired immunodeficiency syndrome (AIDS). To date, the primary focus for therapeutics development has been to target the viral enzymes reverse transcriptase (RT) and protease (PI) with small chemical entities. More recently, a new class of inhibitors, synthetic peptides, that target virus entry into cells have been approved. The HIV-1 life cycle provides at least ten potential target for intervention. Some of these targets have not been approachable with small molecule inhibitors. Double-stranded RNA induces potent and specific gene silencing through a process referred to as RNA interference (RNAi) or posttranscriptional gene silencing (PTGS). RNAi is mediated by RNA-induced silencing complex (RISC), a sequence-specific, multicomponent nuclease that destroys messenger RNAs homologous to the silencing trigger. RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the double-stranded RNA trigger. For a review of the RNAi process, see Paddison & Hannon, Cancer Cell (2002) 2:17- 23. Among other applications, RNAi has great potential for use in therapeutic applications. Virtually any disease that is treatable through silencing of expression of one or more genes may be treated using RNAi therapeutic agents. The target genes may be endogenous genes, including mutant endogenous genes, as well as exogenous genes, e.g., viral pathogen genes. For example, siRNA has been demonstrated to effectively inhibit HIV-1 production in T-cells in vitro. The utility of siRNA as inhibitors of HIV-1 has been validated in vitro by several groups. The targets for siRNA intervention have varied from HIV-1 regulatory proteins such as Tat and Rev, to the cell surface chemokine receptors CCR5 and CXCR4 that are used by HIV-1 to attach to target cells. In addition, both synthetic siRNA added to cells exogenously, as well as vector targeted siRNA intracellular expression has been used to validate the efficacy of RNAi as a therapeutic approach to HIV-1 infection. Relevant Literature Capodici et al., J. Immunol. (2002) 169:5196-5201 ; Cobum et al., J. Virol. (2002) 76:9225-9231 ; Hu et al., Curr. Biol. (2002) 12:1301-1311 ; Jacquet et al., Nature (2002) 418: 435-438; Lawrence, Lancet (2002) 359: 2007; Lee et al., Nat. Biotech. (2002) 20: 446-448; Lin et al., Curr. Cancer Durg Targets (2001) 1 :241- 247; Martinez et al., Trends Immunol. (2002) 23: 559-561 ; Martinez et al., AIDS (2002) 16:2385-2390; Park et al., Nuc. Acids Res (2002) 15: 4830-4835; Park et al., Nuc. Acids Res. (2001) Suppl. 1 :219-20; and Yamamoto et al., Microbiol Immunol. (2002) 46:809-817.

SUMMARY OF THE INVENTION RNAi agents selective for vpr (a gene that encodes the viral protein R (Vpr) that plays a role in viral, e.g., HIV-1, infection and replication) and methods for using the same are provided. In the subject methods, an effective amount of a vpr selective RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is introduced into a cell, resulting in the inhibition of vpr expression. Also provided are vpr selective RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including therapeutic applications, such as the treatment of hosts suffering from HIV infection and related conditions.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 provides the nucleotide sequence of vpr of pNL4.3. Figures 2 to 4 provide graphical representations of results obtained in from the experiments performed in the Experimental Section, below. DEFINITIONS For convenience, certain terms employed in the specification, examples, and appended claims are defined below. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a genomic integrated vector, or "integrated vector", which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, i.e., a nucleic acid capable of extra-chromosomal replication in an appropriate host, e.g., a eukaryotic or prokaryotic host cell. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors". As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides. As used herein, the term "gene" or "recombinant gene" refers to a nucleic acid comprising an open reading frame encoding a polypeptide of the present invention, including both exon and (optionally) intron sequences. A "recombinant gene" refers to nucleic acid encoding such regulatory polypeptides, that may optionally include intron sequences that are derived from chromosomal DNA. The term "intron" refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons. As used herein, the term "transfection" means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. A "protein coding sequence" or a sequence that "encodes" a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from procaryotic or eukaryotic mRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence. Likewise, "encodes", unless evident from its context, will be meant to include DNA sequences that encode a polypeptide, as the term is typically used, as well as DNA sequences that are transcribed into inhibitory antisense molecules. By "reducing expression" is meant that the level of expression of a target gene or coding sequence, e.g. a vpr coding sequence, is reduced or inhibited by at least about 2-fold, usually by at least about 5-fold, e.g., 10-fold, 15-fold, 20-fold, 50-fold, 100-fold or more, as compared to a control. By modulating expression of a target gene is meant altering, e.g., reducing, transcription/translation of a coding sequence, e.g., genomic DNA, mRNA etc., into a polypeptide, e.g., protein, product. The term "expression" with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein coding sequence results from transcription and translation of the coding sequence. "Cells," "host cells" or "recombinant host cells" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein. As used herein, the "transfection" is art recognized and means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. "Transduction", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a dsRNA construct. 'Transient transfection" refers to cases where exogenous DNA does not integrate into the genome of a transfected cell, e.g., where episomal DNA is transcribed into mRNA and translated into protein. A cell has been "stably transfected" with a nucleic acid construct when the nucleic acid construct is capable of being inherited by daughter cells. As used herein, a "reporter gene construct" is a nucleic acid that includes a "reporter gene" operatively linked to at least one transcriptional regulatory sequence. Transcription of the reporter gene is controlled by these sequences to which they are linked. The activity of at least one or more of these control sequences can be directly or indirectly regulated by the target receptor protein. Exemplary transcriptional control sequences are promoter sequences. A reporter gene is meant to include a promoter-reporter gene construct that is heterologously expressed in a cell. The "growth state" of a cell refers to the rate of proliferation of the cell and the state of differentiation of the cell. "Inhibition of gene expression" refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene. "Specificity" refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or

99% as compared to a cell not treated according to the present invention. Lower doses of administered active agent and longer times after administration of active agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell: mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region. DESCRIPTION OF THE SPECIFIC EMBODIMENTS RNAi agents selective for vpr (a gene that encodes the viral protein R (Vpr) that plays a role in viral, e.g., HIV-1, infection and replication) and methods for using the same are provided. In the subject methods, an effective amount of a vpr selective RNAi agent, e.g., an interfering ribonucleic acid (such as an siRNA or shRNA) or a transcription template thereof, e.g., a DNA encoding an shRNA, is introduced into a cell, resulting in the inhibition of vpr expression. Also provided are vpr selective RNAi agent pharmaceutical preparations for use in the subject methods. The subject methods and compositions find use in a variety of different applications, including therapeutic applications, such as the treatment of hosts suffering from HIV infection and related conditions.

Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

In this specification and the appended claims, the singular forms "a," "an" and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, representative methods, devices and materials are now described.

All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the components that are described in the publications that might be used in connection with the presently described invention.

As summarized above, the subject invention provides methods of using vpr selective RNAi agents to selectively modulate gene expression in target, e.g., mammalian, cells. In further describing the subject invention, the subject methods are described first in greater detail, followed by a review of representative pharmaceutical compositions and kits thereof that find use in practicing the subject methods.

METHODS

As indicated above, the subject invention provides methods of employing a vpr selective RNAi agent to modulate expression of a target vpr gene or coding sequence in a target, typicaly mammalian, cell, where the target cell may be present in vitro or in a host. In many embodiments, the subject invention provides methods of reducing expression of a vpr gene or coding sequence in a target mammalian cell, including human cell, or host organism including the same. By reducing expression is meant that the level of expression of the vpr target gene or coding sequence is reduced or inhibited by at least about 2-fold, usually by at least about 5-fold, e.g., 10-fold, 15-fold, 20-fold, 50-fold, 100-fold or more, as compared to a control. In certain embodiments, the expression of the vpr target gene is reduced to such an extent that expression of the vpr target gene/coding sequence is effectively inhibited. By modulating expression of a vpr target gene is meant altering, e.g., reducing, transcription/translation of a coding sequence, e.g., genomic DNA, mRNA etc., into a polypeptide, e.g., protein, product. The subject invention provides methods of modulating expression of a vpr target gene in a mammalian cell or organism comprising the same. By "organism comprising the same" is meant a mammalian organism or host that is not an in vitro cell or cell culture, where the host organism may be a fetus, but is generally a host organism in a post-natal stage of development, e.g., juvenile, adult, etc. In practicing the subject methods, an effective amount of a vpr selective RNAi agent is introduced into the target mammalian cell, e.g., by using conventional methods of introducing nucleic acids into a cell, such as electroporation, liposome mediated uptake, etc., by administration of the agent to the host organism to modulate expression of a target gene in a desirable manner, e.g., to achieve the desired reduction in target cell gene expression. By vpr selective RNAi agent is meant an agent that modulates expression of a vpr target gene by a RNA interference mechanism. The RNAi agents employed in one embodiment of the subject invention are small ribonucleic acid molecules (also referred to herein as interfering ribonucleic acids), i.e., oligoribonucleotides, that are present in duplex structures, e.g., two distinct oligoribonucleotides hybridized to each other or a single ribooligonucleotide that assumes a small hairpin formation to produce a duplex structure. By oligoribonucleotide is meant a ribonucleic acid that does not exceed about 100 nt in length, and typically does not exceed about 75 nt length, where the length in certain embodiments is less than about 70 nt. Where the RNA agent is a duplex structure of two distinct ribonucleic acids hybridized to each other, e.g., an siRNA (such as d-siRNA as described in copending application serial no. 60/377,704; the disclosure of which is herein incorporated by reference), the length of the duplex structure typically ranges from about 15 to 30 bp, usually from about 15 to 29 bp, where lengths between about 20 and 29 bps, e.g., 21 bp, 22 bp, 23 bp are of particular interest in certain embodiments. Where the RNA agent is a duplex structure of a single ribonucleic acid that is present in a hairpin formation, i.e., a shRNA, the length of the hybridized portion of the hairpin is typically the same as that provided above for the siRNA type of agent or longer by 4-8 nucleotides. The weight of the RNAi agents of this embodiment typically ranges from about 5,000 daltons to about 35,000 daltons, and in many embodiments is at least about 10,000 daltons and less than about 27,500 daltons, often less than about 25,000 daltons. In certain embodiments, instead of the RNAi agent being an interfering ribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAi agent may encode an interfering ribonucleic acid, e.g., an shRNA, as described above. In other words, the RNAi agent may be a transcriptional template of the interfering ribonucleic acid. In these embodiments, the transcriptional template is typically a DNA that encodes the interfering ribonucleic acid. The DNA may be present in a vector, where a variety of different vectors are known in the art, e.g., a plasmid vector, a viral vector (such as a lentiviral vector), etc. As the subject RNAi agents are vpr selective, they selectively inhibit the expression of a vpr coding sequence, as described above. By vpr coding sequence is meant a coding sequence of a vpr gene, where the vpr gene is, in certain embodiments, a viral vpr gene, particularly a retroviral vpr gene, such as an HIV vpr gene, e.g., an HIV-1 vpr gene (SEQ ID NO:01). The subject RNAi agents typically include a nucleic acid sequence or found in the target vpr gene, where the sequence may be present in any region or location of the target vpr gene. As indicated above, the RNAi agent can be introduced into the target mammalian cell(s) using any convenient protocol, where the protocol will vary depending on whether the target cells are in vitro or in vivo. Where the mammalian target cells are in vivo, the vpr selective RNAi agent can be administered to the mammalian host using any convenient protocol, where the protocol employed is typically a nucleic acid administration protocol, where a number of different such protocols are known in the art. The following discussion provides a review of representative nucleic acid administration protocols that may be employed. The nucleic acids may be introduced into tissues or host cells by any number of routes, including viral infection, microinjection, or fusion of vesicles. Jet injection may also be used for intra-muscular administration, as described by Furth et al. (1992), Anal Biochem 205:365-368. The nucleic acids may be coated onto gold microparticles, and delivered intradermally by a particle bombardment device, or "gene gun" as described in the literature (see, for example, Tang et al. (1992), Nature 356:152-154), where gold microprojectiles are coated with the DNA, then bombarded into skin cells. Expression vectors may be used to introduce the nucleic acids into a cell. Such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences. Transcription cassettes may be prepared comprising a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region. The transcription cassettes may be introduced into a variety of vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks. For example, the RNAi agent can be fed directly to, injected into, the host organism containing the target gene. The agent may be directly introduced into the cell (i.e., intracellulariy); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, etc. Methods for oral introduction include direct mixing of RNA with food of the organism. Physical methods of introducing nucleic acids include injection directly into the cell or extracellular injection into the organism of an RNA solution. The agent may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of the agent may yield more effective inhibition; lower doses may also be useful for specific applications. In certain embodiments, a hydrodynamic nucleic acid administration protocol is employed. Where the agent is a ribonucleic acid, the hydrodynamic ribonucleic acid administration protocol described in detail below is of particular interest. Where the agent is a deoxyribonucleic acid, the hydrodynamic deoxyribonucleic acid administration protocols described in Chang et al., J. Virol. (2001) 75:3469-3473; Liu et al., Gene Ther. (1999) 6:1258-1266; Wolff et al., Science (1990) 247: 1465- 1468; Zhang et al., Hum. Gene Ther. (1999) 10:1735-1737: and Zhang et al., Gene Ther. (1999) 7:1344-1349; are of interest. Additional nucleic acid delivery protocols of interest include, but are not limited to: those described in U.S. Patents of interest include 5,985,847 and 5,922,687 (the disclosures of which are herein incorporated by reference); WO/11092;. Acsadi et al., New Biol. (1991) 3:71-81 ; Hickman et al., Hum. Gen. Ther. (1994) 5:1477- 1483; and Wolff et al., Science (1990) 247: 1465-1468; etc. In certain embodiments, two or more different vpr selective RNAi agents may be administered to the host, e.g., where each agent is directed to a different location or region of the vpr gene. In certain embodiments, the vpr selective RNAi agent may be delivered with one or more additional RNAi agents, e.g., RNAi agents specific for other viral pathogen targets, e.g., REV, TAT, and the like.

PHARMACEUTICAL PREPARATIONS

Depending on the nature of the RNAi agent, the active agent(s) may be administered to the host using any convenient means capable of resulting in the desired modulation of target gene expression. Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the agents can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration. In pharmaceutical dosage forms, the agents may be administered alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting. For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor(s) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public. Those of skill in the art will readily appreciate that dose levels can vary as a function of the specific compound, the nature of the delivery vehicle, and the like. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. Introduction of an effective amount of an RNAi agent into a mammalian cell as described above results in a modulation of target gene(s) expression, e.g., a reduction of target gene(s) expression, as described above. The above described methods work in any mammalian cell, where representative mammal cells of interest include, but are not limited to cells of: ungulates or hooved animals, e.g., cattle, goats, pigs, sheep, etc.; rodents, e.g., hamsters, mice, rats, etc.; lagomorphs, e.g., rabbits; primates, e.g., monkeys, baboons, humans, etc.; and the like. The above-described methods find use in a variety of different applications, representative types of which are now described in greater detail below.

UTILITY The subject methods find use in a variety of therapeutic applications in which it is desired to selectively inhibit expression of a vpr coding sequence in a subject. In such methods, an effective amount of an RNAi active agent is administered to the subject or target cell thereof. By effective amount is meant a dosage sufficient to selectively inhibit expression of the vpr target gene, as desired. As indicated above, in many embodiments of this type of application, the subject methods are employed to reduce/inhibit expression of a vpr target gene or coding sequence in the host in order to achieve a desired therapeutic outcome. The subject methods find use in the treatment of a variety of different conditions in which the modulation of vpr target gene expression in a host, e.g., mammalian host such as a human, is desired. By treatment is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. In many embodiments, the pathological condition being treated is AIDS. - A variety of hosts are treatable according to the subject methods. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.

KITS Also provided are reagents and kits thereof for practicing one or more of the above-described methods. The subject reagents and kits thereof may vary greatly. Typically, the kits at least include a vpr selective RNAi agent as described above. In addition to the above components, the subject kits will further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e.g., diskette, CD, etc., on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL The vpr gene (figure 1) of HIV-1 encodes for the regulatory protein vpr. Vpr has been described to play an important role in mediating nuclear entry of the HIV genome and in enhancing the ability of the virus to replicate by arresting the infected cell in the G2 phase of the cell cycle prior to cell division. Given the important role Ppr plays in HIV-1 infection and replication, we designed siRNA as short hair-pin RNA (shRNA) that are complementary to two stretches of the vpr gene, shRNA vpr-l and shRNA vpr-ll (figure 1 , single underline and double underline, respectively). The DNA encoding the shRNA vpr-l or -II were cloned into the mammalian expression vector, pTZ, downstream of the polymerase III U6 promoter, or into a lentiviral expression vector also downstream of the U6 promoter. The expression vectors carrying the appropriate shRNA encoding gene were introduced into human embryonic kidney cell line transformed with the SV40 large T antigen (293T). The cells were co-transfected with a HIV-1 infectious clone pNL4.3. Virus production was evaluated on days 1 , 2, and 3 post-transfection by quantifying the extracellular levels of the HIV-1 core protein p24. To validate the activity of the selected shRNA sequences, we used multiple clones of each construct. As shown in figures 2 and 3, both shRNA vpr-l and shRNA vpr-ll, respectively, reduced virus production from cells co-transfected with pNL4.3. An irrelevant or control siRNA was used as a negative control. Figures 2&3 also show that the different clones of the shRNA exerted similar activities confirming that the anti-viral effect was sequence directed. Figure 4 presents the inhibition of HIV-1 production by shRNA vpr-ll encoded by a lentivirus vector. As described for the previous experiments, 293 T cells were co-transfected with the lentivirus vector carrying the shRNA vpr-ll gene and the HIV-1 infectious clone pNL4.3. HIV-1 p24 was quantified from the culture media over the course of three days. As shown in figure 4, the inhibition of p24 production was approximately 1.5 log compared the irrelevant control. Both shRNA clones exhibited similar levels of inhibition. In addition to inhibiting the production of the Vpr protein by catalytic degradation of the vpr mRNA, the shRNA vpr exert their antiviral effect directly by causing the catalytic degradation of the HIV-1 genomic RNA that is used as the template for producing the gag proteins, including p24, that represent the core of the virus. This would explain the efficacy of the vpr-directed shRNA in the in vitro transfection system that does not rely on the classic mechanism of Vpr in the virus life cycle. It is evident from the above results and discussion that the subject invention provides an important new way of inhibiting HIV replication. Accordingly, the invention provides an important new therapeutic agent for the treatment of AIDS. As such, the subject invention represents a significant contribution to the art.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of selectively reducing expression of a vpr coding sequence in a mammalian target cell, said method comprising: introducing into said mammalian target cell an effective amount of a RNAi agent specific for said vpr coding sequence to selectively reduce expression of said coding sequence.

2. The method according to Claim 1, wherein said RNAi agent is an interfering ribonucleic acid.

3. The method according to Claim 2, wherein said interfering ribonucleic acid is a siRNA.

4. The method according to Claim 2, wherein said interfering ribonucleic acid is a shRNA.

5. The method according to Claim 1 , wherein said RNAi agent is a transcription template of an interfering ribonucleic acid.

6. The method according to Claim 5, wherein said transcription template is a deoxyribonucleic acid.

7. The method according to Claim 6, wherein said deoxyribonucleic acid encodes a shRNA.

8. The method according to Claim 1 , wherein said mammalian cell is present in vitro.

9. The method according to Claim 1, wherein said mammalian cell is present in vivo.

10. The method according to Claim 1 , wherein said vpr coding sequence is an HIV vpr coding sequence.

11. A RNAi agent specific for a vpr coding sequence.

12. The RNAi agent according to Claim 11 , wherein said RNAi agent is an interfering ribonucleic acid.

13. The RNAi agent according to Claim 12, wherein said interfering ribonucleic acid is a siRNA.

14. The RNAi agent according to Claim 12, wherein said interfering ribonucleic acid is a shRNA.

15. The RNAi agent according to Claim 11 , wherein said RNAi agent is a transcription template of an interfering ribonucleic acid.

16. The RNAi agent according to Claim 15, wherein said transcription template is a deoxyribonucleic acid.

17. The RNAi agent according to Claim 16, wherein said deoxyribonucleic acid encodes a shRNA.

18. The RNAi agent according to Claim 11 , wherein said vpr coding sequence is an HIV vpr coding sequence.

19. A pharmaceutical preparation comprising an RNAi agent specific for a vpr coding sequence in a pharmaceutically acceptable delivery vehicle.

20. A kit for use in practicing the method of Claim 1 , said kit comprising: (a) a pharmaceutical preparation comprising an RNAi agent specific for a vpr coding sequence in a pharmaceutically acceptable delivery vehicle; and (b) instructions for practicing the method of Claim 1.