CN101180395A - flu treatment - Google Patents

Abstract

The present invention provides compositions comprising an RNAi-inducing entity targeting an influenza virus transcript and any of a variety of delivery agents. The present invention additionally includes methods of inhibiting the biological activity of influenza virus and/or treating or preventing influenza using the composition. The present invention provides RNAi appropriate conserved target partial sequences for RNAi in various influenza virus A strains isolated from human hosts and/or avian hosts, and RNAi-inducing entities targeting said appropriate conserved target portions, such as siRNA and shRNA. The invention provides various nucleic acids comprising sequences identical to or complementary to at least a portion of one or more of the appropriate conserved target partial sequences. The invention additionally provides methods and compositions for delivering an RNAi-inducing agent to an organ or tissue (eg, lung) of a mammalian subject. Also provided are methods for diagnosing influenza and for determining the susceptibility of influenza virus to inhibition by RNAi-inducing agents. Another aspect of the invention is directed to transgenic animals expressing RNAi-inducing agents targeting influenza genes.

Description

flu treatment

Cross References to Related Applications

This application claims priority to U.S. Provisional Patent Application 60/664,580, filed March 22, 2005, and U.S. Patent Application No. 11/102,097, entitled Influenza Therapeutic, filed April 8, 2005, which The case is hereby incorporated by reference.

governmental support

The United States Government provided financial support in the development of this invention. Specifically, the development of this invention was supported by National Institutes of Health grant numbers 5-R01-AI44477, 5-R01-AI44478, 5-ROI-CA60686, and 1-RO1-AI50631. The government has certain rights in this invention.

technical field

Background technique

Influenza is one of the most widespread infections worldwide. In the United States, between 20,000 and 40,000 people die each year from influenza A virus infection or its complications. An estimated 20 to 40 million people died during the 1918 influenza A virus pandemic. During a pandemic, the number of flu-related hospitalizations can exceed 300,000 in the winter alone.

The epidemiological success of influenza viruses has been attributed to several properties. First, influenza viruses are easily transmitted from person to person through aerosols (droplet infection). Second, influenza virus antigens undergo frequent small changes (antigenic drift) such that the virus can easily escape the protective immunity induced by previous exposure to different variants of the virus. Again, new strains of influenza virus can easily be generated by reassortment or mixing of genetic material between different strains (antigenic shift). In the case of influenza A virus, such mixing can occur between subtypes or strains invading different species. The 1918 pandemic is thought to have been caused by a hybrid strain of virus resulting from reassortment between swine and human influenza A viruses.

Despite continued efforts, there are still no satisfactory therapies for influenza virus infection, and existing vaccines are of limited value due in part to the aforementioned properties of antigenic shift and drift. To this end, influenza A virus has been under global surveillance for many years and the US National Institutes of Health has named it one of the highest priority pathogens for biodefense. Although current vaccines based on inactivated viruses are able to prevent disease in approximately 70-80% of healthy individuals under the age of 65, this percentage is much lower in the elderly or immunocompromised. Additionally, the costs and possible side effects associated with vaccine administration make this approach less than optimal. Although the 4 antiviral drugs currently approved in the United States for the treatment and/or prevention of influenza are helpful, their use is limited due to concerns about side effects, compliance, and the possible emergence of resistant strains. Therefore, there is still a need to develop effective therapies for the treatment and prevention of influenza virus infection.

Contents of the invention

The present invention provides novel compositions and methods for treating respiratory viral infections, such as influenza virus infections caused by influenza A, B and/or C viruses. The compositions and methods are based on RNA interference (RNAi). RNAi is a conserved cellular process in which the expression of a target RNA is inhibited in a sequence-specific manner by the presence of a double-stranded RNA that contains a portion that is complementary to the target RNA. Inhibition can be caused by cleaving the target or inhibiting its translation. In contrast to current influenza therapeutics, the RNAi-inducing entities of the invention inhibit the expression of influenza virus transcripts and thus prevent viral protein synthesis. This represents a fundamentally new approach to controlling influenza virus infection.

The present invention provides RNAi-inducing agents that target one or more target transcripts involved in viral production, replication, infection, and/or transcription, etc., of viral RNA, such as short interfering RNA (siRNA) and short hairpin RNA ( shRNA) molecules. In addition, the invention provides vectors whose presence in a cell produces transcription of one or more RNAs that hybridize to each other or to themselves to form siRNAs or shRNAs that inhibit viral production, viral Expression of at least one target transcript for infection, viral replication and/or transcription, etc. Preferably, the virus is a respiratory virus. Preferably the virus is an RNA virus. RNA viruses include positive-strand viruses and negative-strand RNA viruses such as influenza virus. Viral genomes can be segmented or non-segmented. According to certain embodiments of the invention, the target transcript encodes a protein selected from the group consisting of: a polymerase, a nucleocapsid protein, a neuraminidase, a hemagglutinin, a matrix protein, and a nonstructural protein. In particular embodiments, the target transcript encodes an influenza virus protein selected from the group consisting of: hemagglutinin, neuraminidase, membrane protein 1, membrane protein 2, nonstructural protein 1, nonstructural protein 2 , polymerase protein PB1, polymerase protein PB2, polymerase protein PA, nucleoprotein NP.

The invention also provides compositions comprising an RNAi-inducing agent and/or vector (eg, an RNAi-inducing agent and/or vector described herein), wherein the composition further comprises a delivery agent that facilitates delivery of the RNAi-inducing agent or vector. Preferred delivery agents include cationic polymers.

The present invention additionally provides methods of treating or preventing viral diseases, especially diseases caused by respiratory viruses (e.g., influenza) by, prior to exposure to the virus, while exposure occurs, or after exposure, in appropriate A composition of the invention comprising an RNAi-inducing entity is administered to a subject within a time window of , or at any point during which the subject exhibits symptoms of a disease caused by the virus. Compositions can be administered by various routes. Preferred routes include intravenous, or directly into the respiratory system by inhalation, intranasally, in aerosol form and the like.

The present invention provides nucleic acid sequences representing portions of influenza virus transcripts that are preferred targets for RNAi. Certain preferred targeting moieties are functionally preferred targets for RNAi. Certain preferred target moieties are suitably conserved among variants, so that an RNAi-inducing agent designed based on a particular variant will also inhibit variants whose corresponding target moieties differ in sequence. Certain preferred target portions are highly conserved among variants.

The invention provides nucleic acids comprising one or more preferred targeting moieties, complements thereof, and fragments of either. The invention additionally provides nucleic acids that are RNAi-inducing entities, such as RNAi-inducing agents and RNAi-inducing vectors that target one or more of the targeting moieties. In preferred embodiments, the RNAi-inducing entity targets the NP, PA, PB1 or PB2 gene. The present invention provides highly potent RNAi-inducing entities targeting certain preferred targeting moieties. Such highly potent RNAi-inducing entities may be particularly useful in the treatment or prevention of influenza virus infection.

Specifically, the present invention provides an RNAi-inducing agent targeting influenza virus transcripts, wherein the RNAi-inducing agent comprises: a nucleic acid moiety whose sequence comprises a sequence selected from the group consisting of SEQ ID NO: 272-380, its complement body, or any fragment having a length of at least 15 nucleotides. The present invention additionally provides an RNAi inducer targeting an influenza virus gene selected from the group consisting of a polymerase protein PB1 gene, a polymerase protein PB2 gene, a polymerase protein PA gene, and a nucleoprotein NP gene.

The present invention also provides an isolated nucleic acid or its complement, its sequence comprising a sequence selected from the group consisting of SEQ ID NO: 272-380, or comprising a sequence selected from the group consisting of SEQ ID NO: 272-380 has at least 15 nucleotides in length, wherein the nucleic acid has a length of 100 nucleotides or less. In certain embodiments, the length is at least 16 nucleotides.

The present invention provides methods of diagnosing viral infections and determining whether a subject suspected of having a viral infection is infected with a particular type of virus, strain, or the like. In certain embodiments, the methods comprise determining whether a subject is infected with an influenza virus susceptible to inhibition by one or more of the RNAi-inducing entities of the invention. In a preferred embodiment, a patient is diagnosed with an influenza virus infection, and an RNAi-inducing entity targeted to the particular influenza strain that infects the subject is administered. The present invention thus provides a combined method of influenza diagnosis and treatment. Certain diagnostic methods use one or more nucleic acids of the invention.

The invention also provides diagnostic kits for detecting influenza virus infection and/or determining whether influenza virus is susceptible to inhibition by an RNAi-inducing entity. Kits may comprise one or more nucleic acids of the invention and/or probes or primers for detecting preferred targeting moieties of influenza virus transcripts.

The present invention additionally provides methods of delivering an RNAi-inducing entity into the respiratory tract of a mammalian subject. The inventors have discovered that RNAi-inducing entities are effective in inhibiting gene expression in the lung when delivered directly into the airways of mammalian subjects. The present inventors have further discovered that RNAi-inducing agents, such as siRNA, can be delivered directly into the vasculature of mammalian subjects using conventional volumes and administration methods, and can effectively inhibit gene expression in the respiratory system, such as in the lungs. gene expression. For example, siRNA targeting influenza virus transcripts inhibited influenza production in the lung when delivered to mice by the intravenous or inhalation route, suggesting that therapeutically effective inhibition of gene expression in the respiratory system can be achieved by any means. In addition, siRNA targeting the luciferase transcript inhibited luciferase expression when administered by inhalation or intravenous routes to luciferase-expressing mice, suggesting that either approach can be used to inhibit essential expression of any gene. siRNAs targeting endogenous genes were also effective in inhibiting expression in the lung when delivered by inhalation. The present invention thus provides methods that allow the use of RNAi to treat a wide range of diseases that afflict the respiratory system, including infections caused by respiratory viruses. Intravascular delivery methods can also be used to deliver an effective amount of an RNAi-inducing agent to organs or tissues other than the lungs.

In one aspect, the invention provides a method of inhibiting expression of a transcript in an organ or tissue of a mammalian subject comprising introducing an RNAi-inducing entity, such as an RNAi-inducing agent, targeting the transcript directly into the respiratory system of the subject . In a preferred embodiment, the organ or tissue is part of the airway, such as the lungs. Accordingly, the present invention provides methods of inhibiting expression of a transcript in the respiratory system of a mammalian subject comprising introducing an RNAi-inducing entity, such as an RNAi-inducing agent, targeting the transcript directly into the respiratory system of the subject. In other embodiments, the RNAi-inducing entity is delivered directly into the respiratory system, into blood vessels and transported through the vasculature to active sites outside the respiratory system, that is, the respiratory route is for systemic delivery.

The present invention additionally provides a method of inhibiting expression of a transcript in the respiratory system of a mammalian subject comprising introducing an RNAi-inducing agent targeting the transcript directly into the vasculature of the mammalian subject.

In another aspect, the invention provides a method of inhibiting expression of a transcript in an organ or tissue of a mammalian subject comprising introducing an RNAi-inducing agent targeting the transcript directly into the vasculature of the mammalian subject. In another embodiment, the invention provides a method of inhibiting expression of a transcript in a parenchymal organ or tissue of a mammalian subject comprising introducing into the subject an RNAi-inducing entity, such as an RNAi-inducing agent, targeting the transcript in the respiratory system, where the RNAi-inducing agent enters the vasculature and is transported to other locations in the body. In certain embodiments of any of the above methods, the RNAi-inducing agent is administered in an aqueous medium substantially free of lipids or delivery-enhancing polymers. In other embodiments of the invention, the RNAi-inducing agent is administered in a composition comprising a cationic polymer.

The present invention provides compositions suitable for delivery to the respiratory system. Specifically, the present invention provides a breathable aerosol formulation containing liquid or solid particles (such as dry powder), said liquid or solid particles comprising one or more of the RNAi-inducing agents and/or RNAi-inducing carriers of the present invention. RNAi-inducing agent and/or RNAi-inducing carrier. Formulations may contain delivery agents and/or excipients. The present invention also provides a nasal spray comprising an RNAi-inducing agent or an RNAi-inducing carrier. The invention further provides devices for delivering compositions of the invention, for example, devices for delivering dry or liquid aerosol formulations into the respiratory system, such as inhalers or nebulizers. The device can deliver single or multiple doses of the composition. Compositions of the invention may be provided within the device and/or may be provided separately (eg, as a refill). The device can be disposable.

In another aspect, the invention provides a non-human transgenic animal expressing an RNAi-inducing agent targeting an influenza gene.

This application refers to various patents, journal articles, and other publications, all of which are hereby incorporated by reference. In addition, the following standard reference works are incorporated herein by reference: Ausubel, F., et al. (eds.) Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, John Wiley & Sons, N.Y., July 2002 edition; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; The Pharmacological Basis of Therapeutics by Goodman and Gilman, pp. Edition 10. McGraw Hill, 2001.

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 invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not limiting. Other features and advantages of the invention will be apparent from the following description and claims. Where elements are listed in Markush group format, it is understood that the respective subgroups of these elements are also disclosed, and any elements may be removed from the group. Where ranges are given, endpoints are inclusive unless otherwise stated or otherwise evident from context.

Description of drawings

Figure 1A (modified from Julkunen, I. et al., infra) presents a schematic diagram of influenza virus.

Figure IB (modified from Fields' Virology, infra) shows the genome organization of influenza virus and transcripts obtained from the influenza genome. Thin lines at the 5' and 3' ends of the mRNA indicate untranslated regions. Shaded or hatched regions indicate coding regions in 0 or +1 reading frames, respectively. Introns are described by V-shaped lines. The small rectangle at the 5' end of the mRNA represents the heterologous cellular RNA covalently linked to the viral nucleic acid. A _(n) stands for polyA tail.

Figure 2 (modified from Julkunen, I. et al., infra) shows the influenza virus replication cycle.

Figure 3 shows the structures of siRNAs observed in the Drosophila system.

Figure 4 schematically illustrates the steps involved in RNA interference in Drosophila.

Figure 5 shows various exemplary siRNA and shRNA structures effective according to the present invention.

Figure 6 illustrates an alternative repression pathway in which a DICER enzyme cleaves a substrate with a base mismatch in the stem to generate a repression product that binds to the 3'UTR of the target transcript and inhibits translation.

Figure 7 presents an example of a construct that can be used for the direct transcription of both strands of the siRNA of the invention.

Figure 8 depicts an example of a construct that can be used for the direct transcription of single RNA molecules that hybridize to form shRNAs according to the invention.

Figure 9 shows a sequence comparison among six strains of influenza virus A with human host origin. Dark shaded areas were used to design siRNAs tested as described in Example 2. The base sequence is that of strain A/Puerto Rico/8/34. Lightly shaded letters indicate nucleotides that differ from the base sequence.

Figure 10 shows a sequence comparison between 2 strains of influenza virus with human host origin and 5 strains of influenza virus A with animal host origin. Dark shaded areas were used to design siRNAs tested as described in Example 2. The base sequence is that of strain A/Puerto Rico/8/34. Lightly shaded letters indicate nucleotides that differ from the base sequence.

11A-11F show the results of experiments showing that siRNA inhibits influenza virus production in MDCK cells. Six different siRNAs targeting various viral transcripts were introduced into MDCK cells by electroporation, and cells were infected with viruses 8 hours later. Figure 11A is a graph showing the presence or absence of various Time course of virus titers in culture supernatants as measured with siRNA or control siRNA. Figure 11B is a graph showing, as analyzed by hemagglutinin, at various times after infection with influenza virus strain A/WSN/33 (H1N1) (WSN), at an MOI of 0.01, in the presence or absence of various siRNAs or control siRNA Measured, time course of virus titers in culture supernatants. Figure 11C shows plaque analysis showing virus titers in culture supernatants from virus-infected cells transfected with control or with siRNA NP-1496. Figure 1 ID shows the inhibition of influenza virus production at different doses of siRNA. MDCK cells were transfected with indicated amounts of NP-1496 siRNA, and then infected with PR8 virus at an MOI of 0.01. Virus titers were measured 48 hours after infection. Representative data from one of 2 experiments are shown. FIG. 11E shows inhibition of influenza virus production by administration of siRNA after virus infection. MDCK cells were infected with PR8 virus for 2 hours at an MOI of 0.01, and then transfected with NP-1496 (2.5 nmol). Virus titers were measured at indicated times post-infection. Representative data from one of 2 experiments are shown.

Figure 12 shows a sequence comparison between a portion of the 3' region of the NP sequences of 12 influenza A virus subtypes or isolates with human or animal host origin. Shaded areas were used to design siRNAs tested as described in Examples 2 and 3. The base sequence is that of strain A/Puerto Rico/8/34. Shaded letters indicate nucleotides that differ from the base sequence.

Figure 13 shows the position of various siRNAs relative to the influenza virus gene segments in relation to their effectiveness in inhibiting influenza virus.

Figure 14A is a schematic diagram of a developing chicken embryo showing areas for injection of siRNA and siRNA/delivery agent compositions.

Figure 14B shows the ability of various siRNAs to inhibit influenza virus production in developing chicken embryos.

Figure 15 is a schematic diagram showing the interaction of nucleoproteins with viral RNA molecules.

Figures 16A and 16B show schematic diagrams illustrating the differences between influenza virus vRNA, mRNA and cRNA (template RNA) and the relationship between them. The 12 conserved nucleotides at the 3' end and 13 nucleotides at the 5' end of each influenza A virus vRNA fragment are shown in Figure 16B. mRNAs contain m ⁷ GpppN ^m cap structures and an average of 10 to 13 nucleotides obtained from a subset of host cell RNAs. Polyadenylation of the mRNA occurs at a site in the mRNA corresponding to a position 15 to 22 nucleotides before the 5' end of the vRNA fragment. Arrows indicate the positions of primers specific for various RNA species. (modified from reference (1)).

Figure 17 shows the amount of viral NP and NS RNA species at various times after infection with virus in control transfected or siRNA NP-1496 transfected cells 6-8 hours before infection.

Figure 18A shows that inhibition of influenza virus production requires the wild-type (wt) antisense strand in the duplex siRNA. MDCK cells were first transfected with siRNA formed from wt and modified (m) strands, and 8 hours later infected with PR8 virus at an MOI of 0.1. Twenty-four hours after infection, the virus titers in the culture supernatants were analyzed. Representative data from one of 2 experiments are shown. Figure 18B shows that M-specific siRNAs inhibit the accumulation of specific mRNAs. MDCK cells were transfected with M-37, infected with PR8 virus at an MOI of 0.01, and 1, 2 and 3 hours after infection, harvested for RNA isolation. The levels of M-specific mRNA, cRNA and vRNA were measured by reverse transcription using RNA-specific primers followed by real-time PCR. The content of each viral RNA species was normalized to the content of γ-actin mRNA in the same samples (bottom panel). The relative content of RNA is expressed in the form of mean ± S.D. Representative data from one of 2 experiments are shown.

Figures 19A-D show that NP-specific siRNA inhibits the accumulation of not only NP-specific mRNA, vRNA and cRNA but also M and NS-specific mRNA, vRNA and cRNA. MDCK (A-C) and Vero (D) cells were transfected with NP-1496, infected with PR8 virus at an MOI of 0.1, and harvested for RNA isolation 1, 2 and 3 hours after infection. The levels of mRNA, cRNA and vRNA specific for NP, M and NS were measured by reverse transcription using RNA-specific primers followed by real-time PCR. The content of each viral RNA species was normalized to the content of γ-actin mRNA in the same samples (not shown). The relative amount of RNA is shown. Representative data from one of 3 experiments are shown.

Figures 19E-G, on the right side of each panel, show that PA-specific siRNA inhibits the accumulation of not only PA-specific mRNA, vRNA and cRNA but also M and NS-specific mRNA, vRNA and cRNA. MDCK cells were transfected with PA-1496, infected with PR8 virus at an MOI of 0.1, and harvested for RNA isolation 1, 2 and 3 hours after infection. The levels of mRNA, cRNA and vRNA specific for PA, M and NS were measured by reverse transcription using RNA-specific primers followed by real-time PCR. The content of each viral RNA species was normalized to the content of γ-actin mRNA in the same samples (not shown). The relative amount of RNA is shown.

Figure 19H shows that NP-specific siRNA inhibits the accumulation of PB1-specific mRNA (upper panel), PB2-specific mRNA (middle panel) and PA-specific mRNA (lower panel). MDCK cells were transfected with NP-1496, infected with PR8 virus at an MOI of 0.1, and harvested for RNA isolation 1, 2 and 3 hours after infection. The amount of mRNA specific for PB1, PB2, and PA mRNA was measured by reverse transcription using RNA-specific primers, followed by real-time PCR. The content of each viral RNA species was normalized to the content of γ-actin mRNA (not shown) in the same samples. The relative amount of RNA is shown.

Figure 20A shows the sequences of siRNA CD8-61 and its hairpin derivative CD8-61F.

Figure 20B shows inhibition of CD8a expression by CD8-61 and CD8-61F. CD8 ⁺ CD4 ⁺ T cell lines were electroporated and transfected with CD8-61 or CD8-61F. After 48 hours, CD8a expression was analyzed by flow cytometry. Unlabeled cell line, control transfection.

Figure 20C shows a schematic diagram of the pSLOOP III vector in which expression of CD8-61F hairpin RNA is driven by the H1 RNA pol III promoter. Terminator, a termination signal sequence.

Figure 20D presents a graph showing inhibition of CD8a in HeLa cells using pSLOOP III. Untransfected cells do not express CD8a. Cells were transfected with a CD8a expression vector and the promoter-less pSLOOP III-CD8-61F construct, synthetic siRNA, or pSLOOP III-CD8-61F with a promoter.

Figure 21A shows a schematic diagram of NP-1496 and GFP-949 siRNA and their hairpin derivatives/precursors.

Figure 21B shows tandem arrays of NP-1496H and GFP-949H in 2 different orders.

Figure 21C shows the pSLOOP III expression vector. Hairpin precursors to siRNAs were cloned in the pSLOOP III vector alone (upper panel), in tandem arrays (middle panel), or with independent promoter and termination sequences (lower panel).

Figure 22A is a graph showing that siRNA inhibits influenza virus production in mice when administered together with the cationic polymer PEI prior to infection with influenza virus. Solid squares (no treatment); open squares (GFP siRNA); open circles (30 μg NP siRNA); closed circles (60 μg NP siRNA). Individual symbols represent individual animals, p-values between different groups are shown.

Figure 22B is a graph showing that siRNA inhibits influenza virus production in mice when administered with cationic polymer PLL prior to infection with influenza virus. Filled squares (no treatment); open squares (GFP siRNA); filled circles (60 μg NP siRNA). Individual symbols represent individual animals, p-values between different groups are shown.

Figure 22C is a graph showing that siRNA was significantly more effective in inhibiting influenza virus production in mice when administered with the cationic polymer jetPEI prior to infection with influenza virus than when administered in PBS. Open squares (no treatment); open triangles (GFP siRNA in PBS); closed triangles (NP siRNA in PBS); open circles (GFP siRNA with jetPEI); closed circles (NP siRNA with jetPEI). Individual symbols represent individual animals, p-values between different groups are shown.

Figure 22D is a graph showing that siRNA targeting NP inhibits influenza virus production in mice when administered via the intravenous route along with cationic poly(beta amino ester) (J28). Open circles (no treatment); filled squares (NP siRNA and J28).

Figure 22E is a graph showing that siRNA targeting NP inhibits influenza virus production in mice when administered together with cationic poly(β amino ester) (J28 or C32) via the intraperitoneal route, while control RNA (GFP) has no significant effect picture. Open circles (no treatment); open squares (GFP siRNA and J28); filled squares (NP siRNA and J28); open triangles (GFP siRNA and C32); filled triangles (NP siRNA and C32). Shows p-values between control and treatment groups.

Figure 23 is a graph showing that siRNAs targeting influenza virus NP and PA transcripts exhibit additive effects when administered together prior to infection with influenza virus. Filled squares (no treatment); open circles (60 μg NP siRNA); open triangles (60 μg PA siRNA); filled circles (60 μg NP siRNA+60 μg PA siRNA). Individual symbols represent individual animals, p-values between different groups are shown.

Figure 24 is a graph showing that siRNA inhibits influenza virus production in mice when administered after infection with influenza virus. Open squares (no treatment); open squares (60 μg GFP siRNA); open triangles (60 μg PA siRNA); open circles (60 μg NP siRNA); closed circles (60 μg NP+60 μg PA siRNA). Individual symbols represent individual animals, p-values between different groups are shown.

Figure 25A is a schematic diagram of a lentiviral vector expressing shRNA. Transcription of the shRNA is driven by the U6 promoter. EGFP expression is driven by the CMV promoter. SIN-LTR, Ψ, cPPT and WRE are lentiviral components. The sequence of NP-1496 shRNA is shown.

Figure 25B presents a graph of flow cytometry results demonstrating that Vero cells infected with the lentivirus described in Figure 25B expressed EGFP in a dose-dependent manner. Lentivirus was generated by co-transfecting a DNA vector encoding NP-1496a shRNA and a packaging vector into 293T cells. Culture supernatant (0.25 ml or 1.0 ml) was used to infect Vero cells. The resulting Vero cell lines (Vero-NP-0.25 and Vero-NP-1.0) and control (uninfected) Vero cells were analyzed for GFP expression by flow cytometry. Mean fluorescence intensities of Vero-NP-0.25 (upper panel) and Vero-NP-1.0 (lower panel) cells are shown. The shaded curve represents the mean fluorescence intensity of control (uninfected) Vero cells.

Figure 25C is a graph showing inhibition of influenza virus production in Vero cells expressing NP-1496 shRNA. Parental and Vero cells expressing NP-1496 shRNA were infected with PR8 virus at MOIs of 0.04, 0.2, and 1. Forty-eight hours after infection, virus titers in supernatants were determined by hemagglutination (HA) assay.

Fig. 26 is a graph showing inhibition of influenza virus production in mice by administration of a DNA vector expressing siRNA targeting influenza virus transcripts. 60 μg of DNA encoding RSV, NP-1496 (NP) or PB1-2257 (PB1 ) shRNA was mixed with 40 μl Infasurf and administered to mice by instillation. For the no treatment (NT) group, mice were instilled with 60 μl of 5% glucose. Thirteen hours later, mice were infected intranasally with PR8 virus at 12000 pfu per mouse. Twenty-four hours after infection, virus titers in the lungs were measured by MDCK/hemagglutinin assay. Each data point represents one mouse and p-values between groups are shown.

Figure 27A shows the results of an electrophoretic mobility shift assay detecting complex formation between siRNA and poly-L-lysine (PLL). siRNA-polymer complexes were formed by mixing 150 ng of NP-1496 siRNA with increasing amounts of polymer (0-1200 ng) for 30 min at room temperature. The reaction mixture was then run on a 4% agarose gel and visualized for siRNA using ethidium-bromide staining.

Figure 27B shows the results of an electrophoretic mobility shift assay detecting complex formation between siRNA and poly-L-arginine (PLA). SiRNA-polymer complexes were formed by mixing 150 ng of NP-1496 siRNA with increasing amounts of polymer (0-1200 ng) for 30 min at room temperature. The reaction mixture was then run on a 4% agarose gel and visualized for siRNA using ethidium bromide staining.

Figure 28A is a graph showing the cytotoxicity of siRNA/PLL complexes. Vero cells in 96-well plates were treated with siRNA (400 pmol)/polymer complexes for 6 hours. The polymer-containing medium was then replaced with DMEM-10% FCS. After 24 h, the metabolic activity of the cells was measured by using the MTT assay. Square = PLL (MW ~ 8K); Circle = PLL (MW ~ 42K); Solid square = 25%; Open triangle = 50%; Solid triangle = 75%; X = 95%. Data are presented as the mean of three triplicates.

Figure 28B is a graph showing the cytotoxicity of siRNA/PLA complexes. Vero cells in 96-well plates were treated with siRNA (400 pmol)/polymer complexes for 6 hours. The polymer-containing medium was then replaced with DMEM-10% FCS. After 24 h, the metabolic activity of the cells was measured by using the MTT assay. Data are presented as the mean of three triplicates.

Figure 29A is a graph showing that PLL stimulates cellular uptake of siRNA. Vero cells in 24-well plates were incubated with Lipofectamine+siRNA (400 pmol) or with siRNA (400 pmol)/polymer complex for 6 hours. Cells were then washed and infected with PR8 virus at an MOI of 0.04. Virus titers in culture supernatants at different time points post infection were measured by HA assay. The ratio of polymer to siRNA is shown. Open circles = no treatment; filled squares = Lipofectamine; filled triangles = PLL (MW ~ 42K); open triangles = PLL (MW ~ 8K).

Figure 29B is a graph showing that poly-L-arginine stimulates cellular uptake of siRNA. Vero cells in 24-well plates were incubated with siRNA (400 pmol)/polymer complexes for 6 hours. Cells were then washed and infected with PR8 virus at an MOI of 0.04. Virus titers in culture supernatants at different time points post infection were measured by HA assay. The ratio of polymer to siRNA is shown. 0, 25, 50, 75 and 95% refer to the percentage of ε-amino groups on PLL substituted with imidazole acetyl groups. Filled circles = no transfection; Open circles = Lipofectamine; Open and filled squares = 0% and 25% (note that the data points for 0% and 25% are the same); Filled triangles = 50%; Open triangles = 75% ; X = 95%.

Figure 30A is a histogram showing that PEI mediates DNA transfection in the lung following intravenous injection. Luc activity is shown relative to light units in 0.5 mg protein in different organs over a 4-day period. Data are from one of 2 experiments.

Figure 30B is a histogram showing that PEI mediates DNA transfection in the lung following intratracheal administration. This graph shows the mean Luc activity relative to light units in 0.5 mg protein in the indicated organs of 3 mice per group 24 hours after DNA administration. Error histograms indicate standard deviation.

Figure 30C is a histogram showing inhibition of luciferase activity in the lung by intravenous delivery of siRNA. This graph shows the mean Luc activity relative to light units in 0.5 mg protein in the indicated organs of 3 mice per group 24 hours after administration of the luciferase construct. Error histograms indicate standard deviation.

Figure 30D is a histogram showing that delivery of siRNA by inhalation inhibits luciferase activity in the lung. This graph shows mean Luc activity relative to light units in 0.5 mg of protein in mouse lungs 24 hours after administration of luciferase constructs. Error histograms indicate standard deviation. Experiments were performed in duplicate using 3 mice per group and similar results were obtained. The results of one of the experiments are presented here.

Figures 31A and 31B are graphs showing that administration of siRNA in the absence of delivery agent inhibits influenza virus production in mice.

Figures 32A-32J show the sequence of the influenza strain PR8 transcript used to select the target portion of RNAi.

Figure 33 is Table 17 showing the sequences of functional target moieties that inhibit RNAi of influenza virus.

Figure 34 is Table 18 showing the sequences of suitable conserved influenza target moieties for RNAi among influenza strains from humans.

Figure 35 is Table 20 showing the sequences of suitable conserved influenza target moieties for RNAi between influenza strains from humans and avians.

Detailed ways

abbreviation

DNA: deoxyribonucleic acid

RNA: ribonucleic acid

vRNA: virion RNA in the influenza virus genome, negative strand

cRNA: complementary RNA, direct transcript of vRNA, positive strand

mRNA: Messenger RNA transcribed from a vRNA or cellular gene, positive strand, used as a template for protein synthesis

dsRNA: Double-stranded RNA

siRNA: short interfering RNA

shRNA: short hairpin RNA

miRNA: microRNA

RNAi: RNA interference

bp: base pair

nt: nucleotide

definition

As used herein, the term "about" or "approximately" with respect to a number generally includes the term "about" or "approximately" referring to a number in either direction unless otherwise stated or it is obvious from the context to the contrary (except that the stated number will exceed 100% of the possible value). Numbers in the range of 5% (greater or smaller than said number).

The term "avian" as used herein is used to denote any species, subspecies or racial organism of the classification Aves (ava), for example, chickens, turkeys, ducks, geese, quails, pheasants, parrots, finches, Eagle and crow. The term includes Gallus gallus, or chickens (eg, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire Chicken (New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partidge-colored), and Strains from turkeys, pheasants, quails, ducks, ostriches and other commonly reared poultry.

The term "complementarity" is used herein according to its art-recognized meaning, referring to the ability of precise pairing between specific bases, nucleosides, nucleotides or nucleic acids. For example, adenine (A) and uridine (U) are complementary; adenine (A) and thymidine (T) are complementary; and, guanine (G) and cytosine (C) are complementary , and is known in the art as Watson-Crick base pairing. A nucleotide at a position in a first nucleic acid sequence forms a complementary base pair if the nucleotides at a position in a first nucleic acid sequence are complementary to a nucleotide in an opposite orientation in a second nucleic acid sequence when the strands are aligned in an antiparallel orientation , and said nucleic acid is complementary at said position. The percent complementarity of a first nucleic acid to a second nucleic acid can be estimated by aligning the two nucleic acids in an anti-parallel orientation to obtain maximum complementarity over an evaluation window along the second nucleic acid, determining that complementarity is formed within the window The total number of nts in both strands of base pairs, divided by the total number of nts within the window and multiplied by 100. For example, AAAAAAAA and TTTGTTAT are 75% complementary because, out of a total of 16 nts, 12 nts are in complementary base pairs. Nucleic acids that are at least 70% complementary within the assessment window are considered substantially complementary within that window. When calculating the number of complementary nts required to achieve a specific percent complementarity, fractions were rounded to the nearest whole number. A position occupied by a non-complementary nucleotide constitutes a mismatch, ie, the position is occupied by a non-complementary base pair. To achieve maximum complementarity, gaps can be introduced into either or both of the nucleic acids within the evaluation window. The nucleotides opposite the gap are unpaired and constitute a bulge, that is, the opposite nucleotide is not present in another nucleic acid. Typically, percent complementarity is determined over an evaluation window of length at least 15 nt, such as 19 nt, wherein said length does not include gaps. For the determination of percent complementarity, a 1 nt overhang is considered a single non-complementary nt; a bulge between 2 and 5 nts is considered 2 non-complementary nts; a bulge between 6 and 10 nts is considered 3 A non-complementary nt. A bulge of length K nts (where K is greater than 10) is considered as 3+(K-10) non-complementary nts.

"Directly into the respiratory system" means administration through the nose, mouth or trachea, preferably through the nose or mouth so that a substantial portion of the active agent in the composition (eg, greater than 10%, preferably greater than 25%) enters the upper and/or lower respiratory tract.

"Directly into the vasculature" refers to administration into a blood vessel (eg, an artery or vein) by injection or catheter or any other method of entering the vasculature from outside the body (usually involving penetration of the vessel wall). "Indirectly into the vasculature" refers to a mode of administration that does not penetrate the vasculature. A preferred example of indirect delivery of a substance into the vascular system is direct delivery of the substance into the respiratory system followed by passage of the substance through the vessel wall. The substance can then be transported to a target tissue or organ elsewhere in the body (and possibly back to the lungs).

An "effective amount" of an active agent is an amount of the active agent sufficient to elicit the desired biological response. The absolute amount of a particular agent to be effective may vary with such factors as the desired biological endpoint, the agent to be delivered, the target tissue, and the like, as will be appreciated by those of skill in the art. An "effective amount" can be administered in a single dose or in multiple doses. For example, an effective amount of an RNAi-inducing entity may be an amount sufficient to achieve one or more of the following: (i) reduce expression of the target transcript by at least 20%, preferably at least 40%; (ii) ) reduce the viral titer by at least 25%; (iii) reduce the viral titer by at least 2-fold; (iv) delay or prevent the development of a clinically significant viral infection; (v) reduce the duration of at least one symptom of the viral infection or severity etc.

A composition is "substantially free" of a substance if it contains less than 1% by weight of that substance, preferably less than 0.5%, more preferably less than 0.1%. More preferably, said substance is completely absent from said composition. A composition is considered to be substantially free of delivery-enhancing polymers or lipids if such polymers or lipids are not intentionally included in the composition.

As used herein, the term "hybridization" refers to the interaction between two nucleic acid sequences comprising or consisting of complementary parts, so as to form Cellular medium stable duplex structure. Generally, if the Tm of the duplex formed by the first nucleic acid and the second nucleic acid is larger than that of the second nucleic acid and is the same length as the second nucleic acid and is 100% complementary to the second nucleic acid and contains the same type of nucleoside and internucleoside linkages The first nucleic acid is considered to hybridize to the second nucleic acid if the Tm of the duplex formed by the linked third nucleic acid is less than 15°C, preferably less than 10°C. Hybridization conditions suitable for various applications are known in the art and/or can be found in standard reference works, eg Ausubel, supra, and Sambrook, supra. In an exemplary embodiment, stringent hybridization conditions comprise 6X sodium chloride/sodium citrate (SSC) and 0.1% SDS at a temperature 10-15°C lower than the Tm of the fully complementary duplex, followed by The Tm of the duplex was lower than 25°C and washed 1-2 times for 30 minutes in 2X SSC and 0.1% SDS.

"Homology" refers to the degree of identity of two or more nucleic acid sequences. Within the evaluation window, the percent homology between a first nucleic acid and a second nucleic acid can be calculated by aligning the two nucleic acids in a parallel orientation, and determining the ratio of nucleotides opposite to the same nucleotide within the evaluation window. Quantity, divided by the total number of nucleotides in the window and multiplied by 100. When calculating the number of identical nucleotides required to achieve a particular percent homology, fractions are rounded to the nearest whole number. Nucleic acids that are at least 70% homologous (eg, at least 80%, at least 90%, or more than 90%) within the evaluation window are considered substantially homologous within the window. Typically, the evaluation window is at least 15 nt, such as 19 nt, along the length of the second nucleic acid, where the length does not include gaps. For the determination of percent homology, a 1 nt gap is considered a single nt not opposite the same nt; a gap between 2 and 5 nts is considered 2 nts not opposite the same nt; a gap between 6 and 10 nts is considered The gap between is considered as 3 nts not opposite to the same nt. A gap of length K nts (where K is greater than 10) is considered as 3+(K-10) nts not opposite the same nt.

As used herein, the term "influenza virus" refers to any strain of influenza virus that is capable of causing disease in animal or human subjects, or that is a candidate of interest for experimental analysis. Fields, B. et al. describe influenza viruses in Fields' Virology, 4th Edition, Philadelphia: Lippincott Williams and Wilkins; ISBN: 0781718325, 2001 . In particular, the term encompasses any strain of influenza A virus that is capable of causing disease in an animal or human subject, or that is a candidate of interest for experimental analysis. A large number of influenza A isolates have been partially or fully sequenced. Appendix A presents only those that have been deposited in a public database (Influenza Sequence Database (ISD), see Macken, C, Lu, H., Goodman, J., & Boykin, L., "The value of a database in surveillance and Partial list of complete sequences of influenza A genome fragments in vaccine selection. "in Options for the Control of Influenza IV. ADMEOsterhaus, N. Cox & AW Hampson (eds.) Amsterdam: Elsevier Science, 2001, 103-106). The database also contains the complete sequences of influenza B and C genome segments. This database is available at the World Wide Web site URL http://www.flu.lanl.gov/ along with a convenient search engine that allows users to search by genome fragment, by species infected by the virus and by year of isolation. Influenza sequences are also available on Genbank. Accordingly, the sequence of the influenza gene can be readily obtained by, or can be determined by, one of skill in the art.

As used herein with respect to the synthesis, processing or activity of an RNAi-inducing agent, the term "in vivo" generally refers to events that occur within a cell as opposed to a cell-free system. Typically, cells can be maintained in tissue culture or can be part of a whole organism.

"Isolated" as used herein means 1) separated from at least some components normally associated with it in nature; 2) prepared or purified by methods involving man-made; and/or 3) not found in nature. Any of the nucleic acids and nucleic acid structures described herein may be in isolated form.

"Ligand" as used herein means a molecule that specifically binds a second molecule by a mechanism other than antigen-antibody interaction. For example, the term encompasses naturally occurring or synthetic polypeptides, peptides and small molecules, including molecules whose structures have been created by man.

"Nucleic acid-based assay" refers to any assay or method that detects the presence of and/or identifies a nucleic acid. This analysis can be qualitative or quantitative.

"Nucleobase" as used herein means one capable of forming hydrogen bonds, preferably Watson-Crick hydrogen bonds, pairing with complementary nucleic acid bases or nucleobase analogs, such as pairing with purines or pyrimidines nitrogen-containing heterocyclyl moiety. Typical nucleobases are the naturally occurring nucleobases adenine, guanine, cytosine, uracil, thymine and analogs of naturally occurring nucleobases (Fasman, Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394 , CRC Press, Boca Raton, Fla., 1989). The terms "nucleic acid base" and "base" are used interchangeably herein.

"Nucleotides" include nitrogenous bases, sugar molecules, and phosphate groups. Nucleosides comprise a nitrogenous base (nucleic acid base) attached to a sugar molecule. In naturally occurring nucleic acids, phosphate groups covalently link adjacent nucleosides to form polymers. Nucleic acids can include naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6 )-methylguanine and 2-thiocytidine), chemically modified bases, biologically modified bases (eg, methylated bases), inserted bases, modified sugars ( For example, 2'-fluororibose, ribose, 2-deoxyribose, arabinose and hexose). Nucleosides may contain universal bases, that is, bases that may replace one of the naturally occurring bases commonly found in nucleic acids, or preferably, any base (when the base is used in a duplex relative time) (see, eg, 142-149). In certain embodiments of the invention, nucleic acids comprise no basic residues.

"Operably linked" as used herein refers to a relationship between two nucleic acid sequences wherein the expression of one of the nucleic acid sequences is controlled, regulated, adjusted, etc. by the other. For example, transcription of a nucleic acid sequence is directed by an operably linked promoter sequence; post-transcriptional processing of a nucleic acid is directed by an operably linked processing sequence; translation of a nucleic acid sequence is directed by an operably linked translation control sequence; The trafficking or localization sequence directs the trafficking or localization of the nucleic acid or polypeptide; and, the post-translational processing of the polypeptide is directed by an operably linked processing sequence. Preferably, the nucleic acid sequence operably linked to the second nucleic acid sequence is directly or indirectly covalently linked to said sequence, but any effective three-dimensional association is also acceptable.

The term "organ" as used in the art refers to a tissue or group of tissues that constitute a morphologically and functionally distinct part of an organism. Examples include lung, heart, liver, pancreas, breast, kidney, intestine, bladder, bone, skin, and the like. The term "tissue" as used in the art refers to a group of cells generally having a similar structure, often organized to perform one or more same or related functions. Red blood cells, white blood cells, and platelets are considered circulating tissues that contain individual cells or cell fragments.

"Preventing" means preventing a disease, disorder, condition, or aggravation of symptoms or manifestations thereof, or the severity thereof, from occurring. Prevention includes reducing the risk that aggravation of a disease, disorder, condition, or symptoms or manifestations thereof, or its severity, will occur. Thus, a composition or method is said to prevent said composition or method if it reduces the risk, on an individual or population basis, that a disease, disorder, condition, or symptom or manifestation thereof, or worsening of its severity, will occur. Aggravation of a disease, disorder, condition, or symptoms or manifestations thereof, or the severity thereof.

The term "primer" as used herein refers to a primer, whether natural or synthetic, capable of serving as an initiator of nucleic acid synthesis when hybridized to a nucleic acid template under conditions that initiate primer extension (e.g., polymerase-catalyzed primer extension). The starting oligonucleotide. The appropriate length of the primer will depend on the intended use of the primer, but is usually in the range of 15 to 35 nt. In some cases, primers can be longer, for example up to about 60 nt in length. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Primers need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize to the template used for primer extension.

The term "probe" as used herein, when referring to a nucleic acid, refers to a nucleic acid that can hybridize to a complementary nucleic acid and thereby detect the presence of the complementary nucleic acid. The probe should be sufficiently complementary to the nucleic acid being detected such that it will hybridize specifically under the stringency of the hybridization conditions employed. Probes can be modified with labels such as fluorescent moieties, biotin, and the like.

"Purified" as used herein means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure, wherein it is said to be pure when removed from substantially all other compounds or entities, that is, preferably at least about 90%, more preferably Is at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.

The term "regulatory sequence" is used herein to describe a region of a nucleic acid sequence that directs, enhances or inhibits the expression (especially transcription, but in some cases other events such as splicing or other processing) of an operably linked sequence. The term includes expression signals such as promoters, enhancers and other transcriptional control elements. In some embodiments of the invention, regulatory sequences direct constitutive expression of a nucleotide sequence; in other embodiments, regulatory sequences direct tissue-specific and/or inducible expression. Regulatory sequences can direct the expression of a nucleotide sequence only in cells that have been infected with an infectious agent. For example, regulatory sequences may comprise promoters and/or enhancers, such as virus-specific promoters or enhancers recognized by viral proteins, eg, viral polymerases, transcription factors, and the like. Alternatively, the regulatory sequences may comprise promoters and/or enhancers that are active in epithelial cells, such as respiratory epithelial cells. For example, the promoter of a gene encoding a surfactant protein can be used.

"Respiratory system" refers to any upper respiratory tract component (eg, nares, nasopharynx, oropharynx) or lower respiratory tract component (eg, trachea, bronchi, bronchioles, and/or alveoli). The larynx can be considered a component of the upper or lower airway. The terms "respiratory system" and "airway" are used interchangeably herein.

A "respiratory virus" is a virus that infects cells present in the upper and/or lower respiratory tract of a subject. Preferably, the virus infects respiratory epithelial cells. Other cells that may be infected include, but are not limited to, bronchoalveolar macrophages, dendritic cells, and the like. Examples of respiratory viruses include: influenza virus, parainfluenza virus (PIV), pneumovirus, metapneumovirus, coronavirus, adenovirus, rhinovirus, respiratory syncytial virus (RSV), reovirus, herpesvirus, and hantavirus Virus (hantavirus).

The term "RNAi-inducing agent" refers to siRNA, shRNA, and other double-stranded structures (eg, dsRNA) of other small RNA species that can be processed to produce siRNA or shRNA or to inhibit the expression of a target transcript by RNA interference. In certain embodiments of the invention, the RNAi-inducing agent inhibits the expression of a target RNA through an RNA interference pathway involving translational repression.

The term "RNAi-inducing entity" encompasses RNA molecules and vectors, the presence of which in a cell produces RNAi and causes a decrease in the expression of a transcript targeted by the RNAi-inducing entity. The RNAi-inducing entity can be, for example, an RNAi-inducing agent such as siRNA, shRNA, or an RNAi-inducing vector. The use of the terms "RNAi-inducing entity", "RNAi-inducing agent" or "RNAi-inducing vector" is not intended to imply that the entity, agent or vector generally upregulates or activates RNAi (although it may), but simply indicates that the entity The presence of the , agent or vector in the cell produces an RNAi-mediated decrease in expression of the target transcript. An "RNAi-inducing entity" as used herein is an entity that is artificially modified or produced and/or whose presence in a cell is the result of human intervention, e.g., its interaction with endogenous RNA species or during viral infection During natural processes, different RNA species are produced in cells.

An "RNAi-inducing vector" is a vector whose presence within a cell results in the transcription of one or more RNAs that hybridize to each other or to themselves to form an RNAi-inducing agent such as siRNA or shRNA. In various embodiments of the invention, the term encompasses a plasmid or virus, the presence of which within a cell results in the production of one or more RNAs that hybridize to themselves or to each other to form an RNAi-inducing agent. Typically, a vector comprises a nucleic acid operably linked to an expression signal for transcription of one or more RNA molecules that hybridize or self-hybridize to form the RNAi-inducing agent when the vector is present in the cell. Thus, the vector provides a template for the intracellular synthesis of the RNAi-inducing agent. To induce RNAi, the presence of the viral genome within the cell constitutes the presence of the virus within the cell. A vector is considered to be present in a cell if it is introduced into the cell, enters the cell, or is inherited from a parent cell, regardless of whether it is subsequently modified or processed within the cell. An RNAi-inducing vector is said to target a transcript if the vector comprises a template for the transcription of an RNAi-inducing agent that targets the transcript. In addition to transcript repression in cells, the vectors have many other uses. For example, they can be used to produce RNAi-inducing agents in vitro, and/or in cells that may or may not contain the transcripts targeted by the vector.

"Short interfering RNA" includes double-stranded (duplex) RNAs of length between 15 and about 29 nucleotides or any other subrange or specific value in the interval between 15 and 29, e.g., 16- 18, 17-19, 21-23, 24-27, 27-29 nt long and optionally additionally contain one or two single-stranded overhangs, such as 3' overhangs on one or both strands. In certain embodiments, the duplex is about 19 nt long. The overhang can be, for example, 1-6 residues in length, eg, 2 nt. siRNA can be formed from 2 RNA molecules that hybridize together, or, alternatively, can be produced from shRNA. In certain embodiments of the invention, one or both of the 5' ends of the siRNA have a phosphate group, while in other embodiments, one or more of the 5' ends have no phosphate group. In some embodiments of the invention, one or both of the 3' termini have hydroxyl groups, while in other embodiments, none have hydroxyl groups. One strand of the siRNA, referred to as the "antisense strand" or "guide strand," includes a portion that hybridizes to the target transcript. In certain preferred embodiments of the invention, the antisense strand of the siRNA is 100% complementary to a region of the target transcript, that is, it hybridizes to the target transcript between 15 and about 29 nt in length. , preferably at least 16 nt in length, more preferably 18-20 in length, eg 19 nt in the target region without a single mismatch or bulge. The region of complementarity may be any sub-range or specific value within the interval between 17 and 29, eg, 17-18, 19-21, 21-23, 19-23, 24-27, 27-29. In other embodiments, the antisense strand is substantially complementary to the target region, that is, there is one or more mismatches and/or bulges in the duplex formed by the antisense strand and the target transcript. out. The two strands of the siRNA are substantially complementary, preferably 100% complementary to each other in the duplex portion.

The term "short hairpin RNA" refers to an RNA molecule comprising at least 2 double strands (double strands) that have hybridized or are capable of hybridizing to form long enough to mediate RNAi (as described for siRNA duplexes). complementary portion of the duplex) structure, and at least one single-stranded portion forming a loop connecting the regions of the duplex-forming shRNA. This structure is also known as a stem/loop structure, where the stem is part of the duplex. The construct may additionally comprise an overhang on the 5' or 3' end (eg, as described for siRNA). Preferably, the loop is about 1-20, more preferably about 4-10 and most preferably about 6-9 nt long and/or the overhang is about 1-20, and more preferably about 2-15 nt long. The loop can be located 5' or 3' to the region complementary to the target transcript desired to be suppressed (ie, the antisense portion of the shRNA). In certain embodiments, the overhang comprises one or more U residues, eg, between 1 and 5 Us. As described further below, shRNA is processed into siRNA using the conserved cellular RNAi machinery. Thus, shRNAs are precursors to siRNAs and are often also capable of inhibiting the expression of target transcripts that are complementary to the portion of the shRNA known as the antisense or guide strand of the shRNA. In general, the characteristics of the duplex formed between the antisense strand of the shRNA and the target transcript are similar to those of the duplex formed between the guide strand of the siRNA and the target transcript. In certain embodiments of the invention, the 5' end of the shRNA has a phosphate group, while in other embodiments it does not. In certain embodiments of the invention, the 3' end of the shRNA has a hydroxyl group, while in other embodiments it does not.

As used herein, the term "subject" refers to an individual susceptible to a viral infection, eg, influenza virus. The term includes birds and animals, such as domestic birds and animals (such as chickens, mammals including pigs, horses, dogs, cats, etc.) and wild animals, non-human primates, and humans.

For purposes described herein, if (1) the RNAi-inducing agent comprises a target transcript that is at least 80%, preferably at least about 85%, more preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary strands to achieve a length of at least about 15, more preferably at least about 17, still more preferably at least about 18 or 19 to about 21-23, or 24- A stretch of 29 nucleotides; and/or (2) one strand of the RNAi-inducing agent hybridizes to the target transcript, then the RNAi-inducing agent is said to "target" the target transcript. Suitable hybridization conditions are those typically found in the cytoplasm or nucleus of mammalian cells and/or in Drosophila lysates, eg, as described in US Publications 20020086356 and 20040229266 and refs 21 and 28. In certain embodiments of the invention, the GU or UG base pairs in the duplex formed by the antisense strand and the target transcript are not considered to be significant for purposes of determining whether the RNAi-inducing agent targets the transcript. mismatch. An RNA-inducing vector whose presence in the cell results in the production of an RNAi-inducing agent targeting the transcript is also considered to target the transcript. Transcript-targeting RNAi inducers are also thought to target genes that direct transcript synthesis. RNAi inducers that inhibit the expression of target transcripts involved in virus production, viral replication, viral pathogenicity, and/or viral infection are said to inhibit the virus.

A "target portion" is a region of a target transcript that hybridizes to the antisense strand of an RNAi-inducing agent.

The term "target transcript" refers to any RNA that is the target of RNAi. Messenger RNA is a preferred target. The terms "target RNA" and "target transcript" are used interchangeably herein.

As used herein, "treating" includes reversing, slowing down and/or inhibiting the progression of the disease, disorder or condition to which the term applies, and/or reversing, slowing down and/or inhibiting one of the diseases, disorders or conditions one or more symptoms or manifestations.

The term "vector" refers to a nucleic acid molecule capable of mediating entry (eg, transfer, transport, etc.) of a second nucleic acid molecule into a cell. The transferred nucleic acid is typically ligated (eg, inserted) into a carrier nucleic acid molecule. A vector may include sequences that direct spontaneous replication, or may include sequences sufficient to permit incorporation into host cell DNA. Useful vectors include, for example, plasmids (usually DNA molecules, although RNA plasmids are also known), cosmids and viral vectors. As is well known in the art, the term viral vector may refer to a nucleic acid molecule (such as a plasmid) that includes virus-derived nucleic acid elements that typically facilitate the transfer or incorporation of the nucleic acid molecule (examples include retroviral or lentiviral vectors), or to a vector that mediates Nucleic acid transferred viruses or viral particles (examples include retroviruses or lentiviruses). As will be apparent to those of skill in the art, viral vectors can include various viral components in addition to nucleic acid.

I. Influenza virus life cycle and characteristics

Influenza viruses are enveloped, negative-strand RNA viruses of the Orthomyxoviridae family. It is classified as influenza A, B and C, with influenza A being the most pathogenic and considered the only type capable of undergoing reassortment with animal strains. Influenza types A, B, and C can be distinguished by differences in their nucleoproteins and matrix proteins (see Figure 1). As discussed further below, influenza A subtypes are defined by changes in their hemagglutinin (HA) and neuraminidase (NA) genes and are often distinguished by antibodies that bind the corresponding proteins.

The influenza A virus genome is composed of 10 genes distributed in 8 RNA segments. The genes encode 10 proteins: envelope glycoproteins cytohemagglutinin (HA) and neuraminidase (NA); matrix protein (M1); nucleoprotein (NP); 3 polymerases (PB1, PB2 and PA) , are components of RNA-dependent transcriptase, also referred to herein as polymerase or polymerase complex; ion channel protein (M2) and nonstructural proteins (NS1 and NS2). For additional details on influenza A virus and its molecular causative agents, see Julkunen, I., et al., Cytokine and Growth Factor Reviews, 12:171-180, 2001. See also Fields, B., et al., Fields' Virology, 4th ed., Philadelphia: Lippincott Williams and Wilkins; ISBN: 0781718325, 2001. The organization of the influenza B virus genome closely resembles that of influenza A, while the influenza C virus genome contains 7 RNA segments and no NA.

Classification of influenza A viruses is based on the hemagglutinin (H1-H15) and neuraminidase (N1-N9) genes. The World Health Organization (WHO) nomenclature defines various virus strains by their animal host origin (specified to exclude humans), geographic origin, number of strains, year of isolation, and antigenic description of HA and NA. For example, A/Puerto Rico/8/34 (H1N1), known as strain A, isolate 8, arose in humans in Puerto Rico in 1934 and has antigenic subtype 1 of HA and NA. As another example, A/chicken/Hong Kong/258/97 (H5N1) called strain A, isolate 258, was produced in chickens in Hong Kong in 1997 and has antigenic subtype 5 of HA and NA Antigenic subtype 1. Viruses with H1, H2 and H3 types of HA and N1 and N2 types of NA have caused human epidemics.

As mentioned above, in influenza virus A, genetic variation occurs through 2 main mechanisms. Genetic drift, which occurs through point mutations, often at antigenically prominent positions due to selective pressure from the host immune response, and genetic shift (also called reassortment), which involves a subtype A segment of the entire viral genome is replaced by another subtype. Many different types of animal species including humans, pigs, birds, horses, aquatic mammals and other animals that can become infected with influenza A virus. Some influenza A viruses are restricted to specific species and generally do not infect different species. However, some influenza A viruses can infect several different animal species, mainly birds, pigs and humans. This ability is thought to be responsible for the major antigenic shift in influenza A viruses. For example, assume a pig becomes infected with an influenza A virus from a human and at the same time becomes infected with a different influenza A virus from a duck. When the two different viruses are multiplied in pig cells, the genes of the human and duck strains can "mix," producing new viruses with unique combinations of RNA segments. The process is called genetic reassortment.

Like other viruses, influenza viruses replicate inside cells. Influenza A virus replicates in the epithelial cells of the respiratory tract. However, monocytes/macrophages and other white blood cells can also be infected. Many other cell types with cell surface glycoproteins containing sialic acid that serve as viral receptors are susceptible to infection in vitro.

The influenza A infection/replication cycle is schematically depicted in Figure 1 . As shown in Figure 1A, influenza A virion 100 comprises genome 101 consisting of 8 negative-strand RNA segments: PB2 (102), PB1 (103), PA (104), HA (105), NP (106 ), NA (107), M (108) and NS (109). Numbered from 1 to 8 by convention, PB2=1, PB1=2, PA=3, HA=4, NP=5, NA=6, M=7 and NS=8. Genomic RNA fragments are packaged inside a layer of membrane protein M1 120 surrounded by a lipid bilayer 130 from which the extracellular domains of envelope glycoproteins HA 140 and NA 150 and ion channel M2 160 protrude . RNA fragments 102-108 are capped by nucleoprotein MP 170 and contain viral polymerase complex 180 consisting of polymerases PB1, PB2 and PA. The nonstructural protein NS2 190 is also found within the virion. The nonstructural protein NS1 (not shown) was found within infected cells.

Figure IB shows the genome structure of influenza virus and transcripts generated from the influenza genome (not drawn to scale). Six of the eight genomic RNA fragments (PB1 (102), PB2 (103), PA (104), HA (105), NP (106) and NA (107)) each serve as a single, Template for unspliced transcripts. Three mRNA transcripts have been identified from influenza virus A segment M (108): a colinear transcript encoding the _M1 protein 191, a spliced mRNA encoding the _M2 protein and containing a 689 nucleotide intron 192, and another spliced mRNA 193 with the ability to encode a 9 amino acid peptide (M ₃ ) not yet detected in virus-infected cells. The 2 mRNA transcripts are from influenza virus A segment NS: encoding NS ₁ protein The unspliced mRNA 194 and the spliced mRNA 195 encoding the NS ₂ protein and including a 473 nucleotide intron.

The infection cycle (Fig. 2) begins when the virion 100 attaches via its hemagglutinin to the surface of a susceptible cell. The ligated virus is endocytosed into the coated vesicle 200 by clathrin-dependent endocytosis. The low pH in the endosome triggers the fusion of the viral and endosomal membranes, causing the release of the viral ribonucleoprotein (vRNP) complex (nucleocapsid) 210 into the cytoplasm. The viral nucleocapsid is introduced into the nucleus, after which primary viral mRNA synthesis is initiated by the viral RNA polymerase complex consisting of PB1, PB2 and PA polymerase. Primers generated on host cell precursor mRNA (pre-mRNA) by the endonuclease activity of the PB2 protein are used to prime viral mRNA synthesis using viral RNA (vRNA) 220 as a template. The PB1 protein catalyzes the synthesis of virus-specific mRNA 230, which is transported into the cytoplasm and translated.

Transports newly synthesized polymerase into the nucleus and regulates replication and secondary viral mRNA synthesis. PB1 , PB2, PA, and NP prime the synthesis of complementary RNA (cRNA) 240 from viral RNA (vRNA), after which new vRNA molecules are synthesized 250 . The viral polymerase complex uses the vRNA as a template to synthesize secondary mRNA 260. Thus, transcription of vRNA by virus-encoded transcriptases produces mRNA that serves as a template for viral protein synthesis, and also produces complementary RNA (cRNA) that serves as a template for the additional synthesis of vRNA for new virion production. Trafficking of viral mRNA into the cytoplasm where viral structural proteins are produced 270 . The proteins PB1, PB2, PA and NP are transported into the nucleus, the site of assembly of the vRNP complex (nucleocapsid) 280 . Budding and release of virus particles occurs at the plasma membrane.

Influenza A virus replicates rapidly in cells, causing host cell death by cytolysis or apoptosis. Infection causes a wide variety of cellular activities and changes in methods including inhibition of host cell gene expression. The viral polymerase complex binds and cleaves newly synthesized cellular polymerase II transcripts in the nucleus. NS1 proteins block cellular precursor mRNA splicing and inhibit nuclear export of host mRNAs. Translation of cellular mRNA is greatly inhibited, whereas viral mRNA is efficiently translated. Maintenance of efficient translation of viral mRNA is achieved in part by viral downregulation of the cellular interferon (IFN) response, a host response that normally acts to inhibit translation in virus-infected cells. Specifically, the viral NS1 protein binds IFN-induced PKR and inhibits its activity. Thus, it is clear that influenza virus infection causes profound changes in cellular biosynthesis, including changes in the processing and translation of cellular mRNA.

II. Selection, design and synthesis of RNAi-inducing entities

A. Selection and design of RNAi-inducing entities

The invention provides RNAi-inducing entities that target one or more influenza virus transcripts. Various viral RNA transcripts (primary and secondary vRNA, primary and secondary viral mRNA, and viral cRNA) are present in influenza virus-infected cells and play important roles in the viral life cycle. Any of said transcripts are suitable targets for RNAi-mediated inhibition by direct or indirect mechanisms according to the invention. Preferred RNAi-inducing entities targeting any viral mRNA transcript will specifically reduce the level of the transcript itself in a direct manner, for example by causing degradation of the transcript. In addition, RNAi-inducing agents that target certain influenza virus transcripts (eg, NP, PA, PB1 ) will indirectly cause decreased levels of influenza virus transcripts that they do not specifically target.

Viral transcripts that can serve as targets for RNAi-based therapies according to the invention include, for example, 1) any influenza virus genome segment; 2) transcripts encoding any viral protein, including encoding proteins PB1, PB2, PA, NP , NS1, NS2, M1, M2, HA or NA transcripts. Although the inventors have obtained data suggesting that viral mRNA is the sole or primary target of RNAi, it is possible that a single RNAi inducer targets the vRNA, cRNA and/or mRNA form of the transcript. In particularly preferred embodiments, the target transcript encodes an influenza virus protein NP, PA, PB1 or PB2.

The general characteristics of RNAi-inducing agents are known in the art. RNA interference was originally recognized as a phenomenon in which the presence of long dsRNAs (typically hundreds of nts) in a cell causes sequence-specific degradation of mRNA containing a region complementary to one strand of the dsRNA (US Patent No. 6,506,559). siRNAs were first discovered in RNAi studies in Drosophila as described in WO01/75164 and US Publication Nos. 20020086356 and 20030108923. Specifically, it was found that in Drosophila, long dsRNAs are processed by an RNase III-like enzyme called Dicer (Bernstein et al., Nature 409:363, 2001) into smaller dsRNAs consisting of two 21nt strands, each One both has a 5' phosphate group and a 3' hydroxyl group and includes a 19nt region that is exactly complementary to the other strand so that there is a 19nt duplex region flanked by 2nt 3' overhangs. Figure 3 shows a schematic representation of siRNAs found in Drosophila. The structure includes a 19 nucleotide double stranded (DS) portion 300 comprising a sense strand 310 and an antisense strand 315 . Each strand has a 2nt 3' overhang 320.

The short dsRNA (siRNA) acts to inhibit the expression of any gene that includes a region that is complementary to one of the dsRNA strands, presumably because the helicase activity unwinds the 19bp duplex in the siRNA, allowing a ("antisense" or "guide" strand) forms another duplex with the target transcript. The antisense strand is incorporated into an endonuclease complex, RISC, and directed to a complementary target RNA. Enzymatic activity exists at a single location in the RISC split ("slice"), producing unprotected RNA ends that are rapidly degraded by the cellular machinery (Figure 4). As described below, additional repression mechanisms mediated by short RNA species (microRNAs) are also known (see, e.g., Ruvkun, G., Science, 294, 797-799, 2001; Zeng, Y., et al. People, Molecular Cell, 9, 1-20, 2002). It should be understood that the discussion of mechanisms and the diagrams depicting mechanisms are not intended to imply any limitation on the RNA interference mechanisms useful in the present invention.

Dicer enzyme homologues (Sharp, Genes Dev.15; 485, 2001; Zamore, Nat.Struct.Biol.8: 746, 2001) have been found in various species from nematodes (C.elegans) to humans, thereby improving raised the possibility that a mechanism similar to RNAi might be able to suppress gene expression in a variety of different cell types, including mammalian or even human cells. However, long dsRNAs (eg, dsRNAs with double-stranded regions longer than about 30-50 nucleotides) are known to activate interferon responses in mammalian cells. Thus, rather than achieving the specific gene repression observed with the Drosophila RNAi machinery, the presence of long dsRNA in mammalian cells is expected to cause non-specific repression of translation mediated by interferon, possibly resulting in cell death. Long dsRNAs are therefore not considered suitable for inhibiting the expression of specific genes in mammalian cells.

However, the present inventors and others have discovered that siRNAs are effective in reducing the expression of target genes, including viral genes, when introduced into mammalian cells. The present inventors have found that when included in siRNA or shRNA and tested as described below and in co-pending patent application U.S.S.N. A large proportion of sequences proved to be effective inhibitory sequences. About 15% of siRNAs from the initial design group (Example 1) showed robust effects and effectively inhibited virus production in cells infected with PR8 or WSN strains of influenza virus; about 40% showed significant effects (i.e., in cells infected with PR8 There was a statistically significant difference (p<0.05) between virus production in the presence of siRNA versus absence of siRNA in cells infected with WSN and/or in cells infected with WSN; approximately 45% showed no or minimal effect.

Specifically, targeting RNA to the genes encoding RNA-dependent RNA transcriptase and nucleoprotein NP significantly reduced the amount of virus produced in infected mammalian cells (Examples 2, 4, 5, 6). The inventors have also demonstrated that siRNA targeting influenza virus transcripts can inhibit influenza virus replication in vivo in intact organisms (ie chicken embryos infected with influenza virus) (Example 3). In addition, the inventors have demonstrated that siRNA targeting influenza virus transcripts can inhibit virus production in mice when administered either before or after virus infection (Examples 12, 14, 16, 23-26, etc.). Furthermore, the present inventors have demonstrated that administration of a DNA vector useful for siRNA precursor (shRNA) expression inhibits influenza virus production in mice. Additional potent RNAi inducers were designed using a second set of design criteria, including a number of siRNAs that were highly potent when tested for their ability to inhibit influenza virus production in cells and/or mice. Thus, the present invention demonstrates that treatment with RNAi agents, such as siRNA, shRNA, or vectors whose presence in cells causes expression of said agents, is an effective strategy for inhibiting infection and/or replication of respiratory viruses, such as influenza virus . Two highly potent avian influenza strains were subsequently tested against highly pathogenic avian influenza strains and showed inhibition of viral beads, demonstrating the ability of the siRNAs to inhibit a broad range of influenza strains (Tompkins, et al., Proc. Natl. Acad. Sci., 101(23):8682-6, 2004). Accordingly, the present invention provides RNAi-inducing agents that inhibit viral production in cells infected with any of a variety of different influenza virus strains.

While not wishing to be bound by any theory, the inventors suggest that the findings are particularly interesting in view of the profound changes in cellular activities such as metabolism and biosynthetic activity that occur following infection with influenza virus. Influenza virus infection inhibits fundamental cellular processes such as cellular mRNA splicing, trafficking and translation and causes inhibition of cellular protein synthesis. Regardless of the changes, the finding that RNAi-inducing agents targeting influenza virus transcripts inhibit viral replication suggests that the underlying cellular mechanism for RNAi-mediated gene expression suppression continues in influenza-infected function in cells.

For any particular gene target chosen, the design of RNAi-inducing agents for use in accordance with the invention will preferably follow certain guidelines. In general, it is expected that the target sequence will be specific to the virus (compared to the host), and preferably important or essential to the function of the virus. Therefore, it is desirable to avoid portions of the target transcript that may be shared with other transcripts that are not desired to be degraded. A database search can be performed to determine whether any strand is substantially complementary to any sequence in the genome of the organism (eg, human) to which the agent will be delivered, and thus such sequences can be avoided. As described herein, portions of viral transcripts that are conserved among multiple variants are preferred targets.

Preferred RNAi-inducing agents for use in accordance with the invention comprise a base pair region between 15 and about 29 nt long (eg, about 19 nt in length), and optionally have one or more free ends or loops end. Figure 5 presents various structures useful as RNAi inducers according to the present invention. Figure 5A shows constructs found to be active in the Drosophila system described above and in mammalian cells. The present invention contemplates the administration of siRNAs having the structure shown in Figure 5A to mammalian cells to treat or prevent influenza infection. However, it is not required that the administered agent has such a structure. For example, the administered composition can include any structure capable of being processed in vivo into the Figure 5A structure, so long as the administered agent does not cause an undesired or deleterious event, such as induction of an interferon response. The invention can also encompass the administration of agents that are not precisely processed into the structure shown in Figure 5A, so long as the administration of the agent sufficiently reduces viral transcript levels as discussed herein. In some cases, an agent delivered into a cell according to the present invention may undergo one or more processing steps (see below for further discussion) before becoming an active inhibitor; in such cases, those skilled in the art will appreciate that , the relevant agent is preferably designed to include sequences that may be necessary for its processing.

Figures 5B and 5C show additional constructs that can be used to mediate RNAi. The hairpin (stem-loop) structure can function directly as an inhibitory RNA or can undergo intracellular processing (eg, by Dicer) to generate an siRNA structure such as the structure depicted in Figure 5A. FIG. 5B shows an agent comprising an RNA molecule containing two complementary regions that hybridize to each other forming a duplex region represented as stem 400 , loop 410 and overhang 320 . The molecules are said to self-hybridize, and this class of structures is called shRNA. See also Figures 20 and 21 for examples of shRNA constructs. Figure 5C shows an agent comprising an RNA circle comprising sufficient complementary elements to form a stem 400 approximately 19 bp long. The agents can exhibit improved stability compared to various other siRNAs described herein.

In descriptions of RNAi-inducing agents and their activity, as in the case of siRNAs, it is often convenient to consider agents as having 2 strands. Typically, the sequence of the duplex portion of one strand of the RNAi-inducing agent is substantially complementary to the target transcript in that region. The sequence of the duplex portion of the other strand of the RNAi-inducing agent is typically substantially identical to the targeted portion of the target transcript. The strand that contains the portion that is complementary to the target is called the "antisense strand," while the other strand is often called the "sense strand." The portion of the antisense strand that is complementary to the target can be referred to as the "inhibition region."

The duplex structure of shRNA can be considered to comprise an antisense strand and a sense strand, wherein the antisense strand is the first part of the molecule that forms a duplex with or is capable of forming a duplex with the second part of the molecule, and is complementary to the targeted portion of the target transcript. The sense strand is the portion of the molecule that forms a duplex or is capable of forming a duplex with the first moiety. Those of skill in the art will appreciate that an "antisense strand" targeting a vRNA strand from a negative-strand RNA virus will be antisense to the vRNA, but the "sense strand" relative to the viral cRNA. Likewise, an "antisense strand" targeting a cRNA strand from a negative-strand RNA virus would be antisense to the cRNA and a "sense strand" to the vRNA sequence.

For purposes of description, the following discussion will often refer to siRNA. However, as will be apparent to those skilled in the art, the teachings relating to the two strands of an siRNA generally apply, for example, to the sense and reverse strands of the stem portion of any RNAi-inducing agent that can be processed intracellularly to produce the corresponding shRNA of the siRNA. Sense chain. Thus, in general, the discussion below also applies to the design, selection and delivery of RNAi-inducing agents of the invention, eg, shRNAs that undergo intracellular processing to produce RNAs that mediate target cleavage or translational repression.

In general, it is preferred that the antisense strand of the siRNA hybridize to a target site comprising or consisting of an exon sequence in the target transcript. In certain embodiments of the invention, the antisense strand hybridizes to the 5' or 3' untranslated region. In general, any site available for hybridization to the antisense strand to produce cleavage and degradation of the transcript and/or translational inhibition can be utilized.

RNAi-inducing agents can be selected according to various methods. In general, as described above, the RNAi-inducing agents of the invention preferably include a region (the "duplex region") in which one strand contains a portion of the target transcript ("target portion") of a length sufficiently complementary to A suppression region between 15-29 nts to allow in vivo hybridization between the strand and the target transcript. It will be appreciated that the duplex region - also referred to as the "core region" - does not include overhangs. If present, the overhang may, but need not, be complementary to the target transcript. Preferably, the duplex region includes most or all of the duplex structures described in Figures 3, 4 and 5.

Preferably, the inhibitory region is 100% complementary to the target. However, those skilled in the art will recognize that mismatches and bulges may exist in the duplex formed by the antisense strand and the target. Thus, the region of inhibition need only be sufficiently complementary to the target so that hybridization can occur, eg, in cells and/or in in vitro systems providing RNAi such as the Drosophila extract system described above, under physiological conditions. Preferably, the region of inhibition and the target are at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably 100% complementary to each other. Preferably, less than 6 residues, or about 20% (e.g., 1, 2, 3, 4) of the antisense strand residues in the region are within the 15-29 nt (e.g., 19 nt) window In-range mismatch with target. Preferably, less than 4 residues, or about 15% of the antisense strand residues, are mismatched to the target in the region. Preferably, there are only 1 or 2 mismatches. One or more (or all) of the mismatches may be U-G mismatches. For example, if the inhibitory region is 15-16 nt long, there may be 0-3 mismatches; if the inhibitory region is 17 nt long, there may be 0-4 mismatches; if the inhibitory region is 18 nt long, there can be 0-5 mismatches; if the inhibitory region is 19 nt long, there can be 0-6 mismatches. The number of permissible mismatches is increased by one nt for each additional nt present in the suppression region up to the upper limit of the RNAi-inducing agent suppression region, eg, about 30 nt in length. In certain embodiments, the mismatches are not in consecutive positions. In certain embodiments, there are no mismatched segments longer than 2 nt in length. In a preferred embodiment, the evaluation window of 15-19 nt contains 0-1 mismatches (preferably 0), and the evaluation window of 20-29 nt contains 0-2 mismatches (preferably 0-1 , more preferably 0). Those skilled in the art will recognize that duplex structures interrupted by bulges will generally allow for a greater number of unpaired nts. Those skilled in the art will also recognize that mismatches in the central portion of the antisense strand/target RNA duplex may preferably be avoided (see, e.g., Elbashir et al., EMBO J. 20:6877, 2001). For example, the 3' nucleotide of the antisense strand of an siRNA often does not significantly contribute to the specificity of target discrimination and is not critical for target cleavage. In certain embodiments, the antisense strand and the target are complementary at position 10 of the inhibitory region of the antisense strand. In other embodiments, they are not complementary.

Certain RNAi-inducing agents contain a strand that hybridizes to a target site that includes or consists entirely of a 3'UTR sequence. Those skilled in the art will appreciate that the resulting duplexes may tolerate a greater number of mismatches and/or bulges, especially in the central region of the duplex, while still producing effective inhibition. For example, one or both strands may include one or more "extra" nucleotides that form bulges as shown in FIG. 6 . There may be one or more protrusions, eg, 5-10 nt long. Typically, the length of the fully complementary stretch is at least 5 nt, eg 6, 7 or more nt, while the mismatched region may eg be 1 , 2, 3 or 4 nt long. Duplexes often include two perfectly complementary segments separated by a mismatch region. Various configurations are possible. For example, there may be multiple regions of mismatch.

Some mismatches may be required because duplex formation in the 3'UTR or in other positions may repress the expression of the protein encoded by the transcript by a mechanism that is consistent with the cleavage that is a hallmark of classical RNA repression Related but different. As shown in Figure 6, the Dicer enzyme known to produce siRNA in the Drosophila system described above, as well as in various organisms, also processes small, transient RNA (stRNA) substrates into inhibitors that, upon binding to When within the 3'UTR of the target transcript, translation of the transcript is blocked (see Grishok, A. et al., Cell 106, 23-24, 2001; Hutvagner, G. et al., Science, 293, 834-838, 2001; Ketting, R. et al., Genes Dev., 15, 2654-2659). Subsequent discoveries have shown that the genomes of organisms from nematodes to mammals encode a class of short (approximately 19-25 nucleotides) RNAs called microRNAs (miRNAs) that repress endogenous Translation of sexual mRNA. MicroRNAs are discussed in Barrel, DP., Cell, 116(2):281-97, 2004; Novina, C. and Sharp, PA, Nature, 430:161-164, 2004; and US Publication No. 20050059005 . miRNAs bind target mRNA transcripts at partially complementary sites and prevent their translation. RNAi inducers with the structure of siRNA (2 individual short strands that hybridize to each other) can act in a manner similar to miRNAs, that is, by reducing the translation of transcripts rather than their stability (Doench, JG et al. Genes & Development, 17:438-442, 2003). The siRNAs are thought to undergo intracellular processing to produce single-stranded RNAs that act through the miRNA translational repression pathway. For the purposes of the present invention, any partial or complete double chain described herein that binds a target transcript and reduces its expression (i.e., reduces the level of the transcript and/or reduces the synthesis of the polypeptide encoded by the transcript). Short RNA strands are considered RNAi inducers, regardless of whether they act by triggering degradation, inhibiting translation, or by other means. Additionally, any precursor RNA structure that can be processed in vivo (ie, in a cell or organism) to produce the RNAi-inducing agent is suitable for use in the present invention.

In some cases, the sequence of the RNAi-inducing agent is selected so that the entire antisense strand (including, if present, the 3' overhang) is fully complementary to the target transcript. However, the overhang does not have to be complementary or identical to the target transcript. Any desired sequence (eg, UU) can simply be appended to the 3' end of the antisense and/or sense core regions to create 3' overhangs. Typically, overhangs containing one or more pyrimidines, usually U, T or dT, are used. When synthesizing RNAi-inducing agents, it may be more convenient to use T instead of U in the overhang. Using dT instead of T increases stability.

Preferably, the strands of the RNAi-inducing agent are 100% complementary to each other in the core region. However, those skilled in the art will recognize that mismatches and bulges may exist in the duplex formed by the antisense and sense strands. The strands need only be sufficiently complementary to each other so that hybridization can occur, eg, in cells and/or in an in vitro RNAi-providing system such as the Drosophila extract system described above, under physiological conditions. Preferably, the 2 strands of the RNAi-inducing agent are substantially complementary in the core region, eg at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably 100% complementary to each other in the core region. For example, a 15-16 nt long core region may contain 0-3 mismatches; a 17 nt long core region may contain 0-4 mismatches; an 18 nt long core region may contain 0-3 5 mismatches; 19 nt long core region can contain 0-6 mismatches. The number of permissible mismatches is increased by one nt for each additional nt present in the suppression region up to the upper limit of the RNAi-inducing agent suppression region, eg, about 30 nt in length. In a preferred embodiment, the 15-19 nt core region contains 0-1 mismatches (preferably 0), and the 20-29 nt core region contains 0-2 mismatches (preferably 0-1 , more preferably 0). Those skilled in the art will recognize that duplex structures interrupted by bulges will generally allow for a greater number of unpaired nts.

In summary, RNAi-inducing agents can be designed by selecting a portion of the target and designing an RNAi-inducing agent comprising: an antisense strand whose sequence is sufficiently complementary to hybridize to the target, e.g., within 15-29 nucleotides, For example substantially complementary or 100% complementary to the target transcript within 19 nucleotides; sense strand with sufficient sequence complementarity to hybridize with the antisense strand, eg substantially complementary or preferably 100% complementary to the antisense strand. A 3' overhang such as the overhang described above can then be added to the sequence to generate the siRNA construct.

Those skilled in the art will appreciate that RNAi-inducing agents such as siRNA may exhibit a range of melting temperatures (Tm) according to the above principles. The Tm is defined as the temperature at which 50% of the nucleic acid and its perfect complement are in solution in the duplex. Representative examples of putative Tm can be readily determined based on the siRNA sequences disclosed in the Examples herein, using methods well known in the art, either experimentally or using appropriate empirically or theoretically derived equations. In certain embodiments of the invention, the calculated Tm of the duplex formed by the antisense strand and the target transcript is compared to the Tm that would be obtained between the target and an antisense having a fully complementary inhibitory region to the target. The calculated Tm of the duplex formed between the strands is at most 5°C lower, at most 10°C lower or at most 15°C lower. In certain embodiments of the invention, the calculated Tm of the duplex formed by the antisense and sense strands of an RNAi-inducing agent is compared to that of the antisense strand with a fully complementary (excludes overhangs if necessary) The calculated Tm of the duplex formed between the sense strands of ) is at most 5°C lower, at most 10°C lower or at most 15°C lower.

Those skilled in the art will be able to calculate Tm values. Several studies have derived exact equations for Tm using thermodynamic basis sets for nearest neighbor interactions. Thermodynamic parameter values are available in the literature. For RNA see Freier, SM, et al., Proc. Natl. Acad. Sci. 83, 9373-9377, 1986. Rychlik, W., et al., Nucl. Acids Res. 18(21), 6409-6412, 1990. Preferably, use the closer values and methods in Walter, AE, Proc. -940, 1999 for the values and methods described. Computer programs for calculating Tm are widely available. See, eg, the website URL www.basic.nwu.edu/biotools/oligocalc.html . Preferably, a program for calculating relevant parameters such as AG and Tm, available on the mfold web server at the URL www.bioinfo.rpi.edu/applications/mfold , such as Zuker, M., Nucl.Acids. Res., 31(13), 2003.

One aspect of the invention is to identify when multiple strains, subtypes, etc. (commonly referred to as variants) of an infectious agent exist, whose genomes have changes in sequence, often requiring selection and/or design targeting between the different variants RNAi inducers in highly conserved regions among them. By comparing a sufficient number of sequences and selecting highly conserved regions, it is possible to target multiple variants with a single RNAi-inducing agent targeting the highly conserved region, e.g., the antisense strand of the RNAi-inducing agent is of sufficient length. The range is substantially complementary to highly conserved regions such that RNAi-inducing agents mediate RNAi. According to some embodiments of the invention, a region is said to be highly conserved among variants if the region is identical among the variants. According to some embodiments of the present invention, if the region targeted by the RNAi-inducing agent, for example 15-29 nucleotides, preferably 19 nucleotides, differs between variants by at most one nucleotide ( That is, at 0 or 1 nucleotide position), then it is highly conserved. According to certain embodiments of the invention, a region differs between variants if the region differs by at most 2 nucleotides (i.e., at 0, 1 or 2 nucleotide positions) between variants. is highly conservative. According to certain embodiments of the invention, a region differs between variants by at most 3 nucleotides (i.e., at 0, 1, 2 or 3 nucleotide positions), then the region is are highly conserved among individuals. According to certain embodiments of the invention, the RNAi-inducing agent targets a region that is highly conserved among at least 5, 10, 15, 20, 25, 30, 40, 50 or more variants.

To identify regions that are highly conserved among a panel of multiple variants, the following procedure can be used. Selects a member of the sequence group as the base sequence, that is, the sequence to which other sequences will be compared. According to various embodiments of the invention, the base sequence may be one of the sequences in the group being compared, or may be a consensus sequence derived from the sequences in the group, e.g., by determining for each position the The most common nucleotide at that position was obtained.

After the base sequence is selected, the sequences of each member of the plurality of variant panels are compared to the base sequence. The number of differences between the base sequence and any member of the plurality of variant groups within a region of the sequence (e.g., 15-29 nucleotides, such as a region of 19 nucleotides), used to determine a particular region of interest Whether it is highly conserved between the base sequence and the group members being compared to it. As described above, in various embodiments of the invention, if the number of positions at which the sequence differs between two regions is 0; 0 or 1; 0, 1 or 2; or 0, 1, 2 or 3, then This region is believed to be highly conserved. The antisense sequence of the RNAi-inducing agent may be selected to be complementary to the base sequence across a highly conserved region of 15-29 nucleotides, such as 19 nucleotides, or may be selected to be complementary to one of the other sequences across a highly conserved region . Typically, the sense strand sequence is selected to be identical to the base sequence or one of the other sequences across a highly conserved region such that the antisense and sense strands are 100% complementary to each other in the duplex portion of the RNAi-inducing agent.

Typically, the antisense strand sequence is chosen with respect to the base sequence as described above. However, in certain embodiments of the invention, particularly if the nucleotide at a particular position in the second sequence in the set being compared is more present in the sequence being compared than the corresponding position in the base sequence nucleotides are more common, then the antisense strand sequence can be selected with respect to the second sequence (eg, 100% complementary to a highly conserved region present in the second sequence). Additionally, according to some embodiments of the invention, if the consensus nucleotide (the most frequently occurring nucleotide) at the position where there is a difference is different from the nucleotide found in the base sequence, then using consensus nucleotides. Note that this may result in a sequence that differs from any of the sequences being compared (since the consensus sequence may be used as the base sequence).

Example 1 describes the selection of highly conserved targets based on a comparison of sequence panels from 6 influenza A strains with human host origins, and a comparison of sequence panels from 7 influenza A strains with different animal host origins, including humans part, and design siRNA sequences targeting that part. Different methods of selecting highly conserved regions can be used. The invention encompasses RNAi-inducing agents whose duplex portions (and optionally any overhangs) are selected based on highly conserved regions meeting the criteria provided herein, regardless of how the highly conserved regions are selected. It is also understood that the invention encompasses RNAi-inducing agents that target portions of the influenza virus transcript that do not meet the criteria for the preferred target regions described herein. For example, an RNAi inducer that does not target a highly conserved or moderately conserved target portion may still be an effective inhibitor of influenza virus production for certain strains. Less potent RNAi-inducing entities can also be used to assess target specificity, identify determinants of RNAi efficacy, improve RNAi design, etc.

Table 1A lists 21 nucleotide regions that are highly conserved in the set of influenza virus sequences for each of the viral gene segments. Each sequence listed in Table 1A includes a 19nt region (nt3-21); and an initial 2nt sequence that is absent in the sense strand of the corresponding siRNA but is complementary to the 3' overhang of the antisense strand of the siRNA. It will be appreciated that the 19nt region can be used as the sense strand to design various siRNA molecules with different 3' overhangs in either or both of the sense and antisense strands. Accordingly, a variety of sense and antisense siRNA sequences can be obtained from the respective sequences listed in Table 1A. Twenty such siRNA sequences are listed in Table 2.

Table IB lists additional siRNAs designed based on highly conserved regions of influenza viruses. Both strands are shown in the 5' to 3' orientation. A dTdT 3' overhang is appended to each strand. Nucleotides 1 to 19 in each sequence of the sense strand sequences listed in Table 1B have sequences identical to highly conserved regions of influenza virus transcripts. The corresponding antisense sequence is complementary to the sense strand. In certain embodiments of the present invention, "highly conserved region" refers to nt 3-21 in any of the sequences listed in Table 1A, or in any sense strand of the sense strands listed in Table 1B. nt 1-19. This region exists in double-stranded form in certain RNAi-inducing agents of the invention so that the antisense strand of the agent targets a highly conserved portion. The sequence of this region is referred to as "highly conserved sequence" or "highly conserved target portion".

Selection of highly conserved target moieties is one approach to designing RNAi-inducing entities that will successfully inhibit a variety of different influenza virus strains. However, the present invention also provides alternative methods of selecting preferred target moieties that are appropriately conserved in order to increase the expression of RNAi-inducing agents targeting the target moiety to inhibit the expression of the target transcript in multiple different strains The possibility that the different strains differ in the sequence in the target portion. According to the invention, RNAi-inducing agents whose antisense strand comprises an inhibitory region 100% complementary to a target portion found in one or more strains (eg PR8), if the target portion is appropriately conserved, preferably effectively inhibit the expression of corresponding transcripts present in one or more other strains in which the corresponding target portion is less than 100% complementary to the inhibitory region of the antisense strand.

The present invention provides various RNAi-inducing agents that target appropriately conserved portions of influenza A virus transcripts, wherein the appropriately conserved target portions are selected according to the methods of the present invention. In certain embodiments of the invention, the target moiety is suitably conserved across multiple influenza A strains isolated from humans. In certain embodiments of the invention, said portion is suitably conserved across multiple strains of influenza A virus isolated from animals other than humans, eg, birds. In certain embodiments of the invention, the portion is suitably conserved across multiple strains of influenza A virus isolated from humans as well as across multiple strains of influenza A virus isolated from non-human animals such as birds. For example, said portion may be suitably conserved among at least 5, 10, 15, 20 or more variants of human and/or animal origin. In certain embodiments of the invention, target moieties are suitably conserved and highly conserved.

For any viral target, appropriately conserved portions of the target can be identified by first aligning transcript sequences from multiple variants and comparing them to a chosen base sequence to identify differences, i.e. , where the identity of one or more nucleotides in the sequence differs from that in the base sequence. Evaluate the nature of the difference to determine whether the difference is significant. When discussing groups of sequences that are aligned and compared to select conserved regions, "difference" refers to a position in one or more of the sequences that differs from the base sequence, regardless of how many of the sequences differ from the base sequence. The base sequence can be selected in various ways. For example, it may be convenient to select a sequence from strains that are highly prevalent or readily available under laboratory assembly.

Appropriately conserved target portions satisfy the following criteria when comparing corresponding target portions present in within-group variants: (1) allow A to G or C to U differences between the base sequence and the corresponding sequence at any position ; (2) only at one or more of positions 1, 18 and 19, the difference between G being A or C being A between the basic sequence and the corresponding sequence is allowed; (3) between positions 1 and 9, in Between the base sequence and the corresponding sequence, there are 0, 1, 2, or 3 differences; (4) there are no more than 2 consecutive differences between the base sequence and the corresponding sequence; and, (5) between positions 11 and 17 , there is at most 1 difference between the base sequence and the corresponding sequence. Any strain can be selected as the base strain. Preferably, at least 5 variants are compared to identify appropriately conserved target portions.

Influenza viruses circulating in avian and/or other animal hosts sometimes acquire the ability to infect humans. Possibly due in part to the lack of immunity in humans, such strains often result in high mortality. Examples include the 1918 pandemic and deaths from avian influenza infection in humans in Hong Kong (1997) and Vietnam (2004). There is growing concern about the potential for strains with avian or other animal host origins to spread into the human population, while existing vaccines do not protect humans from avian or swine influenza virus infection. The invention identifies appropriately conserved and/or highly conserved target portions across a plurality of strains originally isolated from humans (human-derived strains) and strains originally isolated from non-human animals such as birds (avian-derived strains), and An RNAi-inducing agent targeted to the targeting moiety is provided. The RNAi inducers are capable of conferring protection against a variety of influenza virus strains, including human-derived strains and strains derived from non-human animal hosts.

Example 17 describes the identification of targeted portions of influenza transcripts that are appropriate conserved targets for RNAi. The first step was to identify all possible 19 nucleotide influenza target moieties. The sequence of each of the 19 nucleotide potential target moieties found in PR8 or another influenza virus strain listed in Tables 15A-15H or Tables 19A-19F is contemplated herein, but not specifically stated. The sequences can be readily identified by referring to the sequences of the influenza virus fragments in Figures 32A-32J.

The next step is to identify the preferred functional target portion of RNAi, ie, the region of the gene whose sequence characteristics suggest that an RNAi-inducing agent with an antisense strand that hybridizes to the target will effectively inhibit its expression. Preferably, the functional target moiety meets various criteria regarding GC content and the absence of contiguous stretches of G or C residues. Table 17 (Figure 33) lists the sequences of preferred functional target moieties present in the base strain PR8.

To identify appropriately conserved target moieties, corresponding functional target moieties found in a large number of human-derived influenza virus strains were then aligned. Generally, when the genome sequences are aligned for maximum homology and/or are homologous, for example, at least 50% identical in different strains, the "corresponding target portion" in different strains is usually present in different viral strains. at approximately the same position in the genome. Typically, the degree of homology of corresponding target portions when comparing two strains is at least 60%, 70%, 80% or more. For example, the corresponding targeting moiety can be different from the targeting moiety found at positions 1, 2, 3 or 4 in the base strain. The exact sequence of the preferred homologous target portion is easily identified by accessing the sequence of related influenza virus fragments in a database such as Genbank, aligning it with the base strain sequence, and locating a portion, which is at about At least 80% identical, preferably at least 90% identical at identical nucleotide positions and/or to the target portion found in the base virus strain (eg, PR8). Homologous targeting moieties are an aspect of the invention. Appropriately conserved target moieties are selected using the criteria described above. Table 18 (FIG. 34) lists the sequences of target moieties that are well conserved among human-derived influenza strains, as present in the base strain PR8.

In addition to considering strains isolated from human hosts, isolates from avian hosts are also aligned and compared to identify appropriately conserved and/or highly Conserved target portion. Isolates from one or more other animal hosts can also be used for such comparisons. Appropriately conserved target portions of the base sequence are compared to the aligned avian sequences and selected using the same criteria used to identify appropriately conserved target portions among human isolates. Table 20 (Figure 35) lists the sequences of target moieties that are well conserved between human and avian derived influenza strains, as present in the base strain PR8.

Each of the 19 nucleotide potential target moieties found in PR8 or another influenza strain listed in Tables 15A-15H or Tables 19A-19F is an aspect of the invention. Each of the preferred functional target moieties found in PR8 or another influenza strain listed in Tables 15A-15H or Tables 19A-19F is an aspect of the invention. Each suitably conserved and/or highly conserved target moiety found in PR8 or another influenza strain listed in Tables 15A-15H or Tables 19A-19F is an aspect of the invention. Each of said sequences (that is to say, possible, preferably functional, appropriately conserved sequences between human-derived strains, highly conserved sequences between human-derived strains and avian-derived strains, and/or among human- or avian-derived strains or highly conserved sequences between the two) can be used as the sequence of the inhibitory region of the antisense strand of the RNAi-inducing agent targeting the target moiety. An RNAi-inducing agent having an antisense strand comprising each of said sequences or a fragment thereof at least 15 nucleotides in length is an aspect of the invention. However, as described herein, various RNAi-inducing agents comprising antisense strands that exhibit less than perfect complementarity to the target moiety, or that are shorter or longer, may also be used. The sequence of the targeting moiety can serve as the sense strand of the RNAi-inducing agent. A sense strand that is not fully complementary to the antisense strand can also be used as described herein.

As noted above, the target portions listed in Tables 17, 18 and 20 are identical to those of the influenza A virus PR8 transcript. The corresponding target portions of the respective transcripts that were identical or highly homologous to those listed in Tables 17, 18 and 20 were found in other influenza A strains. In certain embodiments of the invention, the inhibitory region of the antisense strand of an RNAi-inducing agent that targets a potential influenza virus target moiety, such as a target moiety listed in Tables 17, 18, or 20, is not related to the PR8 sequence. 100% complementary, but 100% complementary to the corresponding target portion listed in Tables 15A-15H or Tables 19A-19F found in one or more other strains. The invention provides RNAi-inducing entities, such as RNAi-inducing agents, such as siRNA or shRNA, that target each of the potential targeting moieties, functional targeting moieties, appropriately conserved and highly conserved targeting moieties described herein. The invention also provides RNAi-inducing entities, e.g., RNAi-inducing agents targeting a target moiety comprising at least 15 contiguous nts of a potential targeting moiety, a functional targeting moiety, a suitably conserved moiety or a highly conserved targeting moiety as described herein , such as siRNA or shRNA. The invention also provides RNAi-inducing entities, such as those targeting a target moiety up to about 29 nt in length comprising a potential targeting moiety, a functional targeting moiety, an appropriately conserved targeting moiety, or a highly conserved targeting moiety as described herein. RNAi inducers, such as siRNA or shRNA. The other nucleotides of the targeting moiety are preferably positioned directly 5' and/or 3' from the 19nt targeting moieties listed herein. In other words, some other portions up to about 10 nt of the longer targeting portion may be upstream of the 19nt targeting portion and some may be downstream of the 19nt targeting portion. The exact sequence is readily identifiable by accessing the appropriate entry for the genomic fragment containing the listed or corresponding target moiety from any strain and identifying the 5' and/or 3' directly to the listed or corresponding target moiety. ' Nucleotides. Figures 32A-32J present the genomic fragment sequence of PR8 (positive strand form).

The present invention provides RNAi-inducing agents that have been tested in cell culture and/or in animal models to demonstrate their effectiveness and identify portions of influenza virus transcripts that can be targeted in a highly efficient manner. Example 18 describes a high throughput screen (HTS) for identifying siRNAs effective in reducing the levels of targeted influenza virus transcripts. Examples 19-22 describe high throughput screens for identifying siRNAs effective in reducing influenza virus production in cells. Certain preferred potent siRNAs reduce influenza A virus titers by at least 4-fold when contacted with cells at a concentration of 100 nM. Certain preferred highly potent siRNAs reduce influenza A virus titers by at least 8-fold, or an even greater degree, when contacted with cells at a concentration of 100 nM. Certain preferred potent siRNAs reduce influenza A virus titers by at least 4-fold when contacted with cells at a concentration of 1 nM. Certain preferred effective siRNAs reduce influenza A virus titers by at least 4-fold, at least 8-fold, or at least 16-fold when contacted with cells at a concentration of 100 nM. Certain highly potent siRNAs reduced influenza A virus titers by at least 2-fold when contacted with cells at concentrations of 5 nM or less. Certain even more highly potent siRNAs reduced influenza A virus titers by at least 2-fold when contacted with cells at concentrations of 1 nM or less. Whereas the more highly potent siRNA reduced influenza A virus titers by at least 2-fold when contacted with cells at a concentration of 0.8 pM or less. Concentration refers to the concentration of siRNA present in the medium outside the cells at the time of introduction.

In certain embodiments, the RNAi-inducing agent targets a target moiety within 200 nt of the 3' end of a gene from, for example, a NP, PA, PB1 or PB2 gene. Seven highly potent siRNAs were found to target the target moiety within this region. The inventors observed that the 5' and 3' ends of the influenza gene segment comprise about 10 bases that are highly conserved across different gene segments and across different influenza virus strains. In particular, three siRNAs targeting the 5'UTR of the PB1 transcript showed a 4-fold reduction in viral titer at 100 nM.

B. Synthesis of RNAi-induced entities

The RNAi-inducing agents of the present invention can be prepared according to any available technique including, but not limited to, chemical synthesis, in vivo or in vitro enzymatic or chemical cleavage, or in vivo or in vitro template transcription. For example, RNA can be produced by enzymatic synthesis or by partial/total organic synthesis, and modified nucleotides can be introduced by in vitro enzymatic or organic synthesis. In one embodiment, siRNA is prepared chemically. Synthetic methods of RNA molecules are known in the art, especially chemical synthesis methods as described in Verma and Eckstein, Annu. Rev. Biochem. 67:99-134 (1998). In another embodiment, siRNA is prepared enzymatically. For example, siRNA can be prepared by enzymatic processing of long dsRNA with sufficient complementarity to the desired target RNA. Processing of long dsRNA can be accomplished in vitro, eg, using appropriate cell lysates, and the siRNA can subsequently be purified by gel electrophoresis or gel filtration. For example, RNA can be purified from a mixture by extraction with solvents or resins, precipitation, electrophoresis, chromatography, or combinations thereof. Alternatively, RNA can be used with no or minimal purification to avoid losses due to sample processing.

The RNAi-inducing agents of the present invention can be delivered as a single shRNA molecule or as 2 strands that hybridize to each other. For example, 2 separate 21nt RNA strands can be generated, where each strand contains a 19nt region that is complementary to the other, and the individual strands can hybridize together to produce a structure such as that depicted in Figure 5A.

In some embodiments, each strand of the siRNA is produced by in vitro or in vivo transcription from a promoter. For example, a construct may comprise two separate transcribable regions, where each region produces a 21 nt transcript containing a 19 nt region complementary to the other. Alternatively, a single construct may be utilized comprising opposing promoters P1 and P2 and terminators t1 and t2 positioned so as to produce two different transcripts each at least partially complementary to the other ( Figure 7). In another embodiment, the shRNA is produced as a single transcript, eg, by transcription of a single transcription unit encoding a self-complementary region. Figure 8 depicts one such embodiment. As indicated, templates were used that included first and second complementary regions, and optionally loop regions. Constructs encoding one or more siRNA and/or shRNA strands are encompassed by the invention. In addition to the promoter, the construct optionally includes one or more other regulatory elements, such as terminators.

In vitro transcription can be performed using various available systems including T7, SP6, and T3 promoter/polymerase systems (eg, systems commercially available from Promega, Clontech, New England Biolabs, etc.). When siRNA is synthesized in vitro, it can be hybridized prior to transfection or delivery to a subject. It will be appreciated that siRNA compositions of the invention need not consist entirely of double-stranded (hybridized) molecules. For example, siRNA compositions can include a small proportion of single-stranded RNA. In general, preferred compositions comprise at least about 80% dsRNA, at least about 90% dsRNA, at least about 95% dsRNA, or even at least about 99-100% dsRNA. However, an siRNA composition may contain less than 80% hybridizing RNA as long as it contains sufficient dsRNA to be effective.

Those skilled in the art will appreciate that if the siRNA or shRNA agents of the invention are produced in vivo, it is generally preferred that they be produced by transcription of one or more transcription units. The primary transcript can optionally be processed (eg, using one or more cellular enzymes) to produce the final agent for gene suppression. It will additionally be appreciated that appropriate promoters and/or regulatory elements can be readily selected to allow expression of the relevant transcriptional unit in mammalian cells. In some embodiments of the invention, it may be desirable to utilize a regulatable promoter; in other embodiments, constitutive expression may be desired. The term "expression" as used herein with respect to siRNA or siRNA precursor synthesis (transcription) does not imply translation of the transcribed RNA.

In certain preferred embodiments of the present invention, the promoter used to direct the in vivo expression of one or more siRNA or shRNA transcription units is the promoter of RNA polymerase III (Pol III). Pol III directs the synthesis of small transcripts that terminate after encountering a stretch of 4-5 T residues in the template. Certain Pol III promoters, such as U6 or H1 promoters, do not require cis-acting regulatory elements in the transcribed region (other than the first transcribed nt) and are therefore preferred according to certain embodiments of the invention , as these promoters allow easy selection of the desired siRNA sequence. See, eg, Yu, J. et al., Proc.Natl.Acad.Sci., 99(9), 6047-6052 (2002); Sui, G. et al., Proc.Natl.Acad.Sci., 99( 8), 5515-5520 (2002); Paddison, P. et al., Genes and Dev., 16, 948-958 (2002); Brummelkamp, T. et al., Science, 296, 550-553 (2002); Miyagashi, M. and Taira, K., Nat. Biotech, 20, 497-500 (2002); Paul, C et al., Nat. Biotech, 20, 505-508 (2002); Tuschl, T. et al., Nat. Biotech , 20, 446-448 (2002). The Pol II promoter can also be used, for example, as described in Xia, H. et al., Nat. Biotechnol, 20, pp. 1006-1010, 2002. As described herein, constructs may be used in which a hairpin sequence is juxtaposed near the transcription start site and followed by a polyA cassette, producing minimal to no overhangs in the transcribed hairpin. In certain embodiments of the present invention, tissue-specific, cell-specific or inducible Pol II promoters can be used as long as the above requirements are met. The Pol I promoter can also be used in various embodiments (McCown 2003).

It will be appreciated that in vivo expression of a construct providing a template for the synthesis of siRNA or shRNA, such as that depicted in Figures 7 and 8, can ideally be achieved by introducing the construct into a vector such as a DNA plasmid or viral vector , and introduce the vector into mammalian cells. Any of a variety of vectors may be selected, although in certain embodiments it may be desirable to select a vector that delivers the construct to one or more cells susceptible to influenza virus infection. The invention encompasses vectors containing siRNA and/or shRNA transcriptional units, as well as cells containing such vectors or otherwise engineered to contain transcriptional units encoding one or more siRNA or shRNA strands. In certain preferred embodiments of the invention, the vectors of the invention are gene therapy vectors suitable for delivering constructs expressing siRNA or shRNA to mammalian cells, most preferably human cells. The vectors can be administered to a subject before or after exposure to influenza virus to provide prophylaxis or treatment of diseases and conditions resulting from viral infection.

The invention thus provides various viral and non-viral vectors, the presence of which in a cell results in the transcription of one or more RNAs that hybridize to themselves or to each other to form genes that inhibit the expression of at least one influenza virus transcript in the cell. Expressed RNAi agents. In certain embodiments of the invention, 2 separate, complementary siRNA strands are transcribed using a single vector containing 2 promoters, each of which directs the transcription of a single siRNA strand, i.e. That is, operably linked to a template for siRNA to allow transcription to occur. The two promoters can be in the same orientation, in which case each promoter is operably linked to a template for one of the siRNA strands. Alternatively, the promoter can be in reverse orientation, flanked by a single template so that transcription from the promoter synthesizes 2 complementary RNA strands.

In other embodiments of the invention, vectors containing a promoter driving transcription of a single RNA molecule (eg, shRNA) comprising two complementary regions are used. In certain embodiments of the invention, vectors are used that contain multiple promoters, each of which drives the transcription of a single RNA molecule comprising two complementary regions. Alternatively, multiple different shRNAs can be transcribed from a single promoter or from multiple promoters. Various configurations are possible. For example, a single promoter can direct the synthesis of a single RNA transcript containing multiple self-complementary regions, each of which can hybridize to generate multiple stem-loop structures. The construct can be cleaved in vivo, for example by Dicer, to generate multiple different shRNAs. It will be appreciated that the transcript preferably contains a termination signal at the 3' end of the transcript rather than between individual shRNA units. In another embodiment of the invention, the vector includes a plurality of promoters, each of which directs the synthesis of self-complementary RNA molecules that hybridize to form shRNA. Multiple shRNAs can all target the same transcript, or can target different transcripts. Any combination of viral transcripts can be targeted. See Example 11 and Figure 21.

Those skilled in the art will additionally appreciate that in vivo expression of RNAi-inducing agents according to the present invention can be produced over a prolonged period of time (e.g. greater than a few days, preferably at least a few weeks to a few months, more preferably at least a year or longer, possibly a lifetime) ) cells that produce the drug. The cells can be protected indefinitely from influenza virus.

Preferred viral vectors for use in compositions to provide intracellular expression of RNAi-inducing agents include, for example, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, and the like. See, for example, Kobinger, G.P., et al., Nat Biotechnol 19(3):225-30, 2001, describing a Filovirus (Filovirus) envelope protein-based vector-pseudotyped HIV vector that efficiently converts The apical surface is transduced with intact airway epithelium. See also Lois, C, et al., Science, 295:868-872, Feb. 1, 2002, describing FUGW lentiviral vectors; Somia, N. et al. J. Virol. 74(9):4420-4424, 2000; Miyoshi, H., et al., Science 283:682-686, 1999; and US Patent 6,013,516.

Those skilled in the art will appreciate that agents such as the nucleic acids described herein, including but not limited to nucleic acids having any of the structures depicted in Figure 5, or any other effective structure described herein, can be fully Consists of nucleotides such as those found in naturally occurring nucleic acids, or may instead include one or more analogs of said nucleotides, or may otherwise differ from naturally occurring nucleic acids. Nucleic acids containing modified backbones or non-naturally occurring internucleoside linkages are useful in the present invention.

A modified nucleic acid need not be uniformly modified along the entire length of the molecule. For example, there may be different nucleotide modifications and/or backbone structures at each position in a nucleic acid. In certain embodiments of the invention, it may be desirable to stabilize the siRNA structure, for example, by including nucleotide analogs at one or more free strand ends to reduce digestion by, for example, exonucleases . To this end, deoxynucleotides, eg, pyrimidines such as deoxythymidine, may be included at one or more free ends. Alternatively or additionally, it may be desirable to include one or more nucleotide analogs in order to increase Or reduce the stability of the 19bp stem. It will be appreciated by those skilled in the art that the nucleotide analogs may be located at any position where the target-specific activity, e.g., RNAi-mediated activity, is not substantially affected, for example, at the 5' and/or 3' regions of the RNA molecule middle. For example, in certain embodiments, between 1-5 residues at the 5' and/or 3' ends of the siRNA or shRNA strands are nucleotide analogs. In certain embodiments of the invention, one or more of the nucleic acids in the RNAi-inducing agent of the invention comprises at least 50% unmodified RNA, at least 80% modified RNA, at least 90% unmodified RNA or 100% % unmodified RNA. In certain embodiments of the present invention, one or more nucleic acids of the nucleic acids in the RNAi-inducing agent of the present invention comprise 100% unmodified RNA in the portion involved in duplex formation in the RNAi-inducing agent.

According to certain embodiments of the invention, various nucleotide modifications are selectively used in the sense strand or the antisense strand of the siRNA, shRNA or microRNA precursor. For example, preferably unmodified ribonucleotides may be utilized in the antisense strand, while modified ribonucleotides and/or modified or unmodified deoxyribonucleosides may be used at some or all positions in the sense strand acid. According to certain embodiments of the invention, only unmodified ribonucleotides are used in the duplex portion of the antisense strand and/or the sense strand, while the overhang of the antisense strand and/or the sense strand may include modified Modified ribonucleotides and/or deoxyribonucleotides. In certain embodiments of the invention, one or both siRNA strands comprise one or more O-methylated ribonucleotides.

Many nucleotide analogs and nucleotide modifications are known in the art and have been studied for their effect on properties such as hybridization and nuclease resistance. Many modifications have been shown to alter one or more aspects of an oligonucleotide, such as the ability to hybridize to complementary nucleic acids, stability, bioavailability, nuclease resistance, and the like. For example, 2' modifications include halo, alkoxy and allyloxy. In some embodiments, the 2′-OH group is replaced by a group selected from H, OR, R, halogen, SH, SR ₁ , NH ₂ , _NHR , NR ₂ , or CN, where R is C ₁ -C ₆ alkyl, alkenyl or alkynyl and halogen is F, Cl, Br or I. Examples of modified linkages include phosphorothioate and 5'-N-phosphoramidite linkages. Nos. 6,403,779; 6,399,754; 6,225,460; 6,127,533; 6,031,086; 6,005,087; grooming. See also Crooke, S. (ed.) "Antisense Drug Technology: Principles, Strategies, and Applications" (1st Ed.), Marcel Dekker; ISBN: 0824705661; 1st Ed. (2001) and references therein. For the purposes of this invention, chemical elements are identified according to the Periodic Table of the Elements, CAS Edition, Handbook of Chemistry and Physics, 75th Edition, Inside Enclosure, and specific functional groups are generally defined as described therein. Analogs and modifications can be tested using, for example, the assays described herein or other appropriate assays to select those that are effective in reducing viral gene expression. In certain embodiments, the RNAi-inducing agent comprises one or more modifications to sugars, nucleosides, or internucleoside linkages, such as described in No. 20030175950, No. 20040192626, No. 20040092470, No. 20050020525 , US Publication No. 20050032733, and/or any modification in references 137-139.

In certain embodiments of the invention, nucleic acids are produced by modification with the following properties: increased absorbency (e.g., increased absorbency across the mucus layer, increased oral absorption, etc.), stability in the bloodstream or in cells increased renal system clearance, increased ability to cross cell membranes, increased ability to escape intracellular compartments such as endosomes, etc. As will be appreciated by those skilled in the art, analogs or modifications may result in a change in Tm, which may allow for increased tolerance of mismatches between the guide sequence and the target while still achieving effective inhibition, or may result in increased resistance to the desired target. Transcript specificity increases or decreases.

Those skilled in the art will additionally appreciate that effective RNAi-inducing agents for use in accordance with the present invention may comprise one or more moieties that are not nucleotides or nucleotide analogs. In certain embodiments, nucleic acids consist primarily of nucleotide residues, but also contain one or more residues that are not nucleotides. For example, in certain embodiments, 1, 2, 3, 4, 5, or more of the residues in either strand of the potent inhibitor are not nucleosides. In certain embodiments, the portion of the RNAi-inducing agent involved in duplex formation and/or complementary to the target transcript consists of nucleosides, while the overhang consists of non-nucleoside residues. In certain embodiments of the invention, the sense and antisense strands of the RNAi-inducing agent are linked to each other via a non-nucleoside-containing linker.

III. RNAi Inducing Agents and Other Nucleic Acids Based on Recognition of Preferred Influenza Virus Target Portions

The present invention provides various nucleic acids based on the recognition of probable targeting moieties, functional targeting moieties, moderately conserved targeting moieties, and highly conserved targeting moieties. In particular, the invention provides nucleic acids having sequences comprising or consisting of any of the possible targeting moieties described above, said sequences comprising Tables 1A, 1B, 17, 18, 20 and/or 34 Influenza sequences listed in , or subsequences (fragments) thereof that are at least 15 nucleotides in length. siRNAs targeting certain target moieties unexpectedly showed high potency. To assess efficacy, siRNA was administered to cells 6 hours prior to infection with influenza virus, and influenza virus production was tested 24 hours after infection. In certain embodiments, the sequence is selected from SEQ ID NO: 272-380. SiRNA targeting the target moiety (comprising an antisense strand 100% complementary to the target moiety) showed a 4-fold reduction (75% reduction) in virus production in cells at 100 nM. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NO: 274, 286, 287, 292, 297, 298, 304, 305, 309, 310, 311, 319, 324, 327, 334, 346, 347, 360, 361, 364 and 366. SiRNA targeting the target moiety (comprising an antisense strand 100% complementary to the target moiety) showed a 2-fold reduction (50% reduction) in virus production in cells at 5 nM. In certain embodiments, the sequence is selected from SEQ ID NO: 297, 309, 310, 311, 346, 347, 364 and 366. SiRNAs targeting the target moiety showed a 2-fold reduction (50% reduction) in virus production in cells at 5 nM, even when the target moiety differed in up to 2 positions from the corresponding target moiety in PR8, That is, the same is true when there are up to 2 mismatches between the antisense siRNA strand and the target moiety. Complements of the nucleic acids and fragments are also provided. In some embodiments, the fragments are 16, 17 or 18 nts in length. Nucleic acids can be single-stranded or double-stranded and can be unmodified RNA or DNA or modified versions thereof. The sequence may additionally include a 3' overhang, such as a dTdT overhang. The invention also provides nucleic acids that are substantially identical (e.g., at least 70%) to any of the sequences listed in Tables 1A, 1B, 17, 18, 20, and/or 34, or subsequences thereof that are at least 15 nt in length. , at least 80% identical, at least 90% identical, 100% identical), 100% complementary or substantially complementary (eg, at least 70%, at least 80% complementary, at least 90% complementary, 100% complementary). The present invention further provides vectors comprising one or more of the above-mentioned nucleic acids.

Nucleic acids include RNAi-inducing agents such as (i) siRNA; (ii) shRNA; (iii) single-stranded RNA that hybridizes to complementary single-stranded RNA to form siRNA; and (iv) a template comprising nucleic acid transcription for any of the foregoing nucleic acids Carrier. Where the sequence is represented as RNA, the invention also provides the corresponding DNA sequence (and vice versa). Where a sequence is presented herein, the invention encompasses a double stranded nucleic acid comprising the sequence and its complement. Any of the nucleic acids of the invention may be limited in size. For example, a nucleic acid can be 19 nt or less, 29 or 30 nt or less, 35 nt or less, 50 nt or less, or 100 nt or less in length.

The invention encompasses any nucleobase-containing structure in which residues such as nucleotides are linked together in an ordered fashion, usually in a linear fashion, so that a nucleobase sequence can be assigned to the structure, wherein the sequence is any of the sequences disclosed herein. Various nucleic acid bases and modified nucleotides and backbones are described above, any of which may be used. In various embodiments of the invention, the structure is a nucleic acid, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), or a chimeric molecule, among others. See, eg, WO92/20702, US Patent No. 6,316,230 and references therein. The invention also encompasses structures comprising alternating nucleic acid bases that have the same base pairing specificity, or that can otherwise replace nucleic acid bases present in the sequence.

Single-stranded nucleic acids can be used as the antisense or sense strand (optionally adding one or more nucleotides at the 3' end to form an overhang) of an RNAi-inducing agent such as siRNA or shRNA. The nucleic acids of the invention can also be used, eg, as conventional antisense reagents, as probes (eg, for detecting influenza virus infection), and the like. "Conventional antisense" refers to methods of inhibiting expression of transcripts by administering single-stranded oligonucleotides in vitro or to a subject. The inhibition is thought to operate by a mechanism distinct from the RNAi machinery and does not require a double-stranded RNA molecule (unlike the duplex formed between the antisense oligonucleotide and the target transcript). See, eg, Crooke, S., infra.

The invention thus provides a nucleic acid comprising a target portion of an influenza A virus transcript, wherein the sequence of the nucleic acid comprises at least 15, 16, 17, 18 or 19 adjacent nts. In certain embodiments, the sequence consists of, or is contained within, the sequences listed in one or more of Tables 1A, 1B, 17, 18, 20, and 34. The invention additionally provides a nucleic acid comprising a target portion of an influenza A virus mRNA transcript, wherein the sequence of the nucleic acid comprises at least 15, 16, 17, 18 or 19 contiguous nucleotides of any possible influenza virus target portion. Nucleotides at the 5' and 3' ends are considered contained in the sequence. In certain embodiments, the sequence is 30 nt or less, 35 nt or less, 50 nt or less, or 100 nt or less in length.

The invention additionally provides that the comprising sequence is substantially identical (at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identical) to a portion of the nucleic acid. Certain such portions are at 1, 2, 3, 4, or 5 positions relative to a possible influenza virus target portion (such as listed in any of Tables 1A, 1B, 17, 18, 20, and/or 34). target part) there is a difference. In certain embodiments of the invention, the difference at 1, 2, 3, 4 or 5 positions is by replacing a C in the target moiety with a U in the substantially identical sequence, or by replacing a C in the target moiety in the substantially identical sequence. G replaces A in the target section. The sequences of certain nucleic acids of the invention comprise sequences found in target transcripts with possible influenza virus target moieties (e.g., those listed in Tables 1A, 1B, 17, 18, 20, and/or 34). Sequences adjacent to the sequence, ie, located 5' or 3' to the target moiety.

The present invention provides RNAi-inducing agents having a window range of at least 15 nt, at least 16, at least 17 nt, at least 18 nt, preferably 19 nt, with potential influenza virus target moieties (e.g. Table 1A , 1B, 17, 18, 20 and/or 34) complementary antisense strand. For example, the antisense strand may be 100% complementary to the listed highly conserved target moieties within 15, 16, 17, 18, or 19 nt, or may be substantially complementary, e.g., relative to the target moiety, with Mismatch between 1 and 5. In certain embodiments, one or more mismatches, eg, all mismatches, between the inhibitory region of the antisense strand and the target are U-G mismatches. The sense strand of the RNAi-inducing agent can be 100% or substantially complementary to the antisense strand. The present invention also provides RNAi-inducing agents having a sense strand whose sequence is highly conserved and/or listed in one or more of Tables 1A, 1B, 17, 18, 20, and/or 34. Suitable conserved sequences are 100% identical or substantially identical within 15, 16, 17, 18 or 19 nt.

For example, the present invention provides an RNAi-inducing agent targeting influenza virus transcripts, wherein the RNAi-inducing agent comprises: a nucleic acid moiety whose sequence comprises a sequence selected from the group consisting of SEQ ID NO: 272-380, its complement, Or any fragment having a length of at least 15 nucleotides. The RNAi-inducing agent preferably comprises a second nucleic acid moiety forming a duplex structure with the first nucleic acid moiety. In certain embodiments, the first and second nucleic acid moieties are each 50 nt or less in length, eg, 35 nt or less in length, eg, 21-23 nt in length, etc. In certain embodiments, the sequence is selected from the group consisting of: SEQ ID NO: 274, 286, 287, 292, 297, 298, 304, 305, 309, 310, 311, 319, 324, 327, 334 , 346, 347, 360, 361 , 364, and 366, their complements, or fragments of either having a length of at least 15 nucleotides. In certain embodiments, the sequence is selected from the group consisting of SEQ ID NO: 297, 309, 310, 311, 346, 347, 364, and 366, the complement thereof, or any one having at least 15 Nucleotide-length fragments. In certain embodiments of the invention, RNAi-inducing agents designed based on possible influenza virus target moieties (e.g., target moieties whose sequences are listed in Tables 1A, 1B, 17, 18, 20, and/or 34) The sequence of the antisense strand includes at least 10, at least 12, at least 15, at least 17 or at least 19 contiguous nts that are 100% complementary to the listed sequence. The sense strand of the RNAi-inducing agent may be 100% or substantially complementary to the antisense strand. In certain embodiments of the invention, the sequences of the sense strand of the RNAi-inducing agent designed based on possible influenza virus target moieties (e.g., the sequences presented in Tables 1A, 1B, 17, 18, 20, and/or 34 ), including at least 10, at least 12, at least 15, at least 17 and/or at least 19 consecutive nts of the listed sequence.

In certain embodiments of the invention, the antisense strand of an RNAi-inducing agent designed based on a potential influenza virus target moiety (e.g., a target moiety presented in Tables 1A, 1B, 17, 20, and/or 34) Sequences, except possibly 1 or 2 mismatches, include at least 10, at least 12, at least 15, at least 17 and/or at least 19 contiguous nts that are 100% complementary to the listed sequence. The sense strand of the RNAi-inducing agent may be 100% or substantially complementary to the antisense strand. In certain embodiments of the invention, the sequences of the sense strand of the RNAi-inducing agent designed based on possible influenza virus target moieties (such as those listed in Tables 1A, 1B, 17, 18, 20, and/or 34 sequence) comprising at least 10 contiguous nucleotides, more preferably at least 12 contiguous nts, more preferably at least 15 contiguous nts of the listed sequence, except that 1 or 2 nt may differ from the listed sequence , more preferably at least 17 consecutive nts, and still more preferably 19 consecutive nts.

In embodiments of the invention where the antisense strand is substantially or 100% complementary to said sequence over less than 19 nt, the remainder of the antisense strand may and preferably is external to the listed target moieties. and adjacent influenza sequences are substantially complementary or 100% complementary. Accordingly, the invention encompasses RNAi-inducing agents comprising an antisense strand whose sequence is "shifted" by 1 or more nt, for example up to 9, from the sequence in Tables 1A, 1B, 17, 18, 20 and/or 34 nt of influenza sequence complementary. The adjacent sequences can be seen in Figure 32.

According to certain embodiments of the invention, the RNAi-inducing agent targets regions that are reasonably conserved and/or highly conserved among influenza variants that naturally infect at least 2, 3, 4, 5 or more organisms of different species. Species may include human, equine/horse, avian, porcine, and others. In certain preferred embodiments of the invention, the species includes humans.

The present invention also provides vectors that can be used for the transcription of the RNAi-inducing agent of the present invention, cells containing the vectors, and methods for treating and/or preventing influenza A virus infection.

The invention provides variants of possible influenza virus target moieties, such as variants of any of the nucleic acids listed in Tables 1A, 1B, 17, 18, 20 and/or 34, and nucleic acids comprising the variants (eg, , an RNAi-inducing agent comprising said variant), wherein the sequence of the variant differs from the listed sequence at 1, 2, 3, 4, 5 or 6 positions. In certain preferred embodiments of the invention, the variants of the listed sequences are compatible with those from influenza strains other than PR8 (such as in the influenza A strains listed in Tables 15A-15H and/or Tables 19A-19F). A portion of the sequence of the transcripts of any virus strain) is identical or substantially identical. In addition, subsequences of the various sequences disclosed herein are also contemplated. Preferably the subsequence is between 15-18 nt in length, eg 15, 16, 17 or 18 nt in length. Sequences resulting from nucleotide deletions and/or additions with respect to the particular sequences listed herein are also contemplated. By way of example, nucleic acids resulting from the deletion of 1, 2, 3, 4, 5 or 6 nts from or the addition of 1, 2, 3, 4, 5 or 6 nts to any of the sequences listed herein are contemplated sequence. Additionally, variants (as described above) with deletion of 1, 2, 3, 4, 5 or 6 nts from or additions of 1, 2, 3, 4, 5 or nt to any of the sequences listed herein are contemplated. The resulting sequence of 6 nt. The deleted or added nucleotides may be located contiguously or non-contiguously relative to the original sequence. Can be located internally or at one or both ends. The added nucleotides may be located anywhere within the sequence, or may be appended at one or both termini.

A nucleic acid of the present invention, such as an RNAi inducer or vector, can be introduced into a cell by any available method. For example, nucleic acids or vectors encoding said nucleic acids can be introduced into cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing foreign nucleic acid (such as DNA or RNA) into cells, including calcium phosphate or calcium chloride co-precipitation, DEAE - Dextran-mediated transfection, lipofection, injection or electroporation. Delivery agents, such as those described below, can be used.

The invention encompasses any cell manipulated to contain a nucleic acid of the invention, eg, an RNAi-inducing agent, such as siRNA, shRNA, or a vector that provides a template for the synthesis of an RNAi-inducing agent of the invention. The cell may be a mammalian cell, such as a human cell or a non-human mammalian cell, or a non-mammalian cell. Preferably, the cells are cells found in the nostrils and/or airways or lungs of a mammalian subject and are susceptible to influenza virus infection. Most preferably, the cells are airway epithelial cells. Optionally, the cells also contain influenza virus RNA.

IV. Diagnostic Methods and Kits

The present invention contemplates the identification of RNAi-based therapeutics for infectious diseases, such as infections caused by respiratory viruses, ideally incorporated into a diagnostic step for determining whether a subject in need of treatment is susceptible to one or more Infected with an infectious agent that inhibits the inhibitory effect of more than one RNAi-inducing entity. By "susceptible to inhibition" is meant that one or more biological activities of an infectious agent can be effectively inhibited by administering an RNAi-inducing entity to a subject. Preferably, the replication, pathogenicity, spread and/or production of the infectious agent is inhibited. For example, preferably, when the RNAi-inducing entity is administered to a subject at a tolerated dose, the replication, pathogenicity, transmission and/or production of the agent is inhibited by at least 25%. Preferably, inhibition is sufficient to produce a therapeutically effective effect.

Influenza virus is used as an example to illustrate the diagnostic method of the present invention adapted to select for RNAi-inducing entities in infected subjects. Of course, selected RNAi-inducing entities can also be administered for prophylaxis, eg, to individuals who have come into contact with infected individuals, regardless of whether the individual has developed symptoms of infection.

The present invention thus provides methods for diagnosing influenza virus infection and determining whether a subject is infected with influenza virus. In certain embodiments, the methods comprise determining whether a subject is infected with influenza virus inhibited by one or more of the RNAi-inducing entities of the invention. For example, samples (e.g., sputum, saliva, nasal washes, nasal swabs, throat swabs, bronchial washes, bronchoalveolar lavage ( BAL) fluid, biopsy specimens, etc.). A sample can be subjected to one or more processing steps. Any such processed sample is considered to have been obtained from the subject. The samples are analyzed to determine whether they contain influenza virus-specific nucleic acids. An "influenza virus-specific nucleic acid" is any nucleic acid or its complement that is derived from or obtained from an influenza virus and can be used as an indicator of the presence of an influenza virus in a sample, and optionally for identifying influenza strains and/or sequences of influenza genes. Nucleic acids may be subjected to processing steps after isolation. For example, can be reverse transcribed, amplified, split, etc. Preferably, the sequence is at least 15 nt long, such as 20-25 nt, 25-30 nt or longer. In certain embodiments, the sequence is different from that found in other viruses such that its presence is a clear indication of the presence of influenza virus.

In certain embodiments, the sequence of an influenza virus-specific nucleic acid, or its complement, present in a sample is compared to the sequence of the antisense or sense strand of an RNAi-inducing agent, such as siRNA or shRNA. The term "comparison" is used in a broad sense to refer to any method by which sequences can be evaluated, for example, by which it can be determined whether a sequence is identical or different from a reference sequence at one or more positions, or by which Assess the degree of difference.

Any of a variety of nucleic acid-based assays can be used. In certain embodiments, the diagnostic assay utilizes a nucleic acid comprising a suitably conserved and/or highly conserved target portion or its complement, or a fragment of a suitably conserved and/or highly conserved portion or its complement. In certain embodiments, nucleic acids are used as amplification primers or hybridization probes, for example, in an assay such as that described below.

In certain embodiments, influenza-specific nucleic acid in the sample is amplified. Any suitable amplification method may be used, including exponential amplification, combined linear amplification, ligation-based amplification, and transcription-based amplification. An example of an exponential nucleic acid amplification method is the polymerase chain reaction (PCR), e.g., described in: Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); PCR Cloning Protocols : From Molecular Cloning to Genetic Engineering, Methods in Molecular Biology, White, B.A., Ed., Vol. 67 (1998); Mullis EP 201,184; Mullis et al., U.S. Patent Nos. 4,582,788 and 4,683,195; Erlich et al., EP 50,424, EP 84,7 , EP 258,017, EP 237,362; and Saiki R. et al., US Patent No. 4,683,194. Combined linear amplification is disclosed by Wallace et al. in US Patent No. 6,027,923. Examples of ligation-based amplification are the Ligation Amplification Reaction (LAR) and the Ligase Chain Reaction (EP Application No. 0320308 B1) taught by Wu et al. (Genomics 4:560 (1989)). Hampson et al. (Nucl. Acids Res. 24(23):4832-4835, 1996) describe the directional random oligonucleotide primer amplification (DROP) method.

Isothermal target amplification methods include transcription-mediated amplification (TMA), autonomous sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), and variations thereof. (see Guatelli et al. Proc. NatL Acad. Sci. U.S.A. 87:1874-1878 (1990); No. 5,766,849 (TMA); and No. 5,654,142 (NASBA) U.S. Patents) and others (e.g., as Malek et al., No. 5,130,238 No. 5,399,491; Kacian and Fultz, U.S. Patent No. 5,399,491; Burg et al., U.S. Patent No. 5,437,990).

Detection or comparison can be performed using any of a variety of methods known in the art, for example, amplification-based analysis, hybridization analysis, primer extension analysis (e.g., allele-specific primer extension, where different influenza viruses The corresponding target portion of the strain is similar to different alleles of the gene), oligonucleotide junction analysis (US Patent Nos. 5,185,243, 5,679,524, and 5,573,907), splitting analysis, heteroduplex tracer analysis (heteroduplex tracking analysis, HTA), etc. Examples include Taqman® assays, Applied Biosystems (US Patent No. 5,723,591). In this assay, 2 PCR primers flank the central probe oligonucleotide. Probe oligonucleotides contain 2 fluorescent moieties. During the polymerization step of the PCR method, the polymerase cleaves the probe oligonucleotide. Splitting causes the two fluorescent moieties to become physically separated, thereby changing the fluorescence emission wavelength. As more PCR product is produced, the intensity of the new wavelength increases. Also available is cyclic probe technology (CPT), a nucleic acid detection system based on signal or probe amplification rather than target amplification (Nos. 5,011,769, 5,403,711, 5,660,988 and 4,876,187 US patent). Invasion split assays, such as the Invader_ assay (Third Wave Technologies) described in Eis, P.S. et al., Nat. Biotechnol. 19:673, 2001, can also be used to detect influenza-specific nucleic acids. Molecular beacons (US Patent Nos. 6,277,607; 6,150,097; US Patent Nos. 6,037,130) or fluorescence energy transfer (FRET) based assays can be used. Molecular beacons are oligonucleotide hairpins that undergo a conformational change upon binding to a perfectly matched template. The conformational change of the oligonucleotide increases the physical distance between the fluorophore moiety and the quencher moiety present on the oligonucleotide. The increased physical distance causes a decrease in the effect of the quencher, thus enhancing the signal from the fluorophore. US Publication No. 20050069908 and references therein describe various other methods that can be used to detect nucleic acids. Probes of the invention may thus comprise one or more moieties that hybridize to influenza-specific sequences and one or more moieties designed according to a particular assay. US Patent Nos. 5,854,033, 6,143,495 and 6,239,150 describe compositions and methods for amplification and multiplex detection of molecules of interest involving rolling circle replication. The method is suitable for simultaneous detection of multiple specific nucleic acids in a sample. For example, it can be used to determine the presence of one or more influenza-specific nucleic acids in a sample. Nucleic acid is sequenced as needed. US Publication No. 20050026180 describes a method for multiplexing nucleic acid reactions including amplification, detection, and genotyping, which can be adapted to detect influenza-specific sequences, as well as to identify specific locations of interest The sequence at which to determine the sensitivity to RNAi-inducing entities.

In certain embodiments, the analysis determines whether the influenza-specific nucleic acid in the sample comprises the same or a different portion as the sense or antisense strand of the RNAi-inducing entity. If necessary, identify the exact differences (if any). This information is used to determine whether an influenza virus is susceptible to inhibition by an RNAi-inducing entity. In addition to the assays discussed above, assays suitable for the detection and/or genetic analysis of infectious agents are described in Molecular Microbiology: Diagnostic Principles and Practice, Persing, D.H., et al., (eds.) Washington, D.C.: ASM Press, 2004. type of analysis. Any of the analyzes described can be performed using an automated system. Many systems for performing nucleic acid-based diagnostic assays are known in the art and can be readily employed for the purposes of the present invention. In one embodiment, nucleic acid from a sample is applied to a microarray (aka "chip") to which a number of nucleic acids complementary to various different influenza transcripts or portions thereof are attached. Hybridization patterns are examined and provide sufficient information to determine whether influenza viruses are susceptible to inhibition by RNAi-inducing entities. In certain embodiments, influenza-specific nucleic acid present in the sample is sequenced (usually after amplification). A variety of different assays can be used.

Diagnostic assays may use any of the nucleic acids described in Section III. In certain embodiments of the invention, the nucleic acid comprises a nucleic acid portion that is not substantially complementary or substantially identical to an influenza virus transcript. For example, nucleic acids can comprise primer binding sites (eg, for universal sequencing primers or amplification primers), hybridization labels (eg, useful for isolating nucleic acids from samples comprising other nucleic acids), and the like. In certain embodiments of the invention, nucleic acids comprise non-nucleotide moieties. A non-nucleotide moiety may be attached to a terminal nucleotide of a nucleic acid, for example, at the 3' end. Such moieties protect nucleic acids from degradation. In certain embodiments, non-nucleotide moieties are detectable moieties such as fluorescent dyes, radioactive atoms, members of fluorescence energy transfer (FRET) pairs, quenchers, and the like. In certain embodiments, the non-nucleotide moiety is a binding moiety, such as biotin or avidin. In certain embodiments, the non-nucleotide moiety is a hapten, such as digoxygenin, 2,4-dinitrophenyl (TEG), and the like. In certain embodiments, the non-nucleotide moiety is a label useful for nucleic acid isolation.

In certain embodiments of the invention, the nucleic acid is attached to an optionally magnetic support, eg a microparticle, such as a bead. The invention additionally provides arrays comprising a plurality of nucleic acids (eg, at least 10, 20, 50 nucleic acids, etc.) of the invention. The nucleic acid is covalently or non-covalently attached to a support, eg, a substantially planar support such as a glass slide. See, eg, US Patent Nos. 5,744,305; 5,800,992; 6,646,243.

Information about whether influenza viruses of a particular strain or of influenza viruses with a particular sequence within the target portion are susceptible to inhibition by a particular RNAi-inducing entity, such as an siRNA or shRNA comprising an antisense strand with a particular sequence, is obtained by referred to as "Sensitive Information". Sensitivity information may include quantitative information about the degree of sensitivity. For purposes of the present invention, if an RNAi-inducing entity, such as an siRNA or shRNA, reduces virus production in an infected cell by at least 25% when contacted with the cell or when administered to a subject at a tolerated dose, then Influenza viruses are thought to be susceptible to inhibition by RNAi-inducing entities.

In a preferred embodiment, if the influenza virus transcript comprises 100% identity to any one of SEQ ID NO: 272-380, preferably to SEQ ID NO: 274, 286, 287, 292, 297, 298, 304, Any one of 305, 309, 310, 311, 319, 324, 327, 334, 346, 347, 360, 361, 364 and 366 is 100% identical, still more preferably with SEQ ID NO: 297, 309, 310 , 311, 346, 347, 364, and 366 are 100% identical to any one of the target moieties, then the influenza virus is considered susceptible to an RNAi-inducing entity comprising an antisense strand 100% complementary to the target moiety. In other embodiments, if the influenza virus transcript is comprised at 1, 2 or 3 positions, preferably 1 or 2 positions, more preferably only 1 position different from any of SEQ ID NO: 272-380 Influenza viruses are considered susceptible to RNAi-inducing entities comprising an antisense strand 100% complementary to the target portion. In other embodiments, if the influenza virus transcript is comprised at 1, 2 or 3 positions, preferably 1 or 2 positions, more preferably only 1 position different from SEQ ID NO: 274, 286, 287, 292 , 297, 298, 304, 305, 309, 310, 311, 319, 324, 327, 334, 346, 347, 360, 361, 364 and 366, then the influenza virus is considered susceptible Sensitive to RNAi-inducing entities comprising an antisense strand 100% complementary to the target moiety. In other embodiments, if the influenza virus transcript is comprised at 1, 2 or 3 positions, preferably 1 or 2 positions, more preferably only 1 position different from SEQ ID NO: 297, 309, 310, 311 , 346, 347, 364, and 366, then the influenza virus is considered susceptible to an RNAi-inducing entity comprising an antisense strand 100% complementary to the target portion.

Information obtained from experiments or previous experience with influenza viruses having specific sequences within the target moiety can also be used to determine whether a virus is susceptible to inhibition by a given RNAi-inducing entity or combination thereof. Sensitivity information can also include theoretical predictions based on, for example, the expected effect of any mismatch that exists between the influenza virus sequence and the antisense strand of the inhibitor.

Sensitive information may be stored in a computer-readable form on a computer-readable medium, for example, in an organized manner in a database. The results of the diagnostic tests performed on the samples obtained from the subject are provided to a computerized system which accesses the information and determines the susceptibility profile of the influenza virus infecting the subject. In certain embodiments, the system recommends a particular RNAi-inducing agent or combination and/or dosage thereof. The present invention thus provides a computerized system for determining the susceptibility of a virus, such as influenza virus, to an RNAi-inducing entity. The invention additionally provides databases containing sensitive information. The computerized system and the automated system for performing the analysis can be part of a single integrated automated system or can be provided separately.

The present invention provides a diagnostic kit for detecting influenza virus infection. Certain kits comprise one or more nucleic acids of the invention. Certain kits comprise one or more nucleic acids useful for detecting portions of influenza virus transcripts comprising preferred target portions of RNAi. A kit may comprise one or more items selected from the group consisting of probes, primers, sequence-specific oligonucleotides, enzymes, substrates, antibodies, nucleotide populations, buffers, Positive and negative controls. Nucleotides can be labeled. For example, one or more populations of fluorescently labeled nucleotides, such as dNTPs, ddNTPs, etc., can be provided.

The probe can be a nucleic acid that includes all or part of a target moiety (e.g., a highly conserved or moderately conserved target moiety) or its complement, or is at least 80% identical or complementary (e.g., 100% identical or complementary) to the target moiety. Complementary) nucleic acids. In certain embodiments, multiple probes are provided. The probes differ at one or more positions and can be used to determine the exact sequence of the influenza virus transcript at that position. For example, a probe can hybridize differentially to a transcript (eg, hybridizes only when the probe is 100% complementary to a targeted portion of the transcript).

The primers can be complementary to sites located upstream and downstream of the target moiety, and can be used to amplify a region of influenza virus nucleic acid comprising the target moiety, which is then sequenced or subjected to additional processing. The length of the amplified region can be, for example, 100-200 nt, 200-300 nt or more. Primers are selected that bind a site at a sufficient distance from the target portion to amplify a region of the desired length. Methods for selecting amplification primers are well known in the art. Kits may contain sequence-specific oligonucleotides. Oligonucleotides are sequence-specific because when an oligonucleotide hybridizes to a substantially complementary nucleic acid (such as an influenza virus-specific nucleic acid), if the 3' terminal nucleotide of the oligonucleotide is completely complementary to the nucleic acid, Complementary, the oligonucleotides will only support extension or ligation mediated by polymerases. Preferably, multiple sequence-specific oligonucleotides are provided. The oligonucleotides differ in their 3' positions and thus can be used to establish the nucleotides located relative to that position when the oligonucleotide hybridizes to a nucleic acid of interest (e.g., an influenza virus-specific nucleic acid). identity of.

Kits of the invention may comprise sample collection materials, such as swabs, test tubes, and the like. The components of the kit may be packaged in individual vessels or tubes, which are typically provided in containers, such as plastic or styrofoam containers, suitable for commercial sale, along with instructions for use of the kit.

V. Transgenic animals

The invention encompasses transgenic animals engineered to contain or express an RNAi-inducing agent of the invention. The animal is suitable for studying the function and/or activity of the RNAi agent of the present invention, and/or for studying the influenza virus infection/replication system. As used herein, a "transgenic animal" is a non-human animal in which one or more, preferably most or all, of the animal's cells includes a transgene. A transgene is exogenous DNA, or a rearrangement (eg, deletion) of endogenous chromosomal DNA, preferably incorporated into or occurring in the genome of a transgenic animal cell. Preferably, the transgene comprises a promoter operably linked to the nucleic acid so that expression of the nucleic acid occurs in the cell.

The transgene can direct the expression of the RNAi-inducing agent in one or more cell types or tissues of the transgenic animal. Certain preferred transgenic animals are non-human mammals, eg rodents such as rats or mice. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, birds such as chickens, amphibians, and the like. According to certain embodiments of the invention, transgenic animals are used as animal models (eg, murines, ferrets, or primates) for testing potential influenza therapeutics. Other non-human animals encompassed by the present invention include domesticated animals, including but not limited to livestock and pets, or any animal used or kept for profit. The animals are partially or completely resistant to influenza virus infection. The RNAi inducer can be, for example, siRNA or shRNA. The RNAi-inducing agent can target any possible influenza virus target moiety, for example a target moiety listed in any of Tables 1A, 1B, 17, 18, 20 and/or 34. In certain embodiments, the RNAi-inducing agent targeting sequence is selected from the targeting portion of each of the following sequences: SEQ ID NO: 274, 286, 287, 292, 297, 298, 304, 305, 309, 310, 311, 319 , 324, 327, 334, 346, 347, 360, 361, 364 and 366, such as any one of SEQ ID NO: 297, 309, 310, 311, 346, 347, 364 and 366. For example, in preferred embodiments, the RNAi-inducing agent has an antisense strand that is complementary to any of the aforementioned target moieties and a sense strand that forms a duplex with the antisense strand.

Methods of making transgenic non-human animals are known in the art. Briefly, the method involves (i) introducing by microinjection an appropriate vector containing the transgene into the nucleus of a fertilized egg, followed by transferring the egg into the reproductive tract of a pseudopregnant female; and, (ii) introducing introduction of an appropriate vector into cultured somatic cells (e.g., using any suitable technique such as transection, electroporation), selection of cells in which the transgene has been incorporated into the genomic DNA, transfer of nuclei from the selected cells to oocytes or In fertilized eggs, oocytes or fertilized eggs are cultured in vitro to the morula or blastocyst stage, if desired, and the embryos are transferred to female recipients. According to other methods, retroviral vectors containing the transgene are used. Retroviral vectors are introduced into cells by infection, either as DNA plasmids or as virions. Cytoplasmic microinjection to introduce appropriate vectors into oocytes or embryo cells can also be used. Sperm-mediated gene transfer is also contemplated. Heterozygous or chimeric animals obtained using the methods are identified and bred to produce homozygotes.

The vector is preferably an RNAi-inducing vector targeting influenza virus transcripts. In a preferred embodiment, the vector comprises a template for transcription of an RNAi-inducing agent, such as siRNA or shRNA, targeting a target moiety that is appropriately conserved among influenza viruses from organisms of transgenic animal species and and/or highly conserved, and optionally suitably conserved and/or highly conserved between influenza viruses from another species, such as humans. The vector comprises a promoter operably linked to a template for transcription of the RNAi-inducing agent. The RNAi-inducing agent can be produced as a single RNA molecule comprising complementary moieties or as two RNA molecules that hybridize within a cell as described above. A promoter can, but need not, be obtained from the species of transgenic animal. RNA Pol I, II or III promoters can be used. Promoters can be constitutive or inducible.

In a preferred embodiment, the transgenic animal is an avian, such as a chicken. Methods for making transgenic birds are known in the art and include those described above and variations thereof. Vectors and methods suitable for producing transgenic birds and other transgenic animals are described, for example, in US Patent No. 6,730,822, US Publication Nos. 20020108132 and 20030126629, and references for such patents. In certain embodiments, transgenic birds are produced using retroviral vectors, such as avian leukosis virus vectors. In other embodiments, transgenic birds are produced using eukaryotic vectors other than retroviral vectors, although the vectors may also contain one or more sequences derived from retroviruses. In certain embodiments, the transgenic avian expresses multiple RNAi-inducing agents each comprising an antisense strand having a different inhibitory region sequence. The RNAi-inducing agents can each target a different strain of influenza virus.

The present invention provides transgenic flocks, wherein different members of the flock express one or more different RNAi-inducing agents each comprising an antisense strand having a different inhibitory region sequence. For example, a first portion of the flock expresses a first RNAi-inducing agent comprising an antisense strand that is 100% complementary to a target portion of a first avian influenza strain, and a second portion of the flock expresses an antisense strand that is 100% complementary to a target portion of a second avian influenza strain. A first RNAi inducer of an antisense strand that is 100% complementary to the target portion of the strain, and a third portion of the flock expresses a third RNAi comprising an antisense strand that is 100% complementary to the target portion of a third avian influenza strain inducers, etc. Flocks whose members express different RNAi-inducing agents are less susceptible to the emergence of resistant influenza strains than those in which all members express the same RNAi-inducing agent.

In another preferred embodiment, the transgenic animal is a mammal, such as pig/swine, cow, etc. Methods suitable for making transgenic mammals include those discussed above, which are additionally described in Gordon et al., Proc. Natl. Germline transition in single-cell embryos), Hooper et al., Nature, 326:292-295, 1987; Kuehn et al., Nature, 326:295-298, 1987 (transfer of genetically engineered embryonic stem cells to blastocysts Middle), and, Campbell et al., Nature, 380:64-66, 1996 (Transfer of nuclei from engineered cells into enucleated oocytes). Other references describe the effect of transgenic technology on pigs (U.S. Patent No. 6,558,663; Machaty, Z et al., CloningStem Cells, 4(1):21-7, 2002; Wall et al., Proc.Natl.Acad.Sci.U.S.A., 88 : 1696-1700, 1991), sheep (Wright et al., Biotechnology, 9: 830-834, 1991), goat (Wang, B., et al., Mol ReprodDev., 63(4): 437-43, 2002) Application of Wagyu (Krimpenfort et al., Biotechnology, 9:844-847, 1991; Galli, et al., Theriogenology, 59(2):599-616, 2003).

In another preferred embodiment, the transgenic animal is a rodent, such as a mouse. Mice and rats expressing RNAi inducers have been generated using various methods (see, e.g., Hasuwa et al., FEBS Lett. 2002 Dec 4;532(1-2):227-30, Xia, et al., Nat . Biotechnol., 20(10): 1006-10, 2002; Rubinson et al., Nat Genet, 33(3): 401-6, 2003).

VI. Compositions and methods for delivering RNAi-inducing entities

RNAi-inducing entities can be administered according to various methods. In one embodiment of the invention, a single RNAi-inducing agent is administered to the subject. A non-limiting example is a single siRNA species comprising an antisense strand complementary to an appropriate conserved and/or highly conserved target portion from various influenza strains. In related embodiments, a population of two or more different RNAi-inducing agents is administered to a subject. In one embodiment, the population of two or more RNAi-inducing agents includes agents comprising an antisense strand whose sequence is identical to that of various strains from a particular virus, such as influenza virus. Conserved and/or highly conserved regions are substantially complementary (preferably 100% complementary). In another embodiment, the population of two or more RNAi-inducing agents includes an agent comprising an antisense strand whose sequence is substantially complementary to a different conserved region from the same virus strain (preferably 100 % complementary). In another embodiment, the population of two or more RNAi-inducing agents includes agents comprising an antisense strand whose sequence is identical to that from various strains of a particular virus, such as influenza virus Appropriately conserved and/or highly conserved regions are substantially complementary (preferably 100% complementary), and RNAi-inducing agents include agents comprising an antisense strand whose sequence is substantially complementary to a different highly conserved region from the same strain. Complementary (preferably 100% complementary).

The present inventors have recognized that effective RNAi therapy, generally including the prevention and treatment of influenza virus infection, would be enhanced by the efficient delivery of RNAi-inducing agents and/or RNAi-inducing vectors to cells in intact organisms. In the case of influenza virus, it is necessary to introduce the agent into cells in the respiratory tract where influenza infection normally occurs. For use in humans, non-viral methods that promote intracellular uptake of the RNAi-inducing agent may preferably be used. The present invention thus provides compositions comprising any of a variety of non-viral delivery agents for enhancing intracellular delivery of RNAi-inducing agents and/or vectors in whole organisms, such as mammals and avians. As used herein, the concept of "delivery" includes the transport of an RNAi-inducing agent or RNAi-inducing vehicle from its point of entry into the body to the cellular location where it acts, the cellular uptake and/or production of the agent or any other means available to the intracellular RNAi machinery. Subsequent steps (eg, release of siRNA or shRNA from endosomes). Compositions of the invention may also include components that stabilize the RNAi-inducing agent so long as degradation of the agent is inhibited in vivo or during formulation of the agent for delivery (eg, RNase inhibitors such as RNAsin). In general, any agent that completely or partially inhibits RNase activity can be used. Examples include RNase inhibitors purified from human placenta or recombinant versions thereof. Although delivery agents are primarily used to enhance delivery of RNAi-inducing agents, they can also be used to enhance delivery of RNAi-inducing vectors.

In certain embodiments of the invention, the delivery agent increases stability, inhibits clearance, facilitates cellular uptake of the composition, facilitates intracellular release of RNAi-inducing entities, reduces cytotoxicity, or directs the composition to specific cell types, tissue or organ. "Inhibiting clearance" means reducing the rate at which a composition is removed from the body by the renal system. Delivery agents can inhibit uptake by cells of the reticuloendothelial system, such as macrophages. The RNAi-inducing entity itself can be modified (e.g., covalently) to increase stability, inhibit clearance, facilitate cellular uptake, facilitate release of the RNAi agent and/or carrier from intracellular compartments such as endosomes, and reduce cytotoxicity Or direct the composition to a specific cell type, tissue or organ. For example, the RNAi-inducing agent can be pegylated, and/or an arginine-rich peptide can be conjugated to the RNAi-inducing agent.

The invention thus encompasses compositions comprising (i) an RNAi-inducing agent targeting a transcript, and/or an RNAi-inducing vector whose presence in the cell results in the production of an RNAi-inducing agent targeting a transcript; and (ii) each Any of a variety of delivery agents, including but not limited to cationic polymers, modified cationic polymers, peptide molecular transporters (including arginine- or histidine-rich peptides), carbohydrates, lipids Substances (including cationic lipids, neutral lipids, and combinations thereof), liposomes, lipid-polycation-DNA complexes (lipopolyplexes), non-cationic polymers, surfactants suitable for introduction into the lung, or the above Any mixture of reagents, etc. Certain delivery agents incorporate moieties that increase the delivery or selective delivery of the RNAi-inducing agent or vector into cells in need of repressed transcripts. In certain embodiments, the transcript is a respiratory virus transcript, such as an influenza virus transcript.

While the use of specific delivery agents is preferred in certain embodiments of the invention, in other preferred embodiments, the RNAi-inducing entity, such as an RNAi-inducing agent, is administered in "naked" form, that is, in the absence of Any delivery agent that enhances transfection, cell entry, etc. For example, the RNAi-inducing agent can be administered in an aqueous medium that is substantially free of lipids and substantially free of delivery-enhancing polymers, eg, cationic or non-cationic polymers, such as those described below. RNAi-inducing agents can be administered intravenously in naked form or directly into the respiratory system (eg, by inhalation through the nose or mouth and into the lungs). In certain embodiments, the RNAi-inducing agent is administered in an amount effective to treat or prevent a respiratory viral infection while causing minimal blood absorption and thus minimal systemic delivery of the RNAi-inducing agent.

A. Delivery method

The present invention provides various methods for delivering compositions comprising RNAi-inducing entities to mammalian subjects. In certain embodiments, the composition is delivered directly into the vasculature and achieves inhibition of the target transcript in an organ or tissue (eg, lung) of the subject. In other embodiments, the composition is delivered directly into the respiratory system. Certain methods were used in Examples 16, 22, 23, and 24, wherein influenza virus production and luciferase or cyclophilin B activity in target organs of mammalian subjects using the methods expression is suppressed. These results demonstrate that the method is broadly applicable to inhibit virtually any desired target transcript.

Specifically, the present invention provides a method for inhibiting gene expression in a tissue or organ of a mammalian subject, comprising the steps of: introducing an effective amount of a gene-targeted RNAi-inducing agent into The composition is introduced directly into the subject's vascular system. Preferably, the RNAi-inducing agent inhibits expression of the target transcript in the lung. Tissue may be acirculatory tissue, that is, tissue other than blood. In a related embodiment, the invention additionally provides a method of inhibiting the production of a virus in the respiratory system of a mammalian subject, wherein the virus infects respiratory epithelial cells, the method comprising the step of: Infection technique, a composition comprising an effective amount of an RNAi-inducing agent targeting a viral gene is introduced into the vascular system of a subject by injection.

The present invention additionally provides a method of inhibiting gene expression in the lungs of a mammalian subject comprising the step of: introducing a composition comprising an effective amount of a gene-targeting RNAi inducer and a delivery agent directly into the respiratory system of the subject middle. In a preferred embodiment, the gene is a respiratory virus gene, such as an influenza virus gene. Preferably, the effective amount inhibits the production of influenza virus in the respiratory system of the subject. In certain embodiments of the methods, or any other aspect of the invention, the virus is a respiratory virus other than RSV.

For example, compositions can be administered nasally or orally, usually followed by inhalation. The composition may comprise particles that remain primarily in the upper respiratory tract, eg, nose, pharynx, etc., as in typical nasal or oral sprays. In other embodiments, the particles are inhaled into the lower respiratory tract. Respirable formulations that can be used to deliver the composition directly into the respiratory system are discussed below. In certain embodiments, direct delivery into the respiratory system results in systemic delivery, eg, the RNAi-inducing agent travels from the lungs into the vasculature and is transported to a target organ or tissue elsewhere in the body.

Methods for delivering an effective amount of an RNAi-inducing agent into the respiratory system of a mammalian subject have a variety of uses including, but not limited to, preventing or treating respiratory viral infections. Various other diseases and conditions affecting the respiratory system can also be prevented and/or treated using the methods of the invention to administer RNAi-inducing agents targeting appropriate transcripts. Examples include cancers such as lung cancer, cystic fibrosis (see, eg, U.S.S.N. 10/200,607), asthma (see, eg, U.S.S.N. 11/069,611), pulmonary hypertension, pulmonary fibrosis, emphysema, and the like. Suitable target genes include, for example, oncogenes, genes encoding pro-angiogenic molecules and/or growth factors (such as vascular endothelial growth factor), pro-inflammatory molecules, and the like. Of course, when RNAi-inducing agents are delivered systemically, transcripts that play a role in diseases affecting any part of the body can be targeted.

In some embodiments of the present invention, the effective amount is between 0.1 mg and 5 mg per kilogram of body weight of the subject. In other embodiments, the effective amount is between 0.1 mg and 10 mg per kilogram of subject body weight, or between 0.5 mg and 20 mg per kilogram subject body weight.

Compositions comprising RNAi-inducing entities may or may not include a delivery agent. Delivery agents suitable for use in the present invention include those described below and in co-pending U.S.S.N. 10/674,087. The delivery agents can be used in combination.

B. Cationic Polymers and Modified Cationic Polymers

The inventors have determined that any of the various cationic polymers and modified cationic polymers enhance the delivery of RNAi-inducing agents through a number of different routes. The present invention thus provides compositions comprising (i) an RNAi-inducing entity targeting a target transcript and (ii) a cationic polymer. The invention additionally provides methods of inhibiting expression of a target gene comprising administering to a mammalian subject a composition comprising an RNAi-inducing agent that targets a target transcript. In particular, the present invention provides methods of treating and/or preventing influenza virus infection comprising administering to a mammalian subject a composition comprising an RNAi-inducing agent targeting influenza virus transcripts and a cationic polymer.

Typically, a cationic polymer is a polymer that is positively charged at about physiological pH, eg, at a pH in the range of about 7.0 to 7.6, preferably about 7.2 to 7.6, more preferably about 7.4. The cationic polymers include (but are not limited to): polylysine (PLL), polyarginine (PLA), polyhistidine; polyethyleneimine (PEI) (37), including such as (for example) ( 76) Linear or branched PEI and low molecular weight PEI; polyvinylpyrrolidone (PVP) (38); chitosan (39, 40); protamine; polyphosphates; 20020045263 polyphosphates); poly(N-isopropylacrylamide), etc. Some of the polymers contain primary amine, imine, guanidine and/or imidazole groups. Preferably the cationic polymer has relatively low toxicity. References 85-87; USSN 6,013,240; WO9602655; and US Publication Nos. 20040167087 and 20030157030 provide additional information on PEI and other polymers suitable for use in the practice of the present invention. A commercially available PEI reagent called jetPEI ^™ (Qbiogene, Carlsbad, CA), which is a linear form of PEI (USSN 6,013,240), can be used.

Suitable cationic polymers also include blends of polymers having different molecular weights, copolymers comprising subunits of any of the above polymers (or other polymers), such as lysine-histidine copolymers wait. The percentages of the various subunits in the copolymer need not be equal, but can be selected, for example, to optimize properties such as the ability to form complexes with nucleic acids while minimizing cytotoxicity. Furthermore, the subunits do not have to alternate in a regular fashion. Appropriate assays for evaluating various polymers for the required properties are described in the Examples. Preferred cationic polymers also include polymers such as those described above, additionally incorporating any of the various modifications. Suitable modifications are discussed below and include, but are not limited to, modification with acetyl, succinyl, acyl or imidazolyl (32).

Although cationic polymers have been shown to facilitate DNA plasmid transfection, it is highly uncertain whether cationic polymers are effective in enhancing siRNA uptake given the substantial differences in structure and size between siRNA and shRNA molecules and DNA plasmids. However, as described in Example 12, the inventors have demonstrated that compositions comprising PEI, PLL, or PLA and siRNA targeting influenza virus RNA significantly inhibit small Production of influenza virus in mice. The inhibition was dose-dependent and exhibited an additive effect when using 2 siRNAs targeting different influenza virus RNAs. Thus, when combined with cationic polymers such as PEI, PLL or PLA, siRNA is able to reach the lung, enter cells, and effectively inhibit the viral replication cycle. While the presence of PEI significantly enhanced delivery to the lung, efficient delivery also occurred in the absence of PEI (Example 12; Figure 22C), suggesting that efficient siRNA delivery to the respiratory system can be achieved using "naked" siRNA. The findings are believed to be the first report of the efficacy of using siRNA to inhibit infectious virus production in mammals (eg, relative inhibition of production of viral transcripts or intermediates in the viral replication cycle). As described in Example 16, pulmonary administration of a mixture of siRNA and cationic polymer effectively inhibited the target transcript in lung cells. Examples 23 and 24 and Figure 31 provide further evidence that naked siRNA delivered into the respiratory system of mammalian subjects effectively inhibits the expression of target transcripts therein.

Other studies (see, e.g., McCaffrey 2002; McCaffrey 2003; Lewis, D.L., 2002) for delivering siRNA to solid organs and tissues in vivo by the intravenous route have employed a technique called hydrodynamic transfection, which involves the rapid delivery of large volumes of fluid into the tail vein of mice and has been shown to cause the accumulation of significant amounts of plasmid DNA in solid organs, especially the liver (Liu 1999; Zhang 1999; Zhang 2000). In contrast to the conventional technique (Liu 1999), which involves injecting approximately 200 μl of fluid, which involves delivering a volume of fluid almost equivalent to the animal's whole blood volume, e.g. 1.6 ml for a mouse weighing 18-20 grams, Equivalent to approximately 8-12% of body weight. In addition, injection using the hydrodynamic transfection method occurs within the short time interval (eg, 5 seconds) necessary for efficient expression of the injected transgene (Liu 1999). siRNA has also been delivered intravenously into subcutaneously implanted tumor cells in nude mice (Filleur 2003), but given the system's distinguishing features, the findings have implications for the intravenous delivery of RNAi inducers into native organs and tissues. relevance is unclear. Furthermore, the present invention demonstrates efficient intravenous delivery of RNAi-inducing agents at doses of 5 mg or less, such as 0.1-5 mg, per kilogram of subject body weight.

Although the mechanism by which hydrodynamic transfection achieves transfer and high-level expression of the injected transgene in the liver is not fully understood, it is believed to be due to the reflux of the DNA solution into the liver through the hepatic vein due to transient cardiac hyperemia (Zhang 2000). A comparable approach for human treatment does not seem likely to be feasible. However, the inventors have used conventional volumes of liquid (e.g. 200 μl) and have demonstrated that conditions would be expected to result in minimal expression of the injected transgene even in the liver (the liver being the easiest site to achieve expression using hydrodynamic transfection). siRNA was effectively delivered to the lungs.

The invention thus provides a method of inhibiting the expression of a transcript (e.g., a viral transcript, such as an influenza virus transcript) in a cell of a mammalian subject comprising using conventional injection techniques, such as using conventional pressure and/or conventional liquid A volumetric technique, the step of introducing into the vasculature of a subject a composition comprising an RNAi-inducing agent, such as siRNA or shRNA, targeted to a target transcript. In a preferred embodiment of the invention, the agent is administered intravenously to produce a therapeutically effective dose in a target organ, such as the lung. In certain embodiments of the invention, the composition comprises a cationic polymer. In a preferred embodiment of the invention, the composition is introduced in a liquid volume equivalent to less than 10% of the subject's body weight. In certain embodiments of the invention, the volume of fluid corresponds to less than 5% of the subject's body weight, less than 2% of the subject's body weight, less than 1% of the subject's body weight or less than 0.1% of the subject's body weight. In certain embodiments of the invention, the methods achieve the delivery of an effective amount of an RNAi-inducing agent in cells in bodily tissues or organs other than the liver, such as the lungs. In certain preferred embodiments of the invention, the compositions are introduced into a vein, eg, by intravenous injection. However, the composition can also be administered into an artery, using devices such as catheters, indwelling intravenous lines, and the like for delivery. In certain preferred embodiments of the invention, the RNAi-inducing agent inhibits virus production, eg, in the lung.

As described in Example 15, the inventors have also demonstrated that the cationic polymers PLL and PLA form complexes with siRNA and facilitate the uptake of functional siRNA in cultured cells. Transfection with complexes of PLL and NP-1496 or complexes of PLA and NP-1496 siRNA inhibited influenza virus production in cells. These results and the results in mice discussed above demonstrate the advantages of using a mixture of cationic polymer and siRNA to deliver siRNA to mammalian cells in a subject. The method described in Example 15 can be used to test additional polymers, for example, polymers that have been modified by the addition of groups such as acyl, succinyl, acetyl, or imidazolyl groups to reduce cytotoxicity and optimize the initial effective polymer. Certain preferred modifications result in a reduction in the positive charge of the cationic polymer. Certain preferred modifications convert primary amines to secondary amines. Methods for modifying cationic polymers to incorporate such additional groups are well known in the art. (See, eg, reference 32). For example, the ε-amino groups of various residues can be substituted, for example, by incorporation of desired modifying groups after synthesis of the polymer. In general, the percentage of substitution needs to be selected to be sufficient to achieve an appropriate reduction in cytotoxicity relative to the unsubstituted polymer without causing too much a reduction in the ability of the polymer to enhance delivery of the RNAi-inducing agent. Thus, in certain embodiments of the invention, between 5% and 75%, eg about 50%, of the residues in the polymer are substituted. Similar effects can be achieved by initially forming copolymers with appropriately selected monomeric subunits, that is, some of which have incorporated the desired modification. Cationic polymers used to facilitate delivery of RNAi-inducing agents can be modified so that they incorporate one or more residues that differ from the main monomeric subunits that make up the polymer. For example, one or more substitute residues may be added to the terminus of the polymer, or the polymers may be linked by residues other than the main monomers that make up the polymer.

Various additional cationic polymers may also be used. Examples include poly(β-amino ester) (PAE) polymers (such as those described in U.S.S.N. 09/969,431; 10/446,444; US Publication 20020131951 and References 34 and 93). While some poly(β-aminoester) (PAE) polymers have been shown to facilitate DNA plasmid transfection, would cationic polymers still be effective given the significant differences in structure and size between siRNA and shRNA molecules and DNA plasmids? Enhanced siRNA uptake is very uncertain. However, as described in Example 12, the inventors demonstrated that siRNA targeting NP (NP-1496) inhibited influenza virus production in mice when administered via the intravenous route along with poly(beta amino ester). In addition, the siRNA inhibited influenza virus production in mice when administered by the intraperitoneal route along with a second poly(beta amino ester).

Additional cationic polymers that may also be used to enhance delivery of the RNAi-inducing agents of the invention include polyamidoamine (PAMAM) dendrimers, poly(2-dimethylamino)ethylmethacrylate (pDMAEMA), and its quaternary polymers. Amine analogs poly(2-trimethylamino)ethyl methacrylate (pTMAEMA), poly[a-(4-aminobutyl)-L-glycolic acid (PAGA), and poly(4-hydroxy-1-proline amino acid esters). See Han (2000) for an additional description.

Modified cationic polymers such as poly(L-histidine)-graft-poly(L-lysine) polymers (Benns 2000), polyhistidine-PEG (Putnam 2003), folic acid-PEG can be used - Graft-polyethyleneimine (Benns 2002), polyethyleneimine-dextran sulfate (Tiyaboonchai 2003), etc. The polymers can be in branched or linear form and can be grafted or ungrafted. In certain embodiments, a polymer is complexed with an RNAi-inducing entity of the invention and then administered to a subject. Any of the polymers can be modified to incorporate PEG or other hydrophilic polymers. Cationic polymers can be modified in multiple ways.

The invention contemplates the modification of any of the delivery agents described herein to incorporate moieties that enhance delivery of an agent into a cell and/or enhance selective delivery of an agent into a particular cell. Any of a variety of moieties can be used, such as (i) antibodies or antibody fragments that specifically bind to molecules expressed by cells in need of inhibition (e.g., airway epithelial cells); (ii) specifically bind to molecules expressed by cells in need of inhibition; Molecular ligands. Methods for conjugating antibodies, ligands and/or delivery agents to nucleic acids or the various delivery agents described herein are well known in the art. See, e.g., "Cross-Linking", Pierce Chemical Technical Library, available at URL www.piercenet.com and originally published in the 1994-95 Pierce Catalog and references cited therein, and Wong SS, Chemistry of Protein Conjugation and Crosslinking, CRC Press Publishers, Boca Raton, 1991; and GT Hermanson, Bioconjugate Techniques, Academic Press, Inc., 1995.

C. Additional Reagents for Delivery of RNAi-Inducing Entities into the Respiratory System

The invention encompasses compositions comprising any of a variety of additional agents and an RNAi-inducing entity, wherein the agent enhances delivery of the RNAi-inducing entity, for example, to respiratory epithelial cells.

In certain embodiments, a peptide molecular transporter is included in the composition, the peptide molecular transporter being a peptide that can penetrate the plasma membrane from the cell surface. It usually consists of 11-34 amino acid residues, is highly enriched in arginine, and is often referred to as arginine-rich peptide (ARP) or penetratin (see ref. 42-51, 120, 134 -36).

In other embodiments, the composition comprises a surfactant suitable for introduction into the lung. Examples include the commercial formulations Infasurf_ (ONY, Inc., Amherst, NY); Survanta_ (Ross Labs, Abbott Park, IL) and ExosurfNeonatal_ (GlaxoSmithKline, Research Triangle Park, NC). Nos. 4,338,301; 4,397,839; 4,312,860; 4,826,821; 5,110,806, U.S.S.N. 4,312,860; 4,826,821; In general, any lipid-containing substance that does not cause parenchymal damage to the lungs can be used as a surfactant.

Detergents and thixotropic solutions can also be used for administration. Perfluorinated chemical liquids such as heptacosafluorotributylamine (Fluorinert) and related molecules may also be used. See (74, 126, 150) and US Patent 6,638,767 for additional discussion. In addition, the invention contemplates the use of protein/polyethyleneimine complexes incorporating RNAi-inducing entities of the invention for delivery into, for example, the respiratory system, eg, the lungs. Other delivery agents that may be used include natural and synthetic cyclodextrins and mixtures of said cyclodextrins with other delivery agents. For additional information see Singh, M, et al., Biotechnol Adv. 20(5-6):341-59, 2002; Eastburn, SD and Tao, BY, Biotechnol Adv., 12(2):325-39, 1994; and US Publication No. 20030157030. Various non-cationic polymers can be used such as poly(lactide) (PLA), poly(glycolide) (PLG) and poly(DL-lactide-co-glycolide) (PLGA) (Panyam 2002), polyvinyl alcohol, poly(N-ethyl-4-vinylpyridinium bromide), Pluronic, poly(ether-anhydride). Combinations of cationic and non-cationic polymers are used in certain embodiments, such as poly(lactic-co-glycolic acid) (PLGA) grafted poly(L-lysine) (Jeong 2002), and others Combinations, including any of PLA, PLG or PLGA and a cationic polymer such as those discussed above or a modified cationic polymer. Block copolymers may also be used, which may contain cationic and/or non-cationic monomers. Examples are described in US Patent Nos. 6,800,663; 6,692,770; 6,669,959; 6,616,941; 6,592,899;

VII. Compositions for inhalation delivery of RNAi-inducing entities

The invention provides compositions comprising RNAi-inducing entities for administration by inhalation. Preferably, the RNAi-inducing entity is an RNAi-inducing entity such as siRNA or shRNA. As noted above, the RNAi-inducing agent can be administered naked or with a delivery agent by inhalation through the nose or mouth directly into the respiratory system and into the lungs. In certain embodiments, the RNAi-inducing agent is effective in treating or preventing a condition affecting the respiratory system, such as a respiratory viral infection, while producing minimal absorption into the blood and thus minimal systemic delivery of the RNAi-inducing agent. amount to invest. In certain embodiments of the invention, the extent of absorption into the blood is such that no clinically significant effect is observed in organs or tissues outside the respiratory system when the RNAi agent is administered at a dose effective in the lungs .

Specifically, the present invention provides a dry powder composition comprising an RNAi-inducing entity, preferably an RNAi-inducing agent. The medicament of the invention is preferably delivered as an aerosol spray from a pressurized container or dispenser, or nebulizer, containing a suitable propellant, eg, a gas such as carbon dioxide. In certain embodiments, the delivery system is adapted to deliver the composition into the major airways (trachea and bronchi) and/or deep into the lungs (bronchioles and/or alveoli) of the subject. In certain embodiments, compositions comprising an RNAi-inducing entity are delivered using a nasal spray. Delivery agents can be included in pharmaceutical compositions. However, the present inventors have also found that RNAi inducers are effective in inhibiting influenza virus when delivered through the respiratory tract into the respiratory system in the absence of specific delivery agents (Example 23). In certain embodiments, the RNAi-inducing agent is delivered to the lung in a composition consisting essentially of the RNAi-inducing agent (e.g., naked siRNA or shRNA) in dry form (e.g., dry powder) or in an aqueous medium. ) consisting essentially of water, optionally including salts (eg, NaCl, phosphate), buffers, and/or ethanol.

Aerosol formulations for delivery to the airways and lungs may contain liquid or dry particles of various sizes and properties. Dry particulate compositions containing particles having a diameter of less than about 1 mm are also referred to herein as dry powders. By "dry" is meant that the composition has a relatively low liquid content so that the particles can be readily dispersed, eg, in a dry powder inhaler device to form an aerosol or spray. "Powder" means a composition consisting essentially or entirely of finely divided solid particles that are relatively free-flowing and capable of being easily dispersed in an inhalation device and subsequently inhaled by a subject (e.g., a patient) , (preferably) so that the particles can reach the alveoli of the lung, that is, suitable for pulmonary delivery. Powder compositions can be characterized in terms of various parameters such as fine particle fraction (FPF), injected dose, average particle density, and mass median aerodynamic diameter (MMAD). Suitable methods are known in the art and are found, for example, in references 31 and 58 and US Publication Nos. 20020146373, 20030012742 and 20040092470. In certain embodiments of the invention, particles are used having a mass mean aerodynamic diameter between 1 μm and 25 μm, preferably between 1 μm and 10 μm. In certain embodiments, macroporous particles ( 31 , 58 ) are used having a mean geometric diameter between 3 and 15 μm and a tap density between 0.04 and 0.6 g/cm ³ .

Methods for making dry particles are known in the art. Suitable methods include spray drying, spray-freeze drying, phase separation, single or double emulsion solvent evaporation, solvent extraction, and simple and complex coacervation methods. Particulate compositions can also be produced using granulation, extrusion and/or spheronization methods. Methods suitable for preparing dry powder oligonucleotide formulations are known in the art. See, eg, US Publication No. 20040092470. It is preferred to use methods that do not substantially reduce the physical integrity of the nucleic acid and its ability to repress the target transcript. Temperature or pH extremes known to cause significant degradation of nucleic acids need to be avoided. It will be appreciated that the degree of degradation may generally vary with the particular conditions and time the nucleic acid is exposed to such conditions, thus minimizing exposure time may be desirable and may allow for more extreme value conditions to be used. Compositions can be tested to determine whether the chosen method is appropriate to maintain adequate efficacy. Preferably, the chosen formulation method results in a composition in which the moiety consisting of nucleic acids has an activity level of at least 10%, preferably at least 20%, 50% or more, of the level of activity of the input nucleic acid.

The conditions used to prepare the particles can be selected to produce particles of a desired size or property (eg, hydrophobicity, hydrophilicity, external morphology, "stickiness," shape, etc.). The method of making the particles and the conditions used (eg, solvent, temperature, concentration, air flow rate, etc.) can also depend on the particular active agents and other components included in the composition. If the particles prepared by any of the above methods have a size range outside the desired range, the particles can be classified, for example, using sieves, by grinding, etc. Combinations of methods may be used.

The dry powder may consist essentially of one or more RNAi-inducing agents. In certain embodiments, formulations include one or more additional agents, eg, stabilizers, delivery-enhancing agents such as those described above, excipients, and the like. The term "excipient" as used herein refers to a substance other than an active agent or a delivery-enhancing agent present in the formulations of the invention. Excipients suitable for pulmonary delivery are known in the art. Any of a number of art-recognized compounds can be included in the formulations of the present invention. Typically, compositions of active agent (ie, RNAi-inducing agent) at a concentration of between 0.1% and 100% by weight may be used.

Methods for testing particles for their ability to, for example, reduce the level of target transcripts and/or inhibit influenza virus production are described in Example 10. Similar methods can be used for any of the aerosol formulations of the present invention. Dry particle compositions can be dissolved in a suitable solvent and delivered as a liquid aerosol or by other suitable means of delivery.

Liquid particles can also be delivered, for example, as an aerosol formulation. Typically, the particles may be in a size range similar to that described above for dry particles. In certain embodiments, the liquid particles are between about 0.5-5 μm for respiratory delivery, although smaller or larger particles may also be used. Suitable aqueous vehicles include water or saline, optionally including alcohol. Additional considerations for pulmonary delivery are discussed in Bisgaard, H., et al., (eds.), Drug Delivery to the Lung, Vol. 26 in "Lung Biology in Health and Disease", Marcel Dekker, New York, 2002.

Particles comprising an RNAi-inducing agent of the invention can also be administered intravenously, if desired. For intravenous delivery, a size of about 10 nm-50 [mu]m is generally preferred.

VIII. Therapeutic Applications

Compositions comprising RNAi-inducing entities of the invention are useful for inhibiting or reducing respiratory virus infection or replication.

In such applications, an effective amount of a composition of the invention is delivered to a cell or organism prior to, concurrently with or after exposure to a virus such as influenza virus. Preferably, the amount of the RNAi-inducing entity is sufficient to reduce or delay one or more symptoms of infection. For purposes of description, this section will often refer to the siRNAs of the invention, but the invention encompasses similar applications of other RNAi-inducing entities targeting viral transcripts. It should also be appreciated that influenza virus is used herein as an example, but the methods are applicable to any of a wide range of other respiratory viruses.

Compositions of the invention may comprise a single RNAi-inducing agent targeting a single site in a single influenza transcript, or may comprise an RNAi-inducing agent targeting one or more sites in one or more influenza transcripts. Multiple different species. In some embodiments of the invention, it will be desirable to utilize compositions containing collections of different RNAi-inducing agents (eg, multiple different siRNAs) targeting different influenza genes. For example, it may be desirable to attack the virus at multiple points in its life cycle using various siRNAs against different viral transcripts. According to certain embodiments of the invention, the compositions contain siRNAi targeting each fragment.

In certain embodiments, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 different RNAi-inducing agent species, e.g., 2, 3, 4, 5, 6, 7, 8 , 9 or 10 different siRNAs. In certain embodiments of the invention, the RNAi-inducing agent targets a portion of the influenza virus genome having a sequence selected from the group consisting of SEQ ID NOs: 272-380. In certain embodiments of the invention, the RNAi-inducing agent targets a portion of the influenza virus genome having a sequence selected from the group consisting of any selected subset of SEQ ID NOs: 272-380. Even if not expressly stated, all such subsets are included herein for any and all purposes.

In certain embodiments of the invention, the composition comprises at least one RNAi-inducing agent targeting PA and at least one RNAi-inducing agent targeting another influenza virus gene, eg, NP, PB1 or PB2. In certain embodiments of the invention, the composition comprises at least one RNAi-inducing agent targeting PB1 and at least one RNAi-inducing agent targeting another influenza virus gene, eg, NP, PA, or PB2. In certain embodiments of the invention, the composition comprises at least one RNAi-inducing agent targeting PB2 and at least one RNAi-inducing agent targeting another influenza virus gene, eg, NP, PB1 or PA. In certain embodiments of the invention, the composition comprises at least one RNAi-inducing agent targeting NP and at least one RNAi-inducing agent targeting another influenza virus gene, eg, PA, PB1 or PB2.

According to certain embodiments of the invention, siRNA compositions of the invention may contain more than one siRNA species targeting a single viral transcript. For example, it may be desirable to include at least one siRNA targeting the coding region of the target transcript and at least one siRNA targeting the 3' UTR. The strategy provides additional assurance that products encoded by relevant transcripts will not be produced, since at least one siRNA in the composition will target the transcript for degradation, while at least one other siRNA inhibits any siRNA that avoids degradation. Translation of transcripts.

Accordingly, the invention encompasses combinations of RNAi-inducing entities of the invention, including, but not limited to, methods of administering multiple RNAi-inducing agents, such as multiple siRNAs or shRNAs, and methods of directing siRNAs that inhibit multiple influenza transcripts from a single vector. A method of synthesis of RNA that is or can be processed to produce multiple siRNAs. See Example 11 for additional details. According to certain embodiments of the invention, the composition comprises an RNAi-inducing agent targeting at least one influenza virus A transcript and an RNAi-inducing agent targeting at least one influenza virus B transcript. In certain embodiments, the composition comprises an RNAi-inducing agent that targets influenza A virus transcripts and influenza B virus transcripts. According to certain embodiments of the invention, the composition comprises multiple siRNAs with different sequences targeting the same portion of a particular fragment. According to certain embodiments of the invention, the composition comprises multiple RNAi-inducing agents that inhibit different strains or subtypes of influenza virus.

Influenza viruses undergo antigenic shift and antigenic drift, and may develop resistance to therapeutic agents. It is contemplated that after a composition of the invention has been used for a period of time, mutations and/or reassortments may occur such that variants may arise that are not inhibited by the particular RNAi-inducing agent provided. The invention therefore encompasses evolving treatment regimens. For example, in a particular case, one or more novel RNAi-inducing agents may be selected in response to a particular mutation or reassortment. For example, it is often possible to design new RNAi-inducing agents that are identical to the original agent, just incorporating any mutations that have occurred or targeting newly acquired RNA segments; in other cases, it will be necessary to target novel RNAi in the same transcript sequence; in other cases, the novel transcript will need to be targeted entirely.

It will often be desirable to administer an RNAi inducer of the invention in combination with one or more other antiviral agents to inhibit, reduce or prevent one or more symptoms or characteristics of infection. In some preferred embodiments of the present invention, the RNAi-inducing agent of the present invention is combined with one or more other antiviral agents such as NA inhibitors, M inhibitors and the like. Examples include amantadine or rimantadine and/or zanamivir, oseltamivir, peramivir (BCX-1812, RWJ-270201) Ro64 -0796(GS 4104) or RWJ-270201. However, the administration of the RNAi inducer composition of the present invention may also be administered with one or more than one including, for example, various known influenza vaccines (eg, conventional vaccines using influenza viruses or viral antigens, and DNA vaccines). Any combination of agents in the various agents. For additional information see Palese, P. and Garcia-Sastre, 2002; Cheung and Lieberman, 2002, Leuscher-Mattli, 2000; and Stiver, 2003.

In various embodiments of the invention, the RNAi-inducing agent is present in the same mixture as the other agent, or the treatment regimen for the individual includes the RNAi-inducing agent and the other agent not necessarily delivered in the same mixture or simultaneously. Thus, as used herein, the term "combination" is not intended to mean that the compounds must be present in a single composition, or as a single composition, e.g., as part of the same dosage unit (e.g., in the same aerosol formulation, particle combination medicaments, tablets, capsules, pills, solutions, etc.) to a subject (although this can also be done). Rather, in certain embodiments of the invention, the agents are administered separately but simultaneously. As used herein, the terms "co-administration" or "simultaneous administration" of two or more compounds are not intended to mean that the compounds must be administered at the exact same time. Typically, compounds are co-administered or administered simultaneously if the compounds are also present in the body in less than de minimis amounts. Thus, the compounds can, but need not, be administered together as part of a single composition. In addition, the compounds can (but need not) be administered simultaneously (e.g., within less than 5 minutes, or within less than 1 minute) or within a short time (e.g., less than 1 hour, less than 30 minutes, less than 10 minutes, about 5 minutes apart) relative to each other. minute). According to various embodiments of the invention, compounds administered within said time interval may be considered to be administered substantially simultaneously. Those skilled in the art will readily be able to determine appropriate time intervals between administrations of the compounds so that each will be present in the body in more than trace amounts, or preferably, in effective concentrations.

The RNAi inducers and vectors of the present invention provide a complementary strategy for vaccination and can be administered to individuals who have or have not been vaccinated with any of a variety of vaccines currently in use or in development (Palese, P. and Garcia-Sastre , A., J. Clin. Invest., 110(1): 9-13, 2002, reviewed). Current vaccine formulations in the United States contain inactivated virus and must be administered by intramuscular injection. The vaccine is divided into three parts and contains, in addition to influenza B types, representative strains from the two subtypes (H3N2 and H1N1) of influenza A currently circulating. Each season-specific recommendation identifies specific strains that are suitable for use in that season's vaccine. Other vaccine approaches include cold-adapted live influenza viruses, which can be administered by nasal spray; live influenza virus vaccines genetically engineered to contain deletions or other mutations in the viral genome; replication-deficient influenza viruses, and DNA vaccines, in which Intramuscular or topical administration of plasmid DNA encoding one or more of the viral proteins (see, e.g., Macklin, M.D., et al., J Virol, 72(2):1491-6, 1998; Ilium , L., et al., Adv Drug Deliv Rev, 51(1-3):81-96, 2001; Ulmer, J., Vaccine, 20:S74-S76, 2002). Immunocompromised patients and elderly individuals may gain particular benefit from RNAi-based therapeutics because they may experience reduced efficacy of influenza virus vaccines.

In some embodiments of the invention, it may be desirable to target administration of the compositions of the invention to cells infected with influenza virus, or at least to cells susceptible to infection by influenza virus (e.g., cells expressing sialic acid-containing receptors). Cell). In other embodiments, an effective maximum delivery selection width will be required.

As noted above, the treatment regimens of the invention include administering an effective amount of an RNAi-inducing agent or vector prior to, concurrently with, or after exposure to influenza virus. For example, uninfected individuals can be "immunized" with the compositions of the invention prior to exposure to influenza; at-risk individuals (e.g., elderly, immunocompromised individuals, recently suspected, likely or persons known to be in contact with persons infected with influenza virus, etc.) may be treated substantially at the same time as the exposure, for example within 2 hours or within 2 hours of exposure. In other embodiments, the subject is treated at a later time, eg, 2-12, 12-24, 24-36, or 36-48 hours after the suspected or known exposure. Subjects can be symptomatic or asymptomatic. In certain embodiments, the subject is protected by administering the RNAi-inducing agent or vehicle up to 48 hours, up to 24 hours, up to 12 hours, up to 3 hours, etc. between exposures. Of course, individuals suspected or known to be infected may receive treatment of the invention at any time.

Certain preferred influenza virus inhibitors inhibit viral replication such that the level of replication in cells containing the inhibitor is at least about 2-fold, preferably at least about 4-fold, more preferably at least about 4-fold lower than the level of replication in control cells not containing the inhibitor. At least about 8 times, 16 times, 64 times, 100 times, 200 times or to an even greater degree. Certain preferred influenza virus inhibitors, following administration of the agent and/or infection, prevent (e.g., reduce to undetectable levels) or significantly reduce viral replication (e.g., by 10% relative to the level that would occur in the absence of the RNAi-inducing agent or 10% or less, 25% or less, 50% or less, 75% or less) for at least 24 hours, at least 36 hours, at least 48 hours or about 60 hours.

In certain embodiments of the invention, sustained release formulations are used for prophylactic purposes, e.g., formulations that release a sufficient amount of active agent over a period of time to protect a subject from influenza virus infection, or to alleviate the symptoms of such infection . For example, the formulation can release an effective amount of the agent over a period of days, a week, 1-2 weeks, or longer. Biodegradable polymeric delivery systems comprising RNAi-inducing agents or carriers can be used.

Thus, the RNAi-inducing entities of the invention are therapeutically effective in at least 3 distinct situations: (i) The RNAi-inducing entities can be administered to subjects who are not suspected or known to have been exposed to influenza virus. In such cases, the RNAi-inducing entity preferably prevents the development of, or lessens the severity of, a clinically significant infection; (ii) the RNAi-inducing entity may, for example, be administered within a leading time interval of up to one week to those suspected or known to have been exposed to influenza Virus subject. The RNAi-inducing entity preferably prevents the development or lessens the severity of a clinically significant infection, (iii) the RNAi-inducing entity may be administered to a subject that has become clinically ill. The RNAi-inducing entity inhibits influenza virus replication and preferably reduces the severity and/or duration of at least one symptom of influenza virus infection. Subjects with infections of the upper respiratory tract, lower respiratory tract, or both may be treated. In certain embodiments of the invention, the subject has viral pneumonia caused by influenza virus infection.

In certain embodiments of the invention, gene therapy is used to prevent influenza or to treat individuals who are already sick. A gene therapy regimen may include administering to a subject an effective amount of a gene therapy vector capable of directing expression of an inhibitory RNAi inducer prior to, substantially simultaneously with, or after influenza virus infection.

As mentioned above, influenza viruses infect a variety of species besides humans. The invention includes the use of compositions of the invention for the treatment of non-human species, especially species such as chickens, pigs and horses.

IX. Pharmaceutical formulations

As discussed above, inhalational delivery of RNAi-inducing entities is preferred in certain embodiments of the invention, while intravenous delivery is preferred in other embodiments of the invention. Inhalation delivery may be more suitable for patients who are in relatively good health, while intravenous delivery may be more suitable for patients who are unable to inhale adequately and/or suffer from conditions that may prevent effective delivery via the respiratory route (e.g., excessive mucus production; Situations where multiple parts are solidified due to bacterial infection or clogged with scar tissue, etc.) or where a relatively constant concentration of agent needs to be maintained.

However, the compositions of the present invention may be formulated for delivery by any effective route including, but not limited to, parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial , ophthalmic, transdermal (topical), transmucosal, rectal and vaginal routes. Preferred routes of delivery include parenteral, transmucosal, nasal, bronchial and oral routes. The pharmaceutical composition of the present invention generally includes an RNAi-inducing agent or a carrier that will cause the RNAi-inducing agent to be produced after delivery, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

Pharmaceutical compositions are formulated to be compatible with the intended route of administration. Solutions or suspensions for parenteral (e.g. intravenous), intramuscular, intradermal or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); salts, citrates, or phosphates; and agents for adjusting osmotic pressure, such as sodium chloride or glucose. The 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 compositions suitable for injectable use generally include sterile aqueous solutions (if 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 NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be liquid to the extent that easy syringability exists. Pharmaceutical formulations are preferably stable under the conditions of manufacture and storage and must be protected 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, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired 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 in the composition, for example, sugars, polyols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which 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 that 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 yield a powder of the active ingredient plus any additional required ingredient from a previously sterile-filtered solution thereof. .

Oral compositions generally include an inert diluent or an edible carrier. For oral therapeutic administration, the active compounds can be combined with excipients and used in the form of tablets, buccal tablets or capsules such as gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binders and/or auxiliary substances may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients, such as starch or Lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterote; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as Mint, methyl salicylate, or orange flavoring. Formulations for oral delivery may advantageously incorporate agents to improve stability in the gastrointestinal tract and/or enhance absorption.

Systemic administration of any of the RNAi-inducing entities of the invention may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. 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 generally known in the art. The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In addition to the delivery agents described above, in certain embodiments of the invention, active entities are prepared with carriers that protect the compound from rapid elimination from the body, such as controlled release formulations, including implant systems and microcapsules chemical delivery system. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Sustained-release formulations, which release the active agent over hours, days, weeks, or even longer periods of time, may be particularly useful for prophylactic purposes. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. Such suspensions may be prepared according to methods known to those skilled in the art, for example, as described in US Patent No. 4,522,811.

It is advantageous to formulate oral or parenteral compositions 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 subjects to be treated; each unit containing the pharmaceutical carrier calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. A predetermined amount of active compound.

Toxicity and therapeutic efficacy of the compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, for determining the _LD50 (the dose lethal to 50% of the population) and the _ED50 (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 exhibiting high therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken in designing delivery systems that target the compounds to the site of the affected tissue in order to minimize possible damage to uninfected cells and thereby reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the _ED50 with little or no toxicity. The dosage may vary within that range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, 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 as determined in cell culture that includes the _IC50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine effective doses in humans. Amounts in plasma can be measured, for example, by high performance liquid chromatography.

The therapeutically effective amount of the pharmaceutical composition is usually 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 2 In the range of 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The pharmaceutical composition can be administered at various intervals and over different periods of time as desired, for example, multiple times a day, once a day, once every other day, once a week for about 1 to 10 weeks, 2 to 8 weeks, about 3 weeks By 7 weeks, about 4, 5 or 6 weeks etc. Those skilled in the art will appreciate that certain factors may affect the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the subject's overall State of health and/or age, and the presence of other medical conditions. In general, treatment of a subject with an RNAi-inducing entity as described herein may include a single treatment or, in many cases, may include a series of treatments.

Exemplary doses include milligrams or micrograms of a nucleic acid of the invention, such as siRNA, per kilogram of subject or sample weight (e.g., about 1 μg/kg per kilogram to about 500 mg/kg per kilogram, about 100 mg/kg to about 5 mg/kg , or about 1 mg/kg to about 50 mg/kg). For topical administration (eg, intranasally), much smaller doses than recited may be used.

In addition, it will be appreciated that appropriate dosages of RNAi-inducing agents depend on the potency of the agent, and can be tailored to a particular receptor as necessary, eg, by administering increasing dosages until a preselected desired response is achieved. It is to be understood that the particular dosage level for any particular animal subject may depend on various factors including the activity of the particular compound employed, the age, weight, general health, sex 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 invention includes the use of compositions comprising nucleic acids of the invention, eg, siRNA or shRNA, for the treatment of non-human animals including, but not limited to, horses, pigs, and birds. Accordingly, dosages and methods of administration can be selected according to known principles of veterinary pharmacology and medicine. Guidance can be found, for example, in Adams, R. (ed.), Veterinary Pharmacology and Therapeutics, 8th ed., Iowa State University Press; ISBN: 0813817439; 2001.

The pharmaceutical compositions of the invention can be included in a container, pack or dispenser together with instructions for administration.

illustration

Example 1: Design of siRNAs to Inhibit Influenza A Virus

Genomic sequences from a panel of influenza strains are compared in positive strand form and the most conserved regions within each segment are identified. The group of viruses includes viruses from birds, pigs, horses and humans. To perform the comparison, sequences of individual fragments from 12 to 15 strains of influenza A virus of different animal (non-human) species isolated in different years were compared with individual fragments from 12 to 15 strains of human isolated in different years. Sequence alignment of fragments. Strains were chosen to cover multiple HA and NA subtypes. Regions were selected that differed by 0, 1 or 2 nucleotides between the different strains. As an example, the following strains were used to select siRNAs targeting NP transcripts, the accession number before each strain name refers to the accession number of the NP sequence, and the length of the compared sequence is indicated by the nucleotide number.

The order of entries in the list below is: accession number, strain name, length of compared sequences, year, subtype. Accession numbers for other genomic fragments vary but are readily found in the databases mentioned above. The strains compared were:

NC_002019 A/Puerto Rico/8/34 1565 1934 H1N1

M30746 A/Wilson-Smith/33 1565 1933 H1N1

M81583 A/Leningrad/134/47/57 1566 1957 H2N2

AF348180 A/Hong Kong/1/68 1520 1968 H3N2

L07345 A/Memphis/101/72 1565 1972 H3N2

D00051 A/Udorn/307/72 1565 1972 H3N2

L07359 A/Guangdong/38/77 1565 1977 H3N2

M59333 A/Ohio/201/83 1565 1983 H1N1

L07364 A/Memphis/14/85 1565 1985 H3N2

M76610 A/Wisconsin (wisconsin)/3623/88 1565 1988 H1N1

U71144 A/Akita/1/94 1497 1994 H3N2

AF084277 A/Hong Kong/483/97 1497 1997 H5N1

AF036359 A/Hong Kong/156/97 1565 1997 H5N1

AF250472 A/Waterfowl/Hong Kong/M603/98 1497 1998 H11N1

ISDN13443 A/Sydney/274/2000 1503 2000 H3N2

M63773 A/Duck/Manitoba/1/53 1565 1953 H10N7

M63775 A/Duck/Pennsylvania/1/69 1565 1969 H6N1

M30750 A/Ma/London/1416/73 1565 1973 H7N7

M63777 A/Seagull/Maryland/5/77 1565 1977 H11N9

M30756 A/Seagull/Maryland/1815/79 1565 1979 H13N6

A/Wild Duck/Astrakhan (Astrakhan) (Guri

M63785

...

Gurjev)/263/82

M27520 A/Whale/Maine/328/84 1565 1984 H13N2

M63768 A/Pig/Iowa/17672/88 1565 1988 H1N1

Z26857 A/Turkey/Germany/3/91 1554 1991 H1N1

U49094 A/Duck/Nanchang/1749/92 1407 1992 H11N2

AF156402 A/Chicken/Hong Kong/G9/97 1536 1997 H9N2

AF285888 A/Pig/Ontario/01911-1/99 1532 1999 H4N6

Figure 9 shows an example of selecting certain regions of PA transcripts that are highly conserved among 6 influenza A variants (all with human host origin), where regions are considered to be if they differ by 0, 1 or 2 nucleotides highly conservative. (Note that the sequence is listed as DNA rather than RNA and thus contains a T rather than a U.) The sequence of strain A/Puerto Rico/8/34 (H1N1) was chosen as the base sequence, i.e., the sequence to which the other sequences were compared . Other members of this group are A/WSN/33(H1N1), A/Leningrad/134/17/57(H2N2), A/Hong Kong/1/68(H3N2), A/Hong Kong/481/97(H5N1 ) and A/Hong Kong/1073/99(H9N2). The figure presents a multiple sequence alignment generated by the computer program CLUSTALW (1.4). Nucleotides that differ from the base sequence are shaded.

Figure 10 shows an example of selecting certain regions of the PA transcript that are highly conserved between 5 influenza A variants (all with different animal host origins) and between 2 strains with human host origins, where if the difference 0, 1 or 2 nucleotides, then the region was considered highly conserved. (Note that the sequence is listed as DNA rather than RNA and thus contains a T rather than a U.) The sequence of strain A/Puerto Rico/8/34 (H1N1) was chosen as the base sequence, i.e., the sequence to which the other sequences were compared . Other members of this group are A/WSN/33 (H1N1), A/Chicken/FPV/Rostock/34 (H7N1), A/Turkey/California/189/66 (H9M2), A/Horse/London /1416/73 (H7N7), A/Seagull/Maryland/704/77 (H13N6) and A/Pig/Hong Kong/9/98 (H9N2). Nucleotides that differ from the base sequence are shaded.

Note that in the sequence comparisons in Figures 9 and 10, many different highly conserved regions could be selected, since most of the sequences meet the highly conserved criteria. However, the sequence with AA at the 5' end provides a 19 nucleotide core sequence and a 2 nucleotide 3' UU overhang in the complementary (antisense) siRNA strand. Therefore, a highly conserved region was scanned to identify a 21 nucleotide portion that has an AA at its 5' end so that the complementary nucleotide present in the antisense strand of the siRNA is UU. For example, each of the shaded sequences has an AA at its 5' end. Note that the UU 3' overhang in the antisense strand of the resulting siRNA molecule can be replaced by TT or dTdT as shown in Table 2. However, the 2nt 3' overhang of the antisense strand need not be UU.

To further illustrate the method, Figure 12 shows a sequence comparison between a portion of the 3' region of the NP sequence in 12 influenza A virus subtypes or isolates with human or animal host origin. The underlined sequence and the corresponding part of the sequence below the underlined sequence were used to design siRNA NP-1496 (see below). These sequences are shown in Figure 12. The base sequence is that of strain A/Puerto Rico/8/34. Shaded letters indicate nucleotides that differ from the base sequence.

Table 1A lists the 21 nucleotide regions that are highly conserved among the sets of influenza virus sequences for each segment of the viral gene segment. In the 5' to 3' direction, the sequences in Table 1A are listed according to the sequence present in the viral mRNA, except that T is used instead of U. Numbers indicate the position of the sequence in the viral genome. For example, PB2-117/137 represents the sequence extending from position 117 to position 137 in fragment PB2. Many sequences met the additional criterion that the sequence had an AA at its 5' end to create a 3' UU overhang in the complementary strand. For the PA segment, the siRNA sequences were based on the A/PR8/34 (H1N1) strain in the presence of 1 or 2 nucleotide differences, except that the sequence PA-2087/2107AAGCAATTGAGGAGTGCCTGA (SEQ ID NO: 30) was Based on strains other than A/WSN/33 (H1N1). Note that at position 20, 5 of the 6 sequences contain a G while the base sequence (Accession No. NC_002019) contains an A. Therefore, in said case, the sequence of the base sequence was not used for siRNA design. The terms PA-2087 and PA-2087(G) are used interchangeably herein.

To design siRNAs based on the sequences listed in Table 1A, nucleotides 3-21 were selected as the core region of the siRNA sense strand sequence, and a 2nt 3' overhang consisting of dTdT was added to each resulting sequence. A sequence complementary to nucleotides 1-21 of each sequence was selected as the corresponding antisense strand. For example, to design siRNAs based on the highly conserved sequence PA-44/64, i.e., AATGCTTCAATCCGATGATTG (SEQ ID NO: 22), a 19nt core region with the sequence TGCTTCAATCCGATGATTG (SEQ ID NO: 109) was chosen. Addition of a 2nt 3' overhang consisting of dTdT, (after replacing T with U) yielded the sequence 5'-UGCUUCAAUCCGAUGAUUGdTdT-3' (SEQ ID NO: 79), which is the sequence of the sense strand of the siRNA. The sequence of the corresponding antisense siRNA strand sequence is complementary to SEQ ID NO: 22, i.e., CAAUCAUCGGAUUGAAGCAdTdT (SEQ ID NO: 80), wherein T has been replaced by U except for the 2nt 3' overhang.

Table IB lists siRNAs designed based on additional highly conserved regions of influenza virus transcripts. The first 19 nt sequence of the sequence indicated as "sense strand" in Table 1B is the sequence of the highly conserved region. The sense strand siRNA sequence is shown to have a dTdT overhang at the 3' end, which does not correspond to the influenza virus sequence and is an optional feature of the siRNA. It is also shown that the corresponding antisense strand also incorporates a dTdT overhang at the 3' end as an optional feature. Names are the same as in Table 1B. For example, PB2-4/22 sense indicates an siRNA whose sense strand has the sequence of nucleotides 4-22 of the PB2 transcript. PB2-4/22 antisense indicates the complementary antisense strand corresponding to PB2-4/22 sense. For siRNAs targeting a site in the transcript that spans the splice site, the position within the unspliced transcript is shown. For example, M-44-52/741-750 indicates that in the spliced mRNA, nucleotides corresponding to 44-52 and 741-750 of the genomic sequence are targeted.

The shaded areas in Figures 9 and 10 represent some 21 nucleotide regions that meet the criteria of being highly conserved. siRNAs were designed based on these sequences as described above. The actual siRNA sequences tested are listed in Table 2.

Table 1A. Conserved regions used to design siRNAs that interfere with influenza A virus infection

Fragment 1: PB2

PB2-117/137 AATCAAGAAGTACACATCAGG (SEQ ID NO: 1)

PB2-124/144 AAGTACACATCAGGAAGACAG (SEQ ID NO: 2)

PB2-170/190 AATGGATGATGGCAATGAAAT (SEQ ID NO: 3)

PB2-195/215 AATTCAGCAGACAAGAGGAT (SEQ ID NO: 4)

PB2-1614/1634 AACTTACTCATCGTCAATGAT (SEQ ID NO: 5)

PB2-1942/1962 AATGTGAGGGGATCAGGAATG (SEQ ID NO: 6)

PB2-2151/2171 AAGCATCAATGAACTGAGCAA (SEQ ID NO: 7)

PB2-2210/2230 AAGGAGACGTGGTGTTGGTAA (SEQ ID NO: 8)

PB2-2240/2260 AACGGGACTCTAGCATACTTA (SEQ ID NO: 9)

PB2-2283/2303 AAGAATTCGGATGGCCATCAA (SEQ ID NO: 10)

Fragment 2: PB1

PB1-6/26 AAGCAGGCAAACCATTTGAAT (SEQ ID NO: 11)

PB1-15/35 AACCATTTGAATGGATGTCAA (SEQ ID NO: 12)

PB1-34/54 AATCCGACCTTACTTTTTCTTA (SEQ ID NO: 13)

PB1-56/76 AAGTGCCAGCACAAAATGCTA (SEQ ID NO: 14)

PB1-129/149 AACAGGATACACCATGGATAC (SEQ ID NO: 15)

PB1-1050/1070 AATGTTTCTCAAACAAAATGGC (SEQ ID NO: 16)

PB1-1242/1262 AATGATGATGGGCATGTTCAA (SEQ ID NO: 17)

PB1-2257/2277 AAGATCTGTTCCACCATTGAA (SEQ ID NO: 18)

Fragment 3: PA

PA-6/26 AAGCAGGTACTGATCCAAAAT (SEQ ID NO: 19)

PA-24/44 AATGGAAGATTTTGTGCGACA (SEQ ID NO: 20)

PA-35/55 TTGTGCGACAATGCTTCAATC (SEQ ID NO: 21)

PA-44/64 AATGCTTCAATCCGATGATTG (SEQ ID NO: 22)

PA-52/72 AATCCGATGATTGTCGAGCTT (SEQ ID NO: 23)

PA-121/141 AACAAATTTGCAGCAATATGC (SEQ ID NO: 24)

PA-617/637 AAGAGACAATTGAAGAAAGGT (SEQ ID NO: 25)

PA-711/731 TAGAGCCTATGTGGATGGATT (SEQ ID NO: 26)

PA-739/759 AACGGCTACATTGAGGGCAAG (SEQ ID NO: 27)

PA-995/1015 AACCACACGAAAAGGGAATAA (SEQ ID NO: 28)

PA-2054/2074 AACCTGGGACCTTTTGATCTTG (SEQ ID NO: 29)

PA-2087/2107 AAGCAATTGAGGAGTGCCTGA (SEQ ID NO: 30)

PA-2110/2130 AATGATCCCTGGGTTTTGCTT (SEQ ID NO: 31)

PA-2131/2151 AATGCTTCTTGGTTCAACTCC (SEQ ID NO: 32)

Fragment 4: HA

HA-1119/1139 TTGGAGCCATTGCCGGTTTTA (SEQ ID NO: 33)

HA-1121/1141 GGAGCCATTGCCGGTTTTTATT (SEQ ID NO: 34)

HA-1571/1591 AATGGGACTTATGATTATCCC (SEQ ID NO: 35)

Fragment 5: NP

NP-19/39 AATCACTCACTGAGTGACATC (SEQ ID NO: 36)

NP-42/62 AATCATGGCGTCCCAAGGCAC (SEQ ID NO: 37)

NP-231/251 AATAGAGAGAATGGTGCTCTC (SEQ ID NO: 38)

NP-390/410 AATAAGGCGAATCTGGCGCCA (SEQ ID NO: 39)

NP-393/413 AAGGCGAATCTGGCGCCAAGC (SEQ ID NO: 40)

NP-708/728 AATGTGCAACATTCTCAAAGG (SEQ ID NO: 41)

NP-1492/1512 AATGAAGGATCTTATTTCTTC (SEQ ID NO: 42)

NP-1496/1516 AAGGATCTTATTTCTTCGGAG (SEQ ID NO: 43)

NP-1519/1539 AATGCAGAGGAGTACGACAAT (SEQ ID NO: 44)

Fragment 6: NA

NA-20/40 AATGAATCCAAATCAGAAAAT (SEQ ID NO: 45)

NA704/724 GAGGACACAAGAGTCTGAATG (SEQ ID NO: 46)

NA-861/881 GAGGAATGTTCCTGTTACCCT (SEQ ID NO: 47)

NA-901/921 GTGTGTGCAGAGACAATTGGC (SEQ ID NO: 48)

Fragment 7: M

M-156/176 AATGGCTAAAGACAAGACCAA (SEQ ID NO: 49)

M-175/195 AATCCTGTCACCTCTGACTAA (SEQ ID NO: 50)

M-218/238 ACGCTCACCGTGCCCAGTGAG (SEQ ID NO: 51)

M-244/264 ACTGCAGCGTAGACGCTTTGT (SEQ ID NO: 52)

M-373/393 ACTCAGTTATTCTGCTGGTGC (SEQ ID NO: 53)

M-377/397 AGTTATTCTGCTGGTGCACTT (SEQ ID NO: 54)

M-480/500 AACAGATTGCTGACTCCCAGC (SEQ ID NO: 55)

M-584/604 AAGGCTATGGAGCAAATGGCT (SEQ ID NO: 56)

M-598/618 AATGGCTGGATCGAGTGAGCA (SEQ ID NO: 57)

M-686/706 ACTCATCCTAGCTCCAGTGCT (SEQ ID NO: 58)

M-731/751 AATTTGCAGGCCTATCAGAAA (SEQ ID NO: 59)

M-816/836 ATTGTGGATTCTTGATCGTCT (SEQ ID NO: 60)

M-934/954 AAGAATATCGAAAGGAACAGC (SEQ ID NO: 61)

M-982/1002 ATTTTGTCAGCATAGAGCTGG (SEQ ID NO: 62)

Fragment 8: NS

NS-101/121 AAGAACTAGGTGATGCCCCAT (SEQ ID NO: 63)

NS-104/124 AACTAGGTGATGCCCCATTCC (SEQ ID NO: 64)

NS-128/148 ATCGGCTTCGCCGAGATCAGA (SEQ ID NO: 65)

NS-137/157 GCCGAGATCAGAAATCCCTAA (SEQ ID NO: 66)

NS-562/582 GGAGTCCTCATCGGAGGACTT (SEQ ID NO: 67)

NS-589/609 AATGATAACACAGTTCGAGTC (SEQ ID NO: 68)

Table 1B. Conserved regions used to design siRNAs that interfere with influenza A virus infection

Fragment 1: PB2

PB2-4/22 sense GAAAGCAGGUCAAUUAUAUAUdTdT SEQ ID NO: 190)

PB2-4/22 antisense AUAUAAUUGACCUGCUUUCdTdT SEQ ID NO: 191)

PB2-12/30 meaningful GUCAAUUAUAUUCAAUAUGdTdT SEQ ID NO: 192)

PB2-12/30 antisense CAUAUUGAAUAUAAUUGACdTdT SEQ ID NO: 193)

PB2-68/86 meaningful CUCGCACCCGCGAGAUACUdTdT SEQ ID NO: 194)

PB2-68/86 antisense AGUAUCUCGCGGGUGCGAGdTdT SEQ ID NO: 195)

PB2-115/133 meaningful AUAAUCAAGAAGUACACAUdTdT SEQ ID NO: 196)

PB2-115/133 antisense AUGUGUACUUCUUGAUUAUAUdTdT SEQ ID NO: 197)

PB2-167/185 meaningful UGAAAUGGAUGAUGGCAAUdTdT SEQ ID NO: 198)

PB2-167/185 antisense AUUGCCAUCAUCCAUUUCAdTdT SEQ ID NO: 199)

PB2-473/491 meaningful CUGGUCAUGCAGAUCUCAGdTdT SEQ ID NO: 200)

PB2-473/491 antisense CUGAGAUCUGCAUGACCAGdTdT SEQ ID NO: 201)

PB2-956/974 meaningful UAUGCAAGGCUGCAAUGGGdTdT SEQ ID NO: 202)

PB2-956/974 antisense CCCAUUGCAGCCUUGCAUAdTdT SEQ ID NO: 203)

PB2-1622/1640 sense CAUCGUCAAUGAUGUGGGAdTdT SEQ ID NO: 204)

PB2-1622/1640 antisense UCCCACAUCAUUGACGAUGdTdT SEQ ID NO: 205)

Fragment 2: PB1

PB1-1124/1142 sense AAAUACCUGCAGAAAUGCUdTdT SEQ ID NO: 206)

PB1-1124/1142 antisense AGCAUUUCUGCAGGUAUUUdTdT SEQ ID NO: 207)

PB1-1618/1636 sense AACAAUAUGAUAAACAAUGdTdT SEQ ID NO: 208)

PB1-1618/1636 antisense CAUUGUUUAUCAUAUUGUUdTdT SEQ ID NO: 209)

Fragment 3: PA

PA-3/21 sense CGAAAGCAGGUACUGAUCCdTdT SEQ ID NO: 210)

PA-3/21 antisense GGAUCAGUACCUGCUUUCGdTdT SEQ ID NO: 211)

PA-544/562 sense AGGCUAUUCACCAUAAGACdTdT SEQ ID NO: 212)

PA-544/562 antisense GUCUUAUGGUGAAUAGCCUdTdT (SEQ ID NO: 213)

PA-587/605 sense GGGAUUCCUUUCGUCAGUCdTdT (SEQ ID NO: 214)

PA-587/605 antisense GACUGACGAAAGGAAUCCCdTdT (SEQ ID NO: 215)

PA-1438/1466 has the sense GCAUCUUGUGCAGCAAUGGdTdT (SEQ ID NO: 216)

PA-1438/1466 antisense CCAUUGCUGCACAAGAUGCdTdT (SEQ ID NO: 217)

PA-2175/2193 has GUUGUGGCAGUGCUACUAUdTdT (SEQ ID NO: 218)

PA-2175/2193 antisense AUAGUAGCACUGCCACAACdTdT (SEQ ID NO: 219)

PA-2188/2206 has UACUAUUUGCUAUCCAUACdTdT (SEQ ID NO: 220)

PA-2188/2206 antisense GUAUGGAUAGCAAAUAGUAdTdT (SEQ ID NO: 221)

Fragment 5: NP

NP-14/32 is meaningful UAGAUAAUCACUCACUGAGdTdT 'SEQ ID NO: 222)

NP-14/32 antisense CUCAGUGAGUGAUUAUCUAdTdT SEQ ID NO: 223)

NP-50/68 sense CGUCCCAAGGCACCAAACGdTdT SEQ ID NO: 224)

NP-50/68 antisense CGUUUGGUGCCUUGGGACGdTdT SEQ ID NO: 225)

NP-1505/1523 has a sense AUUUCUUCGGAGACAAUGCdTdT SEQ ID NO: 226)

NP-1505/1523 antisense GCAUUGUCUCCGAAGAAAUdTdT SEQ ID NO: 227)

NP-1521/1539 sense UGCAGAGGAGUACGACAAUdTdT SEQ ID NO: 228)

NP-1521/1539 antisense AUUGUCGUACUCCUCUGCAdTdT SEQ ID NO: 229)

NP-1488/1506 sense GAGTAATGAAGGATCTTATdTdT SEQ ID NO: 230)

NP-1488/1506 antisense 231) ATAAGATCCTTCATTACTCdTdT SEQ ID NO:

Fragment 7: M

M-3/21 meaningful CGAAAGCAGGUAGAUAUUGdTdT SEQ ID NO: 232)

M-3/21 antisense CAAUAUCUACCUGCUUUCGdTdT SEQ ID NO: 233)

M-13/31 meaningful UAGAUAUUGAAAGAUGAGUdTdT SEQ ID NO: 234)

M-13/31 antisense ACUCAUCUUUCAAUAUCUAdTdT SEQ ID NO: 235)

M-150/158 meaningful UCAUGGAAUGGCUAAAGACdTdT SEQ ID NO: 236)

M-150/158 antisense GUCUUUAGCCAUUCCAUGAdTdT SEQ ID NO: 237)

M-172/190 meaningful ACCAAUCCUGUCACCUCUGdTdT SEQ ID NO: 238)

M-172/190 antisense CAGAGGUGACAGGAUUGGUdTdT SEQ ID NO: 239)

M-211/229 meaningful UGUGUUCACGCUCACCGUGdTdT SEQ ID NO: 240)

M-211/229 antisense CACGGUGAGCGUGAACACAdTdT SEQ ID NO: 241)

M-232/250 sense CAGUGAGCGAGGACUGCAGdTdT SEQ ID NO: 242)

M-232/250 antisense CUGCAGUCCUCGCUCACUGdTdT SEQ ID NO: 243)

M-255/273 meaningful GACGCUUUGUCCAAAAUGCdTdT SEQ ID NO: 244)

M-255/273 antisense GCAUUUUGGACAAAGCGUCdTdT SEQ ID NO: 245)

M-645/663 meaningful GUCAGGCUAGGCAAAUGGUdTdT SEQ ID NO: 246)

M-645/663 antisense ACCAUUUGCCUAGCCUGACdTdT SEQ ID NO: 247)

M-723/741 UUCUUGAAAAUUUGCAGGCdTdT SEQ ID NO: 248)

M-723/741 antisense GCCUGCAAAUUUUCAAGAAdTdT SEQ ID NO: 249)

M-808/826 meaningful UCAUUGGGAUCUUGCACUUdTdT SEQ ID NO: 250)

M-808/826 antisense AAGUGCAAGAUCCCAAUGAdTdT SEQ ID NO: 251)

M-832/850 UGUGGAUUUCUUGAUCGUCUdTdT SEQ ID NO: 252)

M-832/850 antisense AGACGAUCAAGAAUCCACadTdT SEQ ID NO: 253)

M-986/1004 UGUCAGCAUAGAGCUGGAGdTdT SEQ ID NO: 254)

M-986/1004 antisense CUCCAGCUCUAUGCUGACAdTdT SEQ ID NO: 255)

M-44-52/741-750 sense GTCGAAACGCCTATCAGAAdTdT SEQ ID NO: 256)

M-44-52/741-750 antisense UUCUGAUAGGCGUUUCGACdTdT SEQ ID NO: 257)

Fragment 8: NS

NS-5/23 sense AAAAGCAGGGUGACAAAGAdTdT SEQ ID NO: 258)

NS-5/23 antisense UCUUUGUCACCCUGCUUUUdTdT SEQ ID NO: 259)

NS-9/27 sense GCAGGGUGACAAAGACAUAdTdT SEQ ID NO: 260)

NS-9/27 antisense UAUGUCUUUGUCACCCUGCdTdT SEQ ID NO: 261)

NS-543/561 meaningful GGAUGUCAAAAAAUGCAGUUdTdT SEQ ID NO: 262)

NS-543/561 antisense AACUGCAUUUUUGACAUCCdTdT SEQ ID NO: 263)

NS-623/641 meaningful AGAGAUUCGCUUGGAGAAGdTdT SEQ ID NO: 264)

NS-623/641 antisense CUUCUCCAAGCGAAUCUCUdTdT SEQ ID NO: 265)

NS-642/660 sense CAGUAAUGAGAAUGGGAGAdTdT SEQ ID NO: 266)

NS-642/660 antisense UCUCCCAUUCUCAUUACUGdTdT SEQ ID NO: 267)

NS-831/849 meaningful UUGUGGAUUCUUGAUCGUCdTdT SEQ ID NO: 268)

NS-831/839 antisense GACGAUCAAGAAUCCACAAdTdT SEQ ID NO: 269)

Example 2: Inhibition of Influenza A Virus Production by siRNA Targeting Viral RNA Polymerase or Nucleoprotein

Materials and methods

Cell culture. Make Madin-Darby canine kidney cells (MDCK) (from NY, New York, Mount SinaiSchool of Medicine, the donation of Dr.Peter Palese), containing 10% heat-inactivated FCS, 2mM L-glutamine, 100 units/ml penicillin (penicillin) and 100 μg/ml streptomycin (streptomycin) in DMEM medium. Cells were grown at 37°C, 5% CO ₂ . For electroporation, cells were maintained in serum-free RPMI 1640 medium. Virus infection was performed in infection medium (DMEM, 0.3% bovine serum albumin (BSA, Sigma, St. Louis, MO), 10 mM Hepes, 100 units/ml penicillin and 100 ug/ml streptomycin).

Viruses. Influenza viruses A/PR/8/34 (PR8) and A/WSN/33 (WSN), subtype H1N1, at 37°C (donation from Mount Sinai School of Medicine, Dr. Peter Palese) , grown in 10-day-old embryonated chicken eggs (Charles River laboratories, MA) for 48h. Allantoic fluid was collected 48 h after virus inoculation and stored at -80°C. The same virus strain and method were used throughout in the examples described herein.

siRNAs. siRNAs were designed as described above. siRNAs were generally designed according to the principles described in Technical Bulletin #003-Revision B, "siRNA Oligonucleotides for RNAi Applications" (DharmaconResearch, Inc., Lafayette, CO 80026), except meeting the selection criteria described in Example 1. Technical bulletins #003 and #004 from Dharmacon contain various information related to siRNA design parameters, synthesis, etc. and are incorporated herein by reference. The sense and antisense sequences tested are listed in Table 2.

Table 2. siRNA sequences

Name SiRNA Sequence (5′-3′)

PB2-2210/2230 (sense) GGAGACGUGGUGUUGGUAAdTdT (SEQ ID NO: 69)

PB2-2210/2230 (antisense) UUACCAACACCACGUCUCCdTdT (SEQ ID NO: 70)

PB2-2240/2260 (sense) CGGGACUCUAGCAUACUACUUAdTdT (SEQ ID NO: 71)

PB2-2240/2260 (antisense) UAAGUAUGCUAGAGUCCCGdTdT (SEQ ID NO: 72)

PB1-6/26 (sense) GCAGGCAAACCAUUUGAAUdTdT (SEQ ID NO: 73)

PB1-6/26 (antisense) AUUCAAAUGGUUUGCCUGCdTdT (SEQ ID NO: 74)

PB1-129/149 (sense) CAGGAUACACCAUGGAUACdTdT (SEQ ID NO: 75)

PB1-129/149 (antisense) GUAUCCAUGGUGUAUCCUGdTdT (SEQ ID NO: 76)

PB1-2257/2277 (sense) GAUCUGUUCCACCAUUGAAdTdT (SEQ ID NO: 77)

PB1-2257/2277 (antisense) UUCAAUGGUGGAACAGAUCdTdT (SEQ ID NO: 78)

PA-44/64 (sense) UGCUUCAAUCCGAUGAUUGdTdT (SEQ ID NO: 79)

PA-44/64 (antisense) CAAUCAUCGGAUUGAAGCAdTdT (SEQ ID NO: 80)

PA-739/759 (sense) CGGCUACAUUGAGGGCAAGdTdT (SEQ ID NO: 81)

PA-739/759 (antisense) CUUGCCCUCAAUGUAGCCGdTdT (SEQ ID NO: 82)

PA-2087/2107(G) (sense) GCAAUUGAGGAGUGCCUGAdTdT (SEQ ID NO: 83)

PA-2087/2107(G) (antisense) UCAGGCACUCCUCAAUUGCdTdT (SEQ ID NO: 84)

PA-2110/2130 (sense) UGAUCCCUGGGUUUUGCUUdTdT (SEQ ID NO: 85)

PA-2110/2130 (antisense) AAGCAAAACCCAGGGAUCAdTdT (SEQ ID NO: 86)

PA-2131/2151 (sense) UGCUUCUUGGUUCAACUCCdTdT (SEQ ID NO: 87)

PA-2131/2151 (antisense) GGAGUUGAACCAAGAAGCAdTdT (SEQ ID NO: 88)

NP-231/251 (sense) UAGAGAGAAUGGUGCUCUCdTdT (SEQ ID NO: 89)

NP-231/251 (antisense) GAGAGCACCAUUCUCUCUAdTdT (SEQ ID NO: 90)

NP-390/410 (sense) UAAGGCGAAUCUGGCGCCAdTdT (SEQ ID NO: 91)

NP-390/410 (antisense) UGGCGCCAGAUUCGCCUUAdTdT (SEQ ID NO: 92)

NP-1496/1516 (sense) GGAUCUUAUUUCUUCGGAGdTdT (SEQ ID NO: 93)

NP-1496/1516 (antisense) CUCCGAAGAAAUAAGAUCCdTdT (SEQ ID NO: 94)

NP-1496/1516a (sense) GGAUCUUAUUUCUUCGGAGAdTdT (SEQ ID NO: 188)

NP-1496/1516a (antisense) UCUCCGAAGAAAUAAGAUCCdTdT (SEQ ID NO: 189)

M-37/57 (sense) CCGAGGUCGAAACGUACGUdTdT (SEQ ID NO: 95)

M-37/57 (antisense) ACGUACGUUUCGACCUCGGdTdT (SEQ ID NO: 96)

M-480/500 (sense) CAGAUUGCUGACUCCCAGCdTdT (SEQ ID NO: 97)

M-480/500 (antisense) GCUGGGAGUCAGCAAUCUGdTdT (SEQ ID NO: 98)

M-598/618 (sense) UGGCUGGAUCGAGUGAGCAdTdT (SEQ ID NO: 99)

M-598/618 (antisense) UGCUCACUCGAUCCAGCCAdTdT (SEQ ID NO: 100)

M-934/954 (sense) GAAUAUCGAAAGGAACAGCdTdT (SEQ ID NO: 101)

M-934/954 (antisense) GCUGUUCCUUUCGAUAUUCdTdT (SEQ ID NO: 102)

NS-128/148 (sense) CGGCUUCGCCGAGAUCAGAdAdT (SEQ ID NO: 103)

NS-128/148 (antisense) UCUGAUCUCGGCGAAGCCGdAdT (SEQ ID NO: 104

NS-562/582 (R) (sense) GUCCUCCGAUGAGGACUCCdTdT (SEQ ID NO: 105)

NS-562/582(R) (antisense) GGAGUCCUCAUCGGAGGACdTdT (SEQ ED NO: 106)

NS-589/609 (sense) UGAUAACACAGUUCGAGUCdTdT (SEQ ID NO: 107)

NS-589/609 (antisense) GACUCGAACUGUGUUAUCAdTdT (SEQ ID NO: 108)

All siRNAs were synthesized by Dharmacon Research (Lafayette, CO) using 2'ACE protection chemistry. The siRNA strands were deprotected according to the manufacturer's instructions, mixed in equimolar ratios and annealed by heating to 95 °C and slowly decreasing the temperature by 1 °C every 30 s until 35 °C and every minute until 5 °C.

siRNA electroporation. Log-phase cultures of MDCK cells were trypsinized, washed and resuspended in serum-free RPMI 1640 at 2 x ¹⁰⁷ cells per ml. 0.5 ml of cells were placed in a 0.4 cm cuvette and electroporated with 2.5 nmol siRNA using a 400 V, 975 μF Gene Pulser (Bio-Rad). Electroporation efficiency was approximately 30-40% of live cells. The electroporated cells were divided into 3 wells of a 6-well plate in DMEM medium containing 10% FCS, and cultured at 37 °C, 5% _CO2 .

Virus infection. Six to eight hours after electroporation, the serum-containing medium was washed away and 100 μl of PR8 or WSN virus was inoculated into wells, each well containing approximately ¹⁰⁶ cells, at an appropriate multiplicity of infection. Cells were infected with 1,000 PFU (one virus per 1,000 cells; MOI=0.001 ) or 10,000 PFU (one virus per 100 cells; MOI=0.01 ) of virus. After incubation for 1 h at room temperature, 2 ml infection medium with 4 μg/ml trypsin was added to each well, and the cells were incubated at 37 °C, 5% _CO2 . At the indicated times, supernatants were collected from infected cultures and virus titers were determined by hemagglutination of chicken erythrocytes (50 [mu]l, 0.5%, Charles River laboratories, MA).

Measurement of virus titers. Supernatants were collected 24, 36, 48 and 60 hours after infection. Viral titers were measured using standard hemagglutinin assays as described in Knipe DM, Howley, PM, Fundamental Virology, 4th ed., pp. 34-35. Hemagglutination assays were performed in V-bottom 96-well plates. Serial 2-fold dilutions of each sample were incubated on ice with an equal volume of 0.5% chicken erythrocyte suspension (Charles River Laboratories). Wells containing an adherent, homogeneous layer of erythrocytes were scored positive. For plaque analysis, serial 10-fold dilutions of each sample were measured for viral titers as described in Fundamental Virology, 4th Edition, p. 32, as is well known in the art.

result

To investigate the feasibility of using siRNA to inhibit influenza virus replication, various influenza virus A RNAs were targeted. Specifically, the MDCK cell line was utilized, which is susceptible to infection and widely used to study influenza viruses. Each siRNA was individually introduced into the MDCK cell population by electroporation. siRNA targeting GFP (sense: 5'-GGCUACGUCCAGGAGCGCAUU-3' (SEQ ID NO: 110); antisense: 5'-UGCGCUCCUGGACGUAGCCUU-3' (SEQ ID NO: 111)) was used as a control. The siRNA is called GFP-949. In subsequent experiments (described in the Examples below), the UU overhangs at the 3' ends of both strands were replaced with dTdT, with no effect on the results. Control electroporation was also performed as a control. Eight hours after electroporation, cells were infected with influenza A virus PR8 or WSN at an MOI of 0.1 or 0.01, and at various time points thereafter (24, 36, 48, 60 hours) were detected using standard hemagglutination assays. Analyze cells for virus production. GFP expression was analyzed by flow cytometry using standard methods.

Figures 11A and 11B compare the results of experiments in which the ability of individual siRNAs to inhibit the replication of influenza virus A strain A/Puerto Rico/8/34 (H1N1) (Figure 11A) or to inhibit the replication of influenza virus A strain A/WSN/33 (H1N1) The ability to replicate ( FIG. 11B ) was determined by measuring the HA titer. Thus, high HA titers indicate lack of inhibition, while low HA titers indicate effective inhibition. MDCK cells were infected at an MOI of 0.01. For the experiment, one siRNA targeting the PB1 fragment (PB1-2257/2277), one siRNA targeting the PB2 fragment (PB2-2240/2260), one targeting the PA fragment (PA-2087/2107 (G)) and three different siRNAs targeting the NP genome and transcripts (NP-231/251, NP-390/410 and NP-1496/1516). Note that the legends in Figures 11A and 11B list only the 5' nucleotides of the siRNA.

Symbols in Figures 11A and 11B are explained as follows: Solid squares represent control cells that did not receive siRNA. Open squares indicate cells that received GFP control siRNA. Filled circles indicate cells that received siRNAPB1-2257/2277. Open circles indicate cells that received siRNA PB2-2240/2260. Open triangles indicate cells that received siRNAPA-2087/2107 (G). Symbol X indicates cells that received siRNA NP-231/251. Symbol + indicates cells that received siRNANP-390/410. Solid triangles indicate cells that received siRNANP-1496/1516. Note that in the diagram, some symbols sometimes overlap. For example, in Figure 1 IB, the hollow and solid triangles overlap. For illustration, refer to Tables 3 and 4, which list the values for each point.

As shown in Figures 11A and 11B (Tables 3 and 4), virus titers increased over time in the absence of siRNA (blank control TF) or presence of control (GFP) siRNA, peaking approximately 48-60 hours after infection . In contrast, virus titers were significantly lower at 60 hours in the presence of either siRNA. For example, in strain WSN, HA titers (reflecting virus content) were approximately half as large in the presence of siRNA PB2-2240 or NP-231 compared to controls. In particular, in both strains, the viral content was below the detection limit (10,000 PFU/ml) in the presence of siRNANP-1496. This represents a factor of over 60-fold reduction in the PR8 strain and a factor of over 120-fold in the WSN strain. In strain WSN, virus content was also below the detection limit (10,000 PFU/ml) in the presence of siRNA PA-2087(G), and was extremely low in strain PR8. Inhibition of virus production by siRNA was evident even from the earliest time points measured. Efficient inhibition, including suppression of virus production to undetectable levels (as measured by HA titers), has been observed at time points up to 72 hours post-infection.

Table 5 summarizes the results of the siRNA inhibition assay at 60 hours in MDCK cells expressed as fold inhibition. Thus, low values indicate lack of inhibition, while high values indicate effective inhibition. The position of siRNA in the viral gene is indicated by the number after the gene name. As elsewhere herein, the numbering indicates the starting nucleotide of the siRNA in the gene. For example, NP-1496 represents an siRNA specific for NP, the first nucleotide starting at nucleotide 1496 of the NP sequence. Values shown (fold inhibition) were calculated as hemagglutinin units from control transfections divided by hemagglutinin units from transfections with the indicated siRNAs; a value of 1 indicates no inhibition.

A total of 20 siRNAs targeting 6 segments of the influenza virus genome (PB2, PB1, PA, NP, M and NS) were tested in the MDCK cell line system (Table 5). Regardless of whether PR8 or WSN virus was used, approximately 15% of the tested siRNAs (PB1-2257, PA-2087G and NP-1496) showed robust effects, in most cases inhibiting virus production more than 100-fold at MOI = 0.001 and Virus production was inhibited 16 to 64 fold at MOI = 0.01. In particular, when siRNA NP-1496 or PA-2087 was used, the inhibition was so pronounced that there was no detectable hemagglutinin activity in the culture supernatant. The potent siRNAs target 3 different viral gene segments: PB1 and PA, both contained in the RNA transcriptase complex; and NP, a single-stranded RNA-binding nucleoprotein. Consistent with findings in other systems, the sequences targeted by the siRNAs were all relatively localized near the 3&apos; end of the coding region (Figure 13).

Approximately 40% of the siRNAs significantly inhibited virus production, but the degree of inhibition varied depending on certain parameters. Regardless of whether PR8 or WSN virus was used, approximately 15% of the siRNAs effectively inhibited virus production. However, in the case of some siRNAs, the degree of inhibition varied somewhat depending on whether PR8 or WSN was used. Some siRNAs, such as PB2-2240, PB1-129, NP-231 and M-37, significantly inhibited virus production only at early time points (24 to 36 hours post infection) or only at low doses of infection (MOI=0.001) . The siRNA targets different viral gene segments, and the corresponding sequences are located near the 3' end or the 5' end of the coding region (Figure 13). Tables 5A and 5B present the results of the analysis. Approximately 45% of the siRNAs had no discernible effect on virus titers, indicating that they were not effective in interfering with influenza virus production in MDCK cells. In particular, none of the four siRNAs targeting NS gene segments showed any inhibitory effect.

For a more accurate estimation of virus titers, blanking with culture supernatant was performed (at 60 hours) from culture supernatants obtained from virus-infected cells that underwent control transfection or transfected with NP-1496. spot analysis. Approximately 6×10 ⁵ pfu/ml was detected in the control supernatant, while no plaques were detected in the undiluted NP-1496 supernatant ( FIG. 11C ). Because the detection limit of the plaque assay is about 20 pfu (plaque forming units)/ml, the inhibition of virus production by NP-1496 is at least about 30,000-fold. Even at an MOI of 0.1, NP-1496 inhibited virus production by approximately 200-fold.

To determine the efficacy of siRNA, graded amounts of NP-1496 were transfected into MDCK cells followed by infection with PR8 virus. Virus titers in culture supernatants were measured by hemagglutinin assay. As shown in Figure 1 ID, as the amount of siRNA decreased, the virus titer in the culture supernatant increased. However, even when transfected with as little as 25 pmol of siRNA, approximately 4-fold inhibition of virus production was detected compared to control transfections, indicating the efficacy of NP-1496 siRNA in inhibiting influenza virus production.

For therapeutics, there is a need for siRNAs capable of effectively inhibiting ongoing viral infection. In a typical influenza virus infection, shedding of new virions begins about 4 hours after infection. To determine whether siRNA can reduce or eliminate infection of newly released virus in the face of existing infection, MDCK cells were infected with PR8 virus for 2 hours and then transfected with NP-1496 siRNA. As shown in Figure 1 IE, virus titers increased steadily over time after control transfection, whereas in NP-1496 transfected cells, virus titers increased only slightly. Therefore, administration of siRNA after virus infection is effective.

Taken together, the results show that (i) certain siRNAs can effectively inhibit influenza virus production; (ii) influenza virus production can be controlled by siRNAs specific for different viral genes, including those encoding NP, PA, and PB1 proteins. and, (iii) In addition to cells infected at the same time as siRNA administration or after siRNA administration, siRNA inhibition also occurs in cells infected before siRNA administration.

Table 3. Inhibition of production by siRNA to strain A/Puerto Rico/8/34 (H1N1)

siRNA control GFP PB1-2257 PB2-2040 PA-2087(G) NP-231 NP-390 NP-1496 24 hours 8 8 1 4 1 1 4 1 36 hours 16 8 4 8 1 4 8 1 48 hours 32 32 4 8 2 4 8 1 60hrs 64 64 8 8 4 8 32 1

Table 4.siRNA inhibits the production of virus strain A/WSN/33 (H1N1)

siRNA control GFP PB1-2257 PB2-2040 PA-2087(G) NP-231 NP-390 NP-1496 24 hours 32 32 1 8 1 8 16 1

36 hours 64 128 16 32 1 64 64 1 48 hours 128 128 16 64 1 64 64 1 60hrs 128 128 32 64 1 64 128 1

Table 5A. Effects of 20 siRNAs on influenza virus production in MDCK cells

siRNA Virus Infection (MOT) PR8(0.001) PR8(0.01) PR8(0.1) WSN(0.001) WSN(0.01) Example 1 GFP-949PB2-2210PB2-2240PB1-6PB1-129PB1-2257 2161284128256 181641664 Example 2 GFP-949PA-44PA-739PA-2087PA-2110PA-2131 22412884   1121642 Example 3 NP-231NP-390NP-1496M-37 164162 42642  42128128 Example 4 M-37M-480M-598M-934NS-128NS-562NS-589NP-1496 222121164 16 12841284211128 Example 5 GFP-949PB2-2240PB1-2257PA-2087NP-1496NP-231 18816648   1241281282

Table 5B. Effect of siRNA on influenza virus production in MDCK cells

10 ³ pfu/well (MOI 0.001) 10 ⁴ pfu/well (MOI

0.01)

Number of Holes HA Units Number of Holes HA Units

48 24 48 48 24

Example 1 24 HR HR HR 24 HR HR HR 48HR

HR

NT 2 5 4 32 NT 6 7 64 128

NP1496 0 0 1 1 1 NP1496 3 5 8 32

M3 1 5 2 32 M3 5 5 7 32 128

M150 1 3 2 8 8 M150 6 7 64 128

M172 0 2 1 4 4 M172 6 7 64 128

PB24 0 3 1 8 8 PB24 5 7 32 128

PB268 1 5 2 32 PB268 6 7 64 128

PB2115 2 5 4 32 PB2115 6 7 64 128

    Number of Holes   HA Unit

48 24 48 48 24

Example 2 24 HR HR HR HR 24 HR HR HR 48 HR

HR

NT 1 4 2 16 NT 7 ＞8 128 ＞256

NP1496 0 1 1 2 NP1496 0 1 1 1 2

NS5 0 3 1 8 8 NS5 6 ＞8 64 ＞256

NS9 1 4 2 16 NS9 6 ＞8 64 ＞256

M211 1 4 2 16 M211 7 ＞8 128 ＞256

M232 0 2 1 4 4 M232 5 ＞8 32 ＞256

PB2167 0 2 1 4 4 PB2167 5 7 32 128

PB2473 1 4 2 16 PB2473 7 ＞8 128 ＞256

    Number of Holes   HA Unit

48 24 48 48 24

Example 3 24 HR HR HR HR 24 HR HR HR 48 HR

HR

NT 1 5 2 32 NT 4 7 16 128

NP1496 0 1 1 2 NP1496 0 3 1 8

NS543 1 5 2 32 NS543 2 6 4 64

NS623 1 5 2 32 NS623 4 7 16 128

M723 1 5 2 32 M723 2 7 4 128

      Number of holes   HA Unit                                                            

48 24 48 48 24

Example 4 24 HR HR HR 24 HR HR HR 48HR

HR

NT 3 6 8 64 NT ＞8 9 ＞256 512

NP1496 0 1 1 2 NP1496 1 4 2 16

NP1501 0 4 1 16 NP1501 6 8 64 256

NS642 3 5 8 32 NS642 8 9 256 512

NS831 3 5 8 32 NS831 8 9 256 512

PB1 3 6 8 64 PB11618 7 9 128 512

1618

PB212 3 5 8 32 PB212 7 9 128 512

M232 2 5 4 32 M232 6 8 64 256

    Number of holes   HA unit   Number of holes   HA unit

48 24 48 48 24

Example 5 24 HR HR HR 24 HR HR HR 48HR

HR

NT 1 4 2 16 NT 5 7 32 128

NP1496 0 1 1 2 NP1496 0 1 1 2

NP14 1 4 2 16 NP14 5 7 32 128

NP1488 0 1 1 2 NP1488 0 3 1 8

PB2 0 2 1 4 PB22283 4 5 16 32

2283

PA2188 1 4 2 16 PA2188 4 6 16 64

    Number of Holes   HA Unit

48 24 48 48 24

Example 6 24 HR HR HR 24 HR HR HR 48 HR

HR

NT 2 4 4 16 NT 7 8 128 256

NP1496 0 0 1 1 NP1496 1 1 2 2

PB2956 2 4 4 16 PB2956 7 8 128 256

M13 2 4 4 16 M13 7 8 128 256

M255 1 2 2 4 4 M255 5 6 32 64

M645 1 2 2 4 4 M645 5 6 32 64

M808 1 3 2 8 M808 6 7 64 128

M832 1 1 2 2 2 M832 5 6 32 64

M986 2 3 4 8 M986 7 7 128 128

NP1488 0 0 1 1 NP1488 1 4 2 16

    Number of Holes   HA Unit

48 24 48 48 24

Example 7 24 HR HR HR 24 HR HR HR 48 HR

HR

NT 2 5 4 32 NT 6 8 64 256

NP1496 0 0 1 NP1496 2 3 4 8

NP1501 0 4 16 NP1501 4 6 16 64

PB24 0 2 4 4 PB24 5 6 32 64

PB2 0 3 8 8 PB22283 4 6 16 64

2283

M172 2 5 4 32 M172 5 7 32 128

M255 0 2 4 4 M255 4 6 16 64

M645 0 3 8 8 M645 4 6 16 64

M832 0 1 2 2 M832 4 6 16 64

Example 3: siRNA targeting viral RNA polymerase or nucleoprotein inhibits influenza A virus production in chicken embryos.

Materials and methods

SiRNA-oligofectamine complex formation and chick embryo inoculation. siRNA was prepared as described above. Eggs were maintained under standard conditions. 30 μl of Oligofectamine (product number: 12252011 from Life Technologies, now Invitrogen) was mixed with 30 μl of Opti-MEM I (Gibco) and incubated at RT for 5 min. 2.5 nmol (10 μl) of siRNA was mixed with 30 μl of Opti-MEM I and added to the diluted oligofectamine. The siRNA and oligofectamine were incubated for 30 min at RT. 10 day old chicken eggs were inoculated with siRNA-oligofectamine complex together with 100 μl of PR8 virus (5000 pfu/ml). Eggs were incubated at 37°C for the indicated times and allantoic fluid was collected. Virus titers in allantoic fluid were tested by HA assay as described above.

result

To confirm the results in MDCK cells, siRNAs were also analyzed for their ability to inhibit influenza virus production in fertilized chicken eggs. Since electroporation cannot be used on eggs, Oligofectamine (a lipid-based agent that has been shown to promote intracellular uptake of DNA oligonucleotides) and siRNA in vitro (25) were used. Briefly, PR8 virus alone (500 pfu) or virus plus siRNA-oligofectamine complexes were injected into the allantoic cavity of 10-day-old chicken eggs as schematically shown in Figure 14A. Allantoic fluid was collected 17 hours later for measurement of virus titer by hemagglutinin assay. As shown in Figure 14B, when virus was injected alone (in the presence of Oligofectamine), high virus titers were readily detectable. Co-injection of GFP-949 did not significantly affect viral titers. (When Oligofectamine was omitted, no significant decrease in viral titer was observed.)

Injection of siRNAs specific to influenza virus showed results consistent with those observed in MDCK cells: the same siRNAs (NP-1496, PA2087 and PB1-2257) that inhibited production of influenza virus in MDCK cells also inhibited Virus production in chicken eggs, and siRNAs (NP-231, M-37, and PB 1-129) that were less effective in MDCK cells were ineffective in fertilized chicken eggs. Therefore, siRNA is also effective in interfering with influenza virus production in fertilized chicken eggs.

Example 4: siRNA inhibits influenza virus production at the mRNA level

Materials and methods

SiRNA preparation was performed as described above.

RNA extraction, reverse transcription and real-time PCR. 1×10 ⁷ MDCK cells were electroporated with 2.5 nmol of NP-1496 or control electroporated (no siRNA). Eight hours later, cells were inoculated with influenza A PR8 virus at MOI = 0.1. At 1, 2, and 3 hour post-infection time points, supernatants were removed and cells were lysed with Trizol reagent (Gibco). Purify RNA according to manufacturer's instructions. Reverse transcription (RT) was performed using 200 ng of total RNA, specific primers (see below) and Omniscript Reverse Transcriptase Kit (Qiagen) in a 20 μl reaction mixture for 1 hour at 37° C. according to the manufacturer's instructions. Primers specific for mRNA, NP vRNA, NP cRNA, NS vRNA or NS cRNA are as follows:

mRNA, dT ₁₈ =5'-TTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 112)

NP vRNA, NP-367: 5'-CTCGTCGCTTATGACAAAGAAG-3' (SEQ ID NO: 113).

NP cRNA, NP-1565R:

5'-ATATCGTCTCGTATTAGTAGAAACAAGGGTATTTTT-3' (SEQ ID NO: 114).

NS vRNA, NS-527: 5'-CAGGACATACTGATGAGGATG-3' (SEQ ID NO: 115).

NS cRNA, NS-890R:

5'-ATATCGTCTCGTATTAGTAGAAACAAGGGTGTTTT-3' (SEQ ID NO: 116).

1 μl of the RT reaction mix (i.e., the sample obtained by performing reverse transcription) and sequence-specific primers were used for real-time PCR using S YBR Green PCRmaster mix (AB Applied Biosystems). PCR cycles were performed in the ABI PRISM 7000 Sequence Detection System (AB applied Biosystems) and analyzed with the ABI PRISM 7000 SDS software (AB Applied Biosystems). The PCR reaction was performed for 50 cycles at 50°C for 2 min, 95°C for 10 min, then at 95°C for 15 sec and 60°C for 1 min. Cycle time was analyzed as a readout of 0.2 fluorescence units. All reactions were performed in duplicate. Cycle times that vary by more than 1.0 between repetitions are discarded. The repeated cycle times were then averaged and the cycle time for β-actin was subtracted therefrom to obtain the standard value.

PCR primers are as follows.

For NP RNA:

NP-367: 5'-CTCGTCGCTTATGACAAAGAAG-3' (SEQ ID NO: 117).

NP-460R: 5'-AGATCATCATGTGAGTCAGAC-3' (SEQ ID NO: 118).

For NS RNA:

NS-527: 5'-CAGGACATACTGATGAGGATG-3' (SEQ ID NO: 119).

NS-617R: 5'-GTTTCAGAGACTCGAACTGTG-3' (SEQ ID NO: 120).

result

As described above, during influenza virus replication, vRNA is transcribed to produce cRNA that serves as a template for additional vRNA synthesis and mRNA that serves as a template for protein synthesis (1). Although RNAi is known to target the degradation of mRNA in a sequence-specific manner (16-18), vRNA and cRNA are also likely targets of siRNA, since vRNA of influenza A virus is sensitive to nucleases (1). To study the effect of siRNA on the degradation of various RNA species, reverse transcription was performed using sequence-specific primers, followed by real-time PCR to quantify the levels of vRNA, cRNA, and mRNA. Figure 16 shows the relationship between influenza virus vRNA, mRNA and cRNA. As shown in Figures 16A and 16B, the cRNA is the exact complement of the vRNA, but the mRNA contains a cap structure at the 5' end plus an additional 10 to 13 nucleotides from the host cell mRNA and the mRNA at the 3' end. contains a polyA sequence starting at a site complementary to a site 15-22 nucleotides downstream from the 5' end of the vRNA fragment. Thus, mRNA lacks 15 to 22 nucleotides at the 3' end compared to vRNA and cRNA. To distinguish between the 3 viral RNA species, primers specific for vRNA, cRNA and mRNA were used in the first reverse transcription reaction (Figure 16B). For mRNA, poly dT18 was used as a primer. For cRNA, a primer complementary to the 3' end of the RNA missing in the mRNA was used. For vRNA, the primer can be almost anywhere along the RNA as long as it is complementary to the vRNA and not too close to the 5' end. cDNA transcribed from only one of the RNAs was amplified by real-time PCR.

After influenza virus infection, by about 4 hours, new virions begin to be assembled and released. To determine the effect of siRNA on the first round of mRNA and cRNA transcription, RNA was isolated early after infection. Briefly, NP-1496 was electroporated into MDCK cells. Control electroporation (no siRNA) was also performed. After 6 to 8 hours, cells were infected with PR8 at MOI=0.1. Cells were then lysed and RNA isolated at 1, 2 and 3 hours post infection. mRNA, vRNA, and cRNA levels were analyzed by reverse transcription using primers for various RNA species, followed by real-time PCR.

Figure 17 shows the amount of viral NP and NS RNA species at various time points after infection with virus in control transfected or siRNA NP-1496 transfected cells approximately 6-8 hours prior to infection. As shown in Figure 17, 1 hour after infection, there was no significant difference in the amount of NP mRNA between samples transfected with or without NP siRNA. As early as 2 hours post-infection, NP mRNA was increased 38-fold in the control transfection group, whereas in siRNA-transfected cells, NP mRNA levels were not increased (or even slightly decreased). At 3 hours post-infection, mRNA transcript levels continued to increase in control transfections, whereas in siRNA-treated cells, a sequential decrease in NP mRNA levels was observed. NP vRNA and cRNA showed a similar pattern, except that the amount of vRNA and cRNA increased significantly in control transfections only at 3 hours post-infection. While not wishing to be bound by any theory, this may be due to the life cycle of influenza virus, where the first round of mRNA transcription occurs prior to cRNA and further vRNA synthesis.

The results show that, consistent with measurements of intact, live virus by hemagglutinin assay or plaque assay, the amount of all NP RNA species was also significantly reduced by treatment with NP siRNA. Although it is known that siRNA mainly mediates the degradation of mRNA, and although the results described below suggest that the reduction of NP protein content caused by the reduction of NP mRNA content leads to a decrease in the stability of NP cRNA and/or vRNA, the results from the experiment The data do not rule out the possibility that siRNA mediates the degradation of NP cRNA and vRNA.

Example 5: Identifying Targets of RNA Interference

Materials and methods

siRNA preparation of unmodified siRNA was performed as described above. Modified RNA oligonucleotides were also synthesized by Dharmacon in which at each nucleotide residue of the sense or antisense strand or both strands a 2'-O-methyl group was substituted for the 2'-hydroxyl group. The modified oligonucleotides are deprotected and annealed to the complementary strand as described for the unmodified oligonucleotides. siRNA duplexes were analyzed for completion of duplex formation by gel electrophoresis.

Cell culture, transfection with siRNA and infection with virus. These procedures were performed essentially as described above. Briefly, for experiments involving modified NP-1496 siRNA, MDCK cells were first transfected with NP-1496 siRNA (2.5 nmol) formed from wild-type (wt) and modified (m) strands, and 8 Hours later, infection with PR8 virus was performed at an MOI of 0.1. Virus titers in culture supernatants were analyzed 24 hours after infection. For experiments involving M-37 siRNA, MDCK cells were transfected with M-37 siRNA (2.5 nmol), infected with PR8 virus at an MOI of 0.01, and collected 1, 2, and 3 hours after infection for use in for RNA isolation. See Table 2 for the M-37 sense and antisense sequences.

RNA extraction, reverse transcription and real-time PCR were performed essentially as described above. Primers specific for mRNA, M-specific vRNA and M-specific cRNA for reverse transcription are as follows:

mRNA, dT ₁₈ =5'-TTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 112)

M vRNA: 5'-CGCTCAGACATGAGAACAGAATGG-3' (SEQ ID NO: 161)

M cRNA: 5'-ATATCGTCTCGTATTAGTAGAAACAAGGTAGTTTTT-3' (SEQ ID NO: 162).

The PCR primers of M RNA are as follows:

M Forward: 5'-CGCTCAGACATGAGAACAGAATGG-3' (SEQ ID NO: 163)

M reverse: 5'-TAACTAGCCTGACTAGCAACCTC-3' (SEQ ID NO: 164)

result

To investigate the possibility that siRNA may interfere with vRNA and/or cRNA in addition to mRNA, NP-1496 siRNA with a modified sense (S or +) or antisense (AS or -) strand was synthesized. The modification of replacing the 2'-hydroxyl group with a 2'-O-methyl group in each nucleotide residue does not affect base pairing for duplex formation, but the modified RNA strand no longer supports RNA interference. In other words, siRNAs with a modified sense strand but wild-type (mS:wtAS) antisense strand will favor the degradation of RNAs with sequences complementary to the antisense strand but not to the sense strand. Conversely, siRNAs with a wild-type sense strand but a modified antisense strand (wtS:mAS) will support the degradation of RNAs with sequences complementary to the sense strand but will not degradation of the RNA.

MDCK cells were transfected as controls, or with NP-1496 siRNA with either the sense strand (mS:wtAS) or the antisense strand (wtS:mAS) modified and the other strand wild-type. Cells were also transfected with NP-1496 siRNA modified on both strands (mS:mAS). Cells were then infected with PR8 virus, and virus titers in the supernatants were measured. As shown in Figure 18A, high virus titers were detected in cultures subjected to control transfections. As expected, very low viral titers were detected in cultures transfected with wild-type siRNA (wtS:wtAS), but not in cultures transfected with siRNA modified on both strands (mS:mAS). High virus titers were detected in . Viral titers were high in cultures transfected with siRNA modified in the antisense strand (wtAS:mAS), whereas in cultures transfected with siRNA modified in the sense strand only (mS:wtAS) , virus titers are low. While not wishing to be bound by any theory, the inventors believe that the requirement for the wild-type antisense (-) strand of the siRNA duplex to inhibit influenza virus production implies that the target of RNA interference is mRNA (+) or cRNA ( +) or both.

To further differentiate the possibilities, the effect of siRNA on the accumulation of corresponding mRNA, vRNA and cRNA was investigated. To follow transcription in a cohort of co-infected cells, siRNA-transfected MDCK cells were harvested for RNA isolation at 1, 2 and 3 hours post-infection (before release of new virions and re-infection). Viral mRNA, vRNA and cRNA are first individually converted to cDNA by reverse transcription using specific primers. Then, the content of each cDNA was quantified by real-time PCR. As shown in Figure 18B, when M-specific siRNA M-37 was used, a small amount of M-specific mRNA was detected 1 or 2 hours after infection. Three hours after infection, M-specific mRNA was readily detectable in the absence of M-37. In M-37 transfected cells, the level of M-specific mRNA was reduced by about 50%. In contrast, the levels of M-specific vRNA and cRNA were not suppressed by the presence of M-37. While not wishing to be bound by any theory, the results suggest that viral mRNA may be the target of siRNA-mediated interference.

Example 6: Effect of Certain siRNAs on Viral RNA Accumulation

Materials and methods

SiRNA preparation was performed as described above.

RNA extraction, reverse transcription and real-time PCR were performed as described in Example 3. Primers specific for mRNA, NP vRNA, NPcRNA, NS vRNA, NS cRNA, M vRNA or M cRNA are as described in Examples 4 and 5. Primers for reverse transcription specific for PB1 vRNA, PB1 cRNA, PB2 vRNA, PB2 cRNA, PA vRNA, or PA cRNA are as follows:

PB1 vRNA: 5'-GTGCAGAAATCAGCCCGAATGGTTC-3' (SEQ ID NO: 165)

PB1 cRNA: 5'-ATATCGTCTCGTATTAGTAGAAACAAGGCATTT-3' (SEQ ID NO: 166)

PB2 vRNA: 5'-GCGAAAGGAGAGAAGGCTAATGTG-3' (SEQ ID NO: 167)

PB2 cRNA: 5'-ATATGGTCTCGTATTAGTAGAAACAAGGTCGTTT-3' (SEQ ID NO: 168)

PA vRNA: 5'-GCTTCTTATCGTTCAGGCTCTTAGG-3' (SEQ ID NO: 169)

PA cRNA: 5'-ATATCGTCTCGTATTAGTAGAAACAAGGTACTT-3' (SEQ ID NO: 170)

The PCR primers of PB1, PB2 and PA RNA are as follows:

PB1 Forward: 5'-CGGATTGATGCACGGATTGATTTC-3' (SEQ ID NO: 171)

PB1 reverse: 5'-GACGTCTGAGCTCTTCAATGGTGGAAC-3' (SEQ ID NO: 172)

PB2 Forward: 5'-GCGAAAGGAGAGAAGGCTAATGTG-3' (SEQ ID NO: 173)

PB2 reverse: 5'-AATCGCTGTCTGGCTGTCAGTAAG-3' (SEQ ID NO: 174)

PA Forward: 5'-GCTTCTTATCGTTCAGGCTCTTAGG-3' (SEQ ID NO: 175)

PA Reverse: 5'-CCGAGAAGCATTAAGCAAAACCCAG-3' (SEQ ID NO: 176)

result

To determine whether NP-1496 specifically targets the degradation of NP gene segments or whether the levels of viral RNA other than NP are also affected, primers specific to NS were used for RT and real-time PCR, as described above (Example 4 ) measures the amount of different NS RNA species (mRNA, vRNA, cRNA). As shown in Figure 19, changes in NS mRNA, vRNA and cRNA showed the same pattern as observed for NP RNA. At 3 hours post-infection, a significant increase in all NS RNA species was seen in control transfected cells, whereas no significant change in NS RNA content was seen in cells receiving NP-1496 siRNA. This result suggests that transcription and replication of different viral RNAs are co-regulated, at least relative to NP RNAs. Comodulation means that the level of one transcript directly or indirectly affects the level of another. No special mechanism is implied. When NP transcripts were degraded by siRNA treatment, the levels of other viral RNAs were also reduced.

To further explore the effect of NP siRNA on other viral RNAs, the accumulation of mRNA, vRNA and cRNA of all viral genes was measured in cells that had been treated with NP-1496. As shown in Figure 19A (upper panel), NP-specific mRNA was low after 1 or 2 hours of infection. Three hours after infection, NP mRNA was readily detectable in the absence of NP-1496, whereas in the presence of NP-1496, the level of NP mRNA remained at background levels, suggesting that siRNA inhibits the accumulation of specific mRNA. As shown in Figure 19A (middle and lower panels), the levels of NP-specific and NS-specific vRNA and cRNA were greatly suppressed by the presence of NP-1496. The results confirm the results described in Example 4. In addition, the accumulation of mRNA, vRNA and cRNA of M, NS, PB1, PB2 and PA genes was also inhibited in NP-1496-treated cells (Fig. 19B, 19C and 19H). In addition, broad inhibition was also observed for PA-2087. The upper, middle and lower panels to the left of Figures 19E, 19F and 19G show the same results as presented in Figures 19A, 19B and 19C, demonstrating that NP-1496 siRNA inhibits viral mRNA transcription and viral vRNA and cRNA replication . The results of the same experiment performed with PA-2087 siRNA at the same concentrations are presented in the upper, middle and lower panels to the right of Figures 19E, 19F and 19G. As shown in Figure 19E, in the upper right panel, middle panel and lower panel, respectively, at 3 hours after infection, PA, M and NS mRNAs were readily detectable in the absence of PA-2087, whereas the presence of PA-2087 inhibited Transcription of PA, M and NS mRNA. As shown in Figure 19F, in the upper right panel, middle panel and lower panel, respectively, 3 hours after infection, PA, M and NS vRNA were easily detected in the absence of PA-2087, while the presence of PA-2087 inhibited PA , M and NS vRNA accumulation. As shown in Figure 19G, in the upper right panel, middle panel and lower panel, respectively, at 3 hours after infection, PA, M and NS cRNAs were readily detected in the absence of PA-2087, whereas the presence of PA-2087 inhibited PA , M and NS cRNA accumulation. Additionally, Figure 19H shows that NP-specific siRNA inhibits the accumulation of PB1 (upper panel), PB2 (middle panel) and PA (lower panel) specific mRNA.

While not wishing to be bound by any theory, the inventors believe that the broad effect of NP siRNAs may be a result of the importance of NPs in binding and stabilizing vRNAs and cRNAs, rather than because NP-specific siRNAs non-specifically target RNA degradation. The NP gene segment in influenza virus encodes a single-stranded RNA-binding nucleoprotein that binds vRNA and cRNA (see Figure 15). During the viral life cycle, NP mRNA is first transcribed and translated. The main function of the NP protein is to encapsidate the viral genome for the purpose of RNA transcription, replication and assembly. Full-length synthesis of vRNA and cRNA is greatly impaired in the absence of NP protein. When NP siRNA induces the degradation of NP RNA, NP protein synthesis is impaired and the resulting lack of sufficient NP protein subsequently affects the replication of other viral gene segments. In this way, NP siRNA may effectively inhibit virus production at an extremely early stage.

It is hypothesized that the number of NP protein molecules in infected cells regulates the level of mRNA synthesis relative to genomic RNA (vRNA and cRNA) replication (1). Using temperature-sensitive mutations of the NP protein, previous studies have demonstrated that cRNA synthesis, but not mRNA synthesis, is temperature-sensitive in vitro and in vivo (70, 71). NP proteins have been shown to be required for elongation and resistance to termination of nascent cRNA and vRNA transcripts (71, 72). The results presented above demonstrate that NP-specific siRNA inhibits the accumulation of all viral RNAs in infected cells. While not wishing to be bound by any theory, it seems possible that in the presence of NP-specific siRNA, newly transcribed NP mRNA is degraded, causing inhibition of NP protein synthesis following viral infection. In the absence of newly synthesized NPs, further viral transcription and replication and the resulting production of new virions are inhibited.

Likewise, in the specific presence of PA, newly transcribed PA mRNA is degraded, causing inhibition of PA protein synthesis. Although 30-60 copies of RNA transcriptase are present per influenza virion (1), in the absence of newly synthesized RNA transcriptase, further viral transcription and replication may be inhibited. Similar results were obtained using siRNA specific for PB1. In contrast, matrix (M) proteins are not required until late in viral infection (1). Thus, M-specific siRNA inhibits the accumulation of M-specific mRNA but not vRNA, cRNA or other viral RNA. Taken together, the findings demonstrate a critical requirement for newly synthesized nucleoprotein and polymerase proteins in influenza virus RNA transcription and replication. The mRNA-specific and virus-specific mechanisms by which NP-specific, PA-specific and PB1-specific siRNAs interfere with mRNA accumulation and transcription of other viral RNAs suggest that the siRNAs may be particularly potent inhibitors of influenza virus infection.

Example 7: Broad inhibition of influenza virus RNA accumulation by certain siRNAs is not due to interferon response or virus-induced RNA degradation.

Materials and methods

RNA content measurement. RNA content was measured using PCR under standard conditions. The following PCR primers were used to measure γ-actin RNA.

γ-actin forward: 5'-TCTGTCAGGGTTGGAAAGTC-3' (SEQ ID NO: 177)

γ-actin reverse: 5'-AAATGCAAACCGCTTCCAAC-3' (SEQ ID NO: 178)

Culturing of Vero cells and measurement of phosphorylated PKR were performed according to standard techniques described in the references cited below.

result

One possible reason for the broad inhibition of viral RNA accumulation described in Example 6 is the interferon response of infected cells in the presence of siRNA (23, 65, 66). Therefore, the experiments above were repeated in Vero cells, in which the entire IFN locus was deleted, including all α, β and ω genes (67, 68) (Q.G. and J.C. unpublished data). As in MDCK cells, the accumulation of NP-specific, M-specific and NS-specific mRNAs were all inhibited by NP-1496 (Fig. 19D). In addition, PCR was used to analyze the effect of siRNA on the content of transcripts from cellular genes including β-actin, γ-actin and GAPDH. No significant difference in transcript content was detected in the absence or presence of siRNA (lower panel of Figure 18C, showing no effect of M-37 siRNA on γ-actin mRNA and data not shown), indicating that the inhibitory effect of siRNA on Viral RNA is specific. The results suggest that the broad inhibition of viral RNA accumulation by certain siRNAs is not the result of cellular interferon responses.

Following influenza virus infection, the presence of dsRNA also activates cellular pathways that target the RNA for degradation (23). To investigate the role of siRNA in the activation of this pathway, the levels of phosphorylated protein kinase R (PKR), the most critical component of the pathway, were analyzed (23). In the absence of viral infection, transfection of MDCK cells with NP-1496 did not affect the levels of activated PKR (data not shown). Infection with influenza virus resulted in increased levels of phosphorylated PKR, consistent with previous studies (65, 66, 69). However, the increase was the same in the presence or absence of NP-1496 (data not shown). Thus, the broad inhibition of viral RNA accumulation was not a result of enhanced virus-induced degradation in the presence of siRNA.

Example 8: Systematic Identification of siRNAs with Superior Ability to Inhibit Influenza Virus Production Alone or in Combination

A high-throughput screen (Example 18) was performed to identify siRNAs with superior ability to inhibit influenza virus production. siRNAs were tested individually in cell culture, and many were further tested in mice. Certain combinations are also tested and demonstrate additive effects. Systematic testing of additional combinations was performed to identify combinations with synergistic (ie, greater than additive) effects. The siRNA and other RNAi-inducing entities comprising the same antisense strand were further tested against additional influenza virus strains, including adult human and avian pathogens.

Example 9: Evaluation of non-viral delivery agents that facilitate cellular uptake of siRNA. Various non-viral delivery agents were tested for their ability to enhance cellular uptake of siRNA. Subsequent examples provide data showing results (eg, inhibition of influenza virus production) when utilizing a number of polymers in cell culture and animals. Additional delivery agents were tested using similar methods.

Example 10: Testing Compositions Containing RNAi Inducing Agents in Mice

Dried particles containing RNAi-inducing agents targeting influenza virus transcripts were prepared as described (58). In the procedure, water-soluble excipients (ie, lactose, albumin, etc.) and therapeutic agent are dissolved in distilled water. The solution was fed into a Niro Atomizer Portable Spray Dryer (Niro, Inc., Colombus, MD) to produce a compound having an average geometric diameter between 3 and 15 μm and between 0.04 and 0.6 g/cm. Dry powder with a tap density between ³ . The dry powder is administered into the respiratory system of the mouse by inhalation or intratracheal administration. Inhalation delivery of dry powder aerosols was accomplished by forced ventilation on anesthetized mice. For intratracheal administration, a solution containing the therapeutic agent is injected through a tube into the lungs of anesthetized mice (54). In other experiments, for the delivery of liquids, liquid aerosols were generated by nebulizers into airtight plastic cages in which mice were placed (52). Pulmonary delivery of dry powders to small animals can be performed using an insufflator (eg, Model IA-IC) such as that available from PermCentury ( URLwww.penncentury.com ).

Example 11: Inhibition of influenza virus infection by siRNA transcribed from templates provided by DNA vectors or lentiviruses

As an alternative to the methods described above, the use of DNA vectors from which siRNA precursors can be transcribed and processed into potent siRNAs was explored as described in Examples 13 and 14. Figures 20A-20C show vectors used in previous studies (27, 59), the results of which are shown in Figure 20D and additionally described in US Patent No. 10/674,159. Figures 21A-21C show additional constructs that can be used to test the efficacy of hairpin precursors, including precursors to a variety of different siRNAs.

Example 12: Inhibition of Influenza Virus Production in Mice by siRNA

This example describes experiments showing that administration of siRNA targeting influenza virus NP or PA transcripts inhibits influenza virus production in mice when administered before or after infection with influenza virus. The inhibition was dose-dependent and showed an additive effect when 2 siRNAs each targeting transcripts expressed from different influenza virus genes were administered together.

Materials and methods

SiRNA Preparation. The preparation was performed as described above.

SiRNA delivery. JetPEI ^™ with oligonucleotide cationic polymer transfection reagent at room temperature in 5% glucose, N/P ratio = 5 (Qbiogene, Inc., Carlsbad, CA; Cat. No. GDSP20130; N/P P refers to the nitrogen number of phosphate per nucleotide in the jetPEI/siRNA mixture) or with poly-L-lysine (MW (vis) 52,000; MW (LALLS) 41,800, Sigma catalog number P2636), the siRNA (30 or 60 μg of GFP-949, NP-1496 or PA-2087) for 20 min. The mixture was injected intravenously into mice, 200 μl per mouse, into the retro-orbital vein, 4 mice per group. 200 [mu]l of 5% glucose was injected into control (no treatment) mice. Mice were anesthetized with 2.5% Avertin prior to siRNA injection or intranasal infection.

Virus infection. Infect B6 mice (maintained under standard laboratory conditions) with PR8 virus intranasally by pipetting virus-containing buffer into the nose of the mouse, 30 μl per mouse (12,000 pfu).

Virus titer determination. Mice were sacrificed at various time points after infection and lungs were collected. Lungs were homogenized and the homogenate was frozen and thawed twice to release virus. Titers of PR8 virus present in infected lungs were measured by infection of MDCK cells. Flat-bottomed 96-well plates were seeded at 3 × ¹⁰⁴ MDCK cells per well, and 24 hours later, the serum-containing medium was removed. Inoculate 25 μl of lung homogenate undiluted or diluted from 1 × ¹⁰ to 1 × ¹⁰ into triplicate wells. After 1 h incubation, 175 μl infection medium with 4 μg/ml trypsin was added to each well. The presence or absence of virus was determined by hemagglutination of chicken RBC from the supernatant of infected cells after 48 h incubation at 37°C. Hemagglutination assays were performed in V-bottom 96-well plates. Serial 2-fold dilutions of the supernatant were mixed with an equal volume of 0.5% (v/v) chicken erythrocyte suspension (Charles River Laboratories) and incubated on ice for 1 h. Wells containing an adherent, homogeneous layer of erythrocytes were scored positive. Virus titers were determined by the method of Reed and Muench ( _TCID50 ) by interpolation of the dilution endpoint at which 50% of the wells were infected, whereby lower _TCID50s reflect lower viral titers. Data from any 2 groups were compared using the Student t test, which was used throughout the experiments described herein to assess significance.

result

Figure 22A shows the results of an experiment demonstrating that siRNA targeting the viral NP transcript inhibits influenza virus production in mice when administered prior to infection. 30 or 60 μg of GFP-949 or NP-1496 siRNA were incubated with jetPEI and injected intravenously into mice as described above in Materials and Methods. Three hours later, mice were infected intranasally with PR8 virus at 12000 pfu per mouse. Lungs were collected 24 hours after infection. As shown in Figure 22A, the mean log _{10 TCID50} of lung homogenates of mice that received no siRNA treatment (NT; closed squares) or received siRNA targeting GFP (GFP 60 μg; open squares) was 4.2. In mice pretreated with 30 μg of siRNA targeting NP (NP 30 μg; open circles) and jetPEI, the mean log ₁₀ TCID ₅₀ of lung homogenates was 3.9. In mice pretreated with 60 μg of siRNA targeting NP (NP 60 μg; solid circles) and jetPEI, the mean log ₁₀ TCID ₅₀ of lung homogenates was 3.2. There was a significant difference in virus titer of lung homogenate between the group not receiving treatment and the group receiving 60 μg NP siRNA, P=0.0002. Data for individual mice are presented in Table 6A (NT = no treatment).

Figure 22B shows the results of another experiment demonstrating that siRNA targeting the viral NP transcript inhibits influenza virus production in mice when administered intravenously in a composition containing cationic polymer PLL prior to infection. 30 or 60 μg of GFP-949 or NP-1496 siRNA were incubated with PLL as described above in Materials and Methods and injected intravenously into mice. Three hours later, mice were infected intranasally with PR8 virus at 12000 pfu per mouse. Lungs were collected 24 hours after infection. As shown in Figure 22B, the mean log _{10 TCID50} of lung homogenates of mice that received no siRNA treatment (NT; closed squares) or received siRNA targeting GFP (GFP 60 μg; open squares) was 4.1. In mice pretreated with 60 μg of siRNA targeting NP (NP 60 μg; solid circles) and PLL, the mean log ₁₀ TCID ₅₀ of lung homogenate was 3.0. There was a significant difference in the viral titer of the lung homogenate between the group receiving 60 μg GFP and the group receiving 60 μg NP siRNA, P=0.001. Data for individual mice are presented in Table 6A (NT = no treatment). The data demonstrate that siRNA targeting the influenza NP transcript reduces viral titers in the lung when administered prior to viral infection. It was also shown that mixtures of siRNA and cationic polymer effectively inhibited influenza virus in the lung when administered by intravenous injection without the need for techniques such as hydrodynamic transfection.

Table 6A. Inhibition of influenza virus production in mice by siRNA and cationic polymer

deal with Log ₁₀ TCID ₅₀ NT(jetPEI experiment)GFP(60μg)+jetPEINP(30μg)+jetPEINP(60μg)+jetPEINT(PLL experiment)GFP(60μg)+PLLNP(60μg)+PLL 4.34.34.03.34.04.33.3 4.34.34.03.34.34.03.0 4.04.33.73.04.04.03.0   4.04.03.73.04.0 (not in progress) 2.7

Figure 22C shows the results of a third experiment demonstrating that siRNA targeting the viral NP transcript inhibits influenza virus production in mice when administered prior to infection, and demonstrating that the presence of cationic polymers significantly increases the inhibitory efficacy of the siRNA. 60 μg of GFP-949 or NP-1496 siRNA were incubated with phosphate buffered saline (PBS) or jetPEI and injected into mice via the intravenous route as described above in Materials and Methods. Three hours later, mice were infected intranasally with PR8 virus at 12000 pfu per mouse. Lungs were collected 24 hours after infection. As shown in Figure 22C, the mean log ₁₀ TCID ₅₀ of lung homogenates from mice that did not receive siRNA treatment (NT; open squares) was 4.1, while those that received siRNA targeting GFP in PBS (GFP PBS; open triangles ) mean log ₁₀ TCID ₅₀ of mouse lung homogenate was 4.4. In mice pretreated with 60 μg of siRNA targeting NP in PBS (NP PBS; filled triangles), the mean log ₁₀ TCID ₅₀ of lung homogenates was 4.2, relative to untreated or treated with siRNA targeting GFP , showing only a modest increase in efficacy. In mice pretreated with 60 μg of siRNA targeting GFP in jetPEI (GFP PEI; open circles), the mean log ₁₀ TCID ₅₀ of lung homogenates was 4.2. However, in mice receiving 60 μg of siRNA targeting NP in jetPEI (NP PEI; filled circles), the mean log ₁₀ TCID ₅₀ of lung homogenate was 3.2. There was a significant difference in the virus titer of lung homogenate between the group receiving GFP siRNA in PBS and the group receiving NP siRNA in PBS, P=0.04, while the group receiving GFP siRNA and jetPEI was significantly different from the group receiving NP siRNA and jetPEI. There was a highly significant difference in virus titers of lung homogenates between groups, P=0.003. Data for individual mice are presented in Table 6B (NT = no treatment).

Table 6B. Inhibition of Influenza Virus Production in Mice by siRNAs Showing Increased Efficacy in the Presence of Cationic Polymers system

deal with Log ₁₀ TCID ₅₀ NT 4.3 4.3 4.0 3.7

GFP(60μg)+PBSNP(60μg)+PBSGFP(60μg)+jetPEINT(60μg)+jetPEI 4.33.74.33.3 4.34.34.33.0 4.74.04.03.7 4.34.03.03.0

Additional experiments were performed to assess the ability of siRNAs to inhibit influenza virus production at various times post-infection when administered at various time points before or after infection.

siRNA was administered as described above, except that 120 μg of siRNA was administered 12 hours prior to viral infection. Table 6C shows the results expressed as log ₁₀ TCID ₅₀ . The P value for the NP-treated group compared to the control group was 0.049.

Table 6C

mouse 1 mouse 2 mouse 3 mouse 4 NT 4.3 4 4 4 GFP-949 4.3 4 4 4 NP-1496 4 3.7 3.7 3.3

In another experiment, siRNA (60 μg) was administered 3 hours prior to infection. 1500 pfu of PR8 virus were administered intranasally. Infected lungs were collected 48 h after infection. Table 6D shows the results expressed as log ₁₀ TCID ₅₀ . The P value for the NP-treated group compared to the control group was 0.03.

Table 6D

mouse 1 mouse 2 mouse 3 mouse 4 NT 4 4 4 4 GFP-949 4.3 4 4 3.7 NP-1496 3 3.7 3.7 3.3

In another experiment, siRNA (120 μg) was administered 24 hours after PR8 (1500 pfu) infection. Fifty-two hours after infection, lungs were harvested and virus titers were measured. Table 6D shows the results expressed as log ₁₀ TCID ₅₀ . The P value for the NP-treated group compared to the control group was 0.03.

Table 6E

mouse 1 mouse 2 mouse 3 mouse 4 GFP-949 2.3 2.7 2 2.7 NP-1496 2 2 1.7 2

Other polymers have also been shown to be effective siRNA delivery agents. Figure 22D is a graph showing that siRNA targeting NP (NP-1496) inhibits influenza virus production in mice when administered via the intravenous route along with poly(beta amino ester) (J28). Figure 22E is a graph showing that siRNA targeting NP (NP-1496) inhibits influenza virus production in mice when administered together with poly(beta amino ester) (J28 or C32) via the intraperitoneal route, while control RNA (GFP) Plots with no significant effect. Experiments were performed essentially as described above, except that the ratio of polymer to siRNA was a weight/weight ratio (eg, 60:1 w/w). Three hours prior to intranasal infection with 12,000 pfu of PR8 virus, the polymer and siRNA were mixed and administered to mice by either the intravenous or intraperitoneal route. Lungs were collected after 24 hours and HA analysis was performed. The amine and bis(acrylate) monomers present in J28 and C32 are described and depicted in U.S.S.N. 10/446,444. Polymer is a donation of Dr. Robert Langer.

Figure 23 shows the results of an experiment demonstrating that siRNAs targeting different influenza virus transcripts show additive effects. 60 μg of NP-1496 siRNA, 60 μg of PA-2087 siRNA, or 60 μg of NP-1496 siRNA + 60 μg of PA-2087 siRNA were incubated with jetPEI and injected into mice via the intravenous route as described above in Materials and Methods. Three hours later, mice were infected intranasally with PR8 virus at 12,000 pfu per mouse. Lungs were collected 24 hours after infection. As shown in Figure 23, the mean log _{10 TCID50} of lung homogenates from mice that did not receive siRNA treatment (NT; filled squares) was 4.2. In mice receiving 60 μg of siRNA targeting NP (NP 60 μg; open circles), the mean log ₁₀ TCID ₅₀ of lung homogenate was 3.2. In mice receiving 60 μg of siRNA targeting PA (PA 60 μg; open triangles), the mean log ₁₀ TCID ₅₀ of lung homogenates was 3.4. In mice receiving 60 μg siRNA targeting NP + 60 μg siRNA targeting PA (NP+PA; filled circles), the mean log ₁₀ TCID ₅₀ of lung homogenate was 2.4. There were significant differences in virus titers of lung homogenate between the group not receiving treatment and the group receiving 60 μg NP siRNA, 60 μg PA siRNA or 60 μg NP siRNA+60 μg PA siRNA, P=0.003, 0.01 and 0.0001, respectively. There was a significant difference in virus titer of lung homogenate between the group receiving 60 μg NP siRNA or 60 μg NP siRNA and the group receiving 60 μg NP siRNA+60 μg PA siRNA, P=0.01. Data for individual mice are presented in Table 7 (NT = no treatment). The data demonstrate that pretreatment with siRNA targeting influenza NP or PA transcripts reduces viral titers in the lungs of mice subsequently infected with influenza virus. The data additionally indicated that combinations of siRNAs targeting different viral transcripts exhibited additive effects, suggesting that therapy with combinations of siRNAs targeting different transcripts would make the various siRNAs dose reduction.

Table 7. Additive effects of siRNA against influenza virus in mice

deal with log ₁₀ TCID ₅₀ NTNP (60μg) PA (60μg) NP+PA (60μg each) 4.33.73.72.7 4.33.33.72.7 4.03.03.02.3 4.03.03.02.0

Figure 24 shows the results of an experiment demonstrating that siRNA targeting the viral NP transcript inhibits influenza virus production in mice when administered post-infection. Mice were infected intranasally with PR8 virus (500 pfu). 60 μg of GFP-949 siRNA, 60 μg PA-2087 siRNA, 60 μg NP-1496 siRNA or 60 μg NP siRNA+60 μg PA siRNA were incubated with jetPEI as described above in Materials and Methods and injected into mice 5 hours later via the intravenous route. Lungs were harvested 28 hours after siRNA administration. As shown in Figure 24, the mean log _{10 TCID50} of lung homogenates from mice that received no siRNA treatment (NT; closed squares) or received the GFP-specific siRNA GFP-949 (GFP; open squares) was 3.0. In mice receiving 60 μg of siRNA targeting PA (PA 60 μg; open triangles), the mean log ₁₀ TCID ₅₀ of lung homogenate was 2.2. In mice receiving 60 μg of siRNA targeting NP (NP 60 μg; open circles), the mean log ₁₀ TCID ₅₀ of lung homogenates was 2.2. In mice receiving 60 μg NP siRNA + 60 μg PA siRNA (PA+NP; filled circles), the mean log ₁₀ TCID ₅₀ of lung homogenate was 1.8. There were significant differences in virus titers of lung homogenate between the group not receiving treatment and the group receiving 60 μg PA, NP siRNA or 60 μg NP siRNA+60 μg PA siRNA, P=0.09, 0.02 and 0.003, respectively. The difference in viral titers of lung homogenates between the group receiving NP siRNA and the group receiving PA+NP siRNA had a P value of 0.2. Data for individual mice are presented in Table 8 (NT = no treatment). The data demonstrate that siRNAs targeting influenza NP and/or PA transcripts reduce viral titers in the lung when administered after viral infection.

Table 8. Inhibition of Influenza Virus Production in Infected Mice by siRNA

deal with log ₁₀ TCTD ₅₀ NTGFP (60 μg) PA (60 μg) NP (60 μg) NP+PA (60 μg each) 3.O3.02.72.72.3 3.03.02.72.32.0 3.03.02.32.31.7 3.02.71.31.71.3

Example 13: Inhibition of Influenza Virus Production in Cells by Administration of Lentiviruses Providing Templates for ShRNA Production

Materials and methods

Cell proliferation. Vero cells were seeded in 24-well plates at 4 x ¹⁰⁵ cells per well in 1 ml DMEM-10% FCS and cultured at 37 °C, 5% _CO2 .

Generation of lentiviruses providing templates for shRNA production. As schematically depicted in Figure 25A, the oligonucleotide used as a template for the synthesis of NP-1496a shRNA (see Figure 25A) was cloned between the U6 promoter and termination sequence of the lentiviral vector pLL3.7 (Rubinson, D. et al., Nature Genetics, Vol. 33, pp. 401-406, 2003). The oligonucleotide was inserted between the Hpal and Xhol restriction sites in the multiple cloning site of pLL3.7. The lentiviral vector also expresses EGFP to facilitate monitoring of transfected/infected cells. Lentiviruses were generated by co-transfecting DNA vectors containing templates for NP-1496a shRNA production, and assembling the vectors into 293T cells. After 48 h, the culture supernatant containing lentivirus was collected, spun at 2000 rpm for 7 min at 4°C and then filtered through a 0.45 μm filter. Vero cells were seeded in 24-well plates at 1 × ¹⁰⁵ per well. After overnight incubation, culture supernatants containing inserts (0.25 ml or 1.0 ml) were added to wells in the presence of 8 μg/ml polybrene. Plates were then centrifuged at 2500 rpm for 1 h at room temperature and returned to the culture. 24 h after infection, the resulting Vero cell lines (Vero-NP-0.25 and Vero-NP-1.0) as well as parental (uninfected) Vero cells were analyzed for GFP expression by flow cytometry. Note that NP-1496a differs from NP-1496 by the unintentional inclusion of an additional nucleotide (A) at the 3' end of the sense portion and the unintentional inclusion of a complementary nucleotide (A) at the 5' end of the antisense portion ( U), thereby forming a duplex portion of 20 nt in length instead of 19 nt as in NP-1496. (See Table 2). According to other embodiments of the invention, the NP-1496 sequence is used instead of the NP-1496a sequence. In addition, the loop portion of NP-1496a shRNA is different from that of NP-1496 shRNA shown in FIG. 21 .

Influenza virus infection and determination of viral titers. Control Vero cells and Vero cells infected with insert-containing lentiviruses (Vero-NP-0.25 and Vero-NP-0.25 and Vero-NP- 1.0). Influenza virus titers in supernatants were determined by HA analysis 48 hours after infection as described in Example 12.

result

The ability of lentivirus containing the template used to produce NP-1496a shRNA to inhibit influenza virus production in Vero cells was tested. NP-1496a shRNA includes two complementary regions capable of forming a stem-loop structure containing a double-stranded portion having the same sequence as the NP-1496a siRNA described above. As shown in Figure 25B, overnight incubation of the lentivirus-containing supernatant with Vero cells resulted in the expression of EGFP, indicating that Vero cells were infected with lentivirus. Shaded curves represent mean fluorescence intensity in control cells (uninfected). When 1 ml of supernatant was used, almost all cells became EGFP positive and the mean fluorescence intensity was higher (1818) (Vero-NP-1.0). When 0.25 ml of supernatant was used, most cells (about 95%) were positive for EGFP and the mean fluorescence intensity was low (503) (Vero-NP-0.25).

Next, parental Vero cells and lentivirus-infected Vero cells were infected with influenza virus at MOIs of 0.04, 0.2, and 0.1, and virus titers were analyzed 48 hours after influenza virus infection. Virus titers in supernatants of parental Vero cell cultures increased with increasing MOI (Fig. 25C). In contrast, virus titers in the supernatants of Vero-NP-1.0 cell cultures remained extremely low, indicating that influenza virus production was inhibited in the cells. Likewise, influenza virus production in Vero-NP-0.25 cell cultures was also partially inhibited. Virus titers are presented in Table 9. The results suggest that NP-1496 shRNA expressed from a lentiviral vector can be processed into siRNA to inhibit influenza virus production in Vero cells. The degree of inhibition appears to be proportional to the degree of virus infection per cell (as represented by EGFP content).

Table 9. Inhibition of Influenza Virus Production by siRNAs Expressed in Cells in Tissue Culture

cell line Virus titer VeroVero-NP-0.25Vero-NP-1.0 1681 64324 128648

Example 14: Inhibition of influenza production in mice by intranasal administration of DNA vectors for transcription of siRNA precursors

Materials and methods

Construction of plasmids used as templates for shRNA. Construction of plasmids for expression of NP-1496a shRNA is described in Example 13. Cloning between the U6 promoter and termination sequence of lentiviral vector pLL3.7 as described in Example 13 and schematically depicted for NP-1496a shRNA in Figure 25A was used to synthesize PB1-2257 shRNA or RSV-specific shRNA. Template oligonucleotides. The sequence of the oligonucleotide is as follows:

NP-1496a has meaning:

5′-TGGATCTTATTTCTTCGGAGATTCAAGAGATCTCCGAAGAAATAAGATCCTTTTTT

C-3' (SEQ ID NO: 179)

NP-1496a antisense:

5'-TCGAGAAAAAAGGATCTTATTTCTTCGGAGATCTCTTGAATCTCCGAAGAAATAA GATCCA-3' (SEQ ID NO: 180)

PB1-2257 has meaning:

5'-TGATCTGTTCCACCATTGAATTCAAGAGATTCAATGGTGGAACAGATCTTTTTTC-3' (SEQ ID NO: 181)

PB1-2257 antisense:

5'-TCGAGAAAAAAGATCTGTTCCACCATTGAATCTCTTGAATTCAATGGTGGAACAGAT CA-3' (SEQ ID NO: 182)

RSV has meaning:

5'-TGCGATAATATAACTGCAAGATTCAAGAGATCTTGCAGTTATATTATCGTTTTTTTC-3' (SEQ ID NO: 183)

RSV antisense:

5'-TCGAGAAAAAACGATAATATAACTGCAAGATCTCTTGAATCTTGCAGTTATATTA TCGCA-3' (SEQ ID NO: 184)

RSV shRNA expressed from vectors containing the above oligonucleotides was processed in vivo to generate siRNAs comprising sense and antisense strands with the following sequences:

Sense: 5′-CGATAATATAACTGCAAGA-3′ (SEQ ID NO: 185)

Antisense: 5'-TCTTGCAGTTATATTATCG-3' (SEQ ID NO: 186)

PA-specific hairpins can be similarly constructed using the following oligonucleotides:

PA-2087 has meaning:

5'-TGCAATTGAGGAGTGCCTGATTCAAGAGATCAGGCACTCCTCAATTGCTTTTTTC-3' (SEQ ID NO: 187)

PA-2087 antisense:

5'-TCGAGAAAAAAGCAATTGAGGAGTGCCTGATCTCTTGAATCAGGCACTCCTCAATTGCA-3' (SEQ ID NO: 270)

Determination of virus infection and virus titer. The determination of virus infection and virus titer was performed as described in Example 12.

DNA Delivery. Plasmid DNA (60 μg each) capable of being used as a template for expression of NP-1496a shRNA, PB1-2257 shRNA, or RSV-specific shRNA was individually mixed with 40 μl of Infasurf® (ONY, Inc., Amherst NY) as described above. Mixed with 20 μl of 5% glucose and administered intranasally to groups of 4 mice. A mixture of 40 μl Infasurf and 20 μl of 5% glucose was administered to mice in the no treatment (NT) group. Thirteen hours later, mice were infected intranasally with PR8 virus at 12000 pfu per mouse as described above. Lungs were harvested 24 hours post-infection and virus titers were determined.

result

The ability of shRNA expressed from a DNA vector to inhibit influenza virus infection in mice was tested. For the experiments, plasmid DNA was mixed with Infasurf, a natural surfactant extract from calf lung used to facilitate gene transfer in the lung similar to the vehicle shown previously (74). The DNA/Infasurf mixture was instilled into the mice by using a pipette to drip the mixture into the nose. Thirteen hours later, mice were infected with PR8 virus at 12000 pfu per mouse. Twenty-four hours after influenza virus infection, lungs were harvested and virus titers were measured by MDCK/hemagglutinin assay.

As shown in Figure 26, virus titers were higher in mice not given any plasmid DNA or given a DNA vector expressing respiratory syncytial virus (RSV) specific shRNA. Lower viral titers were observed when mice were given plasmid DNA expressing NP-1496a shRNA or PB1-2257 shRNA. When mouse influenza-specific plasmid DNAs (one expressing NP-1496a shRNA and the other expressing PB1-2257 shRNA) were co-administered, viral titers were further significantly reduced. The mean log _{10 TCID50} of lung homogenates from mice that received no treatment (NT; open squares) or received a plasmid encoding an RSV-specific shRNA (RSV; closed squares) was 4.0 or 4.1, respectively. In mice receiving a plasmid capable of serving as a template for NP-1496a shRNA (NP; open circles), the mean log ₁₀ TCID ₅₀ of lung homogenates was 3.4. In mice receiving a plasmid capable of serving as a template for PB1-2257 shRNA (PB; open triangles), the mean log ₁₀ TCID ₅₀ of lung homogenates was 3.8. In mice receiving a plasmid that could be used as a template for NP and PB shRNA (NP+PB1; filled circles), the mean log _{10 TCID50} of lung homogenates was 3.2. Differences in viral titers of lung homogenates were 0.049, 0.124, and 0.004 between groups that received no treatment or RSV-specific shRNA plasmids and groups that received NP shRNA plasmids, PB1 shRNA plasmids, or NP and PB1 shRNA plasmids, respectively. P value. Data for individual mice are presented in Table 10 (NT = no treatment). The results show that shRNA expressed from a DNA vector can be processed into siRNA to suppress influenza virus production in mice and demonstrate that Infasurf is a suitable vehicle for the delivery of plasmids expressing shRNA. In particular, the data demonstrate that shRNA targeting influenza NP and/or PB1 transcripts reduces viral titers in the lung when administered after viral infection.

Table 10. Inhibition of influenza virus production by shRNA expressed in mice

deal with log ₁₀ TCID ₅₀ NTRSV (60 μg) NP (60 μg) PB1 (60 μg) NP+PB1 (60 μg each) 4.34.34.04.03.7 4.04.03.74.03.3 4.04.03.03.73.0 4.34.03.03.33.0

Example 15: Cationic polymers promote cellular uptake of siRNA

Materials and methods

Reagents. Poly-L-lysine with 2 different average molecular weights [poly-L-lysine (MW(vis) 52,000; MW(LALLS) 41,800, catalog number P2636), and poly-L-lysine were purchased from Sigma. Lysine (MW(vis)9,400; MW(LALLS)8,400, catalog number P2636], poly-L-arginine (MW15,000-70,000, catalog number P7762) and brominated 3-(4,5-di Methylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT). For the purposes of this description, molecular weights obtained using the LALLS method will be assumed, but it will be appreciated that molecular weights are approximate since polymers exhibit some dimensional unevenness.

Gel retardation assay. siRNA-polymer complexes were formed by mixing 10 μl of siRNA (10 pmol, dissolved in 10 mM Hepes buffer, pH 7.2) with 10 μl of polymer solution containing varying amounts of polymer. Complex formation was performed for 30 min at room temperature, after which 20 μl were run on a 4% agarose gel. Bands were visualized by staining with ethidium bromide.

Cytotoxicity assay. siRNA-polymer complexes were formed by mixing equal amounts (50 pmol) of siRNA in 10 mM Hepes buffer (pH 7.2) with polymer solutions containing varying amounts of polymer for 30 min at room temperature. Cytotoxicity was assessed by MTT assay. Cells were seeded in 96-well plates at 30,000 cells per well in 0.2 ml of DMEM containing 10% fetal calf serum (FCS). After overnight incubation at 37°C, the medium was removed and replaced with 0.18ml OPTI-MEM (GIBCO/BRL). siRNA-polymer complexes in 20 μl of Hepes buffer were added to the cells. After 6 h incubation at 37°C, the polymer-containing medium was removed and replaced with DMEM-10% FCS. After 24 h, the metabolic activity of the cells was measured using the MTT assay according to the manufacturer's instructions. Experiments were performed three times, and data were averaged.

Cell culture, transfection, siRNA-polymer complex formation, and viral titer determination Vero cells were incubated at 37°C in a 5% CO ₂ /95% air atmosphere in the presence of 10% heat-inactivated FCS, 2 mM L-glucose Aminoamide, 100 units/ml penicillin and 100 μg/ml streptomycin in DMEM. For transfection experiments, log-phase Vero cells were seeded in 24-well plates at 4 x ¹⁰⁵ cells per well in 1 ml of DMEM-10% FCS. After overnight incubation at 37°C, siRNA-polymer complexes were formed by adding 50 μl (400 pmol (about 700 ng) of siRNA in 10 mM Hepes buffer, pH 7.2) to a 50 μl polymer vortex. Different concentrations of polymer were used to achieve complete complex formation between siRNA and polymer. The mixture was incubated at room temperature for 30 min to complete complex formation. Cell growth medium was removed and replaced with OPTI-MEM I (Life Technologies) just prior to complex addition.

At 37°C, under 5% CO ₂ , after the cells were cultured with the complex for 6 h, the medium containing the complex was removed, and at MOI=0.04, DMEM, 0.3% BSA (Sigma), 10 mM Hepes, 100 200 μl of PR8 virus in infection medium consisting of 1 unit/ml penicillin and 100 μg/ml streptomycin was added to each well. After incubation for 1 h at room temperature with constant shaking, 0.8 ml infection medium containing 4 μg/ml trypsin was added to each well and the cells were incubated at 37° C. under 5% CO 2 . At various time points after infection, supernatants were collected from infected cultures and virus titers were determined by hemagglutination (HA) assay as described above.

For adherent cell lines, siRNA transfection was performed by Lipofectamine 2000 (Life Technology) according to the manufacturer's instructions. Briefly, Vero cells in log phase were seeded in 24-well plates at 4 x ¹⁰⁵ cells per well in 1 ml of DMEM-10% FCS and incubated at 37 °C under 5% _CO2 nourish. The next day, 50 μl of diluted Lipofectamine 2000 in OPTI-MEM I was added to 50 μl of siRNA (400 pmol, dissolved in OPTI-MEM I) to form a complex. Cells were washed and incubated in serum-free medium. Complexes were applied to cells and cells were incubated at 37°C for 6h before being washed and infected with influenza virus as described above. At various time points after infection, supernatants were collected from infected cultures and virus titers were determined by hemagglutination (HA) assay as described above.

result

Poly-L-lysine (PLL) and poly-L-arginine (PLA) were tested for their ability to form complexes with siRNA and promote uptake of siRNA by cultured cells. To determine whether PLL and/or PLA form complexes with siRNA, fixed amounts of NP-1496 siRNA were mixed with increasing amounts of polymer. Polymer/siRNA complex formation was then visualized by electrophoresis in a 4% agarose gel. As the amount of polymer increased, the electrophoretic mobility of siRNA was retarded (FIGS. 27A and 27B), indicating complex formation. Figures 27A and 27B show complex formation between siRNA and PLL (41.8K) or PLA, respectively. The amount of polymer used in each figure increases from left to right. In each of Figures 27A and 27B, a band can be seen in the left lane, indicating that no complex was formed and thus the siRNA entered the gel and subsequently migrated. When moving to the right, the band disappears, indicating complex formation and the complex cannot enter the gel and migrate.

To study the cytotoxicity of siRNA/polymer complexes, mixtures of siRNA and PLL or PLA at different ratios were added to Vero cell cultures in 96-well plates. The metabolic activity of cells was measured by MTT assay (74). Experiments were performed three times, and data were averaged. Cell viability decreased significantly with increasing amounts of PLL (MW about 42K), while PLL (about 8K) showed significantly lower toxicity, with minimal or no toxicity at PLL/siRNA ratios as high as 4:1 (Fig. 28A ; circles = PLL (MW about 8K); squares = PLL (MW about 42K)). As shown in Figure 28B, cell viability decreased with increasing PLA/siRNA ratio, but remained above 80% at PLA/siRNA ratios as high as 4.5:1. Polymer/siRNA ratios are indicated on the X-axis in Figures 28A and 28B. The data plotted in Figures 28A and 28B are presented in Tables 11 and 12. In Table 11, numbers indicate the percent survival of cells after treatment with polymer/siRNA complexes relative to the percent survival of untreated cells. ND = not performed. In Table 12, numbers represent PLA/siRNA ratios, percent survival and standard deviations as indicated.

Table 11. Cytotoxicity of PLL/siRNA complexes (% survival)

deal with Polymer/siRNA ratio PLL about 8.4KPLL about 41.8K 0.592.26ND 1.083.57100 2.084.39100 4.041.42100 8.032.5182.55 16.0ND69.63

Table 12. Cytotoxicity (% survival) of PLA/siRNA complexes

Polymer/siRNA ratio Survival percentage standard deviation 0.1794.61.74 0.51001.91 1.592.332.92 4.5831.51 13.539.194.12

To determine whether PLL or PLA promoted cellular uptake of siRNA, various amounts of polymer and NP-1496 were mixed at the ratio of all siRNA to polymer complexed. In all cases, equal amounts of siRNA were used. A lower polymer/siRNA ratio was used for PLLs around 42K than for PLLs around 8K because the former proved to be more toxic to cells. Complexes were added to Vero cells, and 6 hours later, the cultures were infected with PR8 virus. At various time points after infection, culture supernatants were collected and analyzed for virus by HA assay. 29A is a graph of virus titers over time in cells receiving various transfection treatments (circles = no treatment; squares = Lipofectamine; filled triangles = PLL (about 42K, PLL/siRNA ratio = 2); open triangles = PLL (about 8K, PLL/siRNA ratio=8)). As shown in Figure 29A, virus titers increased over time in non-transfected cultures. Viral titers were significantly lower in NP-1496/Lipofectamine-transfected cultures, and even lower in cultures treated with PLL/NP-1496 complexes. Data plotted in Figure 29A are presented in Table 13A (NT = no treatment; LF2K = Lipofectamine). PLL:siRNA ratios are indicated in parentheses.

PLA was similarly tested over a range of polymer/siRNA ratios. Figure 29B is a graph of virus titers over time in cells receiving various transfection treatments (filled squares = control transfection; filled circles = Lipofectamine; open squares = PLA at PLA/siRNA ratio = 1; open circles = PLA at PLA/siRNA ratio=2; open triangles=PLA at PLA/siRNA ratio=4; closed triangles=PLA at PLA/siRNA ratio=8). As shown in Figure 29B, virus titers increased over time in control (control transfected) cultures and in cultures treated with PLA/siRNA at a 1:1 ratio. Viral titers were significantly lower in cultures transfected with NP-1496/Lipofectamine, and were significantly lower in cultures treated with PLA/siRNA complexes containing complexes at 4:1 or higher PLA/siRNA ratios. even lower. An increase in the amount of polymer resulted in a greater decrease in virus titer. The data plotted in Figure 29B are presented in Table 13B.

Table 13A. Inhibition of Influenza Virus Production by Polymer/siRNA Complexes

deal with Time (hours) twenty four 36 48 60 Control transfection LF2KPLL about 42K (2:1) PLL about 8K (8:1) 16411 64842 641684 641688

Table 13B. Inhibition of influenza virus production by polymer/siRNA complexes

deal with Time (hours) twenty four 36 48 60 Control transfected with LF2KPLA(1:1)PLA(2:1)PLA(4:1)PLA(8:1) 824411 646161641 128161283281 2563225664162

Thus, the cationic polymer facilitated cellular uptake of siRNA and inhibited influenza virus production in cell lines and was more effective than Lipofectamine, a widely used transfection reagent. The results also suggest that additional cationic polymers can be readily identified to stimulate cellular uptake of siRNA, and methods for their identification are described. PLL and PLA can be used as positive controls for the study.

Example 16A: Inhibition of Luciferase Activity in the Lung by Delivery of siRNA into the Vasculature or Airway

Materials and methods

siRNAs were obtained from Dharmacon and deprotected and annealed as described above. The siRNA sequences for NP (NP-1496), PA (PA-2087), PB1 (PB1-2257) and GFP were provided above. Luc-specific siRNA was as described in (McCaffrey, AP et al., Nature, 418:38-39).

PEI-mediated DNA transfection in mice. pCMV-luc DNA (Promega) was mixed with PEI (Qbiogene, Carlsbad, CA) at a nitrogen/phosphorus molar ratio (N/P ratio) of 10 for 20 min at room temperature. For intravenous (i.v.) administration, 8 week old male C57BL/6 mice (Taconic Farms) were injected retro-orbitally with a 200 μl mixture containing 60 μg DNA. For intratracheal (i.t) administration, 50 μl of the mixture containing 30 μg or 60 μg of DNA was administered into the lungs of anesthetized mice using a Perm Century Model IA-IC insufflator.

PEI-mediated siRNA delivery in mice. siRNA-PEI compositions were formed by mixing 60 μg of luc-specific or GFP-specific siRNA with jetPEI at an N/P ratio of 5 for 20 min at room temperature. For intravenous administration, 200 [mu]l mixtures containing the indicated amounts of siRNA were injected via the retro-orbital route. For pulmonary administration, 50 μl was delivered by the intratracheal route.

Luc analysis. At various time points after pCMV-luc DNA administration, lungs, spleens, livers, hearts and kidneys were collected and homogenized in Cell Lysis Buffer (Marker Gene Technologies, Eugene, OR). Luminescence was analyzed with the Luciferase Assay System (Promega) and measured with an Optocomp_I luminometer (MGM Instruments, Hamden, CT). Protein concentration in the homogenate was measured by BCA assay (Pierce).

result

To determine the tissue distribution of PEI-mediated nucleic acid delivery in mice, pCMV-lucDNA-PEI complexes were injected via the intravenous route, and 24 hours later, Luc activity was measured in various organs. Activity was highest in the lung, where Luc activity was detected for at least 4 days (Fig. 30A), whereas in the heart, liver, spleen and kidney, levels were 100-1,000-fold lower and detected for a shorter time after injection. Significant Luc activity was also detected in the lung when the DNA-PEI complex was instilled via the intratracheal route, albeit at lower levels than after intravenous administration (Fig. 30B).

To test the ability of PEI to promote the lung absorption of siRNA after intravenous administration, mice were first administered the pCMV-luc DNA-PEI complex via the intratracheal route, followed by intravenous injection of Luc-specific siRNA complexed with PEI, and PEI complex. Complexed control GFP-specific siRNA or 5% glucose in the same volume. After 24 hours, Luc activity in the lung was 17-fold lower in mice receiving Luc siRNA than in mice given GFP siRNA or no treatment ( FIG. 30C ). Since Luc siRNA could inhibit Luc expression only in the same lung cells transfected with the DNA vector, the results indicated that intravenous injection of the siRNA-PEI mixture achieved effective suppression of the target transcript in the lung.

To test the ability of PEI to promote pulmonary uptake of siRNA following pulmonary administration, mice were administered pCMVDNA-PEI complexes intravenously, followed immediately by intratracheal administration of Luc-specific siRNA mixed with PEI, siRNA mixed with PEI, and siRNA mixed with PEI. Control GFP-specific siRNA or 5% glucose in the same volume. After 24 hours, lung homogenates were analyzed for luciferase activity. Figure 30D shows luciferase activity in lung homogenates from mice dosed with luciferase-specific or GFP-specific siRNA, normalized to protein amount. Luciferase activity was 6.8-fold lower in mice treated with luciferase siRNA than in mice treated with GFP siRNA. The results indicate that pulmonary administration of the siRNA-PEI mixture achieves potent suppression of the target transcript in lung cells.

Example 16B: Inhibition of Cyclophilin B in the Lung by Delivery of siRNA into the Respiratory System

Cyclophilin B is an endogenous gene widely expressed in mammals. To assess the ability of siRNA delivered directly into the respiratory system to inhibit endogenous gene expression, outbred Blackswiss mice (approximately 30 g or more body weight) were anesthetized by isoflurane/oxygen and siRNA targeting cyclophilin B (Dharmacon, D-001136-01-20 siCONTROL Cyclophilin B siRNA (human/mouse/rat)) or control GFP-949 siRNA (2mg/kg) was administered to mice by intranasal route, for each siRNA, 2 mice as a group. Lungs were collected 24 hours after administration. RNA was extracted from lungs and reverse transcribed using random primers. Real-time PCR was then performed using cyclophilin B and GAPDH Taqman gene expression assays (Applied Biosystems). The results (Table 14) show that 70% inhibition of cyclophilin B was achieved by siRNA targeting cyclophilin B.

Table 14: Inhibition of Cyclophilin B in the Lung

average value average standard value normal value Inhibition percentage PBS-1PBS-2GFP-1GFP-2cyclophilin-1cyclophilin-2 5.3954063.1825622.5473524.9575391.1734441.339901 4.2889843.7524461.256672 12.5096870.7

Example 17: Selection of Appropriately Conserved Target Moieties

To identify appropriately conserved regions of various influenza A transcripts for use as targeting moieties for targeting RNAi inducers to inhibit expression in various strains, isolates from humans from a group of Genomic fragments of the resulting strains were aligned (in their plus-strand form, ie, the sequence found in the mRNA). The strains include many strains in addition to those listed in Example 1. Tables 15A-15H list the Genbank accession numbers (left column), strain names (middle column) and serotypes (right column) of influenza A virus genome segments used to identify appropriately conserved regions. The entire sequence of each fragment is aligned and compared, excluding introns. Includes 5' and 3' untranslated regions. The sets of strains aligned differed for the different fragments, but each set included at least 19 isolates from various years spanning between 1934 and 2004. Strains include all HA and NA types known to circulate in humans (H1, H2, H3, H5, H9, N1, N2).

Table 15A: Influenza A virus NP fragments (strains isolated from humans)

AF389119 A/Puerto Rico/8/34/Mount Sinai (Mount Sinai) H1N1

M63752 A/Singapore/1/57 H2N2

M23976 A/Ann Arbor/6/60 H2N2

AY210103 A/Korea/426/68 H2N2

M76606 A/New Jersey/8/76 H1N1

L07351 A/Memphis/18/78 H3N2

AJ628066 A/Fiji/15899/83 H1N1

L07369 A/Memphis/3/88 H3N2

M63755 A/Wisconsin/3523/88 H1N1

L07373 A/Guangdong/38/89 H3N2

L07357 A/Shanghai/6/90 H3N2

L24394 A/MD/12/91 H1N1

AF038254 A/Kitakyushu/159/93 H3N2

AB019358 A/Nagasaki/48/95 H3N2

AJ291400 A/Hong Kong/156/97 H5N1

AF255749 A/Hong Kong/498/97 H3N2

AF255752 A/Hong Kong/542/97 H5N1

AF038259 A/Shiga/25/97 H3N2

AF255753 A/Hong Kong/97/98 H5N1

AF342819 A/Wisconsin/10/98 H1N1

AJ289871 A/Hong Kong/1073/99 H9N2

AJ458276 A/Switzerland/9243/99 H3N2

ISDN13443 A/Sydney/274/2000 H3N2

AB126624 A/Yokohama/22/2002 H1N2

AY575905 A/Hong Kong/212/03 H5N1

AY526749 A/Vietnam/1196/04 H5N1

Table 15B: Influenza A virus PA transcripts (strains isolated from humans)

AF389117 A/Puerto Rico/8/34/Sinai H1N1

M26078 A/Singapore/1/57 H2N2

M23974 A/Ann Arbor/6/60 H2N2

M26079 A/Korea/426/68 H2N2

AJ605762 A/Fiji/15899/83 H1N1

AF037424 A/Kitakyushu/159/93 H3N2

U71138 A/Shiga/20/95 H3N2

AJ289874 A/Hong Kong/156/97 H5N1

AF257198 A/Hong Kong/498/97 H3N2

AF257201 A/Hong Kong/542/97 H5N1

AF037429 A/Shiga/25/97 H3N2

AF258519 A/Hong Kong/427/98 H1N1

AF257202 A/Hong Kong/97/98 H5N1

AY043028 A/Guangzhou/333/99 H9N2

AF257191 A/Hong Kong/1073/99 H9N2

AJ293922 A/Hong Kong/1774/99 H3N2

AB126627 A/Yokohama/22/2002 H1N2

AY576404 A/Hong Kong/212/03 H5N1

AY526750 A/Vietnam/1196/04 H5N1

Table 15C: Influenza A virus PB1 transcripts (strains isolated from humans)

AF389116 A/Puerto Rico/8/34/Sinai H1N1

M25924 A/Singapore/1/57 H2N2

M23972 A/Ann Arbor/6/60 H2N2

M25935 A/Korea/426/68 H2N2

AJ564807 A/Fiji/15899/83 H1N1

AF037418 A/Kitakyushu/159/93 H3N2

U71130 A/Shiga/20/95 H3N2

AJ404633 A/Hong Kong/156/97 H5N1

AF258823 A/Hong Kong/498/97 H3N2

AF258826 A/Hong Kong/542/97 H5N1

AF037423 A/Shiga/25/97 H3N2

AF258526 A/Hong Kong/427/98 H1N1

AF258827 A/Hong Kong/97/98 H5N1

AY043029 A/Guangzhou/333/99 H9N2

AF258816 A/Hong Kong/1073/99 H9N2

AJ293921 A/Hong Kong/1774/99 H3N2

AJ489539 A/UK/627/01 H1N2

AB126625 A/Yokohama/22/2002 H1N2

AY576392 A/Hong Kong/212/03 H5N1

AY526751 A/Vietnam/1196/04 H5N1

Table 15D: Influenza A virus PB2 transcripts (strains isolated from humans)

AF389115 A/Puerto Rico/8/34/Sinai H1N1

M73521 A/Singapore/1/57 H2N2

M23970 A/Ann Arbor/6/60 H2N2

M73524 A/Korea/426/68 H2N2

M91712 A/Udon Thani/307/72 H3N2

AJ564805 A/Fiji/15899/83 H1N1

AF037412 A/Kitakyushu/159/93 H3N2

U53158 A/Wisconsin/4754/94 H1N1

U71134 A/Shiga/20/95 H3N2

AF036363 A/Hong Kong/156/97 H5N1

AF258842 A/Hong Kong/498/97 H3N2

AF258845 A/Hong Kong/542/97 H5N1

AF037417 A/Shiga/25/97 H3N2

AF258525 A/Hong Kong/427/98 H1N1

AF258846 A/Hong Kong/97/98 H5N1

AF342824 A/Wisconsin/10/98 H1N1

AY043030 A/Guangzhou/333/99 H9N2

AJ404630 A/Hong Kong/1073/99 H9N2

AJ293920 A/Hong Kong/1774/99 H3N2

AJ489485 A/UK/627/01 H1N2

AB126626 A/Yokohama/22/2002 H1N2

AY576380 A/Hong Kong/212/03 H5N1

AY526752 A/Vietnam/1196/04 H5N1

Table 1E: Influenza A virus M transcripts (strains isolated from humans)

AF389121 A/Puerto Rico/8/34/Sinai H1N1

X08093 A/Singapore/1/57 H2N2

M23978 A/Ann Arbor/6/60 H2N2

M63531 A/Korea/426/68 H2N2

J02167 A/Udon Thani/72 H3N2

AJ298947 A/Fiji/15899/83 H1N1

U65562 A/Kitakyushu/159/93 H3N2

U53168 A/Wisconsin/4754/94 H1N1

U65573 A/Shiga/20/95 H3N2

AJ458306 A/South Africa/1147/96 H3N2

AF036358 A/Hong Kong/156/97 H5N1

AF255370 A/Hong Kong/498/97 H3N2

AF255373 A/Hong Kong/542/97 H5N1

AF038274 A/Shiga/25/97 H3N2

AF258523 A/Hong Kong/427/98 H1N1

AF255374 A/Hong Kong/97/98 H5N1

AF342818 A/Wisconsin/10/98 H1N1

AY043025 A/Guangzhou/333/99 H9N2

AF255363 A/Hong Kong/1073/99 H9N2

AJ293925 A/Hong Kong/1774/99 H3N2

AJ458304 A/Saudi Arabia/7971/2000 H1N1

AJ489530 A/UK/627/01 H1N2

AB126629 A/Yokohama/22/2002 H1N2

AY575893 A/Hong Kong/212/03 H5N1

AY526748 A/Vietnam/1196/04 H5N1

Table 15F: Influenza A virus NS transcripts (strains isolated from humans)

AF389122 A/Puerto Rico/8/34/Sinai H1N1

AY210151 A/Singapore/1/57 H2N2

AY210161 A/Ann Arbor/6/60 H2N2

AY210191 A/Korea/426/68 H2N2

V01102 A/Udon Thani/72 H3N2

AJ298950 A/Fiji/15899/83 H1N1

D30676 A/Kitakyushu/159/93 H3N2

U53170 A/Wisconsin/4754/94 H1N1

U65673 A/Shiga/20/95 H3N2

AF036360 A/Hong Kong/156/97 H5N1

AF256183 A/Hong Kong/498/97 H3N2

AF256187 A/Hong Kong/542/97 H5N1

AF038279 A/Shiga/25/97 H3N2

AF258521 A/Hong Kong/427/98 H1N1

AF256188 A/Hong Kong/97/98 H5N1

AF342817 A/Wisconsin/10/98 H1N1

AY043027 A/Guangzhou/333/99 H9N2

AF256177 A/Hong Kong/1074/99 H9N2

AJ293941 A/Hong Kong/1774/99 H3N2

AJ519463 A/Saudi Arabia/7971/2000 H1N1

AJ489552 A/UK/627/01 H1N2

AB126628 A/Yokohama/22/2002 H1N2

AY576368 A/Hong Kong/212/03 H5N1

AY526747 A/Vietnam/1196/04 H5N1

Table 15G: Influenza A virus NA transcripts (strains isolated from humans)

AF389120 A/Puerto Rico/8/34/Sinai H1N1

AY209895 A/Singapore/1/57 H2N2

AY209903 A/Ann Arbor/6/60 H2N2

AY209932 A/Korea/426/68 H2N2

M27970 A/New Jersey/8/76 H1N1

K01017 A/Memphis/10/78 H1N1

AJ006954 A/Fiji/15899/83 H1N1

U42634 A/UK/427/88 H3N2

U47816 A/Wisconsin/3523/88 H1N1

U42636 A/Shanghai/24/90 H3N2

U42774 A/California/271/92 H3N2

AF038260 A/Kitakyushu/159/93 H3N2

AJ457945 A/Nanchang/933/95 H3N2

AF036357 A/Hong Kong/156/97 H5N1

AF102670 A/Hong Kong/542/97 H5N1

AF038264 A/Shiga/25/97 H3N2

AF102661 A/Hong Kong/97/98 H5N1

AF533749 A/Montevideo/2728/98 H3N2

AY043022 A/Shaoguan/408/98 H9N2

AF342820 A/Wisconsin/10/98 H1N1

AJ404629 A/Hong Kong/1073/99 H9N2

AJ293923 A/Hong Kong/1774/99 H3N2

AJ307628 A/Montreal/MTL516/00 H3N2

AF503466 A/Singapore/63/2001 H1N2

AJ457947 A/Ireland/1092/2002 H3N2

AB126623 A/Yokohama/22/2002 H1N2

AY575881 A/Hong Kong/212/03 H5N1

AY555151 A/Thailand/1-KAN-1/2004 H5N1

AY526746 A/Vietnam/1196/04 H5N1

Table 15H: Influenza A virus HA transcripts (strains isolated from humans)

AF389118 A/Puerto Rico/8/34/Sinai H1N1

L20410 A/Singapore/1/57 H2N2

AF270721 A/Ann Arbor/6/60 H2N2

L11133 A/Korea/426/68 H2N2

AY661044 A/Bilthoven/628/76 H3N2

AJ289702 A/Fiji/15899/83 H1N1

AY661055 A/UK/427/88 H3N2

L33755 A/Finland/75/88 H1N1

AY661074 A/Shanghai/24/90 H3N2

AF008817 A/California/271/92 H3N2

D30669 A/Kitakyushu/159/93 H3N2

AF008725 A/Nanchang/933/95 H3N2

AF036356 A/Hong Kong/156/97 H5N1

AF102678 A/Hong Kong/542/97 H5N1

AJ311466 A/Sydney/5/97 H3N2

AF102676 A/Hong Kong/97/98 H5N1

AY271794 A/Memphis/31/98 H3N2

AY043017 A/Shaoguan/408/98 H9N2

AF342821 A/Wisconsin/10/98 H1N1

AJ404626 A/Hong Kong/1073/99 H9N2

AJ293926 A/Hong Kong/1774/99 H3N2

ISDNOS0022 A/Oslo (Oslo)/6391/2000 H3N2

AF503481 A/Singapore/63/2001 H1N2

AY661030 A/Netherlands/120/02 H3N2

AB126622 A/Yokohama/22/2002 H1N2

AY575869 A/Hong Kong/212/03 H5N1

AY555150 A/Thailand/1-KAN-1/2004 H5N1

AY526745 A/Vietnam/1196/04 H5N1

To identify a preferred set of target moieties among a large group of influenza A strains, the sequence of the PR8 strain was chosen as the base sequence. The entire sequence of each fragment was used, excluding introns. The sequences are shown in Figures 32A-32J (SEQ ID NOs: 383-392). These sequences are concatenated to form a single long sequence in the following order: NP, PB2, PB1, PA, M1, M2, NS1, NS2, HA, NA. The first possible target moiety is selected by applying a 19 nucleotide "window" starting at the 5' end, and then moving the window 1 nucleotide at a time in the 3' direction to select the next possible target The target portion, to identify a portion of the sequence having a length of 19 nucleotides. Continue this process until the 3' end of the transcript is reached. Each 19 nucleotide region is considered to represent a possible targeting moiety, although it is recognized that shorter or longer targeting moieties could also be selected. Regions covering parts of 2 different transcripts are excluded. The sequences of the 13,472 possible target moieties resulting from the process can be readily determined by referring to SEQ ID NOs: 383-392 in Figure 32. For example, in each sequence, the position where the target moiety is extended is 1-19, 2-20, 3-21, 4-22, 5-23, 6-24, etc. In the NP transcript (SEQ ID NO: 383), the target moiety is 1-19, 2-20, 3-21, 4-22...1547-1565. In the PB2 (SEQ ID NO: 384) transcript, the targeted moieties were 1-19, 2-20, 3-21, 4-22...2323-2341. In the PB1 transcript (SEQ ID NO: 385), the targeting moieties are 1-19, 2-20, 3-21, 4-22...2323-2341. In the PA transcript (SEQ ID NO: 386), the targeting moieties are 1-19, 2-20, 3-21, 4-22...2215-2233. In the M1 transcript (SEQ ID NO: 387), the targeting moieties are 1-19, 2-3-21, 4-22...1009-1027. In the M2 transcript (SEQ ID NO: 388), the targeting moieties are 1-19, 2-20, 3-21, 4-22...321-339. In the NS1 transcript (SEQ ID NO: 389), the targeting moieties are 1-19, 2-20, 3-21, 4-22...872-890. In the NS2 transcript (SEQ ID NO: 390), the targeting moieties are 1-19, 2-20, 3-21, 4-22...400-418. In the HA transcript (SEQ ID NO: 391), the targeting moieties are 1-19, 2-20, 3-21, 4-22...1767-1775. In the NA transcript, the targeting moiety is 1-19, 2-20, 3-21, 4-22...1395-1413 (SEQ ID NO: 392). For illustrative purposes, the top 6 target portions of NP transcripts are listed in Table 16.

Table 16: Targeted moieties in NP genes

ID number sequence Nucleotide position 1 AGCAAAAAGCAGGGTAGATA (SEQ ID NO: 394) 1-19 2 GCAAAAGCAGGGTAGATAA (SEQ ID NO: 395) 2-20 3 CAAAAGCAGGGTAGATAAT (SEQ ID NO: 396) 3-21 4 AAAAGCAGGGTAGATAATC (SEQ ID NO: 397) 4-22 5 AAAGCAGGGTAGATAATCA (SEQ ID NO: 398) 5-23 6 AAGCAGGGTAGATAATCAC (SEQ ID NO: 399) 6-24

Potential target moieties are subjected to a selection step to identify target moieties with preferred characteristics for inhibition by the RNAi-inducing agent. Criteria applied in the selection step included filtering based on GC content, and filtering based on the presence or absence of consecutive stretches of G or C nucleotides. For example, preferred RNAi-inducing agent inhibitory regions preferably contain less than 70% G or C nucleotides, and preferably do not contain contiguous stretches of more than 3G nucleotides or more than 3C nucleotides. Thus, preferred targeting moieties contain less than 70% G or C nucleotides, and preferably contain no contiguous stretches of more than 3G nucleotides or more than 3C nucleotides. The portion of the target that remains after applying the filter is called the "functional target portion". Table 17 lists 2,244 functional target segments.

The sequence of the functional targeting moiety from PR8 was compared to the corresponding targeting moiety of the other strains listed in Tables 15A-15H. The corresponding target portion in other strains is readily identified based on its position in the fragment and based on its sequence, often substantially identical to the target portion in PR8.

It is recognized that GU base pairing can occur according to "wobble rules". It is also recognized that the importance of maintaining complementarity between the antisense strand and the target varies at different positions. Thus identifying at least 80% of the target portions of the bacterial strain compared to PR8 when the target portion in PR8 is compared to the corresponding target portion in the strain (where both aligned in the 5' to 3' direction) Target moieties that meet the following criteria: (1) A to G or C to U differences between the PR8 sequence and the corresponding sequence are allowed at any position; (2) only at one or more of positions 1, 18, and 19 The position allows G to be A or C to be A difference between the PR8 sequence and the corresponding sequence; (3) between positions 1 and 9, there are 0, 1, 2 or 3 differences between the PR8 sequence and the corresponding sequence; (4) There are no more than 2 consecutive differences between the PR8 sequence and the corresponding sequence; and, (5) Between positions 11 and 17, there is at most 1 difference between the PR8 sequence and the corresponding sequence.

Target portions meeting the above criteria are designated as "properly conserved target portions". Listed in Table 18 are the sequences of 220 target moieties that are well conserved among human-derived influenza strains. Target moieties are from transcripts NP, PB2, PB1, PA, M, NS and HA. The appropriately conserved targeting moiety is a preferred target for RNAi-inducing agents of the invention for inhibiting a variety of different human-derived influenza A strains, and is fully complementary to the inhibitory region of certain preferred antisense strands.

After selection of the appropriate conserved target moieties, the DNA from avian species (including ducks, chickens, gulls, teals, terns, quails, pheasants, turkeys, geese, pigeons, falcons, and various other birds) or from the environment ( Corresponding target portions of influenza A strains isolated assuming avian origin) were aligned and compared to target portions identified from human-derived strains. In addition to the strains listed in Example 1, the strains include a large number of strains. Tables 19A-19F list the Genbank accession number (left column), strain name (middle column) and serotype (right column) of the influenza A virus genome segment (in its plus-strand form) used to identify the appropriate conserved target portion. The sets of strains aligned differed for different fragments, but each set included at least 30 strains isolated in various years spanning 1934-2004. The same selection criteria used to identify appropriately conserved target portions from human-derived strains were applied. The results yielded 138 target segments that were moderately conserved between human and avian-derived strains. Target moieties are from transcripts NP, PB2, PB1, PA, M and NS. Table 20 lists these appropriate conserved target moieties. The targeting moiety is a preferred target of the RNAi-inducing agents of the invention for inhibiting a variety of different human-derived and avian-derived influenza A strains, and is associated with the inhibitory region of certain preferred antisense strands of the RNAi-inducing agents of the invention. Completely complementary.

Table 19A: Influenza A virus PA transcripts (strains isolated from birds)

M21850 A/Chicken/FPV/Rostock/34 H7N1

M26088 A/Seagull/Maryland/704/77 H13N6

M26085 A/Pintail/Alberta/119/79 H4N6

AY633193 A/Mallard/Alberta/203/93 H6N5

AY633305 A/Duck/Albert/76/94 H6N8

AF098606 A/Chicken/Hong Kong/728/97 H5N1

AF156444 A/Chicken/Hong Kong/G9/97 H9N2

AY633393 A/teal/Alberta/16/97 H2N9

AF536676 A/Chicken/Henan/98 H9N2

AJ291397 A/Chicken/Pakistan/2/99 H9N2

AF216731 A/Environment/Hong Kong/437-8/99 H5N1

AY633353 A/Pintail/Alberta/207/99 H4N8

AY180690 A/Bantam/Nanchang/9-366/2000 H3N3

AF508676 A/Chicken/Henan/62/00 H9N2

AY059530 A/Duck/Hong Kong/ww461/2000 H5N1

AY180683 A/Duck/Nanchang/4-191/2000 H4N6

AF523462 A/Duck/Shantou/2030/00 H9N1

AY180693 A/quail/Nanchang/4-034/2000 H4N6

AY180696 A/quail/Nanchang/4-040/2000 H9N2

AF457716 A/Chicken/California/1002a/00 H6N2

AY633153 A/Duck/Alberta/127/00 H3N8

AF509210 A/Chicken/Hong Kong/866.3/01 H5N1

AY180709 A/quail/Nanchang/2-040/2001 H3N6

AF523454 A/Duck/Shantou/4808/01 H9N2

AF457683 A/Chicken/California/6643/01 H6N2

AY180678 A/Chicken/Nanchang/3-201/01 H3N6

AY422033 A/Duck/Hokkaido/86/01 H2N3

AY303660 A/Chicken/Chile/176822/02 H7N3

AY576411 A/Chicken/Hong Kong/61.9/02 H5N1

AY586433 A/Turkey/Italy/220158/2002 H7N3

AY342420 A/Chicken/Netherlands/1/03 H7N7

AY616764 A/Chicken/British Columbia/04 H7N3

AY609311 A/Chicken/Guangdong/174/04 H5N1

Table 19B: Influenza A virus PB1 transcripts (strains isolated from birds)

U48280 A/Duck/Hong Kong/62/76 H11N2

M25933 A/Seagull/Maryland/704/77 H13N6

M25925 A/Turkey/Minnesota/833/80 H4N2

AY633210 A/Duck/Alberta/206/96 H6N8

AY633186 A/Mallard/Alberta/202/96 H2N5

AF098592 A/Chicken/Hong Kong/728/97 H5N1

AF156416 A/Chicken/Hong Kong/G9/97 H9N2

AY633394 A/teal/Alberta/16/97 H2N9

AF536666 A/Chicken/Henan/98 H9N2

AF508626 A/Chicken/Korea/99029/99 H9N2

AF216732 A/Environment/Hong Kong/437-8/99 H5N1

AY633354 A/Pintail/Alberta/207/99 H4N8

AY180888 A/Bantam/Nanchang/9-366/2000 H3N3

AY180885 A/Duck/Nanchang/4-191/2000 H4N6

AF523445 A/Duck/Shantou/2030/00 H9N1

AY180891 A/quail/Nanchang/4-034/2000 H4N6

AY180892 A/quail/Nanchang/4-040/2000 H9N2

AF457698 A/Chicken/California/431/00 H6N2

AY633154 A/Duck/Alberta/127/00 H3N8

AF509184 A/Chicken/Hong Kong/866.3/01 H5N1

AY180893 A/Chicken/Nanchang/1-101/2001 H3N6

AY180864 A/quail/Nanchang/2-040/2001 H3N6

AF523431 A/Duck/Shantou/4808/01 H9N2

AY180872 A/Chicken/Nanchang/3-201/01 H3N6

AY422037 A/Duck/Hokkaido/86/01 H2N3

AY303664 A/Chicken/Chile/4957/02 H7N3

AY576400 A/Chicken/Hong Kong/YU777/02 H5N1

AY586436 A/Turkey/Italy/220158/2002 H7N3

AY340085 A/Chicken/Netherlands/1/03 H7N7

AY616765 A/Chicken/British Columbia/04 H7N3

AY609310 A/Chicken/Guangdong/174/04 H5N1

AY590582 A/Chicken/Nakorn-Pathom/Thailand H5N1

/CU-K2/2004

Table 19C: Influenza A virus PB2 transcripts (strains isolated from birds)

M73514 A/Turkey/Minnesota/833/80 H4N2

M73516 A/Seagull/Astrakhan/227/84 H13N6

AF268115 A/Ruddy Turnstone/Delaware H3N4

(Delaware)/168/94

AY63 3243 A/Mallard/Alberta/232/94 H6N8

AF268119 A/Waterfowl/Delaware/23/96 H10N9

AF508651 A/Chicken/Guangdong/11/97 H9N2

AF098579 A/Chicken/Hong Kong/728/97 H5N1

AF156430 A/Chicken/Hong Kong/G9/97 H9N2

AF536686 A/Chicken/Henan/98 H9N2

AJ410602 A/Duck/Hong Kong/323/98 H6N2

AF508642 A/Chicken/Pakistan/5/99 H9N2

AF216733 A/Environment/Hong Kong/437-8/99 H5N1

AY633355 A/Pintail/Alberta/207/99 H4N8

AY180775 A/Bantam/Nanchang/9-366/2000 H3N3

AY180770 A/Duck/Nanchang/4-191/2000 H4N6

AF523481 A/Duck/Shantou/2030/00 H9N1

AY180772 A/quail/Nanchang/4-034/2000 H4N6

AY180773 A/quail/Nanchang/4-040/2000 H9N2

AF457689 A/Chicken/California/465/00 H6N2

AY633155 A/Duck/Alberta/127/00 H3N8

AF509158 A/Chicken/Hong Kong/866.3/01 H5N1

AY180759 A/quail/Nanchang/2-040/2001 H3N6

AF523464 A/Duck/Shantou/4808/01 H9N2

AF457681 A/Chicken/California/6643/01 H6N2

AY180763 A/Chicken/Nanchang/3-201/01 H3N6

AY422041 A/Duck/Hokkaido/86/01 H2N3

AY576388 A/Chicken/Hong Kong/YU777/02 H5N1

AJ627496 A/Turkey/Italy/220158/2002 H7N3

AY342413 A/poultry/Netherlands/219/03 H7N7

AY342414 A/Chicken/Netherlands/1/03 H7N7

AY616766 A/Chicken/British Columbia/04 H7N3

AY609309 A/Chicken/Guangdong/174/04 H5N1

AY590681 A/Chicken/Nakorn Pathom/Thailand/CU-K2/2004 H5N1

Table 19D: Influenza A virus M transcripts (strains isolated from birds)

M23917 A/Chicken/FPV/Weybridge H7N7

M63538 A/Seagull/Massachusetts/26/80 H13N6

AJ427302 A/curlew sandpiper/Hong Kong H10N5

/208/89

U49117 A/Duck/Nanchang/1749/92 H11N2

AF073180 A/Chicken/New Jersey/15086-3/94 H7N3

AF098562 A/Chicken/Hong Kong/728/97 H5N1

AF156458 A/Chicken/Hong Kong/G9/97 H9N2

AF250487 A/Duck/Hong Kong/P169/97 H3N8

AF250489 A/Duck/Hong Kong/P54/97 H11N9

AF250490 A/Duck/Hong Kong/T25/97 H11N8

AF250492 A/Duck/Hong Kong/Y264/97 H4N8

AY633389 A/teal/Alberta/16/07 H2N9

AF536726 A/Chicken/Henan/98 H9N2

AY241600 A/Chicken/NY/12273-11/99 H7N3

AF216727 A/Environment/Hong Kong/437-8/99 H5N1

AJ427299 A/Waterfowl/Hong Kong/399/99 H3N8

AY633349 A/Pintail/Alberta/207/99 H3N3

AY180518 A/Bantam/Nanchang/9-366/2000 H3N3

AY180509 A/Duck/Nanchang/4191/2000 H4N6

AF523500 A/Duck/Shantou/2030/00 H9N1

AY180507 A/quail/Nanchang/4-034/2000 H4N6

AY180504 A/quail/Nanchang/4-040/2000 H9N2

AY300962 A/poultry/NY/53726/00 H5N2

AY633149 A/Duck/Alberta/127/00 H3N8

AF474055 A/Chicken/California/6643/2001 H6N2

AF509055 A/Chicken/Hong Kong/866.3/01 H5N1

AY180523 A/quail/Nanchang/2-040/2001 H3N6

AF523484 A/wild duck/Shantou/4804/01 H9N2

AY300975 A/blue-winged teal/TX/2/01 H7N3

AY180505 A/Chicken/Nanchang/3-201/01 H3N6

AY422020 A/Duck/Hokkaido/86/01 H2N3

AY241624 A/Turkey/VA/67/02 H7N2

AY300974 A/Duck/NY/191255-59/02 H5N8

AY300971 A/Turkey/CA/D0208651-C/02 H5N2

AJ627497 A/Turkey/Italy/220168/2002 H7N3

AY340091 A/Chicken/Netherlands/1/03 H7N7

AY611525 A/Chicken/British Columbia/04 H7N3

AY609315 A/Chicken/Guangdong/174/04 H5N1

AY590578 A/Chicken/Nakorn Pathom/Thailand/CU-K2/2004 H5N1

Table 19E: Influenza A virus NS transcripts (strains isolated from birds)

J02105 A/Duck/Alberta/60/76 H12N5

U96743 A/Seagull/Maryland/1824/78 H13N9

U96739 A/Chicken/Pennsylvania/1370/83 H13N6

AF007035 A/Duck/Ohio/421/87 H7N8

AF074267 A/Chicken/New Jersey/15086-3/94 H7N3

AF156472 A/Chicken/Hong Kong/G9/97 H9N2

AF319651 A/Chicken/Italy/9097/97 H5N9

AF250495 A/Duck/Hong Kong/P169/97 H3N8

AF250498 A/Duck/Hong Kong/P54/97 H11N9

AF250499 A/Duck/Hong Kong/T25/97 H11N8

AF250501 A/Duck/Hong Kong/Y264/97 H4N8

AY633392 A/teal/Alberta/16/97 H2N9

AF536736 A/Chicken/Henan/98 H9N2

AJ410587 A/Duck/Hong Kong/324/98 H6N2

AY241629 A/Chicken/NY/12273-11/99 H7N3

AF216726 A/Environment/Hong Kong/437-8/99 H5N1

AJ427300 A/Waterfowl/Hong Kong/399/99 H3N8

AY633352 A/Pintail/Alberta/207/99 H4N8

AY180647 A/Bantam/Nanchang/9-366/2000 H3N3

AY180606 A/Duck/Nanchang/4-191/2000 H4N6

AF523502 A/Duck/Shantou/2030/00 H9N1

AY180614 A/quail/Nanchang/4-034/2000 H4N6

AY180617 A/quail/Nanchang/4-040/2000 H9N2

AY300986 A/poultry/NY/53726/00 H5N2

AY180620 A/Duck/Alberta/127/00 H3N8

AY300999 A/Blue-winged Teal/TX/2/01 H7N3

AF457675 A/Chicken/California/905/01 H6N2

AY259220 A/Chicken/Hebei/1/01 H9N2

AY180600 A/Chicken/Nanchang/3-201/01 H3N6

AY422029 A/Duck/Hokkaido/86/01 H2N3

AY241662 A/Turkey/VA/67/02 H7N2

AY300998 A/Duck/NY/191255-59/02 H5N8

AY300995 A/Turkey/CA/D0208651-C/02 H5N2

AY303645 A/Chicken/Chile/4322/03 H7N3

AY342424 A/Chicken/Netherlands/1/03 H7N7

AY611528 A/Chicken/British Columbia/04 H7N3

AY609316 A/Chicken/Guangdong/174/04 H5N1

AY590580 A/Chicken/Nakorn Pathom/Thailand/CU-K2/2004 H5N1

Table 19F: Influenza A virus NP transcripts (strains isolated from birds)

M63779 A/FPV/Dobson/'Netherlands'/27 H7N7

AJ243993 A/FPV/Rostock/34 H7N1

M22576 A/FPV/Rostock/34 H7N1

M21937 A/FPV/Rostock/34 H7N1

M24660 A/FPV/Rostock/34 (mutant strain ts19) H7N1

M24556 A/FPV/Rostock/34 (reversion mutant 19R) H7N1

M24557 A/FPV/Rostock/34 (reversion mutant 81R) H7N1

M24453 A/Chicken/Germany/N/49 H10N7

M63773 A/Duck/Manitoba/1/53 H10N7

M63780 A/Duck/UK/1/56 H11N6

M30762 A/Duck/Czechoslovakia/56 H4N6

M30763 A/Duck/Ukraine/2/60 H11N8

M30767 A/Tern/South Africa/61 H5N3

M63781 A/Duck/UK/1/62 H4N6

AF156415 A/Turkey/California/189/66 H9N2

M63774 A/Turkey/Ontario/7732/66 H5N9

M63775 A/Duck/Pennsylvania/1/69 H6N1

M27298 A/Shearwater/Australia/72 H6N5

M27519 A/Tern/Turkmenia/18/72 H3N3

M22344 A/Parrot/Ulster/73 H7N1

M63776 A/Duck/Memphis/928/74 H3N8

M22573 A/Duck/Hong Kong/7/75 H3N2

M36812 A/Pintail Duck/Primorje/695/76 H2N3

AF523423 A/Duck/Hong Kong/86/76 H9N2

U49093 A/Goose/Hong Kong/8/76 H1N1

M30760 A/Duck/New Zealand/31/76 H4N6

U49097 A/Duck/Hong Kong/193/77 H1N2

M63777 A/Seagull/Maryland/5/77 H11N9

M30765 A/budgerigar/Hokkaido/1/77 H4N6

M22574 A/Duck/Bavaria/2/77 H1N1

M27521 A/Seagull/Maryland/704/77 H13N6

M76603 A/Turkey/UK/647/77 H1N1

M63782 A/Duck/Beijing/1/78 H3N6

AF523421 A/Duck/Hong Kong/289/78 H9N2

AF523424 A/Duck/Hong Kong/366/78 H9N2

D00050 A/Mallard/New York/6750/78 H2N2

M30755 A/Seagull/Maryland/1824/78 H13N9

AF523422 A/Duck/Hong Kong/552/79 H9N2

U49095 A/Duck/Hong Kong/717/79 H1N3

M30761 A/Grey Teal/Australia/2/79 H4N4

M30756 A/Seagull/Maryland/1815/79 H13N6

M63783 A/Duck/Australia/749/80 H1N1

AF079571 A/Duck/Hokkaido/8/80 H3N8

M30752 A/Seagull/Massachusetts/26/80 H13N6

M30757 A/Seagull/Minnesota/945/80 H13N6

M63784 A/teal/Iceland/29/80 H7N7

M30769 A/Turkey/Minnesota/833/80 H4N2

M63778 A/Turkey/Minnesota/I661/81 H1N1

M63785 A/wild duck/Astrakhan (Guriyev)/263/82H14N5

M30764 A/Mall Duck/Astrakhan/244/82 H14

M30768 A/Chicken/Pennsylvania/1/83 H5N2

AY633119 A/Mallard/Alberta/743/83 H9N1

M30753 A/Seagull/Astrakhan/227/84 H13N6

AY633311 A/Mallard/Alberta/98/85 H6N2

AY633319 A/Pintail/Alberta/113/85 H6N2

M30766 A/Turnstone/New Jersey/47/85 H4N6

Z26855 A/Oystercatcher/Germany/87 H1N1

AY633279 A/Duck/Alberta/321/88 H9N2

M76609 A/Turkey/North Carolina (North Carolina) H1N1

/1790/88

AJ427301 A/Snipe/Hong Kong/208/89 H10N5

AY633295 A/Mallard/Alberta/11/91 H9N2

AY633167 A/Mallard/Alberta/17/91 H9N2

Z26857 A/Turkey/Germany/3/91 H1N1

U49094 A/Duck/Nanchang/1749/92 H11N2

AF156410 A/quail/Hong Kong/AF157/92 H9N2

AY633191 A/Mallard/Alberta/203/92 H6N5

AF156414 A/Quail/Arkansas/29209-1/93 H9N2

AY633335 A/Pintail/Alberta/179/93 H6N1

AF156409 A/Chicken/Beijing/1/94 H9N2

AY497120 A/Chicken/Hidalgo/232/94 H5N2

AF098624 A/Chicken/Hidalgo/26654-1368/94 H5N2

AF156408 A/Chicken/Hong Kong/739/94 H9N2

AF098625 A/Chicken/Mexico/26654-1374/94 H5N2

AY497113 A/Chicken/Mexico/31381-7/94 H5N2

AF098627 A/Chicken/Puebla/14585-622/94 H5N2

AY497114 A/Chicken/Pueblo/8623-607/94 H5N2

AF098626 A/Chicken/Pueblo/8623-607/94 H5N2

AY633383 A/Pochard/Alberta/291/94 H6N8

AY633239 A/Duck/Alberta/232/94 H6N8

AY633303 A/Duck/Alberta/76/94 H6N8

AY633327 A/Pintail/Alberta/155/94 H6N8

AF536699 A/Chicken/Beijing/1/95 H9N2

AF213906 A/Chicken/Italy/24/95 H1N1

AY497117 A/Chicken/Pueblo/28159-474/95 H5N2

AF098628 A/Chicken/Queretaro/14588-19/95 H5N2

AF508600 A/Duck/Germany/113/95 H9N2

AF213905 A/Duck/Italy/24/95 H1N1

AF508596 A/ostrich/South Africa/9508103/95 H9N2

AB020778 A/Chicken/Beijing/1/96 H9N2

AF536703 A/Chicken/Hebei/1/96 H9N2

AF156412 A/Chicken/Korea/25232-96006/96 H9N2

AF156411 A/Chicken/Korea/38349-p96323/96 H9N2

AF203787 A/Chicken/Korea/MS96/96 H9N2

AF508613 A/Chicken/Shandong/6/96 H9N2

AF144303 A/Goose/Guangdong/1/96 H5N1

AY633207 A/Duck/Alberta/206/96 H6N8

AF508617 A/quail/Shanghai/8/96 H9N2

AF156413 A/Waterfowl/Delaware/9/96 H9N2

AY633183 A/Mallard/Alberta/202/96 H2N5

AF536700 A/Chicken/Beijing/2/97 H9N2

AF508607 A/Chicken/Guangdong/11/97 H9N2

AF536702 A/Chicken/Guangdong/97 H9N2

AF508609 A/Chicken/Heilongjiang/10/97 H9N2

AF046084 A/Chicken/Hong Kong/220/97 H5N1

AF098618 A/Chicken/Hong Kong/728/97 H5N1

AF098619 A/Chicken/Hong Kong/786/97 H5N1

AF098620 A/Chicken/Hong Kong/915/97 H5N1

AF156403 A/Chicken/Hong Kong/G23/97 H9N2

AF156402 A/Chicken/Hong Kong/G9/97 H9N2

AF098617 A/Chicken/Hong Kong/y388/97 H5N1

AF319644 A/Chicken/Italy/312/97 H5N2

AF319645 A/Chicken/Italy/330/97 H5N2

AF319646 A/Chicken/Italy/367/97 H5N2

AF319647 A/Chicken/Italy/9097/97 H5N9

AF508615 A/Chicken/Shenzhen/9/97 H9N2

AF508612 A/Chicken/Sichuan/5/97 H9N2

AF250473 A/Duck/Hong Kong/P185/97 H3N8

AF250474 A/Duck/Hong Kong/P54/97 H11N9

AF250470 A/Duck/Hong Kong/T25/97 H11N8

AF250471 A/Duck/Hong Kong/T37/97 H11N8

AF156405 A/Duck/Hong Kong/Y280/97 H9N2

AF156406 A/Duck/Hong Kong/Y439/97 H9N2

AF098621 A/Duck/Hong Kong/p46/97 H5N1

AF098622 A/Duck/Hong Kong/y283/97 H5N1

AF508616 A/Duck/Nanjing/1/97 H9N2

ISDN22474 A/Duck/Singapore/645/97 H5N3

AF370122 A/Goose/Guangdong/3/97 H5N1

AF250475 A/Goose/Hong Kong/W217/97 H6N9

AF098623 A/Goose/Hong Kong/w355/97 H5N1

AF508603 A/pheasant/Ireland/PV18/97 H9N2

AF156404 A/Pigeon/Hong Kong/Y233/97 H9N2

AF156407 A/Quail/Hong Kong/G1/97 H9N2

AF250480 A/teal/Hong Kong/W312/97 H6N1

AF057293 A/Chicken/Hong Kong/258/97 H5N1

AY633135 A/Mallard/Alberta/117/97 H3N8

AB049161 A/parakeet/Chiba H9N2

/1/97

AY633343 A/Pintail/Alberta/156/97 H3N8

AY633367 A/Pintail/Alberta/22/97 H2N9

AY633391 A/teal/Alberta/16/97 H2N9

AF250472 A/Waterfowl/Hong Kong/M603/98 H11N1

AF508605 A/Chicken/Beijing/8/98 H9N2

AF508599 A/Chicken/Germany/R45/98 H9N2

AF536704 A/Chicken/Hebei/2/98 H9N2

AF536705 A/Chicken/Hebei/3/98 H9N2

AF508608 A/Chicken/Hebei/4/98 H9N2

AF536706 A/Chicken/Henan/98 H9N2

AY497116 A/Chicken/Pueblo/231-5284/98 H5N2

AF536708 A/Chicken/Shandong/98 H9N2

AY253753 A/Chicken/Shanghai/F/98 H9N2

AY497115 A/Chicken/Aguascalientes H5N2

/124-3705/98

AY633199 A/Mallard/Alberta/205/98 H2N3

AY633215 A/Mallard/Alberta/211/98 H1N1

AY633231 A/Mallard/Alberta/226/98 H2N3

AY633247 A/Duck/Alberta/242/98 H3N8

AY633255 A/Duck/Alberta/279/98 H3N8

AY633263 A/Mallard/Alberta/295/98 H4N6

AY633271 A/Duck/Alberta/30/98 H4N6

AY633287 A/Mallard/Alberta/47/98 H4N1

AB049162 A/parakeet/Narita/92A/98 H9N2

AF536701 A/Chicken/Beijing/3/99 H9N2

AF222619 A/Chicken/Hong Kong/FY20/99 H9N2

AF222620 A/Chicken/Hong Kong/KC12/99 H9N2

AF222616 A/Chicken/Hong Kong/NT16/99 H9N2

AF186272 A/Chicken/Hong Kong/SF2/99 H9N2

AF508601 A/Chicken/Iran/11T/99 H9N2

AF508604 A/Chicken/Korea/99029/99 H9N2

AF536707 A/Chicken/Liaoning/99 H9N2

AF508611 A/Chicken/Ningxia/5/99 H9N2

AJ291394 A/Chicken/Pakistan/2/99 H9N2

AF508597 A/Chicken/Pakistan/4/99 H9N2

AF508598 A/Chicken/Pakistan/5/99 H9N2

AF508602 A/Chicken/Saudi Arabia/532/99 H9N2

AF508614 A/Chicken/Shijiazhuang/2/99 H9N2

AF216736 A/Environment/Hong Kong/437-10/99 H5N1

AF216712 A/Environment/Hong Kong/437-4/99 H5N1

AF216720 A/Environment/Hong Kong/437-6/99 H5N1

AF216728 A/Environment/Hong Kong/437-8/99 H5N1

AF222618 A/pheasant/Hong Kong/SSP11/99 H9N2

AF222615 A/Pigeon/Hong Kong/FY6/99 H9N2

AF222614 A/Quail/Hong Kong/A17/99 H9N2

AF186270 A/Quail/Hong Kong/NT28/99 H9N2

AF222617 A/Quail/Hong Kong/SSP10/99 H9N2

AF186271 A/Silky Chicken/Hong Kong H9N2

/SF43/99

AF222621 A/silk feather chicken/Hong Kong/SF44/99 H9N2

AY038019 A/Turkey/MO/24093/99 H1N2

AJ427298 A/Waterfowl/Hong Kong/399/99 H3N8

AF261750 A/Chicken/Taiwan/7-5/99 H6N1

AY585429 A/Duck/Guangxi/07/1999 H5N1

AJ410555 A/Duck/Hong Kong/3096/99 H6N2

AJ410556 A/Duck/Hong Kong/3461/99 H6N1

AY633127 A/Duck/Alberta/111/99 H4N6

AY633175 A/Duck/Alberta/199/99 H3N6

AY633223 A/Duck/Alberta/215/99 H6N8

AJ410548 A/pheasant/Hong Kong/SH39/99 H6N1

AY633351 A/Pintail/Alberta/207/99 H4N8

AY633359 A/Pintail/Alberta/210/99 H4N6

AY633375 A/Pintail/Alberta/37/99 H3N8

AJ410549 A/quail/Hong Kong/1721-20/99 H6N1

AJ410550 A/quail/Hong Kong/1721-30/99 H6N1

AJ627488 A/Turkey/Italy/4603/1999 H7N1

AY180580 A/Bantam/Nanchang/9-366/2000 H3N3

AF474069 A/Chicken/California/650/00 H6N2

AF508606 A/Chicken/Guangdong/10/00 H9N2

AF508610 A/Chicken/Henan/62/00 H9N2

AY180581 A/Chicken/Nanchang/1-0016/2000 H9N2

AY180545 A/Chicken/Nanchang/12-220/2000 H3N6

AY180527 A/Chicken/Nanchang/12-301/2000 H3N6

AY180554 A/Chicken/Nanchang/2-0527/2000 H4N6

AY180539 A/Chicken/Nanchang/3-0128/2000 H4N6

AY180531 A/Chicken/Nanchang/4-008/2000 H4N6

AY180562 A/Chicken/Nanchang/4-010/2000 H9N2

AY180529 A/Chicken/Nanchang/7-010/2000 H3N6

AY180535 A/Chicken/Nanchang/9-220/2000 H3N6

AY059497 A/Duck/Hong Kong/2986.1/2000 H5N1

AY059494 A/Duck/Hong Kong/ww381/2000 H5N1

AY059495 A/Duck/Hong Kong/ww461/2000 H5N1

AY180584 A/Duck/Nanchang/1-0070/2000 H9N2

AY180549 A/Duck/Nanchang/10-096/2000 H3N6

AY180553 A/Duck/Nanchang/10-383/2000 H3N6

AY180583 A/Duck/Nanchang/10-389/2000 H9N2

AY180542 A/Duck/Nanchang/11-197/2000 H9N2

AY180544 A/Duck/Nanchang/11-290/2000 H9N2

AY180537 A/Duck/Nanchang/11-392/2000 H9N2

AY180586 A/Duck/Nanchang/12-280/2000 H3N6

AY180524 A/Duck/Nanchang/2-0147/2000 H4N6

AY180557 A/Duck/Nanchang/2-0485/2000 H2N9

AY180558 A/Duck/Nanchang/2-0486/2000 H2N9

AY180573 A/Duck/Nanchang/2-0492/2000 H2N9

AY180574 A/Duck/Nanchang/3-090/2000 H2N9

AY180572 A/Duck/Nanchang/4-165/2000 H4N6

AY180559 A/Duck/Nanchang/4-173/2000 H4N6

AY180568 A/Duck/Nanchang/4-184/2000 H2N9

AY180571 A/Duck/Nanchang/4-190/2000 H2N9

AY180570 A/Duck/Nanchang/4-191/2000 H4N6

AY180534 A/Duck/Nanchang/7-092/2000 H9N2

AY180547 A/Duck/Nanchang/8-174/2000 H3N6

AY180546 A/Duck/Nanchang/8-197/2000 H3N6

AY180532 A/Duck/Nanchang/8-198/2000 H3N6

AY180526 A/Duck/Nanchang/9-091/2000 H3N6

AY180579 A/Duck/Nanchang/9-385/2000 H3N6

AF523413 A/Duck/Shantou/1042/00 H9N2

AF523410 A/Duck/Shantou/1043/00 H9N2

AF523425 A/Duck/Shantou/1588/00 H9N1

AF523420 A/Duck/Shantou/1881/00 H9N2

AF523426 A/Duck/Shantou/2030/00 H9N1

AF523415 A/Duck/Shantou/2102/00 H9N2

AF523411 A/Duck/Shantou/2134/00 H9N2

AF523419 A/Duck/Shantou/2143/00 H9N2

AF523417 A/Duck/Shantou/2144/00 H9N2

AF523416 A/Duck/Shantou/830/00 H9N2

AY059498 A/Goose/Hong Kong/3014.8/2000 H5N1

AF398419 A/Goose/Hong Kong/385.3/2000 H5N1

AF398420 A/Goose/Hong Kong/385.5/2000 H5N1

AY059492 A/Goose/Hong Kong/ww26/2000 H5N1

AY059493 A/Goose/Hong Kong/ww28/2000 H5N1

AY059496 A/Goose/Hong Kong/ww491/2000 H5N1

AY180530 A/pigeon/Nanchang/11-045/2000 H3N6

AY180538 A/pigeon/Nanchang/11-145/2000 H9N2

AY180525 A/pigeon/Nanchang/2-0461/2000 H9N2

AY180560 A/pigeon/Nanchang/7-058/2000 H9N2

AY180536 A/pigeon/Nanchang/8-142/2000 H3N6

AY180582 A/pigeon/Nanchang/9-058/2000 H3N3

AY180550 A/quail/Nanchang/10-028/2000 H3N6

AY180543 A/quail/Nanchang/12-340/2000 H1N1

AY180575 A/quail/Nanchang/2-0460/2000 H9N2

AY180576 A/quail/Nanchang/2-0579/2000 H4N6

AY180540 A/quail/Nanchang/4-026/2000 H4N6

AY180541 A/quail/Nanchang/4-034/2000 H4N6

AY180563 A/quail/Nanchang/4-040/2000 H9N2

AY180548 A/quail/Nanchang/7-026/2000 H3N6

AY180564 A/Duck/Nanchang/2-0480/2000 H9N2

AF457701 A/Chicken/California/431/00 H6N2

AF457693 A/Chicken/California/465/00 H6N2

AY496851 A/Chicken/Mudanjiang/0823/2000 H9N2

AJ410554 A/European stone bird (chukka)/Hong Kong/FY295/00H6N1

AJ410553 A/Ou Stone Bird/Hong Kong/NT261/00 H6N1

AY585423 A/Duck/Fujian/19/2000 H5N1

AY585425 A/Duck/Guangdong/07/2000 H5N1

AY585426 A/Duck/Guangdong/12/2000 H5N1

AY585428 A/Duck/Guangdong/40/2000 H5N1

AY585439 A/Duck/Zhejiang/11/2000 H5N1

AY585440 A/Duck/Zhejiang/52/2000 H5N1

AY633143 A/Mallard/Alberta/119/00 H4N6

AY633151 A/Duck/Alberta/127/00 H3N8

AY633159 A/Mallard/Alberta/136/00 H4N6

AJ421064 A/pheasant/Hong Kong/FY294/00 H6N1

AJ427309 A/pheasant/Hong Kong/FY294/00 H6N1

AJ427864 A/quail/Hong Kong/FY298/00 H6N1

AJ410551 A/quail/Hong Kong/SF550/00 H6N1

AJ410552 A/quail/Hong Kong/SF595/00 H6N1

AF474070 A/Chicken/California/139/01 H6N2

AY497118 A/Chicken/El Salvador/102711-1/01 H5N2

AF509126 A/Chicken/Hong Kong/715.5/01 H5N1

AF509127 A/Chicken/Hong Kong/751.1/01 H5N1

AF509128 A/Chicken/Hong Kong/822.1/01 H5N1

AF509129 A/Chicken/Hong Kong/829.2/01 H5N1

AF509130 A/Chicken/Hong Kong/830.2/01 H5N1

AF509131 A/Chicken/Hong Kong/858.3/01 H5N1

AF509132 A/Chicken/Hong Kong/866.3/01 H5N1

AF509133 A/Chicken/Hong Kong/867.1/01 H5N1

AF509135 A/Chicken/Hong Kong/873.3/01 H5N1

AF509136 A/Chicken/Hong Kong/876.1/01 H5N1

AF509134 A/Chicken/Hong Kong/879.1/01 H5N1

AF509137 A/Chicken/Hong Kong/891.1/01 H5N1

AF509138 A/Chicken/Hong Kong/893.2/01 H5N1

AF509120 A/Chicken/Hong Kong/FY150/01 H5N1

AY221551 A/Chicken/Hong Kong/FY150/01 H5N1

AY221550 A/Chicken/Hong Kong/FY150/01- H5N1

AF509117 A/Chicken/Hong Kong/FY77/01 H5N1

AY221549 A/Chicken/Hong Kong/NT873.3/01 H5N1

AY221548 A/Chicken/Hong Kong/NT873.3/01- H5N1

AF509125 A/Chicken/Hong Kong/SF219/01 H5N1

AY221556 A/Chicken/Hong Kong/YU562/01 H5N1

AF509118 A/Chicken/Hong Kong/YU562/01 H5N1

AF509119 A/Chicken/Hong Kong/YU563/01 H5N1

AY221555 A/Chicken/Hong Kong/YU822.2/01 H5N1

AY221554 A/Chicken/Hong Kong/YU822.2/01- H5N1

AY180585 A/Chicken/Nanchang/1-020/2001 H3N6

AY180565 A/Chicken/Nanchang/1-101/2001 H3N6

AY180533 A/Chicken/Nanchang/2-120/2001 H3N6

AY180566 A/Chicken/Nanchang/2-220/2001 H3N6

AY180555 A/Chicken/Nanchang/3-120/2001 H3N2

AY180578 A/Chicken/Nanchang/4-301/2001 H9N2

AY180551 A/Chicken/Nanchang/4-361/2001 H9N2

AF468842 A/Duck/Anyang/AVL-1/2001 H5N1

AF509141 A/Duck/Hong Kong/573.4/01 H5N1

AF509142 A/Duck/Hong Kong/646.3/01 H5N1

AY233394 A/Duck/NC/91347/01 H1N2

AY180577 A/Duck/Nanchang/1-100/2001 H3N6

AY180528 A/Duck/Nanchang/1-161/2001 H3N6

AY180552 A/Duck/Nanchang/1-181/2001 H3N6

AY180556 A/Duck/Nanchang/2-182/2001 H3N6

AF523414 A/Duck/Shantou/1605/01 H9N2

AF523418 A/Duck/Shantou/2088/01 H9N2

AF509139 A/Goose/Hong Kong/76.1/01 H5N1

AF509140 A/Goose/Hong Kong/ww100/01 H5N1

AF509121 A/pheasant/Hong Kong/FY155/01 H5N1

AY221553 A/pheasant/Hong Kong/FY155/01 H5N1

AY221552 A/pheasant/Hong Kong/FY155/01- H5N1

AF509124 A/Pigeon/Hong Kong/SF215/01 H5N1

AF509123 A/quail/Hong Kong/SF203/01 H5N1

AY180569 A/quail/Nanchang/2-040/2001 H3N6

AY180567 A/quail/Nanchang/3-140/2001 H3N6

AF509122 A/silk feather chicken/Hong Kong/SF189/01 H5N1

AF523412 A/Duck/Shantou/4808/01 H9N2

AF457709 A/Chicken/California/139/01 H6N2

AF457685 A/Chicken/California/6643/01 H6N2

AF457676 A/Chicken/California/905/01 H6N2

AY180561 A/Chicken/Nanchang/3-201/01 H3N6

AY268949 A/Chicken/Wangcheng (Wangcheng)/4/2001 H9N2

AY585422 A/Duck/Fujian/17/2001 H5N1

AY585424 A/Duck/Guangdong/01/2001 H5N1

AY585430 A/Duck/Guangxi/22/2001 H5N1

AY585431 A/Duck/Guangxi/35/2001 H5N1

AY585432 A/Duck/Guangxi/50/2001 H5N1

AY422023 A/Duck/Hokkaido/107/01 H2N3

AY422024 A/Duck/Hokkaido/17/01 H2N3

AY422025 A/Duck/Hokkaido/86/01 H2N3

AY422026 A/Duck/Hokkaido/95/01 H2N2

AY585434 A/Duck/Shanghai/08/2001 H5N1

AY585435 A/Duck/Shanghai/13/2001 H5N1

AY585438 A/Duck/Shanghai/38/2001 H5N1

AY586423 A/Duck/Italy/33/01 H7N3

AY586424 A/Duck/Italy/43/01 H7N3

AY497119 A/Chicken/Guatemala/194573/02 H5N2

AY651511 A/Ck/HK/31.2/2002 H5N1

AY651522 A/Ck/HK/3169.1/2002 H5N1

AY651521 A/Ck/HK/3176.3/2002 H5N1

AY651512 A/Ck/HK/37.4/2002 H5N1

AY651514 A/Ck/HK/YU22/2002 H5N1

AY575908 A/Eg/Hong Kong/757.3/02 H5N1

AY575909 A/G.H/Hong Kong/793.1/02 H5N1

AY651510 A/Gf/HK/38/2002 H5N1

AY651513 A/SCk/HK/YU100/2002 H5N1

AY303658 A/Chile/Chile/176822/02 H7N3

AY303659 A/Chicken/Chile/4957/02 H7N3

AY575911 A/Chicken/Hong Kong/31.4/02 H5N1

AY575915 A/Chicken/Hong Kong/409.1/02 H5N1

AY575912 A/Chicken/Hong Kong/61.9/02 H5N1

AY575914 A/Chicken/Hong Kong/96.1/02 H5N1

AY575913 A/Chicken/Hong Kong/YU777/02 H5N1

AY585420 A/Duck/Fujian/01/2002 H5N1

AY585421 A/Duck/Fujian/13/2002 H5N1

AY585427 A/Duck/Guangdong/22/2002 H5N1

AY585433 A/Duck/Guangxi/53/2002 H5N1

AY575910 A/Duck/Hong Kong/821/02 H5N1

AY585436 A/Duck/Shanghai/35/2002 H5N1

AY585437 A/Duck/Shanghai/37/2002 H5N1

AY651524 A/Wild Pigeon/HK/862.7/2002 H5N1

AY575907 A/Goose/Hong Kong/739.2/02 H5N1

AY651526 A/Grey Heron/HK/861.1/2002 H5N1

AY575916 A/pheasant/Hong Kong/sv674.15/02 H5N1

AY651527 A/teal/China/2978.1/2002 H5N1

AY651525 A/Tree Sparrow/HK/864/2002 H5N1

AY586426 A/Turkey/Italy/214845/02 H7N3

AJ627486 A/Turkey/Italy/214845/2002 H7N3

AJ627495 A/Turkey/Italy/220158/2002 H7N3

AY586425 A/Turkey/Italy/220158/2002 H7N3

AY651515 A/Ck/HK/2133.1/2003 H5N1

AY651519 A/Ck/HK/FY157/2003 H5N1

AY651516 A/Ck/HK/NT93/2003 H5N1

AY651517 A/Ck/HK/SSP141/2003 H5N1

AY651518 A/Ck/HK/WF157/2003 H5N1

AY651520 A/Ck/HK/YU324/2003 H5N1

AY651490 A/Ck/Indonesia/2A/2003 H5N1

AY651485 A/Ck/Indonesia/BL/2003 H5N1

AY651487 A/Ck/Indonesia/PA/2003 H5N1

AY651532 A/Ck/ST/4231/2003 H5N1

AY651529 A/Dk/HN/5806/2003 H5N1

AY651534 A/Dk/ST/4003/2003 H5N1

AY651535 A/Dk/YN/6255/2003 H5N1

AY651536 A/Dk/YN/6445/2003 H5N1

AY342426 A/poultry/Netherlands/033/03 H7N7

AY342425 A/poultry/Netherlands/219/03 H7N7

AY651523 A/Black-headed Seagull/HK/12.1/2003 H5N1

AJ620352 A/Chicken/Germany/R28/03 H7N7

AY342427 A/Chicken/Netherlands/1/03 H7N7

AY518364 A/Duck/China/E319-2/03 H5N1

AY576929 A/Chicken/Vietnam/CM/2004 H5N1

AY651488 A/Ck/Indonesia/4/2004 H5N1

AY651489 A/Ck/Indonesia/5/2004 H5N1

AY651491 A/Ck/Thailand/1/2004 H5N1

AY651492 A/Ck/Thailand/73/2004 H5N1

AY651493 A/Ck/Thailand/9.1/2004 H5N1

AY651502 A/Ck/Vietnam/33/2004 H5N1

AY651503 A/Ck/Vietnam/35/2004 H5N1

AY651504 A/Ck/Vietnam/36/2004 H5N1

AY651505 A/Ck/Vietnam/37/2004 H5N1

AY651506 A/Ck/Vietnam/38/2004 H5N1

AY651507 A/Ck/Vietnam/39/2004 H5N1

AY651508 A/Ck/Vietnam/C57/2004 H5N1

AY651538 A/Ck/YN/115/2004 H5N1

AY651537 A/Ck/YN/374/2004 H5N1

AY651531 A/Dk/HN/101/2004 H5N1

AY651530 A/Dk/HN/303/2004 H5N1

AY651486 A/Dk/Indonesia/MS/2004 H5N1

AY651496 A/Dk/Thailand/71.1/2004 H5N1

AY651509 A/Dk/Vietnam/11/2004 H5N1

AY650273 A/GSC Chicken/British Columbia/04 H7N3

AY648290 A/GSC Chicken B/British Columbia/04 H7N3

AY651497 A/Gs/Thailand/79/2004 H5N1

AY651533 A/Ph/ST/44/2004 H5N1

AY651494 A/Qa/Thailand/57/2004 H5N1

AY651495 A/Bird/Thailand/3.1/2004 H5N1

AY611527 A/Chicken/British Columbia/04 H7N3

AY646081 A/Chicken/British Columbia/GSC Human B/04 H7N3

AY609313 A/Chicken/Guangdong/174/04 H5N1

AY684707 A/Chicken/Hubei/327/2004 H5N1

AY653196 A/Chicken/Jilin/9/2004 H5N1

AY590579 A/Chicken/Nakorn Pathom/Thailand/CU-K2/2004 H5N1

AY574189 A/Chicken/Vietnam/HD1/2004 H5N1

AY574192 A/Chicken/Vietnam/HD2/2004 H5N1

AY576931 A/American duck/Vietnam/MdGL/2004 H5N1

AY651528 A/peregrine falcon H5N1

/HK/D0028/2004

Example 18: Identification of highly potent siRNAs by high-throughput screening

Cell culture. Human lung epithelial cells A-549 (ATCC) were cultured in DMEM containing 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin grow in the base. Other cell lines derived from airways, such as Calu-3 or HBE cells (ATCC), or other cell lines may also be used. Cells were grown at 37 °C in a humidified incubator with 5% _CO2 .

siRNA. Various siRNAs were designed and synthesized, each siRNA containing an antisense strand with a 19 nucleotide inhibitory region fully complementary to the highly conserved target moieties listed in Table 16 and a sense strand complementary to the antisense strand. siRNAs contain 3'dTdT overhangs on both strands. All siRNAs were synthesized by Dharmacon Research (Lafayette, CO) using 2'ACE protection chemistry. The siRNA was deprotected, desalted and annealed by the manufacturer.

Highly potent siRNAs were identified by dual-luciferase assays. According to the manufacturer's instructions (see Promega Technical Bulletin No. 329, available at www.promega.com/tbs/tb329/tb329.pdf ), siRNAs corresponding to relevant influenza Viral transcripts, full-length cDNAs of the NP, PA, PB1, PB2, M, NS, NA or HA genes from the PR8 virus were cloned into the psiCHECK ^™ -2 vector (cat# C8021, Promega, Madison, WI). The cDNA was inserted into the 3'UTR of a synthetic Renilla luciferase gene (hLuc) optimized for expression in mammalian cells. DNA and siRNA (or non-specific sicontrol, Dharmacon) targeting the cDNA insert were co-transfected into A-549 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection, a fusion of the Renilla gene and the influenza virus gene was transcribed. Luciferase activity was measured 24 hours after transfection as a function of the amount of each siRNA. In this assay, RNAi mediated by siRNA or shRNA that hybridizes to the targeted portion of the fusion transcript (i.e., the influenza-specific portion of the fusion transcript) causes degradation of the fused Renilla:influenza virus gene transcript, thereby reducing Luciferase signal. Firefly luciferase expressed from the same vector was used as transfection control and specificity control as described in Promega Technical Bulletin 329. Specifically, 24 hours after transfection, lysates and substrate buffer (Dual-Glo luciferase assay system) were added to the cells, and luciferase activity was read. After 10 min, Stop and Glo for Renilla was added and Renilla luciferase activity was read. Each sample was run three times. Firefly luciferase activity from each well was used as a transfection control for Renilla in the same well. The ratio of Renilla/firefly luciferase activity was used as the final value of luciferase activity from each well. The mean of ratios from triplicate influenza siRNAs was compared to the mean of triplicate sicontrol. Percent inhibition was calculated as 100 x (1 - influenza siRNA/sicontrol).

Table 26 presents data for many of the highly potent siRNAs identified in the screen. This table lists the siRNA target genes (NP, PA, PB1, PB2), siRNA concentration and ID number. The siRNA ID column lists the average percent inhibition for each concentration tested. Blanks indicate no experiments were performed. The results showed that many siRNAs inhibited transcript expression by at least 80% even at concentrations as low as 0.6 nM.

Table 26

Target gene: NP

Mean inhibition (%) Concentration (nM) siRNAID 154 758 1121 1313 NP-1496 1499 0.00480.0240.120.631575 9.3248.265.7184.3790.0990.4690.03 010.5635.2566.7671.9674.2169.94 10.8834.7266.5484.7589.0889.1386.96 11.7246.9375.2887.4391.0591.6789.82 29.2368.6284.7490.4190.96 12.0866.1378.9984.7185.97

Target gene: PA

Mean inhibition (%) Concentration (nM) 7736 7803 8282 8286 0.00480.024 11.429.3 27.2744.88 11.9418.78 11.6237.57

0.120.631575 60.1478.2585.7685.7485.42 77.3785.1386.7887.8286 40.3485.9592.1893.34 69.0383.238989.890.64

Target gene: PB1

Mean inhibition (%) Concentration (nM) PB1- 4276 5018 5457 5773 6124 2257 0.00480.0240.120.631575 4.6160.8187.9892.1792.07 022.9872.9985.8389.1289.0793.4 9.1123.9260.6782.6387.6389.8188.57 022.2153.4675.0984.7684.9487.56 8.427.0357.6776.6580.8483.93 13.734.266.3281.2984.8886.0582.29

Target gene: PB2

Mean inhibition (%) Concentration (nM) PB2- 2327 3276 3807 3817 2240 0.00480.0240.120.631575 7.7951.4981.9288.7990.8490.8290.95 030.4966.5382.0987.1987.0488.39 017.1542.3670.4479.6585.0883.26 14.5145.2673.0480.3583.7683.5382.06 5.7635.4455.8377.4880.6682.6585.19

Certain siRNAs and/or shRNAs identified as highly effective using the above screens were tested against influenza virus as described elsewhere herein, additionally in tissue culture and/or animal models as described in Example 19.

Example 19: Identification of highly potent siRNAs that inhibit viral replication by screening

Cell culture. Vero cells (ATCC) were grown in DMEM medium containing 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin. Cells were grown at 37 °C in a humidified incubator with 5% _CO2 . Virus infection was performed in DMEM containing 0.3% bovine serum albumin (BSA, Sigma, St. Louis, MO), 10 mM Hepes, 100 units/ml penicillin and 100 μg/ml streptomycin.

siRNA. Designed and synthesized siRNAs containing an antisense strand with a 19 nucleotide inhibitory region fully complementary to each of the highly conserved target moieties listed in Table 18 and a sense strand complementary to the antisense strand . siRNAs contain 3'dTdT overhangs on both strands. All siRNAs were synthesized by Dharmacon Research (Lafayette, CO) using 2'ACE protection chemistry. The siRNA was deprotected, desalted and annealed by the manufacturer.

siRNA transfection. Log-phase cultures of Vero cells were trypsinized, washed and seeded in 96-well plates at 20,000 cells per well and incubated at 37 °C in a humidified incubator with 5% _CO2 overnight. For the first screen, 10 pmol of siRNA (19 bp duplex region with dTdT 3' overhang) was added to 25 μl of Opti-MEM I (Invitrogen). 0.75 μl of Lipofectamine 2000 (Invitrogen) and 0.19 μl of SuperRNAsin (Ambion) were diluted in 25 μl of Opti-MEMI and mixed gently. Diluted lipids were combined with diluted siRNA (total volume 50 μl) and the mixture was incubated at room temperature for 20 min to form siRNA-Lipofectamine 2000 complexes. At the end of the incubation, 50 μl of the transfection complex was pipetted into each well containing the cells and 50 μl of DMEM medium containing 10% FCS. The final concentration of each siRNA was 100 nM. Each siRNA was tested in triplicate and the results were averaged. NP-1496 (100 nM) and sicontrol (Dharmacon) were used as positive and negative controls, respectively. The second screening for siRNA transfection was performed in the same manner except using siRNA at a concentration of 1 nM.

Virus infection. After 6 h of incubation at 37 °C in a humidified incubator with 5% _CO , the supernatant was removed and the cells in each well were infected with PR8 virus in 25 μl of PBS containing 0.3% BSA. Infections were performed at an MOI of 0.1 for the first and second screens. The plate was shaken gently for 1 h at room temperature, after which 175 μl of DMEM containing 0.3% BSA, 4 μg trypsin, 10 mM HEPES was added to each well. The plates were then incubated at 37 °C in a humidified incubator with 5% _CO2 .

Measurement of virus titers. Supernatants were collected 24 hours after infection. Virus titers were measured as described above.

result

To identify siRNAs with higher activity from a panel of 215 selected siRNAs targeting highly conserved target parts of the influenza genome, a series of high-throughput screens (HTS) were performed. Rather than directly testing whether siRNAs could inhibit the expression of their specific target genes, it was decided to focus on assessing the ability of siRNAs to inhibit influenza virus production in cells. Therefore, viral titer was used as a readout, although it is recognized that some siRNAs that do not significantly reduce viral titer may still possess the ability to degrade target mRNA and thus be suitable for some purposes.

Of the 215 siRNAs tested in the first HTS, 90 showed at least a 4-fold reduction in PR8 virus titers relative to no treatment (NT) or sicontrol. When vero cells were transfected with 100nM sicontrol, no non-specific inhibition was seen. Transfection efficiency was about 80%. The results are summarized in Table 21 and expressed in terms of fold inhibition of viral titers. Actual levels of inhibition may be greater than those presented in the table.

Table 21: Results of High Throughput Screen #1

viral titer suppression siRNAID (multiple) >8 752, 754, 758, 1121, 1122, 1223, 1225, 1254, 1313, 1316, 1381, 1383, 1488, 1499, 1527, NP1496 ≥4 109, 119, 154, 222, 224, 301, 302, 359, 375, 688, 692, 1490, 1568, 1714, 1720, 1728, 2054, 2283, 2327, 3225, 3231, 3276, 3277, 3693, 3700, 3880, 3882, 3887, 4120, 4163, 4276, 4283, 4913, 4915, 4980, 5018, 5359, 5360, 5457, 5460, 5574, 5578, 5580, 5584, 5680, 5773, 5791, 5793, 5795, 5902, 5942, 5945, 5947, 5997, 6081, 6087, 6089, 6351, 6436, 6559, 6696, 6732, 6744, 7202, 7424, 75177580, 7581, 7648, 7736, 7803, 7807, 8172, 8271, 85, 2, 82 8286, 8289, 8299, 8300, 8709, 9331

A second high-throughput screen was performed using a lower concentration of siRNA (1 nM) to more precisely identify highly potent siRNAs from siRNAs that showed at least a 4-fold reduction in viral titers in the first HTS. Thirty siRNAs showed an approximately 3- to 4-fold reduction in PR8 virus titers in Vero cell cultures when tested at the lower concentrations. The results of the second HTS are presented in Table 22.

Table 22: Results of High Throughput Screen #2

siRNAID average value siRNAID average value siRNAID average value 1091191542222243013023593756886927527547581121sicontrol535953605457546055745578558055845680 21.67121.671.671.6721.671.672.6721.671.33141.671.671.331.331.671.671.6713.33 112212231225125413131316138113831488149915271590171422832327NT594759976081608760896351643665596696 11.671.331.331121.671.671.671.6721.671.671.3341.671.671.331.331.331.67221.67 327632773693370038803882388741204163427642834913491549805018764877367803780781728271828282858286 1.331.6743.334222211.3321.3321.3311.6711.671.333.33141

5773579157935795590259425945 121.672.6722.672 6732674472027424751775807581 1.671.671.6741.671.331.33 8289829983009331sicontrolNT 141.67444

Example 20: Confirmation of siRNA screens

Materials and methods

Thirty siRNAs selected from the second HTS and some siRNAs from the first HTS were tested in MDCK cells to confirm the results. Ten million MDCK cells in serum-free RPMI 1640 medium were mixed with siRNA at a final concentration of 100 nM and electroporated at 400 V and 975 μF using an electroporator (Bio-Rad). Electroporated cells were divided into 3 wells of a 6-well plate and cultured in DMEM containing 10% fetal calf serum for 6 hours. The medium was then removed and 100 microliters of PR8 virus in infection medium consisting of DMEM, 0.3% BSA, 10 mM Hepes, 100 units/ml penicillin and 100 μg/ml streptomycin was added to each well. Infections were performed at MOIs of 0.2, 0.02 and 0.002 for each of the 3 wells. After incubation for 1 h at room temperature, 2 ml infection medium containing 4 μg/ml trypsin was added to each well, and cells were incubated at 37 °C under 5% _CO2 . At various time points after infection, supernatants were collected from infected cultures and virus titers were determined by hemagglutination analysis.

result

To confirm the ability of the highly potent siRNAs identified in the first and second screens to inhibit influenza virus production, a third screen was performed in MDCK cells. Compared to Vero cells, MDCK cells were susceptible to infection by even smaller amounts of experimental strains of influenza virus. Virions replicate much faster in MDCK cells than in Vero cells. The third screen in MDCK cells additionally compared the efficiency of siRNAs in viral suppression and confirmed the results of the first two screens with an accuracy of about 85-90%.

At MOI = 0.2, the three NP siRNAs reduced virus titers by at least 16-fold 24 hours after infection. Four NP siRNAs (including the above three NP siRNAs), two PB2, three PB1 and four PA siRNAs reduced virus titers by at least 8-fold. The following siRNAs were the 13 most potent siRNAs tested: 154, 758, 1121, 1313, 2327, 3276, 4276, 5018, 5457, 7736, 7803, 8282, and 8286. At MOI=0.02, 8 NP, 2 PB2, 3 PB1 and 8 PA siRNAs (including the siRNA effective at MOI=0.2) reduced virus titers at least 8-fold 24 hours after infection. At MOI=0.002, 10 NP, 8 PB2, 11 PB1, 10 PA siRNAs (including effective siRNAs effective at MOI=0.02) reduced virus titers at least 4-fold 24 hours after infection. No viral inhibition was observed for sicontrol siRNA.

The results of the screening are presented in Table 23. Columns show HA units at MOI=0.2/0.02/0.002 at time 24, 36, 48 and in some cases 60 hours post-infection, respectively. Plates were tested in groups and sicontrol was the control siRNA tested in each group. NT = no treatment. The most effective siRNAs are shown in bold.

Table 23: Results of High Throughput Screen #3

siRNA 24 hours 36h 48h 60h  sicontrol131314882283232732763277siRNAsicontrol154112111221316149915901714369337008289siRNAsicontrol7585018673275807581764877367803828282867807siRNANTsicontrol42764283491554575460558457736081 512/256/832/4/1128/32/1128/64/264/32/264/32/1128/64/224h1024/256/1632/4/164/4/1256/16/1256/32/2256/ 32/1512/256/4512/128/2512/256/2512/256/2512/128/224h1024/256/4128/16/1128/16/1256/32/1256/16/1256/32/1256/32/ 1128/32/164/16/1128/16/1128/32/1256/64/124h1024/256/161024/256/16128/32/1512/64/2512/128/4128/32/1512/64/4512 128/2256/32/1512/128/2 1024/512/32128/16/1256/64/2256/128/2256/64/2128/64/2256/128/436 128/41024/256/161024/256/81024/512/81024/512/161024/128/1636 4256/64/4256/64/1512/32/2256/128/8512/128/836h1024/512/321024/512/32256/64/8512/128/16512/128/16256/64/4512/128/16512/ 128/16256/64/4512/128/16 1024/512/32256/64/2512/256/8256/256/4256/128/4256/128/8256/128/1648 128/82148/256/321024/256/81024/512/161024/512/161024/256/3248 321024/512/161024/512/81024/512/81024/512/161024/512/3248h1024/512/641024/512/64256/64/8512/128/16512/128/16256/64/8512/128/16 128/16256/64/4512/128/16 1024/512/64512/64/41024/256/16256/256/8512/256/8256/128/16512/128/16

60876089817212251254 512/64/2512/128/4512/128/8512/64/4256/64/4 512/128/16512/256/32512/256/16512/64/16256/128/32 512/128/16512/256/32512/256/32512/64/32512/128/32

Example 21: siRNA Dose Response Test

Materials and methods

The dose response of 13 siRNAs selected from the third HTS and some siRNAs from the second HTS were tested in MDCK cells. Ten million MDCK cells in serum-free RPMI 1640 medium were mixed with siRNA at various siRNA concentrations ranging from 0.8 nM to 100 nM. siRNA was electroporated into cells by using an electroporator (Bio-Rad) at 400V and 975[mu]F. Electroporated cells were divided into 3 wells of a 6-well plate and cultured in DMEM containing 10% fetal calf serum for 6 hours. The medium was then removed, and 100 microliters of PR8 virus in infection medium consisting of DMEM 0.3% BSA, 10 mM Hepes, 100 units/ml penicillin, and 100 μg/ml streptomycin were added to each well to MOIs of 0.2, 0.02 and 0.002 for each of the 3 wells were obtained. After incubation for 1 h at room temperature, 2 ml of infection medium containing 4 μg/ml trypsin was added to each well, and the cells were incubated at 37 °C under ₅ % CO. At various times after infection, supernatants were collected from infected cultures and virus titers were determined by hemagglutination assay as described above.

Results: The minimum concentration of siRNA that inhibited viral production 2-fold at 24 hours post-infection at MOI=0.2 and MOI=0.02 is as follows, where asterisks indicate siRNAs identified as described in Example 1.

NP gene fragments as targets:

154 0.8nM

758 1nM

1121 1nM

1313 0.8nM

1499 5nM

NP1496 ^* 4nM

PB2 gene fragments as targets:

2327 1nM

3276 1nM

PB1 gene fragments as targets:

4276 1nM

5018 5nM

5457 5nM

5773 5nM

PB1-2257 ^* 5nM

PA gene fragments as targets:

7736 5nM

7803 1nM

8282 5nM

8286 5nM

The results are shown in Table 24 in terms of HA units after 24, 36 or 48 hours of infection at MOIs of 0.2/0.02/0.002 for various siRNA concentrations.

Table 24: Dose Response Screening Results

siRNA 24h 36h 48h  NTSicontrol 100nMNP1496 0.8nMNP1496 4nMNP1496 20nM1313 0.8nM1313 4nM1313 20nM1313 25nM154 0.8nM154 4nM154 20nM154 25nM8172 100nM8289 100nMsiRNANTSicontrol 100nM7803 1nM7803 5nM7803 25nM8282 1nM8282 5nM8282 25nM6696 100nMsiRNANTSicontro1 25nM2327 1nM2327 5nM2327 25nM3276 1nM3276 5nM3276 25nM 1024/128/21024/128/21024/128/2512/64/1512/64/1512/64/1512/64/1256/32/1128/8/1512/64/1512/64/1256/32/1128/ 8/1512/128/1256/32/124h1024/128/21024/128/2512/64/1512/32/1256/32/11024/64/2512/64/1512/64/1512/32/124h1024/128/ 21024/128/2512/64/1512/64/1512/32/11024/64/2512/64/1512/64/1 2048/256/42048/256/41024/256/41024/256/41024/256/41024/256/21024/128/2512/64/1256/32/11024/256/21024/128/2512/64/1256/ 32/11024/256/4512/128/236h2048/256/42048/256/41024/64/21024/64/2512/64/21024/128/41024/128/41024/128/41024/128/436h2048/256/ 42048/256/41024/128/21024/128/21024/64/21024/128/41024/128/21024/64/2 2048/256/82048/256/82048/256/82048/256/42048/256/42048/256/42048/256/41024/128/2512/64/12048/256/42048/128/21024/128/2512/ 64/12048/256/82048/256/448h2048/256/82048/256/82048/128/41024/128/41024/128/42048/256/82048/256/81024/128/42048/256/848h26/048/2 82048/256/82048/128/82048/128/81024/64/82048/128/82048/128/82048/64/4

 758 1nm758 5nM758 25nMsiRNANTSicontrol 25nM4276 1nM4276 5nM4276 25nM5018 1nM5018 5nM5018 25nM5457 1nM5457 5nM5457 25nM7736 1nM7736 5nM7736 25nMsiRNANTSicontrol 25 nM1121 1nM1121 5nM1121 25nM5773 1nM5773 5nM5773 25nMPB1-2257 1nMPB1-2257 5nMPB1-2257 25nMSiRNANTsicontrol 25nM1499 1nM1499 5nM1499 25nM8286 1nM8286 5nM8286 25nM1499+82862.5+2.5 512/64/1512/64/1256/32/124h1024/128/21024/128/2512/64/1512/32/1256/16/11024/64/2512/32/1256/16/11024/128/2512/ 32/1512/32/11024/64/2512/64/1512/64/124h512/128/4512/128/4256/128/4256/64/1128/32/1256/64/2256/64/1128/32/ 1512/64/1256/64/1256/64/124h1024/256/41024/256/41024/256/4512/64/2512/32/11024/128/2512/64/2512/64/21024/256/2 1024/128/21024/128/21024/64/236h2048/256/42048/256/41024/128/21024/64/2512/32/12048/128/21024/64/2512/32/12048/256/41024/ 64/21024/64/21024/128/41024/128/2512/64/136h1024/256/81024/256/8512/256/8512/128/4256/64/1512/256/8512/128/2256/64/ 11024/128/4512/128/4512/128/436h2048/512/162048/512/162048/256/82048/128/42048/64/22048/256/42048/128/42048/128/42048/256/4 1024/128/42048/128/82048/64/848h2048/512/82048/512/81024/128/41024/128/4512/64/22048/256/41024/128/21024/64/12048/512/81024/ 128/41024/128/42048/256/82048/256/42048/256/448 41024/128/8512/128/8512/128/848h2048/1024/322048/1024/322048/512/162048/256/82048/64/42048/256/82048/256/82048/256/82048/256/8

Example 22: Effective inhibition of influenza virus using siRNA combinations

Combinations of certain siRNAs selected from the third HTS and some siRNAs from the second HTS were tested against influenza. Transfection, infection and viral titer testing were performed as described in Example 21. Transfection of 1499 and 4276 together with each siRNA at 12.5 nM inhibited virus production slightly more effectively than 1499 or 4276 alone at 25 nM.

Table 25: Effects of siRNAs in combination

siRNA 24h 36h 48h NTsicontrol 50nM1499+8282 12.5+12.51499+4276 12.5+12.58282+4276 12.5+12.51499+8282 25+251499+4276 25+258282+4276 25+251499 25nM1499 50nM8282 25nM8282 50nM4276 25nM4276 50nM 1024/128/41024/128/4512/32/1256/16/1512/32/1512/32/1256/16/1256/32/1512/32/1512/16/1512/64/1512/32/1256/ 32/1256/16/1 2048/256/82048/256/81024/64/2512/32/11024/64/21024/64/2512/32/11024/64/21024/64/21024/64/21024/128/41024/64/2512/ 64/2512/32/1 2048/512/162048/512/161024/128/8512/64/41024/128/81024/128/8512/64/21024/128/41024/128/81024/128/81024/128/81024/128/81024/ 128/8512/64/2

Example 23: Inhibition of Influenza Virus by Direct Delivery of Naked siRNA into the Respiratory System

Materials and methods

siRNA preparation, virus infection, lung collection and influenza virus titer analysis were performed as described in Example 12. Mice were anesthetized using isoflurane (administered by inhalation). siRNA was delivered by intranasal drip in a volume of 50 μl. p-values were calculated using Student's T-test.

result

siRNA (NP-1496) in phosphate buffered saline (PBS) was administered to groups of mice (5 mice per group). Mice were infected with influenza virus (2000 PFU) 3 hours after siRNA administration. Lungs were harvested 24 hours post-infection and virus titers were measured. In preliminary experiments, mice were anesthetized with avertin and 2 mg/kg of siRNA was administered by intranasal drip. A decrease in viral titer was observed relative to the control group, although this did not reach statistical significance (data not shown).

In the second experiment, black Swiss mice were anesthetized using isoflurane/ _O . Various amounts of siRNA in PBS were administered intranasally to mice, 50 μl per mouse. Three different groups (5 mice each) received doses of 2 mg/kg, 4 mg/kg or 10 mg/kg of siRNA in PBS by intranasal instillation. A fourth group that received only PBS was used as a control. Three hours later, the mice were anesthetized again with isoflurane/O ₂ , and 30 μl of PR8 virus (2000 pfu=4×lethal dose) were administered intranasally to the mice. 24 h after infection, mouse lungs were harvested, homogenized and virus titers were measured by estimating _TCID50 as described above. Serial 5-fold dilutions were performed on lung homogenates instead of 10-fold dilutions.

Significant and dose-dependent differences in viral titers were seen between each of the 3 treatment groups and control mice. In groups receiving doses of 2 mg/kg, 4 mg/kg and 10 mg/kg, respectively, virus titers were reduced by 3.45-fold (p=0.0125), 4.16-fold (p=0.0063) and 4.62-fold (p=0.0057) relative to the control group . Data for individual mice ( _TCID50 ) are presented in Table 27 and shown in Figure 31A.

Taken together, these results demonstrate the efficacy of siRNA delivered to the respiratory system in aqueous media in the absence of specific agents that enhance delivery.

Table 27: Intranasal delivery of naked siRNA inhibits influenza virus production

deal with log ₁₀ TCID ₅₀ average value P value PBS 26718.37 45687.78 45687.78 15625 26718.37 32087.46 NP (2mg/kg) 15625 15625 3125 3125 9137.56 9327.51 0.008 NP (4mg/kg) 9137.56 9137.56 5343.68 9137.56 5343.68 7620 0.004 NP (10mg/kg) 9137.56 9137.56 9137.56 3125 3125 6732.53 0.003

Example 24: Inhibition of influenza virus production in mice by direct delivery of naked siRNA into the respiratory system

This example confirms the results of Example 23 and demonstrates that influenza virus production in the lung is inhibited by administration of siRNA targeting NP to the respiratory system in the absence of delivery-enhancing agents in aqueous media. 6 μg, 15 μg, 30 μg and 60 μg of NP- 1496 siRNA or 60 μg of GFP-949 siRNA were instilled into mice via intranasal route. Lungs were collected 24 hours after infection. As shown in Table 28 and Figure 3 IB, NP-specific siRNA effectively inhibited influenza virus when administered by intranasal instillation in aqueous media in the absence of delivery agent. Significant and dose-dependent differences in viral titers were seen between mice in each of the 3 treatment groups and the control group.

Table 28: Inhibition of Influenza Virus Production in the Lung Using Naked siRNA

deal with TCID ₅₀ average value P value PBS 125 365.5 213.7 365.5 125 239.95 GFP (60μg) 125 213.7 213.7 213.7 365.5 226.32 NP (6μg) 213.7 213.7 125 213.7 42.7 161.8 0.263 NP (15μg) 125 125 42.7 25 73.1 78.17 0.024 NP (30μg) 8.5 125 42.7 125 14.6 63.18 0.019 NP (60μg) 73.1 14.6 25 25 25 32.54 0.006

Example 25: siRNAs Targeting Influenza Virus Transcripts Permit Mismatches in Target Regions

This example demonstrates that siRNAs whose antisense strand is less than 100% complementary to the targeted transcript within the region of repression (eg, within the 19 base pair region complementary to the target transcript) mediate potent repression. The results demonstrate that the RNAi agents described herein will effectively inhibit a broad range of influenza strains whose sequences differ within the target portion from that of PR8.

Materials and methods

The ability of siRNAs to inhibit expression of influenza genes that are not 100% complementary to the antisense strand of the siRNA within the 19 nucleotide inhibition region was assessed using a dual luciferase assay as described in Example 18. Mismatches resulting from the alignment of human and avian influenza strains (using PR8 as a standard) were introduced into the DNA vector (psiCHECK) using a site-directed mutagenesis kit (Stratagene), that is, modifications of the influenza target sites To include 1 or 2 differences relative to the PR8 sequence, a particular difference corresponds to a difference seen in one or more of the human or avian influenza strains listed in Table 15.

Table 29 shows the results of experiments demonstrating that changes in the viral NP target (the target of NP-1496) did not substantially reduce RNAi activity. (Data shown are average of triplicate). Mismatches at positions near the 5' or 3' end of the antisense strand or near the middle were tested.

Table 29: Effect of mismatches between antisense strand and target region on NP-1496 inhibition

original A3 to G3 T9 to C9 C12 to T12 C15 to T15 A18 to G18 Renilla luciferase inhibition (%) 85.6 81.8 58.3 67.8 72.9 54.7 Residual inhibition compared to original (%) 100 91.3 65.1 75.7 81.4 61.1

Table 30 shows the results of experiments demonstrating that changes in viral PA targets (targets of PA-2087 or PA-8242) did not substantially reduce RNAi activity. (Data shown are average of triplicate). However, the G18 to A18 mutation seen in seven of the 157 human influenza strains substantially affected RNAi activity. (Data shown are average of triplicate). Mismatches at positions near the 5' or 3' end of the antisense strand or near the middle were tested. The presence of 2 mismatches between the inhibitory region of the antisense strand and the target reduced the inhibition by approximately 70-75%, but a significant degree of inhibition was still observed.

Table 30: Effect of mismatches between antisense strand and target region on PA-2087 or PA-8242 inhibition

original A4 to G4 T6 to A6 T6 to C6 C15 to T15 G18 to A18 A19 to G19 T6 to C6 and C15 to T15 Renilla luciferase inhibition (%) 91.7 80.8 75.9 88.8 87.5 7.0 89.3 26.8 Residual inhibition compared to original (%) 100 88.1 82.8 96.9 95.5 7.6 97.4 29.3

Table 31 shows the results of experiments demonstrating that changes in the viral PB2 target (the target of PB2-3817) did not substantially reduce RNAi activity. (Data shown are average of triplicate).

Table 31: Effect of mismatches between antisense strand and target region on inhibition by PB2-3817

original A17 to G17 A18 to T18 Renilla luciferase inhibition (%) 86.7 73.4 75.8

Residual inhibition compared to original (%) 100 100 87.4

Table 32A shows the results of experiments demonstrating that changes in the viral PB1 target (the target of PB1-6124) did not substantially reduce RNAi activity. (Data shown are average of triplicate). Mismatches at positions near the 5' or 3' end of the antisense strand or near the middle were tested. The presence of 2 mismatches between the inhibitory region of the antisense strand and the target reduced the inhibition by about 70-75%, but a significant degree of inhibition was still observed.

Table 32A: Effect of mismatches between antisense strand and target region on inhibition by PB1-6124

original A1 to T1 A5 to G5 T8 to C8 T12 to C12 T8 to C8 and C15 to T15 Renilla luciferase inhibition (%) 82.2 83.9 77.8 63.3 83.2 26.5 Residual inhibition compared to original (%) 100 100 94.7 77 100 32.2

Table 32B shows the results of another experiment demonstrating that changes in the viral PB1 target (the target of PB1-6124) did not substantially reduce RNAi activity. (Data shown are average of triplicate). Mismatches at positions near the 5' or 3' end of the antisense strand were tested. The presence of 2 mismatches between the inhibitory region of the antisense strand and the target does not substantially reduce RNAi activity.

Table 32B: Effect of mismatches between antisense strand and target region on inhibition by PB1-6124

PB1 (Lab ID# ^/ 6124siRNA) original CI5 to T15 A1 to G1 T2 to C2 G3 to A3 A1 to T1 and G3 to A3 Renilla luciferase inhibition (%) Residual inhibition compared to original (%) 84.1100 71.885.4 82.898.5 84.5100 74.989.1 73.987.9

Table 32C shows the results of another experiment demonstrating that changes in the viral PB1 target (the target of PB1-6129) did not substantially reduce RNAi activity. (Data shown are average of triplicate). Mismatches at positions near the 5' or 3' end of the antisense strand or near the middle were tested. A mismatch at position 10 (G:U wobble), which is demonstrated herein to not substantially reduce RNAi activity, has only a relatively small effect on inhibition. The presence of 2 mismatches between the inhibitory region of the antisense strand and the target moderately reduced inhibition, but approximately 60-70% of the original inhibition was still observed.

Table 32C: Effect of mismatches between antisense strand and target region on inhibition by PB1-6129

PB1 (Lab ID# original 6129 siRNA) T3 to C3 T7 to C7 T2 to C2 C10 to T10 T3 to C3 and C10 to T10

Renilla luciferase inhibition 86.4 (%) compared with the original remaining 100 inhibition (%) 87.3100 84.497.8 84.5100 81.394.2 59.068.3

Example 26: Modified siRNA mediates potent inhibition

To explore the inhibitory potential of siRNAs containing modified nucleotides, NP-1496 siRNAs containing sense and antisense strands with 2′-O-methyl modifications at alternating ribonucleotides in each strand were synthesized , and compared with the unmodified NP-1496siRNA test. The 2′-O-methyl modified NP1496 siRNA sequence is as follows: (2′-O-methyl is shown as an “m” in front of the modified nucleotide):

Sense: 5′-GmGA mUCmU UmAU mUUmC UmUC mGGmA G dTdT-3′ (SEQ ID NO: 381)

Antisense: 5′-mCUmC CmGA mAGmA AmAU mAAmG AmUC mC dTdT-3′ (SEQ ID NO: 382)

2'-O-methyl-modified NP1496 siRNA and unmodified NP1496 siRNA were transfected into Vero cells in 24-well plates using lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Six hours after transfection, the medium was aspirated. Cells were inoculated with 200 μl of PR8 at an MOI of 0.1. Culture supernatants were collected 24, 36 and 48 hours after infection. Virus titers were determined as described above. 2'-O-methyl modified NP1496 showed slightly greater inhibition of viral growth than unmodified NP1496. The results are shown in Table 33.

Table 33: Effective inhibition of influenza virus production using modified siRNA

HA unit 24 hours 36h 48 hours No siRNA control 4 8 16 Unmodified NP1496 (400μM) 1 2 8 Modified NP1496 (100μM) 1 2 8 Modified NP1496 (200μM) 1 2 4 Modified NP1496 (400μM) 1 1 4

Example 27: Summary of Screening and Testing for Identifying Highly Effective siRNAs

The results of the above-described screens and in vitro and in vivo testing were collected and combined to generate a comprehensive list of influenza virus sequences that were highly conserved and were targets of highly effective siRNAs. A list of target moieties is presented in Table 34. These sequences are also the complement of the inhibitory region of the antisense strand of certain highly potent RNAi inducers such as siRNA.

Table 34: Highly conserved influenza virus sequences that are targets of highly potent RNAi-inducing entities

sequence SEO ID NO: target gene Laboratory ID NO: GCCACTGAAATCAGAGCAT 272 NP 109 TCAGAGCATCCGTCGGAAA 273 NP 119 GGACGATTTCTACATCCAAA 274 NP 154 CAGCTTAACAATAGAGAGA 275 NP 222 GCTTAACAATAGAGAGAAT 276 NP 224 AATAGAGAGAATGGTGCTC 277 NP 231 GGGAAAGATCCTAAGAAAA 278 NP 301 GGAAAGATCCTAAGAAAAC 279 NP 302 TGAGAGAACTCATCCTTTA 280 NP 359 TTATGACAAAGAAGAAATA 281 NP 375 ACAAGAATTGCTTATGAAA 282 NP 688 GAATTGCTTATGAAAGAAT 283 NP 692 AAGCAATGATGGATCAAGT 284 NP 752 GCAATGATGGATCAAGTGA 285 NP 754 TGATGGATCAAGTGAGAGA 286 NP 758 CCACTAGAGGAGTTCAAAT 287 NP 1121 CACTAGAGGAGTTCAAATT 288 NP 1122 GAGGAAACACCAATCAACA 289 NP 1223 GGAAACACCAATCAACAGA 290 NP 1225 GGGCCAAATCAGCATACAA 291 NP 1254 CAACCATTATGGCAGCATT 292 NP 1313 CCATTATGGCAGCATTCAA 293 NP 1316 AGGATGATGGAAAGTGCAA 294 NP 1381 GATGATGGAAAGTGCAAGA 295 NP 1383 GAGTAATGAAGGATCTTAT 296 NP 1488 GGATCTTATTTCTTCGGAG 297 NP 1498 GATCTTATTTCTTCGGAGA 298 NP 1499 TCTTATTTCTTCGGAGACA 299 NP 1501 GGAGTACGACAATTAAAGA 300 NP 1527 GAACTAAGAAATCTAATGT 301 PB2 1590 TGAAATGGATGATGGCAAT 302 PB2 1714 GGAACATGCTGGGAACAGA 303 PB2 2283 GAATGATGATGTTGATCAA 304 PB2 2327 AATGGAATTTGAACCATTT 305 PB2 3276 ATGGAATTTGAACCATTTC 306 PB2 3277 GCACTAAGCATCAATGAAC 307 PB2 3693 GCATCAATGAACTGAGCAA 308 PB2 3700 GGAGACGTGGTGTTGGTAA 379 PB2 3759 CGGGACTCTAGCATACTTA 309 PB2 3789 ACTGACAGCCAGACAGCGA 310 PB2 3807 AGACAGCGACCAAAAGAAT 311 PB2 3817 GAATTCGGATGGCCATCAA 312 PB2 3832 GCAGGCAAACCATTTGAAT 313 PB1 3878 AGGCAAACCATTTGAATGG 314 PB1 3880 GCAAACCATTTGAATGGAT 315 PB1 3882 CCATTTGAATGGATGTCAA 316 PB1 3887 CAGGATACACCATGGATAC 380 PB1 4001 GACAATGAACCAAGTGGTT 317 PB1 4120

AAGCAATGGCTTTCCTTGA 318 PB1 4163 ACCTATGACTGGACTCTAA 319 PB1 4276 ACTGGACTCTAAATAGAAA 320 PB1 4283 CTCCAATAATGTTCTCAAA 321 PB1 4913 CCAATAATGTTTCCAAACA 322 PB1 4915 GAAACTTAGAACTCAAATA 323 PB1 4980 GCATCGATTTGAAATATTTT 324 PB1 5018 ACATTTGAATTCACAAGTT 325 PB1 5359 CATTTGAATTCACAAGTTTT 326 PB1 5360 GGACATGAGTATTGGAGTT 327 PB1 5457 CATGAGTATTGGAGTTACT 328 PB1 5460 ATGCCATAGAGGTGACACA 329 PB1 5574 CATAGAGGTGACACACAAA 330 PB1 5578 TAGAGGTGACACACAAATA 331 PB1 5580 GGTGACACACAAATACAAA 332 PB1 5584 CCAAATTTATACAACATTA 333 PB1 5680 CCACTGAACCCATTTGTCA 334 PB1 5773 AGCCATAAAGAAATTGAAT 335 PB1 5791 CCATAAAGAAATTGAATCA 336 PB1 5793 ATAAAGAAAATTGAATCAAT 337 PB1 5795 AGAAATCGATCCATCTTGA 338 PB1 5902 TTGAAGATGAACAAATGTA 339 PB1 5942 AAGATGAACAAATGTACCA 340 PB1 5945 GATGAACAAATGTACCAAAA 341 PB1 5947 CAGCAGTTCATACAGAAGA 342 PB1 5997 TTTCGAATCTGGAAGGATA 343 PB1 6081 ATCTGGAAGGATAAAGAAA 344 PB1 6087 CTGGAAGGATAAAGAAAGA 345 PB1 6089 ATGAAGATCTGTTCCACCA 346 PB1 6124 GATCTGTTCCACCATTGAA 347 PB1 6129 ACCTGAAAATCGAAACAAA 348 PA 6351 GCACAGATTTGAAATAATC 349 PA 6436 GAATAGATTCATCGAAATT 350 PA 6559 ACTACACTCTCGATGAAGA 351 PA 6696 AAACCAGACTATTCACCAT 352 PA 6732 TCACCATAAGACAAGAAAT 353 PA 6744 GGAATAAATCCAAATTATC 354 PA 7202 CTAGCAAGTTGGATTCAGA 355 PA 7424 CCAATTGAACACATTGCAA 356 PA 7517 GCCACAGAATACATAATGA 357 PA 7580 CCACAGAATACATA ATGAA 358 PA 7581 GGATGATTTCCAATTAATT 359 PA 7648 GGAAGATCCCACTTAAGGA 360 PA 7736 ACCCAAGACTTGAACCACA 361 PA 7803 AAGACTTGAACCACATAAA 362 PA 7807 CTCCACAACTAGAAGGATT 362 PA 8172 GGCTATATGAAGCAATTGA 363 PA 8271 GCAATTGAGGAGTGCCTGA 364 PA 8282 ATTGAGGAGTGCCTGATTA 365 PA 8285

TTGAGGAGTGCCTGATTAA 366 PA 8286 AGGAGTGCCTGATTAATGA 367 PA 8289 GATTAATGATCCCTGGGTT 368 PA 8299 ATTAATGATCCCTGGGTTT 369 PA 8300 TGATCCCTGGGTTTTGCTT 370 PA 8305 CCGAGGTCGAAACGTACGT 371 m 8447 ACCAATCCTGTCACCTCTG 372 m 8580 CAGTGAGCGAGGACTGCAG 373 m 8640 GACGCTTTGTCCAAAATGC 374 m 8663 TGGCTGGATCGAGTGAGCA 375 m 9008 TGTGGATTCTTGATCGTCT 376 m 9240 GTCTATGAGGGAAGAATAT 377 m 9331 GTCAGGCTAGGCAAATGGT 378 m M645

egalitarianism

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not limited by the above description, but is defined by the appended claims.

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Claims (105)

1. An RNAi-inducing agent targeting influenza virus transcripts, wherein the RNAi-inducing agent comprises: a nucleic acid portion whose sequence comprises a sequence selected from the group consisting of SEQ ID NO: 272-380, its complement, or Any fragment having a length of at least 15 nucleotides. 2. The RNAi inducing agent according to claim 1, wherein said sequence is selected from the group consisting of the following sequences: SEQ ID NO: 274, 286, 287, 292, 297, 298, 304, 305, 309, 310, 311, 319, 324, 327, 334, 346, 347, 360, 361, 364, and 366, their complements, or any fragment having a length of at least 15 nucleotides. 3. The RNAi inducing agent according to claim 1, wherein said sequence is selected from the group consisting of the following sequences: SEQ ID NO: 297, 309, 310, 311, 346, 347, 364 and 366, its complement, Or any fragment having a length of at least 15 nucleotides. 4. The RNAi-inducing agent according to claim 1, which is an siRNA comprising individual nucleic acid strands, one or both of which comprise a single-stranded overhang located 3' thereto. 5. The RNAi-inducing agent according to claim 1, which is an shRNA comprising first and second nucleic acid parts of a single nucleic acid forming a duplex, wherein the first and second nucleic acid parts are linked by a single-stranded nucleic acid part together, and wherein the duplex additionally comprises a single-stranded overhang. 6. An RNAi-inducing agent which is identical to the RNAi-inducing agent except that the sequence of the nucleic acid portion differs at 1 or 2 positions from the nucleic acid portion of the RNAi-inducing agent according to claim 1. 7. A vector comprising a template for transcription of the RNAi-inducing agent according to claim 1, wherein the template is operably linked to a promoter. 8. A method for treating or preventing influenza virus infection, comprising administering the RNAi inducer according to claim 7 to a subject in need. 9. A composition comprising the RNAi inducer according to claim 1 and a delivery agent. 10. The composition of claim 9, wherein the delivery agent is a cationic polymer. 11. The composition of claim 10, wherein the cationic polymer is selected from the group consisting of PEI, PLL, PLA, and poly(beta)urethane. 12. A method for treating or preventing influenza virus infection, comprising administering the RNAi inducer according to claim 1 to a subject in need. 13. A method for diagnosing influenza, comprising the steps of: Determining whether a subject is infected with an influenza virus sensitive to the inhibitory effect of the RNAi inducer according to claim 1. 14. The method of claim 13, further comprising the step of administering the RNAi-inducing agent to the subject. 15. An RNAi inducer targeting an influenza virus gene selected from the group consisting of: polymerase protein PB1 gene, polymerase protein PB2 gene, polymerase protein PA gene, and nucleoprotein NP gene. 16. The RNAi inducer according to claim 15, wherein the gene is PB1. 17. The RNAi inducer according to claim 15, wherein the gene is PB2. 18. The RNAi inducer according to claim 15, wherein the gene is PA. 19. The RNAi inducer according to claim 15, wherein the gene is NP. 20. The RNAi-inducing agent of claim 15, wherein the agent targets a target portion of a gene that is a functionally preferred target portion of RNAi. 21. The RNAi-inducing agent of claim 15, wherein the agent targets an appropriate conserved target portion of the gene. 22. The RNAi-inducing agent of claim 15, wherein the agent targets a highly conserved target portion of the gene. 23. The RNAi-inducing agent according to claim 15, wherein the RNAi-inducing agent comprises: A nucleic acid portion that is 100% complementary over at least 15 contiguous nucleotides to a target portion of said influenza virus gene, wherein said target portion is a functionally preferred target of RNA. 24. An RNAi-inducing agent which is the same as the RNAi-inducing agent except that the nucleic acid portion differs in 1 or 2 positions from that of the RNAi-inducing agent according to claim 23. 25. A vector comprising a template for transcription of the RNAi-inducing agent according to claim 15. 26. An isolated nucleic acid or its complement, its sequence comprising a sequence selected from the group consisting of SEQ ID NO: 272-380, or comprising a sequence selected from the group consisting of SEQ ID NO: 272-380 has at least 16 nucleotides in length, wherein the nucleic acid has a length of 100 nucleotides or less. 27. The nucleic acid according to claim 26, wherein said sequence comprises a sequence selected from the group consisting of SEQ ID NO: 274, 286, 287, 292, 297, 298, 304, 305, 309, 310, 311, 319, 324, 327, 334, 346, 347, 360, 361, 364 and 366 consisting of a sequence of the group, or comprising a sequence selected from the group consisting of SEQ ID NO: 274, 286, 287, 292, 297, 298, 304, 305, 310, 311, 319, Sequences of the group consisting of 324, 327, 334, 346, 347, 360, 361, 364 and 366 have fragments of at least 16 nucleotides in length, wherein the nucleic acid has 100 nucleotides or less acid length. 28. The nucleic acid according to claim 26, wherein said sequence comprises a sequence selected from the group consisting of SEQ ID NO:297,309,310,311,346,347,364 and 366, or comprises a sequence selected from the group consisting of SEQ ID NO:297,309,310,311,346,347,364 and 366 NO: The sequence of the group consisting of 297, 310, 311, 346, 347, 364 and 366 has a fragment of at least 16 nucleotides in length, wherein the nucleic acid has a fragment of 100 nucleotides or less length. 29. The nucleic acid according to claim 26, wherein said sequence comprises a sequence selected from the group consisting of SEQ ID NO: 272-380. 30. The isolated nucleic acid of claim 26, further comprising a detectable moiety attached to the nucleic acid. 31. An RNAi-inducing agent comprising the nucleic acid according to claim 26. 32. A vector comprising a template for transcription of the RNAi-inducing agent according to claim 26. 33. A sequence with An isolated nucleic acid of n1-n2-n3-n4-n5-n6-n7-n8-n9-n10-n11-n12-n13-n14-n15-n16-n17-n18-n19 (N19), said sequence being selected from the group consisting of The group consisting of the following genes share a portion of the influenza virus genes: polymerase protein PB1 gene, polymerase protein PB2 gene, polymerase protein PA gene, and nucleoprotein NP gene, or differ by: Allow A to G or C to U differences in any position; Only differences from G to Yes or C to Yes are allowed at positions n1, n18, and/or there are 0, 1, 2 or 3 differences between N19 and said influenza virus, There are no more than 2 consecutive differences; and There is at most 1 difference between N19 and the influenza virus. 34. An RNAi-inducing agent targeting the nucleic acid of claim 33. 35. An RNAi inducing agent comprising the nucleic acid according to claim 33. 36. A vector comprising a template for transcription of the RNAi-inducing agent according to claim 33. 37. A method of diagnosing influenza in a subject comprising the steps of: Determining whether the subject is infected with an influenza virus strain susceptible to inhibition by the RNAi-inducing entity. 38. The method of claim 37, further comprising the steps of: The RNAi-inducing entity is administered to the subject. 39. The method of claim 37, wherein the determining step comprises performing a nucleic acid-based diagnostic assay. 40. A diagnostic kit comprising: A primer or probe that detects at least part of a target portion of an influenza virus gene that is an appropriate conserved target portion for RNAi. 41. The diagnostic kit of claim 40, wherein the appropriate conserved target moiety comprises a sequence selected from the group consisting of SEQ ID NO: 272-380. 42. A method of inhibiting gene expression in a tissue or organ of a mammalian subject comprising the steps of: A composition comprising an effective amount of an RNAi-inducing agent targeted to the gene is introduced directly into the subject's vasculature without using hydrodynamic transfection techniques. 43. The method of claim 42, wherein the organ is the lung. 44. The method of claim 42, wherein the composition comprises a cationic polymer. 45. The method of claim 42, wherein the gene is a gene of a virus that infects respiratory epithelial cells. 46. The method of claim 45, wherein the virus is influenza virus. 47. The method of claim 42, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of subject body weight. 48. The method of claim 42, wherein the effective amount reduces expression of the gene by at least 2-fold. 49. A method of inhibiting the production of a virus in the respiratory system of a mammalian subject, wherein said virus infects respiratory epithelial cells, said method comprising the steps of: A composition comprising an effective amount of an RNAi-inducing agent targeting the viral gene is introduced into the subject's vasculature by injection without using hydrodynamic transfection techniques. 50. The method of claim 49, wherein the composition comprises a cationic polymer. 51. The method of claim 49, wherein the virus is influenza virus. 52. The method of claim 49, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of subject body weight. 53. The method of claim 49, wherein the composition is administered to the subject prior to infection of a subject not infected with the virus, and wherein when the subject is subsequently infected, The composition effectively inhibits the production of the virus. 54. A method of inhibiting gene expression in the lung of a mammalian subject comprising the steps of: A composition comprising an effective amount of an RNAi-inducing agent targeting the gene and a delivery agent is introduced directly into the respiratory system of the subject. 55. The method of claim 54, wherein the composition is administered intranasally. 56. The method of claim 54, wherein the composition is administered by inhalation. 57. The method of claim 54, wherein the composition comprises a cationic polymer. 58. The method of claim 54, wherein the gene is a gene of a virus that infects respiratory epithelial cells. 59. The method of claim 58, wherein the virus is influenza virus. 60. The method of claim 54, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of subject body weight. 61. The method of claim 54, wherein the effective amount reduces expression of the gene by at least 2-fold. 62. The method of claim 54, wherein the composition is substantially free of delivery enhancing polymers and lipids. 63. A method of inhibiting the production of a virus in the respiratory system of a mammalian subject, wherein said virus infects respiratory epithelial cells, said method comprising the steps of: A composition comprising an effective amount of an RNAi-inducing agent targeted to the viral gene is introduced directly into the respiratory system of the subject. 64. The method of claim 63, wherein the virus is influenza virus. 65. The method of claim 63, wherein the composition is administered intranasally. 66. The method of claim 63, wherein the composition is administered by inhalation. 67. The method of claim 63, wherein the composition comprises a cationic polymer. 68. The method of claim 63, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of subject body weight. 69. The method of claim 63, wherein the effective amount reduces production of the virus by at least 25%. 70. The method of claim 63, wherein the composition is substantially free of delivery enhancing polymers and lipids. 71. The method of claim 63, wherein the composition is administered to the subject prior to infection of a subject not infected with the virus, and wherein when the subject is subsequently infected, The composition effectively inhibits the production of the virus. 72. A transgenic non-human animal expressing an RNAi-inducing agent targeting an influenza virus transcript. 73. The transgenic non-human animal of claim 72, wherein said animal is an avian. 74. A use of an RNAi-inducing agent for the manufacture of a medicament for treating or preventing influenza virus infection, comprising administering the RNAi-inducing agent to a subject in need, wherein the RNAi-inducing agent comprises: a nucleic acid moiety, Its sequence comprises a sequence selected from the group consisting of SEQ ID NO: 272-380, its complement, or any fragment having a length of at least 15 nucleotides, and wherein said template is operably linked to a promoter. 75. A use of an RNAi-inducing agent for the manufacture of a medicament for treating or preventing influenza virus infection, comprising administering the RNAi-inducing agent to a subject in need, wherein the RNAi-inducing agent comprises: a nucleic acid moiety, Its sequence comprises a sequence selected from the group consisting of SEQ ID NO: 272-380, its complement, or any fragment having a length of at least 15 nucleotides. 76. Use of an RNAi inducing agent for the manufacture of a medicament for inhibiting gene expression in a tissue or organ of a mammalian subject, comprising an effective amount of an RNAi targeting the gene without using a hydrodynamic transfection technique The step of introducing the composition of RNAi-inducing agent directly into the vasculature of said subject. 77. The use of the RNAi inducer according to claim 76, wherein the organ is the lung. 78. The use of an RNAi-inducing agent according to claim 76, wherein the composition comprises a cationic polymer. 79. The use of the RNAi inducer according to claim 76, wherein the gene is a viral gene that infects respiratory epithelial cells. 80. The use of the RNAi inducer according to claim 76, wherein the virus is influenza virus. 81. The use of the RNAi-inducing agent according to claim 76, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of body weight of the subject. 82. The use of the RNAi inducer according to claim 76, wherein the effective amount reduces the expression of the gene by at least 2-fold. 83. Use of an RNAi-inducing agent for the manufacture of a medicament for inhibiting the production of a virus in the respiratory system of a mammalian subject, wherein the virus infects respiratory epithelial cells, the use comprising passing Injecting a step of introducing into the subject's vasculature a composition comprising an effective amount of an RNAi-inducing agent targeting the viral gene. 84. The use of an RNAi-inducing agent according to claim 83, wherein the composition comprises a cationic polymer. 85. The use of the RNAi inducer according to claim 83, wherein the virus is influenza virus. 86. The use of the RNAi-inducing agent according to claim 83, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of body weight of the subject. 87. The use of the RNAi-inducing agent according to claim 83, wherein the composition is administered to the subject before infection of a subject not infected with the virus, and wherein when the subject is subsequently When infected, the composition is effective to inhibit the production of the virus. 88. Use of an RNAi inducer for the manufacture of a medicament for inhibiting gene expression in the lungs of a mammalian subject, comprising directly directing a composition comprising an effective amount of an RNAi inducer targeting said gene and a delivery agent A step is introduced into the respiratory system of the subject. 89. The use of the RNAi inducer according to claim 88, wherein the composition is administered intranasally. 90. The use of the RNAi inducer of claim 88, wherein the composition is administered by inhalation. 91. The use of an RNAi-inducing agent according to claim 88, wherein the composition comprises a cationic polymer. 92. The use of the RNAi inducer according to claim 88, wherein the gene is a gene of a virus that infects respiratory epithelial cells. 93. The use of the RNAi inducer according to claim 92, wherein the virus is influenza virus. 94. The use of the RNAi-inducing agent according to claim 88, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of body weight of the subject. 95. The use of the RNAi inducer according to claim 88, wherein the effective amount reduces the expression of the gene by at least 2-fold. 96. The use of an RNAi-inducing agent according to claim 88, wherein the composition is substantially free of delivery-enhancing polymers and lipids. 97. A use of an RNAi inducer for the manufacture of a medicament for inhibiting the production of a virus in the respiratory system of a mammalian subject, wherein the virus infects respiratory epithelial cells, the use comprising comprising an effective amount of the targeted The step of introducing the composition of the RNAi-inducing agent of the viral gene directly into the respiratory system of the subject. 98. The use of the RNAi inducer according to claim 97, wherein the virus is influenza virus. 99. The use of the RNAi inducer according to claim 97, wherein the composition is administered intranasally. 100. The use of the RNAi inducer of claim 97, wherein the composition is administered by inhalation. 101. The use of claim 97, wherein the composition comprises a cationic polymer. 102. The use according to claim 97, wherein the effective amount is between about 0.1 mg and 5 mg per kilogram of subject body weight. 103. The use according to claim 97, wherein said effective amount reduces said virus production by at least 25%. 104. The use according to claim 97, wherein the composition is substantially free of delivery enhancing polymers and lipids. 105. The use of claim 97, wherein the composition is administered to the subject prior to infection of a subject not infected with the virus, and wherein when the subject is subsequently infected, The composition effectively inhibits the production of the virus.

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