WO2011123945A1 - Compostions, methods and uses for treating respiratory syncytial virus infection - Google Patents

Abstract

Provided herein is a method of treating Respiratory Syncytial Virus (RSV) infection in a cell. The method involves administering a nucleolin peptide, a nucleotide antibody, or a nucleolin RNAi to the cell.

Description

COMPOSTIONS, METHODS AND USES FOR TREATING RESPIRATORY

SYNCYTIAL VIRUS INFECTION

RELATED APPLICATIONS

[0001] This application claims priority to United States provisional patent applications 61/321,382 filed 6 April 2010 and 61/419,147 filed 2 December 2010.

TECHNICAL FIELD

[0002] The invention is in the field of treatment of Respiratory Syncytial Virus (RSV) infection, and particularly methods, uses, and compositions for treating RSV infection.

BACKGROUND

[0003] Human respiratory syncytial virus (RSV) is an enveloped, single-stranded, negative-polarity RNA Pneumovirus of the family Paramyxoviridae. It is a common cause of respiratory tract infections worldwide, including bronchiolitis and other serious illnesses (see, for e.g., Collins and Graham, 2008; Wu et al, 2008). Control and prevention of RSV infection is a global health priority; almost all children are infected with RSV during the first two years of life (see, for e.g., Domachowske and Rosenberg, 1999). In the United States alone, over 2 million RSV-infected infants require medical attention annually (Hall et al, 2009). Infants hospitalized for RSV are also at risk for developing recurrent wheezing and asthma (Escobar et al, 2010).

[0004] Polarized, ciliated respiratory epithelial cells are a major target for RSV infection in vivo (see, for e.g., Johnson et al, 2007). RSV infection of ciliated respiratory epithelial cells in vitro occurs on the apical (luminal) aspect (see, for e.g., Zhang et al, 2002). Viral replication in a host cell initiates with attachment of the virus to the plasma membrane via receptor-mediated binding (see, for e.g., Marsh and Helenius, 2006). Candidate RSV receptors have been proposed (see, for e.g., Krusat and Streckert, 1997; Behera et al, 2001 ; and, Malhotra et al, 2003). Laboratory-adapted strains of RSV show increased efficiency of infection by binding to cell surface glycosaminoglycans (GAGs); however, cells deficient in GAGs are permissive to infection (see, for e.g., Feldman et al, 2000) and the role of GAGs in cellular infection by wild-type, community isolates of RSV is unclear (see, for e.g., Hallak et al., 2007).

[0005] There is no efficacious treatment or vaccine available, and passive prophylactic treatment using anti-RSV antibodies (e.g., palivizumab) is expensive, is not 100% effective, and is limited to those at high risk for severe infection (Wu et al, 2008).

[0006] The RSV envelope contains three proteins: small hydrophobic (SH), glycoprotein (G) and fusion (F) (Collins and Graham, 2008). SH protein is not required for virus binding (see, for e.g., Techaarpornkul et al, 2001). RSV G, the heterogeneity of which characterizes RSV subtypes A and B, binds to cell surface glycosaminoglycans (GAGs) at high affinity (see, for e.g., Hallak et al, 2007) but is not an absolute requirement for infection, since mutant RSV deficient in G glycoprotein (RSV AG) remains infectious (see, for e.g., Techaarpornkul et al, 2002). Moreover, cells deficient in cell surface GAGs or with chemically modified GAGs are permissive to RSV, albeit at lower levels than cells expressing abundant GAGs (see, for e.g., Techaarpornkul et al, 2002; and Hallak et al, 2000).

[0007] Nucleolin is a ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth (see, for e.g., Tuteja et al, 1998; and Chen et al, 2008). Nucleolin has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Studies have demonstrated that surface nucleolin may serve as a receptor for various extracellular ligands, for instance those implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis. Nisole et al. (1999), US20040002457A1, and US20020076693A1 disclose that nucleolin is involved in binding of HIV virus to host cells.

SUMMARY

[0008] The present invention is based in part on the discovery that nucleolin plays a role in RSV infection, and that the administration of nucleolin peptide, nucleolin antibody, or nucleolin RNAi to a cell may be useful in the treatment of RSV infection. Such a treatment may result from interference with the interaction between RSV protein F and a cells endogenous nucleolin.

[0009] Furthermore, the present invention is also based in part on the discovery that Sf9 cells are not permissive to RSV infection. This discovery is utilized to develop an assay system to test the infectivity of Sf9 cells based on the structure of the nucleolin being expressed by the Sf9 cell.

[0010] In a first aspect, a method is provided for of treating Respiratory Syncytial Virus (RSV) infection in a cell, the method including administering a nucleolin peptide, a nucleolin antibody, or a nucleolin interference RNA (RNAi) to the cell.

[0011] In a further aspect, there is provided a RNAi molecule having a sequence of SEQ ID NO:2 or SEQ ID NO:5.

[0012] In a further aspect, there is provided a use of a RNAi having a sequence of SEQ ID NO:2 or SEQ ID NO: 5 for the treatment of RSV infection.

[0013] In a further aspect, there is provided a use of a RNAi having a sequence of SEQ ID NO:2 or SEQ ID NO:5 in the preparation of a medicament for the treatment of RSV infection.

[0014] In a further aspect, there is provided a Sf9 cell line expressing a heterologous nucleolin.

[0015] In a further aspect, there is provided a Sf9 cell line expressing a heterologous nucleolin mutant.

[0016] In a further aspect, there is provided a Sf9 cell line expressing a nucleolin mutant for use in screening nucleolin mutations associated with RSV infectivity. [0017] In a further aspect, there is provided a method for screening nucleolin mutations associated with RSV infection, the method comprising: (a) expressing a nucleolin mutant in a Sf9 cell; (b) exposing the cell derived from step (a) with RSV; and (c) determining whether the cell exposed in step (b) becomes infected with RSV. The RSV infection of the Sf9 cells may be compared to infection of Sf9 cells expressing wildtype nucleolin.

[0018] In a further aspect, there is provided a method for screening nucleolin mutations associated with RSV infection, the method comprising: (a) exposing a Sf9 cell, that is expressing a nucleolin mutant, with RSV; and (b) determining whether the cell exposed in step (a) becomes infected with RSV. The RSV infection of the Sf9 cells may be compared to infection of Sf9 cells expressing wildtype nucleolin.

[0019] In a further aspect, there is provided a commercial package, comprising: (a) Sf9 cells expressing a heterologous nucleolin; and (b) RSV.

[0020] In a further aspect, there is provided a vector including a DNA template which encodes an RNA which is homologous to a nucleolin gene and is capable of promoting RNA interference of said nucleolin gene.

[0021] In a further aspect, there is provided a siRNA molecule, wherein the siRNA molecule comprises a sense region and an antisense region, wherein said sense region and said antisense region together form a duplex region comprising 19-30 base pairs and said antisense region comprises a sequence that is the complement of SEQ ID NO:2 for use in the treatment of RSV infection.

[0022] In a further aspect, there is provided a Sf9 cell line expressing heterologous nucleolin is provided in which the Sf9 cell line is permissive to RSV infection.

[0023] In another aspect, there is provided a Sf9 cell line expressing a nucleolin mutant. The Sf9 cell line expressing a nucleolin mutant may be used in screening nucleolin mutations associated with RSV infectivity. [0024] In further aspect, there is provided a method for screening nucleolin mutations associated with RSV infection, the method may include expressing a nucleolin mutant in a Sf9 cell; exposing the cell expressing the nucleolin mutant with RSV; and determining whether the infected cell is infected with RSV. Optionally, RSV infection of the Sf9 cells is compared to infection of Sf9 cells expressing wildtype nucleolin.

[0025] The RNAi may be an siRNA comprising SEQ ID NO: 2 or SEQ ID NO: 5. The RNAi may be an siRNA molecule and the siRNA molecule may include a sense region and an antisense region, wherein the sense region and the antisense region together form a duplex region comprising 19-30 base pairs and the antisense region includes a sequence that is the complement of SEQ ID NO:2 or SEQ ID NO:5. The antisense region may include a sequence that is the complement of SEQ ID NO:2. The antisense region and said sense region may each be 19-29 nucleotides in length. The antisense region and said sense region may each be 19-28 nucleotides in length. The antisense region and said sense region may each be 19-27 nucleotides in length. The antisense region and said sense region may each be 19-26 nucleotides in length. The antisense region and said sense region may each be 19-25 nucleotides in length. The antisense region and said sense region may each be 19-24 nucleotides in length. The antisense region and said sense region may each be 19-23 nucleotides in length. The antisense region and said sense region may each be 19-22 nucleotides in length. The antisense region and said sense region may each be 19-21 nucleotides in length. The antisense region and said sense region may each be 19-20 nucleotides in length. The antisense region and said sense region may each be 20-30 nucleotides in length. The antisense region and said sense region may each be 21-30 nucleotides in length. The antisense region and said sense region may each be 22-30 nucleotides in length. The antisense region and said sense region may each be 23-30 nucleotides in length. The antisense region and said sense region may each be 24-30 nucleotides in length. The antisense region and said sense region may each be 25-30 nucleotides in length. The antisense region and said sense region may each be 26-30 nucleotides in length. The antisense region and said sense region may each be 27-30 nucleotides in length. The antisense region and said sense region may each be 28-30 nucleotides in length. The antisense region and said sense region may each be 29-30 nucleotides in length. The antisense region and said sense region may each be 20-25 nucleotides in length. The antisense region and said sense region may each be 21-25 nucleotides in length. The antisense region and said sense region may each be 22-25 nucleotides in length. The antisense region and said sense region may each be 23-25 nucleotides in length. The antisense region and said sense region may each be 24-25 nucleotides in length. The antisense region and said sense region may each be 21-26 nucleotides in length. The antisense region and the sense region may each be 21 nucleotides in length. The siRNA molecule may include at least one overhang region, wherein the overhang region includes six or fewer nucleotides. The siRNA molecule may include at least one overhang region, wherein the overhang region includes five or fewer nucleotides. The siRNA molecule may include at least one overhang region, wherein the overhang region includes four or fewer nucleotides. The siRNA molecule may include at least one overhang region, wherein the overhang region includes three or fewer nucleotides. The siRNA molecule may include at least one overhang region, wherein the overhang region includes two or fewer nucleotides. The siRNA molecule may include at least one overhang region, wherein the overhang region includes one nucleotide. The siRNA molecule may have no overhang regions.

[0026] The cell may be a human cell. The human cell may be an epithelial cell. The human cell may be a mucosal cell. The human cell may be a cell of the respiratory tract. The human cell may be a ciliated respiratory epithelial cell. The cell may be in a subject having or at risk of developing an RSV infection. The RNAi molecule may be administered intravenously. The RNAi molecule may be topically administered to a mucosal membrane of the subject. The RNAi molecules may be mixed with lipid particles prior to administration. The RNAi molecules may be encapsulated in liposomes prior to administration. The siRNA molecule may be administered intravenously. The siRNA molecule may be topically administered to a mucosal membrane of the subject. The siRNA molecules may be mixed with lipid particles prior to administration. The siRNA molecules may be encapsulated in liposomes prior to administration. [0027] The nucleolin peptide may include a peptide having at least 85% sequence identity to SEQ ID NO: l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 86% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 87% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 88% sequence identity to SEQ ID NO.l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 89% sequence identity to SEQ ID NO:l , wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 90% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 91% sequence identity to SEQ ID NO: l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 92% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 93% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 94% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 95% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 96% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 97% sequence identity to SEQ ID NO: l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 98% sequence identity to SEQ ID NO: l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 99% sequence identity to SEQ ID NO: l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include a peptide having at least 100% sequence identity to SEQ ID NO: l, wherein the nucleolin peptide inhibits RSV infection. The nucleolin peptide may include SEQ ID NO:l . The nucleolin peptide may be soluble. The nucleolin peptide may be bound to a delivery or targeting moiety. The nucleolin peptide may be formulated for delivery to the respiratory tract. The nucleolin peptide may be formulated for aerosol delivery to the respiratory tract. The nucleolin peptide may be formulated for inhalation into the respiratory tract. The nucleolin peptide may be formulated as a spray for delivery to the respiratory tract. The nucleolin peptide may be formulated as a gel.

[0028] The antibody may be a monoclonal antibody. The monoclonal antibody may be a humanized monoclonal antibody. The monoclonal antibody may be a chimeric antibody. The antibody may be selected from one or more of the following: a polyclonal antibody; a monoclonal antibody; or a fragment thereof; a single chain Fc region (scFc); or an intrabody. The antibody may block the interaction between RSV protein F and nucleolin.

[0029] The treatment may be of a human subject. The treatment may be of a bovine subject. The treatment may be of an ovine subject. The treatment may be of an equine subject. The treatment may be of a porcine subject. The treatment may be of a murine subject.

[0030] The RNAi may 19-30 base pairs and may include a sequence that includes SEQ ID NO:2 or SEQ ID NO:5. The RNAi may include a sequence that is the complement of SEQ ID NO:2. The RNAi may be 19-29 nucleotides in length. The RNAi may be 19-28 nucleotides in length. The RNAi may be 19-27 nucleotides in length. The RNAi may be 19-26 nucleotides in length. The RNAi may be 19-25 nucleotides in length. The RNAi may be 19-24 nucleotides in length. The RNAi may be 19-23 nucleotides in length. The RNAi may be 19- 22 nucleotides in length. The RNAi may be 19-21 nucleotides in length. The RNAi may be 19-20 nucleotides in length. The RNAi may be 20-30 nucleotides in length. The RNAi may be 21-30 nucleotides in length. The RNAi may be 22-30 nucleotides in length. The RNAi may be 23-30 nucleotides in length. The RNAi may be 24-30 nucleotides in length. The RNAi may be 25-30 nucleotides in length. The RNAi may be 26-30 nucleotides in length. The RNAi may be 27-30 nucleotides in length. The RNAi may be 28-30 nucleotides in length. The RNAi may be 29-30 nucleotides in length. The RNAi may be 20-25 nucleotides in length. The RNAi may be 21-25 nucleotides in length. The RNAi may be 22-25 nucleotides in length. The RNAi may be 23-25 nucleotides in length. The RNAi may be 24- 25 nucleotides in length. The RNAi may be 21-26 nucleotides in length. The RNAi may be 21 nucleotides in length. The RNAi may be 21 nucleotides in length. The RNAi molecule may include at least one overhang region, wherein the overhang region includes six or fewer nucleotides. The RNAi molecule may include at least one overhang region, wherein the overhang region includes five or fewer nucleotides. The RNAi molecule may include at least one overhang region, wherein the overhang region includes four or fewer nucleotides. The RNAi molecule may include at least one overhang region, wherein the overhang region includes three or fewer nucleotides. The RNAi molecule may include at least one overhang region, wherein the overhang region includes two or fewer nucleotides. The RNAi molecule may include at least one overhang region, wherein the overhang region includes one nucleotide. The RNAi molecule may have no overhang regions.

[0031 ] The heterologous nucleolin may be human nucleolin and the RS V may be a human strain of RSV. The heterologous nucleolin may be bovine nucleolin and the RSV may be a bovine strain of RSV. The heterologous nucleolin may be ovine nucleolin and the RSV may be an ovine strain of RSV. The heterologous nucleolin may be equine nucleolin and the RSV may be an equine strain of RSV. The heterologous nucleolin may be porcine nucleolin and the RSV may be a porcine strain of RSV.

[0032] The Sf9 cell line may be permissive to RSV infection. The Sf9 cell line may not be permissive to RSV infection. The Sf9 cell line may express a nucleolin mutant.

[0033] The vector may be a lentiviral vector. The vector may be an adenoviral vector. The vector may be an adeno-associated viral vector.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1(a) is a photograph of a co-immunoprecipitation of nucleolin with F glycoprotein of RSV A2. (b) is a photograph of a co-immunoprecipitation of nucleolin with F glycoprotein of different RSV strains.

[0035] Figure 2(a) is a graph showing the percentage (%) of HEp-2 cells infected with RSV following incubation with an anti-nucleolin antibody as compared to controls, (b) is a graph showing the percentage (%) of lHAEo- cells infected with RSV in competition experiments following incubation with nucleolin as compared to controls, (c) is a photograph of nucleolin protein expression under RNAi silencing conditions as compared to control siRNAs. (d) is a graph showing the percentage (%) of cells infected with RSV following RNAi silencing as compared to controls.

[0036] Figure 3(a) is a photograph from a RSV VOPBA of proteins extracted from HEp- 2 cells and Sf9 cells and shows no -100 kDa signal for Sf9 cells (β-actin: gel loading control), (b) is a photograph of an immunoblot with anti-Ncl Ab for both Sf9 cells transfected with human nucleolin (Ncl) or pCMV-X6 empty vector (Control), (c) is a bar graph showing significantly increased percentage of RSV infected cells in Ncl-transfected Sf9 compared with control cells.

[0037] Figure 4(a) is a photograph of a cell lysate immunoblot for nucleolin (Ncl) and β- actin and relative quantity determined by densitometry. A 3.5 ± 0.8 fold knockdown in Ncl expression obtained with oligo 3 vs. 3Δ3 treatment (P = 0.05; n = 2) is shown, (b) is a graph quantifying nucleolin staining in epithelial cells over the total number of airway epithelial cells counted per stained mouse lung section, (c) is a graph quantifying the effect of siRNA knockdown of Nucleolin (Ncl) expression on lung titer.

[0038] Figure 5(a) is a graph demonstrating the effect of incubation of RSV with purified nucleolin on subsequent virus replication. Incubation of RSV with pure nucleolin resulted in decreased infection in a dose-dependent manner. This effect was not observed when AdV5 was incubated with similar concentrations of purified nucleolin (b), or when RSV was incubated with similar concentrations of purified transferrin (c).

DETAILED DESCRIPTION

[0039] Any terms not directly defined herein shall be understood to have the meanings commonly associated with them as understood within the present field of art. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions, devices, methods and the like of embodiments, and how to make or use them. It will be appreciated that the same thing may be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein. No significance is to be placed upon whether or not a term is elaborated or discussed herein. Some synonyms or substitutable methods, materials and the like are provided. Recital of one or a few synonyms or equivalents does not exclude use of other synonyms or equivalents, unless it is explicitly stated. Use of examples in the specification, including examples of terms, is for illustrative purposes only and does not limit the scope and meaning of the embodiments described herein.

[0040] A method is provided for "treating" Respiratory Syncytial Virus (RSV) infection in a cell, wherein treating is meant to encompass preventing RSV infection, ameliorating RSV infection, and eradicating RSV infection. The term "treating" as used herein is also meant to include the prophylactic administration of a compound prior to infection of RSV or in anticipation of RSV infection, the administration of a compound when there is an ongoing infection with RSV, or if the RSV is in a latent state. Those skilled in the art would appreciate that the term "preventing" would include the avoidance of infection with RSV or minimizing RSV infection once an exposure to RSV occurs. A person of skill in the art would appreciate that the term "ameliorating" is meant to include the prospect of making an infection more tolerable for a subject afflicted with an RSV infection (for example, by reducing viral load in a subject). A person of skill in the art would also appreciate that the term "eradication" with regards to RSV infection would include elimination of the RSV from a subject or removal of the infection (for example by the treatment itself or in conjunction with another treatment(s) and/or the subjects immune response). Accordingly, as used herein "treatment" may refer to the prevention of an RSV infection, the amelioration of an RSV infection, the eradication of RSV from a subject, or combinations thereof. Furthermore, a "treatment" may be prophylactic, whereby the treatment is administered prior to infection or the appearance of symptoms in a subject or in anticipation of an exposure to RSV. Similarly, "treatment" may be administered following RSV infection or the onset of RSV infection symptoms.

[0041] As used herein, the term "RSV" refers to Respiratory Syncytial Virus, which is an enveloped, single-stranded, negative-polarity RNA Pneumovirus of the family Paramyxoviridae. Human RSV strains are significant causes of bronchiolitis and other serious lower respiratory tract infections worldwide, especially in children, the elderly, and in immunocompromised adults. The term RSV may include, without limitation, respiratory syncytial viruses of the Pneumovirus genus which have specificity for other host species, such as bovine respiratory syncytial virus, ovine respiratory syncytial virus and murine pneumonia virus, which is also known in the art as PVM (see, for e.g., Bern et al, 2009). As used herein, the term "infection" refers to a state in which an infectious agent, such as a virus, and including without limitation RSV, is established within a cell or within a subject, as the case may be. In certain embodiments, the compounds may be used for treating RSV infection. Such methods and compounds are described herein.

[0042] Human RSV strains may be classified as A or B group viruses (i.e., RSV-A and RSV-B) based on MAb binding to the RSV glycoprotein (G protein) (see, for e.g., (Anderson et al, 1985; Coates et al, 1966; Hendry et al., 1986; Mufson et al, 1985) and by genetic analysis (see, for e.g., Garcia et al, 1994; Sullender et al., 1991 ; Sullender et al., 1993). The RSV G protein is thought to be the most variable of the RSV proteins (see, for e.g., Garcia- Barreno et al, 1989; Johnson et al, 1987; Mufson et al., 1985), and its C-terminal region (the second hypervariable region) accounts for strain-specific epitopes (see, for e.g., Cane, 1997; Cane, 2001 ; Cane and and Pringle, 1995; Garcia et al, 1994; Johnson et al, 1987; Peret et al, 1998; Rueda et al, 1991). By contrast the F protein of RSV is much less variable between strains and is thus a more attractive target for a broad based RSV treatment.

[0043] Nucleolin is a ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth (see, for e.g., Tuteja et al, 1998; and Chen et al, 2008). Nucleolin has also been described as a shuttling molecule between nucleus, cytosol and the cell surface. Studies have demonstrated that surface nucleolin may serve as a receptor for various extracellular ligands, for instance those implicated in cell proliferation, differentiation, adhesion, mitogenesis and angiogenesis.

[0044] The methods described herein may encompass the administration to a cell of one or more of a nucleolin peptide, a nucleolin antibody, and a nucleolin RNAi. The term "nucleolin" refers, in part, to the polypeptide expression product of the nucleolin gene, the human orthologue corresponding to EntrezGene # 4691, GenPept Protein Accession #P 19338.3. Homologous nucleolin proteins are found in other species which bear sequence similarity to human nucleolin and may thus find utility herein, including but not limited to mouse (EntrezGene # 17975; GenPept # P09405.2) and rat (EntrezGene # 25135; GenePept # P13383.3). The term "nucleolin" also refers to the nucleolin gene itself, and includes reference to the nucleolin gene as it is present in a variety of genomes including, without limitation, human, bovine, ovine, equine, porcine, and murine. The term "nucleolin" also includes gene products (e.g., mRNA, protein) produced from the underlying nucleolin gene. It will be appreciated by a person skilled in the art that the term "nucleolin peptide" qualifies the understanding of the term "nucleotide" by making specific reference to a "peptide" as it is defined herein. For example, SEQ ID NO:l provides an amino acid sequence of human nucleolin.

[0045] The term "peptide" as used herein refers to short polymers of amino acids linked by peptide bonds. Persons skilled in the art will understand that a peptide bond, which is also know in the art as an amide bond, is a covalent chemical bond formed between two molecules when the carboxyl group of one molecule reacts with the amine group of the other molecule, thereby releasing a molecule of water (H₂0).

[0046] Amino acids are molecules containing an amine group, a carboxylic acid group and a side chain that varies between different amino acids. An amino acid may be in its natural form or it may be a synthetic amino acid. An amino acid may be described as, for example, polar, non- polar, acidic, basic, aromatic or neutral. A polar amino acid is an amino acid that may interact with water by hydrogen bonding at biological or near-neutral pH. The polarity of an amino acid is an indicator of the degree of hydrogen bonding at biological or near-neutral pH. Examples of polar amino acids include serine, proline, threonine, cysteine, asparagine, glutamine, lysine, histidine, arginine, aspartate, tyrosine and glutamate. Examples of non-polar amino acids include glycine, alanine, valine leucine, isoleucine, methionine, phenylalanine, and tryptophan. Acidic amino acids have a net negative charge at a neutral pH. Examples of acidic amino acids include aspartate and glutamate. Basic amino acids have a net positive charge at a neutral pH. Examples of basic amino acids include arginine, lysine and histidine. Aromatic amino acids are generally nonpolar, and may participate in hydrophobic interactions. Examples of aromatic amino acids include phenylalanine, tyrosine and tryptophan. Tyrosine may also participate in hydrogen bonding through the hydroxyl group on the aromatic side chain. Neutral, aliphatic amino acids are generally nonpolar and hydrophobic. Examples of neutral amino acids include alanine, valine, leucine, isoleucine and methionine. An amino acid may be described by more than one descriptive category. In a "conservative" substitution amino acids sharing a common descriptive category may be substitutable for each other in a peptide without a loss of the desired function. An amino acid residue may be generally represented by a one-letter or three-letter designation, corresponding to the trivial name of the amino acid, in accordance with Table A herein. Amino acids comprising the nucleolin peptides described herein will be understood to be in the L- or D- configuration. Amino acids described herein, may be modified by methylation, amidation, acetylation or substitution with other chemical groups which may change the circulating half-life of the peptide without adversely affecting their biological activity. Additionally, a disulfide linkage may be present or absent in the nucleolin peptides described herein. Nonstandard amino acids may occur in nature, and may or may not be genetically encoded. Examples of genetically encoded nonstandard amino acids include selenocysteine, sometimes incorporated into some proteins at a UGA codon, which may normally be a stop codon, or pyrrolysine, sometimes incorporated into some proteins at a UAG codon, which may normally be a stop codon. Some nonstandard amino acids that are not genetically encoded may result from modification of standard amino acids already incorporated in a peptide, or may be metabolic intermediates or precursors, for example. Examples of nonstandard amino acids include 4-hydroxyproline, 5- hydroxylysine, 6-N-methyllysine, gamma-carboxyglutamate, desmosine, selenocysteine, ornithine, citrulline, lanthionine, 1-aminocyclopropane-l-carboxylic acid, gamma-aminobutyric acid, carnitine, sarcosine, or N-formylmethionine. Synthetic variants of standard and nonstandard amino acids are also known and may include chemically derivatized amino acids, amino acids labeled for identification or tracking, or amino acids with a variety of side groups on the alpha carbon. Examples of such side groups are known in the art and may include aliphatic, single aromatic, polycyclic aromatic, heterocyclic, heteronuclear, amino, alkylamino, carboxyl, carboxamide, carboxyl ester, guanidine, amidine, hydroxyl, alkoxy, mercapto-, alkylmercapto-, or other heteroatom-containing side chains. Other synthetic amino acids may include alpha-amino acids, non-alpha amino acids such as beta-amino acids, des-carboxy or des-amino acids. Synthetic variants of amino acids may be synthesized using general methods known in the art, or may be purchased from commercial suppliers, for example RSP Amino Acids LLC (Shirley, MA).

[0047] The hydropathy index of an amino acid is a scale indicating the tendency of an amino acid to seek out an aqueous environment (negative value) or a hydrophobic environment (positive value) (Kyte and Doolittle, 1982). Hydropathy indices of the standard amino acids include alanine (1.8), arginine (-4.5), asparagine (-3.5), aspartic acid (-3.5), cysteine (2.5), glutamine (-3.5), glutamic acid (-3.5), glycine (-0.4), histidine (-3.2), isoleucine (4.5), leucine (3.8), lysine (-3.9), methionine (1.9), phenylalanine (2.8), proline (-1.6), serine (-0.8), threonine (-0.7), tryptophan (- 0.9), tyrosine (-1.3), and valine (4.2). Amino acids with similar hydropathy indices may be substitutable for each other in a nucleolin peptide.

Table A. Nomenclature and abbreviations of the 20 standard L-amino acids

[0048] Nucleolin peptides may be modified in a variety of conventional ways well known to the skilled artisan. Examples of modifications include the following. The terminal amino group and/or carboxyl group of the peptide and/or amino acid side chains may be modified by alkylation, amidation, or acylation to provide esters, amides or substituted amino groups. Heteroatoms may be included in aliphatic modifying groups. This is done using conventional chemical synthetic methods. Other modifications include deamination of glutamyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of serine or threonine; and methylation of amino groups of lysine, arginine, and histidine side chains (see, for e.g., Creighton, 1983).

[0049] In another aspect, one or both, usually one terminus of the nucleolin peptide, may be substituted with a lipophilic group, usually aliphatic or aralkyl group, which may include heteroatoms. Chains may be saturated or unsaturated. Conveniently, commercially available aliphatic fatty acids, alcohols and amines may be used, such as caprylic acid, capric acid, lauric acid, myristic acid and myristyl alcohol, palmitic acid, palmitoleic acid, stearic acid and stearyl amine, oleic acid, linoleic acid, docosahexaenoic acid, etc. (see, for e.g.: U.S. Pat. No. 6,225,444). Preferred are unbranched, naturally occurring fatty acids between 14-22 carbon atoms in length. Other lipophilic molecules include glyceryl lipids and sterols, such as cholesterol. The lipophilic groups may be reacted with the appropriate functional group on the oligopeptide in accordance with conventional methods, frequently during the synthesis on a support, depending on the site of attachment of the oligopeptide to the support. Lipid attachment is useful where oligopeptides may be introduced into the lumen of the liposome, along with other therapeutic agents for administering the nucleolin peptides and agents into a host.

[0050] Depending upon their intended use, particularly for administration to mammalian hosts, which is specifically contemplated herein, the nucleolin peptides may also be modified by attachment to other compounds for the purposes of incorporation into carrier molecules, changing peptide bioavailability, extending or shortening half-life, controlling distribution to various tissues or the blood stream, diminishing or enhancing binding to blood components, and the like. The prior examples serve as examples and are non-limiting. Alternatively, the nucleolin peptides may be soluble nucleolin peptides for administration to a cell or to a subject (for example, to the subjects respiratory tract).

[0051] Nucleolin peptides may be prepared in a number of ways. Chemical synthesis of peptides is known to those persons skilled in the art. Solid phase synthesis is commonly used and various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc.™, Foster City, Calif; Beckman™; etc. Solution phase synthetic methods may also be used, particularly for large-scale productions.

[0052] Nucleolin peptides may also be present in the form of a salt, generally in a salt form which is pharmaceutically acceptable. These include inorganic salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and the like. Various organic salts of the peptide may also be made with, including, but not limited to, acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benozic acid, cinnamic acid, salicylic acid, etc.

[0053] Optionally, the nucleolin peptide may have 90% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 91% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 92% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 93% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 94% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 95% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 96% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 97% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 98% sequence identity to SEQ ID NO: 1. Alternatively, the nucleolin peptide may have 99% sequence identity to SEQ ID NO: 1. Optionally, the nucleolin peptide may comprise a sequence identical to SEQ ID NO: 1. Determining sequence identity would be understood to a person skilled in the art. For example, sequence alignment techniques can be used to arrange peptide sequences to identify regions of similarity that may be a consequence of functional, structural or evolutionary relationships between the sequences. Determining the percentage of similarity between the sequences can readily be ascertained by those skilled in the art using, for example, sequence alignment programs (for e.g., CLUSTAL). Further, amino acid sequence similarity or identity may be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0 algorithm. Techniques for computing amino acid sequence similarity or identity are well known to those skilled in the art, and the use of the BLAST algorithm is described in the art (see, for e.g., Altschul et al, 1990). It is also contemplated that % identity may be determined based on the default settings of the particular alignment tool.

[0054] Further, it will be appreciated that a substantially similar sequence may be an amino acid sequence that differs from a reference sequence only by one or more substitutions, additions, deletions or inversions etc., but which may, for example, be functionally homologous to another substantially similar sequence. It will be appreciated by a person of skill in the art the aspects of the individual amino acids in the nucleolin peptides described herein may be substituted. In particular, where substitution(s) are "conservative" numerous substitutions may be made as described herein without reducing the ability to treat RSV infection.

[0055] Further, the term "antibody" refers to a composition comprising a protein that binds specifically to a corresponding antigen and has a common, general structure of immunoglobulins. The term "antigen" is to be construed broadly and refers to any molecule, composition, or particle that can bind specifically to an antibody. The term "nucleolin antibody" refers to a protein that binds specifically to nucleolin. The term antibody specifically covers polyclonal antibodies, monoclonal antibodies, dimers, multimers, multispecific antibodies {e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity. Antibodies may be murine, human, humanized, chimeric, or derived from other species, these terms and concepts are understood by those persons skilled in the art. Typically, an antibody will comprise at least two heavy chains and two light chains interconnected by disulfide bonds, which when combined form a binding domain that interacts with an antigen. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH I , CH2 and CH3, and may be of the mu (μ), delta (δ), gamma (γ), alpha (a) or epsilon (ε) isotype. Similarly, the light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL, which may be of the kappa or lambda isotype. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The heavy chain constant region mediates binding of the immunoglobulin to host tissue or host factors, particularly through cellular receptors such as the Fc receptors (e.g., FcyRI, FcyRII, FcyRIII, etc.). As used herein, antibody also includes an antigen binding portion of an immunoglobulin that retains the ability to bind antigen. These include, as examples, F(ab), a monovalent fragment of VL CL and VH CH antibody domains; and F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. The term antibody also refers to recombinant single chain Fv fragments (scFv) and bispecific molecules such as, e.g., diabodies, triabodies, and tetrabodies (see, e.g., U.S. Patent No. 5,844,094). The term antibody also refers intrabodies. An intrabody, as it is known to those persons of skill in the art, refers to an antibody that operates within a cell to bind an intracellular protein. An anti-nucleolin intrabody is specifically contemplated herein as a composition for preventing RSV entry into a cell; specifically, the treatment of a cell with an anti-nucleolin intrabody, when such intrabody results in a failure for nucleolin to be expressed on the cell's surface, would result in prevention of nucleolin-RSV-F binding, and subsequent RSV viral entry. Methods for producing intrabodies are known in the art (see, for e.g., Chen et al, 1994; Cohen et al, 1998). [0056] The term "monoclonal antibody" refers to monospecific antibodies that are the same because they are made by clones of a unique parent cell. As detailed above, the term "antibody" includes a "monoclonal antibody." Methods for producing monoclonal antibodies are known (see, for e.g., Kohler and Milstein, 1975). In another embodiment, the nucleolin antibody may be a monoclonal antibody. Nucleolin monoclonal antibodies are also commercially available (see, for e.g., anti-nucleolin monoclonal antibody (clone 4E2); Pierce Antibodies™; and anti-nucleolin monoclonal antibody (clone MS-3); Santa Cruz Biotechnology, Inc.™). For example, the MS-3 clone has been used to bind nucleolin in human fibroblasts (see, for e.g., Kegel et al, 2002).

[0057] In embodiments described herein, interfering RNA (RNAi) compounds are disclosed. RNAi is capable of preventing a gene from producing a functional protein by ensuring that the molecular intermediate, the messenger RNA copy of the gene, is destroyed (see, for e.g., Sharp, 2001). "Small interfering RNAs" are small RNA molecues which are part of the RNAi pathway. Typically, RNAi's, such as siRNA (short interfering RNA) compounds are short double stranded RNA compounds of between 4 and 50 nucleotides in length. It will be appreciated that siRNA compounds are disclosed in this specification and have a length within the range detailed above. The siRNA compounds may include short nucleotide "overhangs" on each end, which are single stranded extensions which are not paired with a complementary base on the opposite strand. Alternatively, the overhangs would be on the 3' end of each strand of the siRNA compound, and are typically 1 -3 nucleotides in length. The siRNA compounds described herein may be synthesized as individual strands which are subsequently annealed to produce the double stranded siRNA compound. Alternately, the siRNA compounds may derived from a short hairpin RNA (shRNA) molecule, or from a longer RNA compound, which has been processed by the cellular enzyme called dicer, which processes the longer RNA compounds to produce siRNA compounds. Generally, siRNA compounds mediate RNA interference via an enzyme-dependent process in which the target mRNA is degraded, such that it can no longer be translated into its associated protein product. Not being bound by theory, the double stranded siRNA compounds are separated into single stranded molecules and integrated into an activated "RISC complex". After integration into the RISC, siRNAs base-pair to their target mRNA and induce cleavage of the mRNA, thereby preventing it from being used as a translation template. As detailed herein, nucleolin siRNAs are provided. In embodiments disclosed herein, the RNAi may be optionally an siRNA comprising SEQ ID NO: 2 or SEQ ID NO: 5, as detailed herein. Many different RNAi molecules exist and are known to those persons skilled in the art; these include, without limitation: siRNA, as described herein; sisiRNA; tsiRNA; RNA-DNA chimeric duplex; tkRNA; Dicer-substrate dsRNA; short hairpin RNA (shRNA); tRNA-shRNA; aiRNA; pre- miRNA; pri-miRNA mimic; pri-miRNA mimic cluster; transcriptional gene silencing (TGS); and combinations thereof. Furthermore, RNAi molecules are commercially available from numerous suppliers. For example, from Novus Biologicals, LLC™ (Littleton, CO - Cat. # H00004691-R01-10nmol); Ori-gene™ (Rockville, MD - Cat. # SR303090 (NCL (Human) - 3 unique 27mer siRNA duplexes - 2 nmol each and Cat. # SR302169 (HNRNPF (Human) - 3 unique 27mer siRNA duplexes - 2 nmol each); and tebu-bio™ (France - Cat. # 01-00049 Nucleolin HeLa SilenciX).

[0058] Design of gene specific antisense RNA compounds, including nucleotide sequence selection and additionally appropriate alterations, would be known to one of skill in the art. Specific targeting of siRNA compounds to modulate expression of a desired gene is generally related to the degree of homology between the siRNA compound and the target gene. Design features to optimize the efficacy and specificity of an antisense RNA compound may depend on the specific sequence chosen for the design of the RNA compound. Numerous examples of methods for designing and optimizing antisense RNA compounds are known (see, for e.g., Pan and Clawson, 2006). There are also many computer based tools for designing antisense RNA compounds, which may, for instance, use algorithms or other rule-based formulae to determine optimal antisense RNA compounds. It would thus be within the skill of one in the art to design a large number of different antisense RNA compounds to test for nucleolin inhibition. Perfect sequence complementarity is often not necessary for the siRNA compound to modulate expression of the target gene. In some embodiments described herein, the antisense RNA compounds include any RNA compounds which bear sequence homology to the nucleolin gene and which are capable of modulating the expression of nucleolin protein. Provided herein are non-limiting examples of RNA compounds which modulate the expression of nucleolin and are thus nucleolin inhibitors. [0059] Inhibitory RNA compounds may also comprise additional features which improve therapeutic properties of the compounds, for instance by increasing the inhibitory effect of the RNA compounds. As an example, sticky overhangs may be added to RNA interference molecules, for instance siRNA molecules to improve gene silencing. One or more overhanging nucleotides can achieve this effect. The addition of multiple overhanging nucleotides, for instance, dT or dA residues has been shown in the literature to improve silencing of target genes (see, for e.g., Bolcato-Bellemin et al, 2007). Thus, according to some embodiments described herein, the RNA compounds may include overhanging "sticky" ends comprising one or more overhanging nucleotide residues.

[0060] In another embodiment, a RNAi having a sequence of SEQ ID NO: 2 or SEQ ID NO: 5 is provided. In another aspect, use of a RNAi having a sequence of SEQ ID NO: 2 or SEQ ID NO: 5 is provided to prevent, treat or ameliorate RSV infection.

[0061] In another aspect, a Sf9 cell line expressing heterologous nucleolin is provided in which the Sf9 cell line is permissive to RSV infection. An Sf9 cell line is known to those persons skilled in the art. It is derived from Spodoptera fragiperda and is often used as an expression system for the production of recombinant proteins or peptides. The term "heterologous" will be understood by those persons skilled in the art as referring to a generalized cell biology concept that the transferred protein was initially cloned from or derived from a different cell type from the recipient cell {e.g., Sf9 cell). Further, it will be appreciated that a cell line that "is permissive to RSV infection" is a cell line that can be infected by RSV. As should be appreciated by reading the accompanying examples, an Sf9 cell line was made permissive to RSV infection through the expression of heterologous nucleolin. Sf9 cells that do not express nucleolin are not permissive to RSV infection. No other such cell line was known. As shown herein, Sf9 cells were found not to be permissive to RSV infection without expressing heterologous nucleolin. Such a system is particularly useful because it provides a unique model system in which eukaryotic cells may be tested for RSV infectivity by altering the structure of the nucleolin expressed by the cells without the confounding effects of an endogenous or native nucleolin or of a nucleolin knockout. [0062] In another aspect, a Sf9 cell line expressing a nucleolin mutant is provided. A person skilled in the art would understand that a nucleolin mutant would refer to any deviation from the wild-type sequence of nucleolin. In another aspect, a Sf9 cell line expressing a nucleolin mutant is provided for use in screening nucleolin mutations associated with RSV infectivity. The term "RSV infectivity" means in one sense determining the level of infection that can be obtained in a cell line based on the expression of a nucleolin mutant. Determining the level of infection, for example, the level of RSV infection, can be obtained through numerous methods and techniques understood to those persons skilled in the art. For example, viral plaque assays as described herein and as known to those persons skilled in the art can be used to determine relative RSV infectivity.

[0063] In another aspect, a method for screening nucleolin mutations associated with RSV infection, the method involves expressing a nucleolin mutant in a Sf9 cell; exposing the cell expressing the nucleolin mutant with RSV; and determining whether the infected cell is infected with RSV. Optionally, RSV infection of the Sf9 cells is compared to infection of Sf9 cells expressing wildtype nucleolin. The term "wildtype" is understood by those persons skilled in the art as referring to the normal, standard sequence of a gene and its products (e.g., RNA, protein). The Sf9 system described herein may be used to determine which amino acids in the nucleolin protein are important for RSV viral entry into a cell and/or attachment to nucleolin. For example, an experimental situation may arise where a first mutant form of nucleolin is prepared and is introduced into the Sf9 cells and allows for RSV viral entry. Therafter, a second mutant form of nucleolin may be prepared, and introduced into the Sf9 cells, whereby RSV viral entry does not occur. A comparison between the mutations present in the first and second mutant forms of nucleolin can readily be undertaken to begin to focus on what amino acids play a role in mediating nucleolin-based RSV infectivity. Those persons skilled in the art will appreciate that the preceding example is non-limiting and may be used to determine the amino acids in nucleolin associated with for nucleolin-mediated RSV infectivity. [0064] The compounds, as described herein, may be in isolation, or may be linked to or in combination with tracer compounds, liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one of skill in the art. In alternate embodiments, such compounds may further comprise an additional medicament, wherein such compounds may be present in a pharmacologically effective amount.

[0065] The term "medicament" as used herein refers to a composition that may be administered to a patient or test subject and is capable of producing an effect in the patient or test subject. The effect may be chemical, biological or physical, and the patient or test subject may be human, or a non-human animal, such as a rodent or transgenic mouse, or a dog, cat, cow, sheep, horse, hamster, guinea pig, rabbit or pig. The medicament may be comprised of the effective chemical entity alone or in combination with a pharmaceutically acceptable excipient.

[0066] The term "pharmaceutically acceptable excipient" may include any and all solvents, dispersion media, coatings, antibacterial, antimicrobial or antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. An excipient may be suitable for intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, topical or oral administration. An excipient may include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersion. Use of such media for preparation of medicaments is known in the art.

[0067] Compositions or compounds according to some embodiments described herein may be administered in any of a variety of known routes. Examples of methods that may be suitable for the administration of a compound include orally, intravenous, inhalation, intramuscular, subcutaneous, topical, intraperitoneal, intra-rectal or intra-vaginal suppository, sublingual, and the like. The compounds described herein may be administered as a sterile aqueous solution, or may be administered in a fat-soluble excipient, or in another solution, suspension, patch, tablet or paste format as is appropriate. A composition comprising the compounds described herein may be formulated for administration by inhalation. For instance, a compound may be combined with an excipient to allow dispersion in an aerosol. Examples of inhalation formulations will be known to those skilled in the art. Other agents may be included in combination with the compounds described herein to aid uptake or metabolism, or delay dispersion within the host, such as in a controlled-release formulation. Examples of controlled release formulations will be known to those of skill in the art, and may include microencapsulation, embolism within a carbohydrate or polymer matrix, and the like. Other methods known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.

[0068] The dosage of the compositions or compounds of some embodiments described herein may vary depending on the route of administration (oral, intravenous, inhalation, or the like) and the form in which the composition or compound is administered (solution, controlled release or the like). Determination of appropriate dosages is within the ability of one of skill in the art. As used herein, an "effective amount", a "therapeutically effective amount", or a "pharmacologically effective amount" of a compound refers to an amount of the nucleolin peptide, nucleolin Ab, or nucleolin RNAi present in such a concentration to result in a therapeutic level of the compound delivered over the term that the compound is used. This may be dependent on mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the compound. Methods of determining effective amounts are known in the art. It is understood that it could be potentially beneficial to restrict delivery of the compounds described herein to the target tissue or cell in which inhibition of RSV infection is desired. It is also understood that it may be desirable to target the compounds described herein to a desired tissue or cell type. The compounds described herein may thus be coupled to a targeting moiety. The compounds may be coupled to a cell uptake moiety. The targeting moiety may also function as the cell uptake moiety.

[0069] Delivery of bioactive molecules such as nucleolin peptides, to a cell or cells in a reasonably efficient manner may require more than just the "dumping" of the naked peptide onto the cell, or administering the naked peptide into the patient or test subject. Agents that enable delivery of bioactive molecules into cells in a suitable manner so as to provide an effective amount, such as a pharmacologically effective amount are known in the art, and are described in, for example, Dietz et al, 2004. Examples of such agents include liposomes, antibodies or receptor ligands that may be coupled to the bioactive molecule, viral vectors, and protein transduction domains (PTD). Examples of PTDs include Antennapedia homeodomain (Perez et al, 1992); transportan (Pooga et al, 1998); the translocation domains of diphtheria toxin (see, for e.g., Stenmark et al, 1991); anthrax toxin (Ballard et al, 1998) and Pseudomonas exotoxin A (Prior et al, 1992); protegrin derivatives such as dermaseptin S4, HSV-1 VP22, PEP-1, basic peptides such as poly-L and poly-D-lysine, HSP70, and HIV- TAT (see, for e.g., Demarchi et al, 1996). Other examples and related details of such protein transduction domains are described in Dietz, supra and references therein.

[0070] Provided herein are pharmaceutical compositions comprising one or a cocktail of RNAi (dsRNA) or antisense RNA (ssRNA) molecules that inhibit nucleolin gene expression and a pharmaceutically acceptable carrier.

[0071] Delivery of a nucleic acid-lipid particle that targets nucleolin gene expression may be accomplished with a lipid particle. The nucleic acid-lipid particle may comprise one or more unmodified and/or modified interfering RNA that silence nucleolin gene expression, a cationic lipid, and a non-cationic lipid. In certain instances, the nucleic acid-lipid particle may further comprise a conjugated lipid that inhibits aggregation of particles as known in the art. Alternatively, the nucleic acid-lipid particle comprises one or more unmodified and/or modified interfering RNA that silence nucleolin gene expression, a cationic lipid, a non- cationic lipid, and a conjugated lipid that inhibits aggregation of particles.

[0072] The present application demonstrates specific nucleolin binding to laboratory- adapted strains and wild- type human RSV. The application also specifically demonstrates: a) decreased RSV infection after anti-nucleolin antibody blocking; b) competition experiments using purified nucleolin; c) nucleolin knock-down by RNA interference; and d) enhanced RSV infection following nucleolin reconstitution in Sf9 cells. Nucleolin, expressed on the apical aspect of polarized epithelial cells, has numerous functions within various cellular compartments, including binding and chaperone capability, and has been implicated in other viral systems. However, as shown herein nucleolin is not a cellular receptor for all viruses - results presented herein for adenovirus (AdV5) clearly show that nucleolin is not a cellular receptor AdV5. Additionally, a very surprising and significant reduction in infectivity of epithelial cells by RSV is shown upon addition of compounds which inhibit nucleolin function, by either interfering with interaction of nucleolin with the viral receptor (for instance, using soluble nucleolin or anti-nucleolin antibodies) or by reducing the cellular expression of nucleolin (for instance, using anti-nucleolin RNA interference compounds). A 98% reduction in infectivity by administration of soluble nucleolin at a concentration of approximately 30 nM is shown. The RSV result comparable to the results for the HPIV-3 viral system, (Bose et a , 2004).

METHODS AND MATERIALS

[0073] The following methods and materials were employed with respect to the Examples described herein.

[0074] Cells. HEp-2, MDCK (American Type Culture Collection (ATCC), Rockville, MD), and 1HAE cells (gift from Dr. D. Gruenert, University of Vermont, Burlington, VT) were maintained in Dulbecco's modified essential medium (Invitrogen™, Carlsbad, CA) supplemented with 1% L-glutamine (Invitrogen™) and 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Invitrogen™), at 37°C. CHO-K1 and pgsA-745 cells (ATCC) were maintained at 37°C in Nutrient Mixture F-12 Ham (Sigma, St. Louis, MO) containing 1% L- glutamine. Sf9 cells (Invitrogen™) were maintained at 27°C in Grace's supplemented media (Invitrogen™) supplemented with 10% (v/v) heat-inactivated FBS. All cell cultures were kept in a humidified incubator containing 5% C0₂.

[0075] Viruses. Working stocks of RSV A2 and RSV B (ATCC), RSV lacking G protein (RSV AG) (gift of Dr. P.L. Collins, National Institutes of Health, Bethesda, MD), RSV expressing GFP (Hallak et al, 2000) (rgRSV224) (gift of Dr. M.E. Peeples, Children's Research Institute, Columbus, OH), and a community isolate of RSV (HLIl) (provided by Drs. R. Tan and E. Thomas, Children's and Women's Health Centre of British Columbia, Vancouver, BC) were prepared as previously described (Kaan and Hegele, 2003). AdV5-GFP (Vector Biolabs™, Philadelphia, PA) was cultured in HEK 293T cells as per manufacturer's instructions.

[0076] MS analysis. Extracted peptides underwent trypsin digestion and LC-MS/MS using an Applied Biosystems™ QSTAR Pulsar I Quadrupole™ Time-of-Flight Mass Spectrometer equipped with nanoflow high performance liquid chromatography. Samples were separated by reverse-phase chromatography over a minimum 120 min gradient while spraying into the mass spectrometer. MS/MS data were analyzed using a protein identification search engine algorithm (MASCOT). Data were searched against all species sequences in MSDB and in IPI Human.

[0077] Neutralization. To each well of -80% confluent HEp-2 cells in 24-well plates, 200 of 10 μg/mL (1 :50 dilution) anti-nucleolin antibody IgG C23 (H-250) or control rabbit IgG (Santa Cruz Biotechnology™) were added and cells kept for 1 h at 37°C. rgRSV224 or AdV5-gfp (MOI=l, 33 per well) was added to cells and after 24 h, cells underwent 0.1% trypsin digestion (5 min) and fixation in 10% formalin.

[0078] VOPBA and Gel Excision. Biotinylated cellular proteins, enriched for cell surface proteins, were isolated by use of Cell Surface Protein Isolation Kit™ (Pierce™, Rockford, IL) and loaded symmetrically on Novex™ Tris-Glycin gels (Invitrogen™). After electrophoresis, gels were bisected, with one-half fixed (50% methanol, 10% acetic acid) for 20 min and kept in 10 mM Tris pH 8.5 at 4°C, and the other half transferred onto Hybond ECL™ (GE Healthcare Bio-Sciences™ Corporation, Piscataway, NJ) nitrocellulose membranes. Membranes were incubated in 5% milk-PBS for 1 h at room temperature (RT), rinsed twice with PBS, incubated with RSV (10⁶-10⁷ plaque forming units/mL of 2.5% milk- PBS) for 2 h at RT, followed by three 10-min washes with PBS, incubation with either 1 :1,000 diluted goat anti-RSV polyclonal antibody (Biodesign International, Saco, ME) or 1 :2,000 diluted rabbit AdV5 antibody (Abeam™, Cambridge, UK) in 2.5% PBS (1 h, RT) and three 10-min washes with PBS. Horse radish peroxidise (HRP)-conjugated, 1 : 1,000 diluted donkey anti-goat or goat anti-rabbit antibodies (Santa Cruz Biotechnologies™, Santa Cruz, CA) were added in 2.5% PBS for 1 h, followed by three 10-min washes with PBS. During imaging with SuperSignal West Pico™ (Pierce) and Chemigenius™ imaging system (Syngene™, Frederick, MD), each membrane was placed on a grid, the locations of bands were marked, and the membrane was wrapped in cellophane. The fixed half of the gel was placed on glass, with alignment of protein size markers between gel and membrane. The portion of the gel corresponding to the membrane VOPBA signal was excised, placed in an Eppendorf tube containing 10 mM Tris pH 8.5, and sent to University of Victoria- Genome British Columbia Proteomics Centre (Victoria, BC, Canada) at ambient temperature for MS analysis. For VOPBA using purified nucleolin (Vaxron™ Corporation, Rockaway, NJ), 10 μΙ_, of 10 μg/μL of nucleolin in buffer provided by the company was diluted with 5X SDS PAGE sample buffer (250 mM Tris base pH 6.8, 0.05% Bromophenol blue, 50% Glycerol, 10% SDS) and water, to obtain IX SDS PAGE sample buffer that was added to individual wells for gel electrophoresis and transfer to nitrocellulose membranes.

[0079] Competition. Identical concentrations (300 pM) of purified nucleolin (Vaxron™ Corp, Rockaway, NJ) or transferrin (Sigma™) were incubated with rgRSV224 or AdV5-gfp (MOI=l) for 3 h at room temperature. Protein pre-incubated viruses were added to -80% confluent HEp-2 monolayers in 24-well plates and kept at 37°C for 90 min with occasional rocking. After removal of unbound viruses, infection was continued for 24 h, after which cells underwent 0.1% trypsin digestion (20 min) and fixation in 10% formalin.

[0080] RNAi. RNAi of cell surface nucleolin was conducted using Oligofectamine (Invitrogen™), as per manufacturer's instructions. RNA compounds were by default synthesized with dTdT overhangs on the 3 ' end. RNA oligonucleotides included:

Human Scramble control (CUUCCUCUCUUUCUCUCCCUUGUGAdTdT) [SEQ ID NO: 4];

Human Nucleolin (UUUCUCAAACGAAGUAAGCUUdTdT) [SEQ ID NO: 2]; Human 3 -nucleoti de-substituted (Δ3) nucleolin

(UUUCUCUAAGGAAGUAAGCGUdTdT) [SEQ ID NO: 3]; and

Human DAF (CCAUCUCCUUCUCAUGUAATTdTdT) [SEQ ID NO: 7]. [0081] 1HAE cells were transfected with oligonucleotides for RNAi, washed twice with fresh growth media the following day and infected with virus or harvested for Western blot 24 h later. Expression of GFP was detected by flow cytometry the following day to determine infection by GFP-RSV.

[0082] Transfection. Sf9 cells were transfected with Lipofectamine 2000 (Invitrogen™) as per manufacturer's instructions.

[0083] Flow cytometry. For enumeration of RSV- or AdV5-associated GFP in neutralization, competition, RNAi and reconstitution experiments, fixed cell suspensions were loaded into a Beckman Coulter Epics-XL (Beckman Coulter™, Miami, FL) flow cytometer and the percentage of positively-staining cells (or fluorescence intensity) was determined using Summit analysis software (Dako Cytomation™, Carpinteria, CA). Quantification of cell surface nucleolin in RNAi experiments live cells were incubated with primary monoclonal anti-nucleolin MS-3 antibody (Santa Cruz Biotechnology™) for 1 hr at room temperature, followed by secondary Alexa 594-conjugated anti-mouse antibody. After fixation with 3% formol saline, cells underwent flow cytometry and results were expressed as percentage of positively-staining cells and GFP-associated fluorescence.

[0084] In vitro testing of mouse siRNA on mouse cells. In preparation for in vivo studies with mouse nucleolin siRNA "Oligo 3" (SEQ ID NO: 5) and control "Oligo 3Δ3" (SEQ ID NO: 6), NIH 3T3 cells where grown in 6-well plates and transfected with test siRNAs in the same method described above and harvested for Western blot 4 days post- transfection. Highly purified siRNAs for in vivo work was purchased from Dharmacon, Inc.:

Mouse nucleolin siRNA (Oligo 3) (GGCUCUGUUCGUGCAAGAAdTdT ) [SEQ ID NO: 5]; and

3-nucleotide-substituted mouse nucleolin (Oligo 3Δ3)

(GUCUCUGAUCGUGCAAGCAdTdT) [SEQ ID NO: 6]. [0085] RSV propagation (in vivo experiments). Cells involved with RSV propagation were routinely screened for mycoplasma and LPS. 2 x 10⁷ HEp-2 cells (ATCC) were seeded in 10% FBS EMEM in a T-150 tissue culture flask. Cells were left to grow in 37°C, 5% C0₂ incubator overnight. The next day (Day 0), cells were rinsed twice with clean PBS. RSV-A2 (ATCC) was added in 12 mL of serum free EMEM at an MOI - 0.1. Cells/RSV underwent incubation for 2 to 4 h while rotating flask every 15 to 30 min. Twenty-eight mL 6% FBS EMEM were added to flasks and cells incubated for 3 to 4 days. By day 4, 50% cells usually detached and virus was harvested and purified.

[0086] RSV harvesting and purification. Infected HEp-2 cells were scraped and collected with media into 50mL Falcon tubes, and then underwent centrifugation at 820 x g for 10 min. All solutions and cells were kept on ice or at 4°C unless otherwise stated. Supernatant was decanted and placed in a cold 50 mL falcon tube. The cell pellet was resuspended with 5 mL medium, then quick frozen in liquid nitrogen, and melted at 37°C with constant agitation. This freeze-thaw cycle was repeated once. The resuspended pellet underwent sonication on ice for 20 s twice and centrifugation at 820 x g for 10 min. The cell pellet was discarded and the supernatant was combined with the original decanted media for ultracentrifugation (SW28 rotor) at 110,000 x g for 45 min, over a sucrose cushion (30% sucrose in 0.1 M sodium chloride, 0.01 M Tris-HCl, 0.001 M EDTA, 1 M urea, pH = 7.5). After this spin a virus pellet was clearly visible and supernatant was decanted and disposed. The pellet was resuspended in 100-150 LPS-free PBS per T-150 flask (yield: 2x108 PFU RSV/mL) and stored at -80°C or in liquid nitrogen.

[0087] RSV titer quantification (viral plaque assays). HEp-2 cells were propagated in 6-well plates in 10% FBS DMEM-F12, to >90% confluence. Media was aspirated and cells were washed twice with PBS or serum free media. Serial dilutions of RSV stock or homogenized sample were obtained using cold PBS or serum free media and 400μ1 of the diluted RSV solution added to each well of the 6-well plates, with at least 1 well per plate left uninfected as negative control. Samples were plated in duplicate or triplicate. Plates were incubated for 90 min at 37°C in a 5% C0₂-containing incubator, with rocking manually every 15 min. Inoculants were removed and the wells were overlayed with 4 mL of 1 :1 4% FBS DMEM-F12/1% agarose and left at room temperature for several minutes before putting back into the incubator. Plates were incubated for 4-7 days at 37°C. Monolayer holes were seen with a dissecting scope after 4-5 days. At this point, 2 mL of 1% formaldehyde (made up in 0.15 M saline) were added to each well for incubation overnight. Agarose was flicked off and the plate rinsed gently with running tap water to remove remaining agarose. 2-3 mL of 0.05% neutral red added to each well for 1 h and excess stain was removed with running tap water. Plates were dried and plaques counted under a dissecting scope or with scanner and computer.

[0088] In vivo RSV experiments. Animal studies were approved by the Hospital for Sick Children Animal Care Committee in accordance with regulations of the Canadian Council on Animal Care. Female Balb/C mice (6-8 weeks old) were purchased from Charles River Laboratories™ (St. Constant, QC, Canada). Animals were kept in a specific pathogen-free environment and fed food and water ad libitum. For intranasal instillation, lightly sedated mice (isoflurane) inhaled 100μL of instillate, applied to the nares with a P-200 pipetter while their mouth was held closed. On day -2, mice received 15 nmol (ΙΟΟμί) siRNA or vehicle control via nasal instillation (n = 6-

7 per group, repeated twice). The siRNA was delivered in optimem: transiTKO (volume ratio 7: 1). On day 0 mice received 5 x 106 pfu (ΙΟΟμί) of RSV via intranasal installation. Mice were monitored daily and on Day 2 were euthanized. The right lung was removed, weighed, resuspended in EMEM (100 mg mL) and homogenized. Serial dilutions of clarified lung homogenates were plated for viral titre (plaque assays). After the right lung was removed, the left atrium was cut and the right ventricle flushed with 5 mL clean PBS to flush the left lung vasculature. The trachea was cannulated and 10% formalin used to inflate the lungs at 20 cm ¾0 pressure. The lung was then removed, placed in formalin for fixation and subsequent histological sectioning.

[0089] Immunohistochemistry. Formalin-fixed, paraffin-embedded mouse lung sections were deparaffinized with xylene and rehydrated before performing antigen retrieval with 10 mM Citrate buffer heated for 20 min in a microwave. Sections were then treated with 3% hydrogen peroxidase for 30 min to inhibit endogenous peroxidase activity and blocked with Dako™ protein block (cat# X0909). The primary antibody was anti-nucleolin rabbit polyclonal (Abeam™; ab22758) used at 1 :1000 dilution and the secondary antibody was a biotinylated goat anti-rabbit IgG used at 1 :200 dilution. The "ABC kit" from Vector Labs™ (PK=6100) was used for subsequent diaminobenzadine (DAB) staining as per manufacturer's instructions. For double immunostaining, a second round of blocking and antigen retrieval was done and the anti-RSV rabbit polyclonal (Abnova™; PAB13816) was substituted as the primary antibody. The "ABC kit" was used for alkaline phosphatase permanent red color staining and slides were counterstained with hematoxylin.

[0090] Statistical analysis. Unless otherwise indicated, results were expressed as mean + SE. Group means were compared by either two-tailed Student t-tests or one way analysis of variance with post hoc Tukey tests (Minitab version 15 software, Minitab Inc., State College, PA). In confocal microscopy image stacks, Pearson's correlation of green (RSV) and red (nucleolin) pixels was used determine the extent of RSV-nucleolin co-localization. A P value < 0.05 was considered statistically significant. All reported means are derived from at least 2 independent experiments in all cases. All reported "n" values are for each group in the experiment unless otherwise stated.

[0091] The following examples are provide for illustrative purposes and are not intended to be limiting, as such:

EXAMPLES

EXAMPLE 1: Virus Overlay Protein Binding Assay (VOPBA) and Co- immunoprecipitation Studies

[0092] A virus overlay protein binding assay (VOBPA) (see, for e.g., Cao et al, 1998) was used to identify candidate receptor molecules. VOPBA using RSV A2 on proteins extracted from human (HEp-2, 1HAE), dog (MDCK), and hamster (CHO- 1 and pgsA-745) cell lines revealed a reproducible signal of molecular weight -100 kDa, which was also seen for other laboratory-adapted and community RSV isolates. In order to locate the signal on unstained polyacrylamide gels, fixed gels were overlaid on membranes; the portion of the gel overlying the signal was excised and subject to mass spectrometry (MS) analysis. Mass spectrometry analysis revealed that nucleolin was common to all samples tested, with Mowse scores (see, for e.g., Pappin et al, 1993) ranging from 161-843 (score > 52 implies a significant MS "hit", P < 0.05), corresponding to 15-24% sequence coverage. Direct RSV- nucleolin binding was confirmed by VOPB A with soluble nucleolin as the only protein target.

[0093] With respect to which RSV-encoded protein was interacting with nucleolin, experiments using lHAEo- cell lysates revealed RSV A2 Fl -nucleolin coimmunoprecipitation (see, for e.g., Figure la). Similar findings were obtained using RSV AG and RSV B (Figure lb). No co-immunoprecipitation of nucleolin and protein G was seen.

[0094] As shown in Figure la, lHAEo- cells were incubated with RSV A2 and immunoprecipitated (IP lanes), with anti-Ncl antibody (left) or RSV F (middle) and G protein antibodies (right). Western blot analysis of control lysates of virus-inoculated cells (L lanes) using antibodies for RSV F, G and nucleolin. Note RSV Fi -nucleolin coimmunoprecipitation (left and middle) and lack of G-nucleolin interaction.

[0095] As shown in Figure 1, immunoprecipitation using anti -nucleolin antibody H-250 and lHAEo- protein showed the presence of RSV Fi and nucleolin (but not RSV G). (N: nucleocapsid protein; P: phosphoprotein; M: matrix protein; G: RSV G).

EXAMPLE 2: Nucleolin and RSV Colocalization Studies

[0096] Confocal microscopy of surface binding of RSV to lHAEo- cells, under conditions where cells are polarized and virus binds to the cell surface without entry (see, for e.g., Srinivasakumar et al, 1991; and Zhang et al, 2002 for general methods) revealed to the inventors that there is a colocalization of nucleolin and RSV on the apical aspect.

[0097] Pre-incubation of cells with anti-nucleolin antibody was associated with significantly decreased RSV-nucleolin colocalization in comparison to cells pre-incubated with serotype-matched, irrelevant antibody (Pearson's correlation, P = 6.83 x 10^"12), providing strong evidence of specific RSV-nucleolin binding at the cell surface. In blocking experiments, HEp-2 cells incubated with anti-nucleolin antibody had significantly decreased RSV infection (Figure 2a, P < 0.01). In competition experiments (Figure 2b), incubation of RSV with nucleolin prior to inoculation of IHAEo- cells was associated with significantly decreased infection (P < 0.001). RNAi silencing of nucleolin showed that treatment of IHAEo- cells with nucleolin-specific (siNcl - SEQ ID NO:2) oligonucleotide had 12.4 ± 1.0- fold reduction in nucleolin protein expression compared to control (siDAF; Decay Accelerating Factor - SEQ ID NO:7) or nucleolin with 3-nucleotide substitution (siNclA3 - SEQ ID NO:3) oligonucleotides (P < 0.02; Figure 2c), and significant reduction of RSV infection (P < 0.01 ; Figure 2d).

[0098] As shown in Figure 2a, HEp-2 cells were incubated with anti-nucleolin antibody (anti-Ncl Ab), or irrelevant isotype-matched antibody, prior to RSV A (RSV-GFP) exposure. At 24 h, flow cytometry shows decreases in RSV-positive cells treated with anti-Ncl Ab (*P < 0.01, n = 3, By ANOVA P = 0.0024).

[0099] As shown in Figure 2b, RSV A2 (RSV-GFP) was incubated with nucleolin or transferrin and added to IHAEo cells. The percent of cells infected at 24 h was determined by flow cytometry (†P < 0.001, n = 3. By ANOVA P = 0.0002) ("ns": no significant difference). In Figure 2c, a nucleolin immunoblot of IHAEo- cells incubated with control (siDAF), nucleolin with 3-nucleotide substitution (siNclA3) or nucleolin siRNA (siNcl) is shown.

[00100] Figure 2d shows IHAEo- cells inoculated with RSV-GFP and enumerated by flow cytometry 24 h later. Note significant decrease in RSV infection after RNAi silencing of nucleolin was shown (siNcl) {%P < 0.01, n = 4, By ANOVA P = 0.0007).

EXAMPLE 3: Sf Cell Experiments

[00101] Spodoptera frugiperda Sf9 insect cells have been proposed to lack a cellular RSV receptor (see, for e.g., Osiowy and Anderson, 1995). When tested, no RSV-associated VOPBA signal was observed (Figure 3a). The human nucleolin gene was transfected into Sf9 cells; protein expression was confirmed by Western blot (Figure 3b) and cell surface expression was observed by confocal microscopy. Transfection efficiency was determined to be 55.6 ± 7.6%. Figure 3c demonstrates significantly higher RSV infection in nucleolin- transfected Sf9 cells compared to control cells transfected with empty pCMV-X6 plasmid vector (P < 0.001).

[00102] As shown in Figure 3, Sf9 cells were made to be permissive to RSV infection by heterologous expression of human nucleolin. As shown in Figure 3 a, native Sf9 cells have no RSV VOPBA signal. More specifically, RSV VOPBA of proteins extracted from HEp-2 cells and Sf9 cells showed no -100 kDa signal for Sf9 cells (β-actin: gel loading control). As shown in Figure 3b, expression of human nucleolin is shown in Sf9 cells. More specifically, Sf9 cells were transfected with human nucleolin (Ncl) or pCMV-X6 empty vector (Control), and extracted cellular proteins were immunoblotted with anti-Ncl Ab. Note positive signal in the nucleolin-transfected sample at -100 kDa. In additional experiments, nucleolin- transfected Sf9 cells were found to express nucleolin on the cell surface. More specifically, fluorescence confocal microscopy (72 h post-transfection) was used to demonstrate nucleolin expression on the cell surface of Ncl-transfected, Sf9 cells (results not shown). Nuclear staining was carried out using blue Hoechst nuclear stain. Further, Sf9 cells were transfected with nucleolin or pCMV-X6 empty vector for 3 days and inoculated with RSV A2-GFP, with cellular infection (assessed by GFP fluorescence) enumerated 24 h later by flow cytometry. Imaging of RSV A2-GFP infected Sf9 cells, 24 h post-infection showed virus-associated GFP visible only in the Ncl transfected Sf9 cells. Phase-contrast and fluorescence images show intracellular localization of virus-associated GFP. Further, and as shown in Figure 3(c), the percentage of RSV infected cells in Ncl-transfected Sf9 cells was significantly increased.

EXAMPLE 4: Mouse Model Experiments

[00103] A RSV mouse model was used in this set of experiments (see, for e.g., Taylor et al, 1984). Briefly, mouse nucleolin siRNAs were tested for their ability to knockdown nucleolin in mouse NIH 3T3 fibroblasts (see: Figure 4a). There was a 3.5 + 0.8 fold knockdown of nucleolin expression in vitro with specific siRNA (Oligo 3 - SEQ ID NO:5) compared to control siRNA containing 3 nucleotide substitutions (Oligo 3Δ3 - SEQ ID NO:6) designed to address potential off-target effects (P = 0.05). [00104] Further, immunohistochemical staining of mouse lung for nucleolin showed intranuclear signals and importantly, positive immunostaining at the apical aspect of airway epithelial cells, which is the point of entry of RSV into these cells. Further, double immunostaining of RSV-infected mouse lung showed overlap of RSV and nucleolin signals (results not shown).

[00105] Further, as a strategy to decrease lung nucleolin expression prior to RSV inoculation of mice we used RNAi. Briefly, mice were given Ncl siRNA or vehicle at day -2 and 5 x 10⁶ pfu of RSV intranasally at day 0. At day 2, animals were sacrificed, with one lung fixed and processed for nucleolin immunohistochemical staining and the

other lung was homogenized for quantitative plaque assays.

[00106] In immunohistochemically-stained sections, airway epithelial cells were counted and scored for the presence or absence of apical nucleolin signal and results expressed as the percentage of the total number of cells counted (Figure 4b). Results showed that the vehicle and control (Oligo 3Δ3) groups had equivalent levels of apical nucleolin positivity compared to the siNcl (Oligo 3) group, which had 57 + 4% Ncl signal (P < 0.001). Correspondingly, -55% reduction in RSV titer per gram of lung tissue was obtained with Oligo 3 (45 + 7% pfu/gram lung; P < 0.001) as compared to vehicle or Oligo 3Δ3 (Figure 4c). Overall, these findings implicate nucleolin as a functional cellular receptor to RSV in vitro and in vivo.

EXAMPLE 5: Competition Experiments

[00107] Competition experiments were performed to confirm the specificity of RSV- nucleolin binding. Different concentrations of purified nucleolin and purified transferrin, (negative control protein) were incubated with rrRSV-BNl or AdV5gfp. The viruses were then used for infecting 80% confluent Hep-2 monolayers. The infectivity at 24 h was quantified by flow cytometry and calculated as infectious units per mL (IU/mL). Figure 5 shows a dose-dependent decrease in RSV infection of Hep-2 cells after incubation of virus with purified nucleolin (p<0.001). [00108] Although embodiments described herein have been described in some detail by way of illustration and example for the purposes of clarity of understanding, it will be readily apparent to those of skill in the art in light of the teachings described herein that changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

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SEQ ID NO: 1; Description: human nucleolin

1 MVKLAKAG N QGDPKKMAPP PKEVEEDSED EEMSEDEEDD SSGEEVVIPQ KKGKKAAATS 61 AKKVVVSPTK KVAVATPAKK AAVTPGKKAA ATPAKKTVTP AKAVTTPGKK GATPGKALVA 121 TPGKKGAAIP AKGAKNGKNA KKEDSDEEED DDSEEDEEDD EDEDEDEDEI EPAAMKAAAA 181 APASEDEDDE DDEDDEDDDD DEEDDSEEEA METTPAKGKK AAKWPVKAK NVAEDEDEEE 241 DDEDEDDDDD EDDEDDDDED DEEEEEEEEE EPVKEAPGKR KKEMAKQKAA PEAKKQKVEG 301 TEPTTAFNLF VGNLNFNKSA PELKTGISDV FAKNDLAVVD VRIGMTRKFG YVDFESAEDL 361 EKALELTGLK VFGNEIKLEK PKGKDSKKER DARTLLAKNL PYKVTQDELK EVFEDAAEIR 421 LVSKDGKSKG IAYIEFKTEA DAEKTFEEKQ GTEIDGRSIS LYYTGEKGQN QDYRGGKNST 481 WSGESKTLVL SNLSYSATEE TLQEVFEKAT FIKVPQNQNG KSKGYAFIEF ASFEDAKEAL 541 NSCNKREIEG RAIRLELQGP RGSPNARSQP SKTLFVKGLS EDTTEETLKE SFDGSVRARI 601 VTDRETGSSK GFGFVDFNSE EDAKAAKEAM EDGEIDGNKV TLDWAKPKGE GGFGGRGGGR 661 GGFGGRGGGR GGRGGFGGRG RGGFGGRGGF RGGRGGGGDH KPQGKKTKFE

SEQ ID NO: 2; Description: Human siRNA targeting human nucleolin

5 '-UUUCUC AAACG AAGUAAGCUU-3 '

SEQ ID NO: 3; Description: 3 -nucleotide substituted human siRNA targeting human nucleolin

5'-UUUCUCUAAGGAAGUAAGCGU-3'

SEQ ID NO: 4; Description: scramble siRNA for human nucleolin experiments

5 '-CUUCCUCUCUUUCUCUCCCUUGUGA-3 '

SEQ ID NO: 5; Description: mouse siRNA targeting mouse nucleolin (Oligo 3)

5 '-GGCUCUGUUCGUGC AAGAA-3 '

SEQ ID NO: 6; Description: 3-nucleotide-substituted mouse siRNA targeting mouse nucleolin (Oligo 3Δ3)

5 '-GUCUCUG AUCGUGC AAGC A-3 '

SEQ ID NO: 7; Description: human DAF RNA oligonucleotide

5 '-CCAUCUCCUUCUCAUGUAAUUUU-3 '

Claims

WHAT IS CLAIMED IS;

1. A method of treating Respiratory Syncytial Virus (RS V) infection in a cell, the

method comprising administering a nucleolin peptide, a nucleolin antibody, or a nucleolin interference RNA (RNAi) to the cell.

2. The method of claim 1, wherein the RNAi is an siRNA molecule and said siRNA

molecule comprises a sense region and an antisense region, wherein said sense region and said antisense region together form a duplex region comprising 19-30 base pairs and said antisense region comprises a sequence that is the complement of SEQ ID NO:2 or SEQ ID NO:5.

3. The method of claim 2, wherein the antisense region comprises a sequence that is the complement of SEQ ID NO:2.

4. The method of claim 3, wherein said antisense region and said sense region are each 19-25 nucleotides in length.

5. The method of claim 3 or 4, wherein said antisense region and said sense region are each 21 nucleotides in length.

6. The method of claim 2, wherein said siRNA molecule comprises at least one overhang region, wherein said overhang region comprises six or fewer nucleotides.

7. The method of claim 2, wherein said siRNA molecule comprises no overhang regions.

8. The method of any one of claims 3-7, wherein the cell is a human cell.

9. The method of claim 8, wherein the human cell is an epithelial cell.

10. The method of claim 8 or 9, wherein the human cell is a ciliated respiratory epithelial cell.

11. The method of any one of claims 8-10, wherein the cell is in a human subject having or at risk of developing an RSV infection.

12. The method of claim 11 , wherein said siRNA molecule is administered intravenously.

13. The method of claim 11 , wherein said siRNA molecule is topically administered to a mucosal membrane of the subject.

14. The method of claim 11, wherein said siRNA molecules are mixed with lipid particles prior to administration.

15. The method of claim 11 , wherein said siRNA molecules are encapsulated in liposomes prior to administration.

16. The method of claim 1 , wherein the nucleolin peptide comprises a peptide having at least 85% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection.

17. The method of claim 1 or 16, wherein the nucleolin peptide comprises a peptide

having at least 90% sequence identity to SEQ ID NO:l, wherein the nucleolin peptide inhibits RSV infection.

18. The method of any one of claims 1 , 16 or 17, wherein the nucleolin peptide comprises a peptide having at least 95% sequence identity to SEQ ID NO: 1, wherein the nucleolin peptide inhibits RSV infection.

19. The method of claim 1 , wherein the nucleolin peptide comprises SEQ ID NO: 1.

20. The of any one of claims 1, or 16-19, wherein the nucleolin peptide is soluble.

21. The method of claim 1 , wherein the antibody is a monoclonal antibody.

22. The method of claim 21 , wherein the monoclonal antibody is a humanized monoclonal antibody.

23. The composition of claim 1, wherein the antibody is selected from one or more of the following: a polyclonal antibody; a monoclonal antibody; or a fragment thereof; a single chain Fc region (scFc); or an intrabody.

24. A RNAi having a sequence of SEQ ID NO:2 or SEQ ID NO:5.

25. A siRNA molecule, wherein said siRNA molecule comprises a sense region and an antisense region, wherein said sense region and said antisense region together form a duplex region comprising 19-30 base pairs and said antisense region comprises a sequence that is the complement of SEQ ID NO:2 for use in the treatment of RSV infection.

26. Use of a RNAi having a sequence of SEQ ID NO:2 or SEQ ID NO:5 for the treatment of RSV infection.

27. Use of a RNAi having a sequence of SEQ ID NO:2 or SEQ ID NO:5 in the

preparation of a medicament for the treatment of RSV infection.

28. The use of claim 26 or 27, wherein the treatment is of a human subject.

29. The use of claim 28, wherein the RNAi is a siRNA molecule and wherein said siRNA molecule comprises a sense region and an antisense region, wherein said sense region and said antisense region together form a duplex region comprising 19-30 base pairs and said antisense region comprises a sequence that is the complement of SEQ ID NO:2.

30. The use of claim 29, wherein said antisense region and said sense region are each 19- 25 nucleotides in length.

31. The use of claim 29 or 30, wherein said antisense region and said sense region are each 21 nucleotides in length.

32. The use of claim 29, 30 or 31 , wherein said siRNA molecule comprises at least one overhang region, wherein said overhang region comprises six or fewer nucleotides.

33. The use of claim 29, 30 or 31 , wherein said siRNA molecule comprises no overhang regions.

34. A Sf9 cell line expressing a heterologous nucleolin.

35. The Sf9 cell line of claim 34, wherein the Sf9 cell line is permissive to RSV infection.

36. The Sf9 cell line of claim 34, wherein the Sf9 cell line is not permissive to RSV

infection.

37. The Sf9 cell line of claim 34, wherein the Sf9 cell line expresses a nucleolin mutant.

38. A Sf9 cell line expressing a heterologous nucleolin mutant.

39. A Sf9 cell line expressing a nucleolin mutant for use in screening nucleolin mutations associated with RSV infectivity.

40. A method for screening nucleolin mutations associated with RSV infection, the

method comprising: a. expressing a nucleolin mutant in a Sf9 cell;

b. exposing the cell derived from step (a) with RSV; and

c. determining whether the cell exposed in step (b) becomes infected with RSV.

41. The method of claim 40, wherein RSV infection of the Sf9 cells is compared to

infection of Sf9 cells expressing wildtype nucleolin.

42. A method for screening nucleolin mutations associated with RSV infection, the

method comprising:

a. exposing a Sf9 cell, that is expressing a nucleolin mutant, with RSV; and b. determining whether the cell exposed in step (a) becomes infected with RSV.

43. The method of claim 42, wherein RSV infection of the Sf9 cells is compared to

infection of Sf9 cells expressing wildtype nucleolin.

44. A commercial package, comprising:

a. Sf9 cells expressing a heterologous nucleolin; and

b. RSV.

45. The commercial package of claim 44, wherein the heterologous nucleolin is human nucleolin and the RSV is a human strain of RSV.

46. The commercial package of claim 44, wherein the heterologous nucleolin is bovine nucleolin and the RSV is a bovine strain of RSV.

47. The commercial package of claim 44, wherein the heterologous nucleolin is ovine nucleolin and the RSV is an ovine strain of RSV.

48. The commercial package of claim 44, wherein the heterologous nucleolin is equine nucleolin and the RSV is an equine strain of RSV.

49. The commercial package of claim 44, wherein the heterologous nucleolin is porcine nucleolin and the RSV is a porcine strain of RSV.

50. A vector comprising a DNA template which encodes an RNA which is homologous to a nucleolin gene and is capable of promoting RNA interference of said nucleolin gene.

51. The vector of claim 50, wherein said vector is a lentiviral vector.

The vector of claim 50, wherein said vector is an adenoviral vector.

The vector of claim 50, wherein said vector is an adeno-associated viral vector.