Respiratory syncytial virus (RSV) a major viral respiratory pathogen and is the leading cause of lower respiratory tract infection in infant, young children and the elderly with immunocompromise (Collins, P. L. et at Respiratory syncytial virus. In: D. M. Knipe, P. M. Howley and D. E. Griffin, Editors, 4th ed., Fields Virology Vol. 1, Lippincott-Raven, Philadelphia, 2001, pp. 1443-1485), and is also a risk factor for the development of asthma (Behera, A. K. et al. J Biol Chem, 2002, 277:25601-25608). RSV produces an annual epidemic of respiratory illness, causing bronchitis and otitis media in infants and young children (Sigurs, N. et al. Am J Respir Crit Care Med., 2000, 161:1501-1507; Sigurs, N. et al. Pediatrics, 1995, 95:500-505) and pneumonia in adults and the elderly (Shay, D. K. et al. JAMA, 1999, 282:1440-1446; Hall, C. B. et al. Clin Infect Dis., 2001, 33:792-796). Immunodeficiency, cardiac arrhythmia, and congenital heart disease are risk factors for more severe diseases with RSV infection (Sly, P. D. et al. Pediatr. Pulmonol., 1989, 7:153-158; Brandenburg, A. H. et al. Vaccine, 2001, 19:2769-2782; Coffin, S. E. and Offit, P. A., Adv. Pediatr. Infect. Dis., 1997, 13:333-348).
Previous RSV infection does not prevent subsequent infections, even in sequential years (Bartz, H. et al. Immunology, 2003, 109:49-57). In the Unites States alone, the severe viral bronchiolitis and pneumonia results in approximately 100,000 hospitalizations and 4500 deaths in infants and young children each year (Carbonell-Estrany, X. and Quero, J. Pediatr Infect Dis J, 2001, 20:874-879; Hall, C. B. Clin Infect Dis., 2000, 31:590-596). During the period of 1991-1998, RSV was associated annually with over 17,000 deaths (Thompson. W. W. et al., JAMA, 2003, 289:179-186). To date, there are no specific antiviral treatments available. Although many different approaches are being taken to develop prophylactic vaccines, none have been licensed for public health use to prevent diseases associated with RSV infection.
RSV is the prototypic member of the Pneumovirus genus of the Paramyxoviridae family and is an enveloped nonsegmented negative-stranded RNA virus. The RSV genome of approximately 15,200 nucleotides is transcribed into 10 transcripts, which encodes 11 distinct viral proteins in the order: NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L. Three RSV envelope glycoproteins involves the fusion F protein, the attachment glycoprotein G and the small hydrophobic SH protein. An unglycosylated matrix M protein is present as an inner virion protein. And the nucleocapsid is composed of the major nucleocapsid protein N, P phosphoprotein, large L polymerase subunit and M2-1 protein. Two nonstructural proteins NS1 and NS2 are expressed from separate mRNAs encoded by the first and second genes, respectively, that follow the 44-nt leader region (Collins, P. L. et al Respiratory syncytial virus. In: D. M. Knipe, P. M. Howley and D. E. Griffin, Editors, 4th ed., Fields Virology Vol. 1, Lippincott-Raven, Philadelphia, 2001, pp. 1443-1485; Collins, P. L. and Wertz, G. W. Virology, 1985, 143:442-451). As their promoter-proximal location, these two mRNAs are the most abundant of the RSV transcripts in a linear start-stop-restart mode (Collins, P. L. et al Respiratory syncytial virus. In: D. M. Knipe, P. M. Howley and D. E. Griffin, Editors, 4th ed., Fields Virology Vol. 1, Lippincott-Raven, Philadelphia, 2001, pp. 1443-1485). Deletion of either NS gene severely attenuates RSV infection in vivo and in vitro, indicating that NS proteins play an important role in viral replication cycle (Jin, H. et al. Virology, 2000, 273:210-208; Teng, M. N. and Collins, P. L. J Virol, 1999, 73:466-473; Teng, M. N. et al. J Virol, 2000, 74:9317-9321; Murphy, B. R. and Collins, P. L. J Clin Invest., 2002, 110:21-27).
Clinical studies have shown that RSV infection in infants is associated with a predominantly Th2-like response (Roman, M. et al. Am J Respir Crit Care Med., 1997, 156:190-195). Hence, RSV is considered a predisposing factor for the development of allergic diseases and asthma (Matsuse, H. et al. J Immunol., 2000, 164:6583-6592; Behera, A. K. et al., Hum. Gene Ther., 2002, 13:1697-1709).
Interferons (IFNs) attenuate RSV replication and also have therapeutic value against allergic diseases, including asthma (Kumar, M. et al. Vaccine, 1999, 18:558-567; Kumar, M. et al. Human Gene Ther., 2002, 13:1415-1425; Kumar, M. et al. Genetic Vaccines and Ther., 2003, 1:3-12). In addition, in vivo intranasal gene delivery approaches have been developed using nanoparticles composed of chitosan, a natural, biocompatible, and biodegradable polymer (Kumar, M. et al. Human Gene Ther., 2002, 13:1415-1425; Kumar, M. et al. Genetic Vaccines and Ther., 2003, 1:3-12; Mohapatra, S. S. Pediatr Infect Dis J., 2003, 22:S100-S103; Hellerman, G. and Mohapatra, S. S. Genetic Vaccines and Ther., 2003, 1:1-3). Since bovine and human RSV NS1 appear to antagonize the Type-I interferon-mediated antiviral response (Bossert, B. and Conzelmann, K. K. J. Virol., 2002, 76:4287-4293; Bossert, B. et al. J. Virol., 2003, 77:8661-8668; Schlender, J. et al. J. Virol., 2000, 74:8234-8242; Spann, K. M. et al. J. Virol., 2004, 78:4363-4369), it was reasoned that blocking NS gene expression might attenuate RSV replication and provide an effective antiviral and immune enhancement therapy.
A naturally occurring gene-silencing mechanism triggered by double-stranded RNA (dsRNA), designated as small interfering RNA (siRNA), has emerged as a very important tool to suppress or knock down gene expression in many systems. RNA interference is triggered by dsRNA that is cleaved by an RNAse-III-like enzyme, Dicer, into 21-25 nucleotide fragments with characteristic 5′ and 3′ termini (Provost, P. D. et al. Embo J, 2002, 21:5864). These siRNAs act as guides for a multi-protein complex, including a PAZ/PIWI domain containing the protein Argonaute2, that cleaves the target mRNA (Hammond, S. M. et al. Science, 2001, 293:1146-1150). These gene-silencing mechanisms are highly specific and potent and can potentially induce inhibition of gene expression throughout an organism. The short interference RNA (siRNA) approach has proven effective in silencing a number of genes of different viruses (Fire, A. Trends Genet., 1999, 15:358-363).
RNA interference (RNAi) is a polynucleotide sequence-specific, post-transcriptional gene silencing mechanism effected by double-stranded RNA that results in degradation of a specific messenger RNA (mRNA), thereby reducing the expression of a desired target polypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164; U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp, Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001); Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated by double-stranded polynucleotides, such as double-stranded RNA (dsRNA), having sequences that correspond to exonic sequences encoding portions of the polypeptides for which expression is compromised. RNAi reportedly is not effected by double-stranded RNA polynucleotides that share sequence identity with intronic or promoter sequences (Elbashir et al., 2001). RNAi pathways have been best characterized in Drosophila and Caenorhabditis elegans, but “small interfering RNA” (siRNA) polynucleotides that interfere with expression of specific polynucleotides in higher eukaryotes such as mammals (including humans) have also been considered (e.g., Tuschl, 2001 Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al., 2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science 296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001 Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107).
According to a current non-limiting model, the RNAi pathway is initiated by ATP-dependent, cleavage of long dsRNA into double-stranded fragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, etc.) nucleotide base pairs in length, called small interfering RNAs (siRNAs) (see review by Hutvagner et al., Curr. Opin. Gen. Dev. 12:225-32 (2002); Elbashir et al., 2001; Nyknen et al., Cell 107:309-21 (2001); Zamore et al., Cell 101:25-33 (2000)). In Drosophila, an enzyme known as “Dicer” cleaves the longer double-stranded RNA into siRNAs; Dicer belongs to the RNase III family of dsRNA-specific endonucleases (WO 01/68836; Bernstein et al., Nature 409:363-66 (2001)). Further, according to this non-limiting model, the siRNA duplexes are incorporated into a protein complex, followed by ATP-dependent unwinding of the siRNA, which then generates an active RNA-induced silencing complex (RISC) (WO 01/68836). The complex recognizes and cleaves a target RNA that is complementary to the guide strand of the siRNA, thus interfering with expression of a specific protein (Hutvagner et al., supra).
In C. elegans and Drosophila, RNAi may be mediated by long double-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire et al., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO 01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells, however, transfection with long dsRNA polynucleotides (i.e., greater than 30 base pairs) leads to activation of a non-specific sequence response that globally blocks the initiation of protein synthesis and causes mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfection of human and other mammalian cells with double-stranded RNAs of about 18-27 nucleotide base pairs in length interferes in a sequence-specific manner with expression of particular polypeptides encoded by messenger RNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164; Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001)); Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr. Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol. Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 Nature Cell Biol. 4:317).
siRNA polynucleotides may offer certain advantages over other polynucleotides known to the art for use in sequence-specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product. These advantages include lower effective siRNA polynucleotide concentrations, enhanced siRNA polynucleotide stability, and shorter siRNA polynucleotide oligonucleotide lengths relative to such other polynucleotides (e.g., antisense, ribozyme or triplex polynucleotides). By way of a brief background, “antisense” polynucleotides bind in a sequence-specific manner to target nucleic acids, such as mRNA or DNA, to prevent transcription of DNA or translation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat. No. 5,190,931; U.S. Pat. No. 5,135,917; U.S. Pat. No. 5,087,617; see also, e.g., Clusel et al., 1993 Nucl. Acids Res. 21:3405-11, describing “dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides can be targeted to any RNA transcript and are capable of catalytically cleaving such transcripts, thus impairing translation of mRNA (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S. Ser. No. 2002/193579). “Triplex” DNA molecules refers to single DNA strands that bind duplex DNA to form a colinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Pat. No. 5,176,996, describing methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Such triple-stranded structures are unstable and form only transiently under physiological conditions. Because single-stranded polynucleotides do not readily diffuse into cells and are therefore susceptible to nuclease digestion, development of single-stranded DNA for antisense or triplex technologies often requires chemically modified nucleotides to improve stability and absorption by cells. siRNAs, by contrast, are readily taken up by intact cells, are effective at interfering with the expression of specific polynucleotides at concentrations that are several orders of magnitude lower than those required for either antisense or ribozyme polynucleotides, and do not require the use of chemically modified nucleotides.
Due to its advantages, RNAi has been applied as a target validation tool in research and as a potential strategy for in vivo target validation and therapeutic product development (Novina, C. D. and Sharp, P. A., Nature, 2004, 430:161-164). In vivo gene silencing with RNAi has been reported using viral vector delivery and high-pressure, high-volume intravenous (i.v.) injection of synthetic iRNAs (Scherr, M. et al. Oligonucleotides, 2003, 13:353-363; Song, E. et al. Nature Med., 2003, 347-351). In vivo gene silencing has been reported after local direct administration (intravitreal, intranasal, and intrathecal) of siRNAs to sequestered anatomical sites in various models of disease or injury, demonstrating the potential for delivery to organs such as the eye, lungs, and central nervous system (Reich, S. J. et al. Mol. Vis., 2003, 9:210-216; Zhang, X. et al. J. Biol. Chem., 2004, 279:10677-10684; Dorn, G. et al. Nucleic Acids Res., 2004, 32, e49). Silencing of endogenous genes by systemic administration of siRNAs has also been demonstrated (Soutschek, J. et al. Nature, 2004, 432:173-178).