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WO2007127487A2 - Materials and methods for reducing inflammation by inhibition of the atrial natriuretic peptide receptor - Google Patents

Materials and methods for reducing inflammation by inhibition of the atrial natriuretic peptide receptor Download PDF

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Publication number
WO2007127487A2
WO2007127487A2 PCT/US2007/010572 US2007010572W WO2007127487A2 WO 2007127487 A2 WO2007127487 A2 WO 2007127487A2 US 2007010572 W US2007010572 W US 2007010572W WO 2007127487 A2 WO2007127487 A2 WO 2007127487A2
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WIPO (PCT)
Prior art keywords
npra
polynucleotide
cells
sirna
subject
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PCT/US2007/010572
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French (fr)
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WO2007127487A3 (en
Inventor
Shyam S. Mohapatra
Weidong Xu
Xiaoyuan Kong
Xiaoqin Wang
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University Of South Florida
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Priority to CA002656990A priority Critical patent/CA2656990A1/en
Priority to EP07794463A priority patent/EP2051585A4/en
Publication of WO2007127487A2 publication Critical patent/WO2007127487A2/en
Publication of WO2007127487A3 publication Critical patent/WO2007127487A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • NP natriuretic hormone peptides
  • Atrial natriuretic factor a 126 amino acid prohormone gives rise to four peptides: long acting natriuretic peptide (LANP, amino acids 1-30), vessel dilator (VD, residues 31-67), kaliuretic peptide (KP, residues 79-98) and atrial natriuretic peptide (ANP, residues 99-126, also referred to here as NP99-126) (Vesely, DL Cardiovasc Res, 2001 51 :647-58).
  • renal tubular cells produce urodilatin, a 32 amino acid peptide (residues 95-126 of ANF), which is realeased to circulation following differential processing of ANF (Forssmann et al. Cardiovassc Res, 2001,51 :450-62.).
  • BNP pro-brain natriuretic peptide
  • the third type of natriuretic hormone, the C-type (CNP) comprises two peptides, 53 and 22 amino acids in length, which are produced by many cell types (Levin, ER et al. N Eng J Med, 1998, 339321-8). Of these peptides, the C-terminal pro-ANF, ANP, has been studied most extensively.
  • NP receptors there are three NP receptors (Misono, KS MoI Cell Biochem, 2002, 230(l-2):49-60; Tremblay, J et al. MoI Cell Biochem, 2002, 230(l-2):31-47).
  • NPRa and NPRb which are coupled to guanylyl cyclase, and the cGMP-independent receptor NPRc.
  • ANP and BNP signal primarily through NPRa, which increases cGMP and activates cGMP-dependent protein kinase (PKG).
  • NPRC functions as a clearance receptor but also appears to signal phospholipase C activation and a decrease in adenylyl cyclase activity (Silberbach, M et al Cell Signal, 2001 13:221-31). Numerous tissues of various organ systems including the lung express these receptors in diverse cells.
  • the NPs are produced in various tissues of the mucosa (lung, gastrointestinal and genitourinary systems), cental nervous system and cardiovascular systems and released into the circulation.
  • the signaling mechanisms underlying ANP' s growth inhibitory effects are poorly understood, although a number of reports suggest that ANP affects signaling via activation of mitogen-activated protein kinases (Silberbach, M et al. Cell Signal, 2001 13:221-31).
  • the potential effects may include inhibition of ERK activation of epidermal growth factor, PKG-induced uncoupling of Ras/Rafl interaction, or induction of MKP- 1 , a MAPK phosphatase that inactivates signaling through a number of growth factors such as endothelin, EGF and FGF (Clark, AR J Endocrinol, 2003, 178: 5- 12).
  • ANP has been shown to mediate anti-inflammatory (Kiemer, AK and Vollmar J Biol Chem, 1998 273:134444-51) and cytoprotective (Kiemer, AK et al, J Immunol, 2000, 165:175-81; Sprenger, H et al, Immunobiology, 1991, 183:94-101) effects. It has been shown to decrease cytokine and stress stimulated activation of NFKB in various cell types, leading to a decrease in pro-inflammatory cytokine production (Kiemer, AK and Vollmar J 5/ ⁇ / Chem, 1998 273:134444-51; Kiemer, AK et al., J Immunol, 2000, 165:175-81; Morita.
  • ANP can reduce tumor necrosis factor- ⁇ (TNF- ⁇ )-stimulated production of adhesion molecules in endothelium.(Kiemer, AK and Vollmar J Biol Chem, 1998 273:134444-51). It has also been shown to attenuate TNF- ⁇ - induced actin polymerization, through activation of MAPK phoshatase-1 (MKP-I) and inhibition of p38 activity, leading to decreased permeability (Clark, AR J Endocrinol, 2003, 178(1):5-12).
  • TNF- ⁇ tumor necrosis factor- ⁇
  • ANP stimulates migration of human neutrophils (Izumi, T et al. J Clin Invest, 2001, 108(2):203-21345), and inhibits nitric oxide (NO) and TNF-a production by murine macrophages (Vesely, DL et al Chest, 1990, 97(6): 1295- 1298, Vesely, DL Am J Obstet Gynecol, 1991, 165(3):567-573).
  • Human peripheral blood monocytes do not express ANP receptors nor do they respond to ANP (Sprenger, H et al. Jmmunobiology, 1991, 183(l-2):94-101).
  • the NP system acting via cells of the innate immune system, modulates the immune response to antigens.
  • 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 5 2002, 21 :5864).
  • 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).
  • siRNA short interference RNA
  • RNA interference 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. Patent No. 6,506,559; Fire et al, Nature 391:806-11 (1998); Sharp, Genes Dev. 13: 139-41 (1999); Elbashir et al Nature 41 1. -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.
  • dsRNA double-stranded RNA
  • 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 investigated (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 Na/. Struct. Biol. 8:746; Matzke et al, 2001 Science 293:1080; Scadden et al, 2001 EMBO Rep. 2:1107).
  • siRNA small interfering RNA
  • 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)).
  • siRNAs small interfering RNAs
  • Dicer 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).
  • RISC RNA-induced silencing complex
  • 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. ScL USA 97:6499-6503 (2000); Kisielow et al, Biochem. J. 363:1-5 (2002); see also WO 01/92513 (RNAi-mediated silencing in yeast)).
  • dsRNA polynucleotides i.e., greater than 30 base pairs
  • 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.
  • mRNA messenger RNAs
  • siRNA polynucleotides may offer certain advantages over other polynucleotides known in the art for use in sequence- specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product.
  • siRNA polynucleotide oligonucleotide lengths relative to such other polynucleotides (e.g., antisense, ribozyme or triplex polynucleotides).
  • 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. Patent No. 5,168,053; U.S. Patent No. 5,190,931; U.S. Patent No.
  • 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. Patent 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 anti sense 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 in vitro and as a potential strategy for in vivo target validation and therapeutic product development (Novina, CD. and Sharp, P.A., Nature, 2004, 430:161-164; Lieberman, J. et al Trends MoI. Med., 2003, 9(9):397-403).
  • RNAi In vivo gene silencing with RNAi has been reported using viral vector delivery, liposomal delivery, and high- pressure, high-volume intravenous (i.v.) injection of synthetic iRNAs (Haider, J. et al Clin. Cancer Res., 2006, 12( 16) :4916-4924; Landen, CN. et al, Cancer Biol. Ther., 2006, 5(12):1708-1713; Scherr, M. et al Oligonucleotides, 2003, 13:353-363; Song, E. et al. Nature Med, 2003, 347-351).
  • siRNAs Silencing of endogenous genes by systemic administration of siRNAs has also been demonstrated (Zimmerman, T.S. et al, Nature, 2006, 441(7089):l l 1-114; Soutschek, J. et al Nature, 2004, 432:173-178).
  • ANP small interference RNA
  • siRNA small interference RNA
  • the present invention pertains to a method for treating inflammatory diseases, respiratory allergies, such as allergic rhinitis and asthma, viral infections, and cancers using a polynucleotide (e.g., an siRNA, antisense nucleotide sequence, and/or ribozyme) or other agent that reduces expression of the atrial natriuretic peptide receptor A, NPRA, or otherwise reduces activity of the receptor (referred to herein as an NPRA inhibitor).
  • a polynucleotide e.g., an siRNA, antisense nucleotide sequence, and/or ribozyme
  • NPRA inhibitor e.g., an siRNA, antisense nucleotide sequence, and/or ribozyme
  • the method of the present invention comprises administering a therapeutically effective amount of an NPRA inhibitor to a subject in need of such treatment.
  • the NPRA inhibitor is an interfering RNA molecule, such as siRNA, specifically targeted to NPRA.
  • siRNA interfering RNA molecule
  • NHP refers to atrial natriuretic factor (ANF) hormone, or a biologically active fragment or homolog thereof.
  • siRNAs comprise an oligonucleotide sequence deduced from selected nucleotide sequence from the NPRA gene.
  • the siRNA is targeted to a sequence within the mRNA sequence encoded by SEQ ID NO:4.
  • the method of the present invention comprises administering an effective amount of at least one nucleic acid molecule encoding an siRNA specifically targeted to NPRA (siNPRA) to a patient in need of such treatment.
  • siNPRA siRNA specifically targeted to NPRA
  • the present inventor has determined that introduction of a nucleic acid molecule encoding siNPRA is capable of inhibiting NPRA expression when introduced via a plasmid vector or a virus, in association with a desirable carrier molecule such as a lipid or polymer-based system.
  • the siRNA delivery method of the present invention permits long-term expression of siNPRA encoding nucleic acid sequences in vivo, thereby conferring bronchoprotective effect and/or anti-inflammatory effect against respiratory allergies, such as asthma.
  • the siNPRA is targeted to a sequence within the mRNA sequence encoded by SEQ ID NO:4.
  • a therapeutically effective amount of at least one nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or biologically active homologs of any of the foregoing are administered to the subject.
  • the nucleic acid molecule(s) can be administered with other inhibitors of NPRA and/or other agents having therapeutic efficacy in treatment of the disease.
  • the present invention concerns synthetic oligonucleotide having the sequence that acts as the interfering RNA (SEQ ID NOs: 1-3) or a biologically active homolog of the foregoing.
  • the present invention concerns a pharmaceutical composition comprising a nucleic acid sequence encoding an siNPRA and a pharmaceutically acceptable carrier, which can be administered by an accepted route.
  • Figures IA and IB show a diagram depicting that overexpression of ANP in the lung augments inflammation and cytokine production in splenocytes.
  • FIG. 1 shows cloning of siNPRA sequences in the pU6 vector.
  • the siNPRA sequences were designed as shown in Sequence IDs and cloned in pSilencer 1(U6) vector using standard procedures. The transformants were tested by digestion with Apa I and EcoR I to release the siRNA inserts. Lanel, lOObp ladder; lane 2:pSilencerl(U6), Lane3- 6, siNPRA ⁇ , Lane7-10, siNPRA9 are shown for illustration.
  • Figures 3 A-3C show the inhibitor y effect of transfected siRNA plasmids on
  • NPRA expression HEKGCA cells grown in 6-well plates were transfected with psiNPRA (2ug). Forty eight hours later, total protein was extracted and Western blotted using an antibody to NPRA. Plasmids encoding ANP, Kp73-102 and VD were used as controls, since they have been shown to downregulate NPRA expression.
  • HEKGCA cells grown in 6-well plates were transfected with psiNPRA (2ug), as indicated and forty eight hours later total protein were extracted western blotted using an antibody to NPRA (Figure 3C). Untransfected cells and cells transfected with U6 vector plasmid without any siNPRA were used as control. Also, filters were stripped and reprobed with antibody to beta-actin.
  • Figures 4 A and 4B show inhibitory effect of siRNA in vitro and in vivo.
  • FIGS 5 A, 5B-1, and 5B-2 show that SiNPRA treatment appears to reduce cytokine production in BALB/c mice.
  • cytokine secreting cells were treated with GolgiStop (BD Pharmingen) and stained for surface and intracellular cytokines (Antibodies obtained from BD Pharmingen). Percent cytokine secreting cells were quantified by intracellular cytokine staining using flow cytometry.
  • FIGS. 6A and 6B show that administration of siNPRA decreases inflammation of the lung in BALB/c mice.
  • Figures 7A-7C show that administration of siNPRA ⁇ by the transdermal route decreases NPRA expression, eosinophilia of the lung and BAL IL-4 cytokine.
  • FIG. 8 A and 8B show that administration of siNPRA decreases inflammation of the lung in BALB/c mice.
  • Figure 9 shows that administration of siNPRA inhibits NPRA expression in the respiratory syncytial virus (RSV) infected lung.
  • psiNPRA9 was encapsulated with chitosan nanoparticles and intranasally delivered to mice. Twenty-four hours later, mice were infected with RSV (5x10 6 pfu/mouse). Four days later, mice were sacrificed and lung were collected for RNA extraction.
  • NPRA fragment were amplified by RT-PCR and analyzed in 1% agarose gel.
  • Figures 1OA and 1OB show that administration of siNPRA inhibits the Respiratory syncytial virus infection of A549 cells.
  • FIG. 11 shows that NPRA-deficient mice are resistant to melanoma tumor formation and metastasis in the B 16 mouse model.
  • B 16 melanoma cells (1.3x10 5 ) were injected subcutaneously into twelve-week-old female C57BL/6 mice and NPRA-deficient mice. Mice were observed for tumor formation for one month, then sacrificed on day-22. Tumors were then removed and weighed.
  • Figures 12A-12E show that siNPRA treatment decreases melanoma tumor formation in bl6 mouse model.
  • Bl 6 melanoma cells (1.3x10 5 ) were injected subcutaneously into twelve-week old female C57BL/6 mice.
  • B16 melanoma cells The tumor size (Figure 13A) over several days post injection and tumor burden (Figure 13B) at day 18 were measured.
  • Figure 13C shows that siNPRA treatment decreases melanoma tumor formation in the B16 mouse model.
  • B16 melanoma cells (1.3xlO 5 ) were injected subcutaneously into twelve-week old female C57BL/6 mice. These mice were then treated with 33 ⁇ g of siNPRA-oligos, siNPRA plasmid, or scrambled oligos. All of these were mixed with chitosan at a ratio of 1:2.5. Mixed chitosan and plasmid or oligos were mixed again with cream before application to the injection area. The control group was given cream only. These treatments were given twice a week. Mice were sacrificed on day-22, and tumors were removed and weighed.
  • Figures 14A and 14B show that siNPRA treatment decreases lewis lung carcinoma.
  • Figure 15 shows that siNPRA treatment decreases ovarian cancer.
  • SEQ ID NO:1 is the nucleotide sequence of an siRNA for NPRA (siNPRAlV. (targeting position 33): 5'-CAT ATG ggg ccc GGG CGC TGC TGC TGC TAC Cct cga aat GGT AGC AGC AGC GCC CTT gaa ttc CCA TGG-3'.
  • SEQ ID NO:2 is the nucleotide sequence of an siRNA for NPRA (siNPRA2) (targeting position 72): 5'-CAT ATG ggg ccc GCG GCC ACG CGA GCG ACC Tct cga aat AGG TCG CTC GCG TGG CCG CTTgaa ttc CCA TGG-3'.
  • SEQ ID NO:3 is the nucleotide sequence of an siRNA for NPRA (siNPRA3): (targeting position 33)siNPRA187top (silO): 5'-CAT ATG ggg ccc GGC TCG GCC GGA CTT GCT Get cga aat CAG CAA GTC CGG CCG AGC CTT gaa ttc CCA TGG-3'.
  • SEQ ID NO:4 is the nucleotide sequence encoding human NPRA (NCBI Accession # AF190631:
  • the present invention provides a method for reducing atrial natriuretic peptide receptor A (NPRA) gene expression and/or function within a subject by administering an effective amount of an NPRA inhibitor to the subject.
  • the NPRA inhibitor is a polynucleotide that is specific for one or more target NPRA genes such that the polynucleotide decreases NPRA gene expression within the subject.
  • the NPRA inhibitor is a chemical compound, such as an oxindole (e.g., isatin).
  • the method of the invention is useful for treating inflammatory diseases in human subjects and non-human subjects suffering from, or at risk for developing, inflammatory reactions.
  • Embodiment 1 an isolated polynucleotide targeted to a target nucleic acid sequence within a natriuretic peptide receptor A (NPRA) gene or NPRA transcript, wherein said polynucleotide inhibits expression of said NPRA gene or transcript.
  • NPRA natriuretic peptide receptor A
  • Embodiment 2 the polynucleotide of embodiment 1, wherein the NPRA is human NPRA (e.g., encoded by SEQ ID NO:4).
  • Embodiment 3 the polynucleotide of embodiment 1, wherein the target nucleic acid sequence is at least a portion of the human NPRA gene or transcript.
  • Embodiment 4 the polynucleotide of any of embodiments 1 to 3, wherein the target nucleic acid sequence is located in a region selected from the group consisting of the 5' untranslated region GJTR), transcription start site, translation start site, and 3' UTR.
  • Embodiment 5 the polynucleotide of any of embodiments 1 to 4, wherein the polynucleotide is a small interfering RNA (siRNA).
  • Embodiment 6 the polynucleotide of any of embodiments 1 to 4, wherein the polynucleotide is an antisense molecule.
  • Embodiment 7 the polynucleotide of any of embodiments 1 to 4, wherein the polynucleotide is a ribozyme.
  • Embodiment 8 the polynucleotide of embodiment 1, wherein the polynucleotide comprises SEQ ID NO: 1 , or SEQ ID NO:2, or SEQ ID No:3.
  • Embodiment 9 the polynucleotide of embodiment 1 , wherein the NPRA gene or NPRA transcript is at least a portion of the mammal gene or transcript.
  • Embodiment 10 a method for reducing NPRA function in a subject, comprising administering an NPRA inhibitor to the subject, such as the polynucleotide of any of embodiments 1 to 9, wherein the polynucleotide is administered in an effective amount to reduce expression of the NPRA gene or transcript.
  • an NPRA inhibitor such as the polynucleotide of any of embodiments 1 to 9, wherein the polynucleotide is administered in an effective amount to reduce expression of the NPRA gene or transcript.
  • Embodiment 11 the method of embodiment 10, wherein the subject is suffering from an inflammatory disease, respiratory allergy, viral infection (such as respiratory virus infection), or cancer (such as melanoma, lung cancer, or ovarian cancer).
  • Embodiment 12 the method of embodiment 10, wherein the subject is not suffering from an inflammatory disease, respiratory allergy, viral infection, or cancer.
  • Embodiment 13 the method of any one of embodiments 10 to 12, wherein the subject is human.
  • Embodiment 14 the method of any one of embodiments 10 to 12, wherein the subject is a non-human mammal.
  • Embodiment 15 the method of any one of embodiments 10 to 14, wherein the NPRA inhibitor is delivered to cells within the subject selected from the group consisting of respiratory epithelial cells, dendritic cells, and monocytes.
  • Embodiment 16 the method of any one of embodiments 10 to 15, wherein the NPRA inhibitor is administered to the subject intranasally.
  • Embodiment 17 the method of any one of embodiments 10 to 16, wherein the NPRA inhibitor is administered intranasally as drops or as an aerosol, or orally or transdermally.
  • Embodiment 18 the method of any one of embodiments 10 to 17, wherein step of administering comprises administering a combination of NPRA inhibitors that reduce the function of NPRA within the subject (such as a combination of polynucleotide, e.g., an siRNA pool).
  • a combination of NPRA inhibitors that reduce the function of NPRA within the subject (such as a combination of polynucleotide, e.g., an siRNA pool).
  • Embodiment 19 the method of any one of embodiments 10 to 18, wherein the NPRA inhibitor is a siRNA and wherein the siRNA reduces expression of NPRA within the subject
  • Embodiment 20 the method of any one of embodiments 10 to 18, wherein the NPRA inhibitor is an oxindole, such as 5-hydroxyoxindole or isatin, or a pharmaceutically acceptable salt thereof (Cane, A. et al. Biochem. Biophy. Res Comm, 2000, 276:379-384; Vine, K.L. et al. Bioorg Med Chem, 2007, 15(2):931-938; Abadi, A.H. et al. Eur J Med Chem, 2006, 41(3):296-305; Igosheva, N. et al. Neurochem Int, 2005, 47(3):216-224; Liu, Y. et al.
  • the NPRA inhibitor is an oxindole, such as 5-hydroxyoxindole or isatin, or a pharmaceutically acceptable salt thereof
  • Isatin also known as lH-indole-2 9 3-dione
  • Isatin is an indole derivative (Sumpter, W.C. Chem Rev, 34(3):393-434; Ogata, A. et al J Neurol Sci, 2003, 206(l):79-83; Glover, V. et al. J Neurochem, 1988, 51(2):656-659; Filomeni, G. et al J Biol Chem, 2007, 282(16):12010- 12021).
  • nucleoside refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar.
  • exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine.
  • nucleotide refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety.
  • nucleotides include nucleoside monophosphates, diphosphates and triphosphates.
  • polynucleotide and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3' carbon atoms.
  • RNA or “RNA molecule” or “ribonucleic acid molecule” refers generally to a polymer of ribonucleotides.
  • DNA or “DNA molecule” or deoxyribonucleic acid molecule” refers generally to a polymer of deoxyribonucleotides.
  • DNA and RNA molecules can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA molecules can be post- transcriptionally modified. DNA and RNA molecules can also be chemically synthesized. DNA and RNA molecules can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively).
  • RNA or “RNA molecule” or “ribonucleic acid molecule” can also refer to a polymer comprising primarily (i.e., greater than 80% or, preferably greater than 90%) ribonucleotides but optionally including at least one non-ribonucleotide molecule, for example, at least one deoxyribonucleotide and/or at least one nucleotide analog.
  • nucleotide analog also referred to herein as an "altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non- naturally occurring ribonucleotides or deoxyribonucleotides. Preferred nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function.
  • RNA analog refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA.
  • the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages.
  • Exemplary RNA analogues include sugar- and/or backbone- modified ribonucleotides and/or deoxyribonucleotides.
  • alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA).
  • RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate (mediates) RNA interference or otherwise reduce target gene expression.
  • flanking sequence operably-linked refers to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function.
  • a flanking sequence operably-linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence.
  • a coding sequence is operably-linked to a promoter when the promoter is capable of directing transcription of that coding sequence.
  • a flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably-linked" to the coding sequence.
  • Each nucleotide sequence coding for a siRNA will typically have its own operably-linked promoter sequence.
  • vector or “vehicle” is used to refer to any molecule (e.g. , nucleic acid, plasmid, or virus) used to transfer coding information (e.g., a polynucleotide of the invention) to a host cell.
  • expression vector refers to a vector that is suitable for use in a host cell (e.g., a subject's cell) and contains nucleic acid sequences which direct and/or control the expression of exogenous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
  • the vectors of the present invention can be conjugated with chitosan or chitosan derivatives.
  • chitosan conjugates can be administered to hosts according to the methods of the present invention.
  • polynucleotide chitosan nanospheres can be generated, as described by Roy, K. et ⁇ l. (Nat Med, 1999, 5:387).
  • Chitosan allows increased bioavailability of the nucleic acid sequences because of protection from degradation by serum nucleases in the matrix and thus has great potential as a mucosal gene delivery system.
  • Chitosan also has many beneficial effects, including anticoagulant activity, wound-healing properties, and immunostimulatory activity, and is capable of modulating immunity of the mucosa and bronchus-associated lymphoid tissue.
  • the vectors are conjugated with chitosan-derived nanoparticles.
  • RNA interference refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.
  • small interfering RNA refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.
  • RNAi target-specific RNA interference
  • mRNA single-stranded RNA that specifies the amino acid sequence of one or more RSV polypeptides. This information is translated during protein synthesis when ribo somes bind to the mRNA.
  • cleavage site refers to the residues, e.g. , nucleotides, at which RISC* cleaves the target RNA, e.g., near the center of the complementary portion of the target RNA, e.g., about 8-12 nucleotides from the 5' end of the complementary portion of the target RNA.
  • mismatch refers to a basepair consisting of non- complementary bases, e.g., not normal complementary G:C, A:T or A:U base pairs.
  • isolated molecule e.g., isolated nucleic acid molecule
  • the term “isolated” molecule refers to molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • the NPRA inhibitors of the invention are administered in an isolated form.
  • in vitro has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts.
  • in vivo also has its art recognized meaning, e.g., involving living cells in an organism, e.g., immortalized cells, primary cells, and/or cell lines in an organism.
  • a gene "involved in” or “associated with” a disorder includes a gene, the normal or aberrant expression or function of which affects or causes a disease or disorder or at least one symptom of the disease or disorder.
  • NPRA protein has been found to have a significant role in pulmonary inflammation and immune modulation. Without being bound by theory, it has been found that signaling through the NPRA protein results in increased cGMP production and activation of protein kinase G 3 leading to regulation of transcription of many genes involved in the cell cycle, apoptosis, and inflammation.
  • polynucleotides, genetic constructs, pharmaceutical compositions, and methods of the invention are useful in decreasing expression of NPRA gene, in vitro or in vivo, consequently causing decreased production of the NPRA protein and decreased inflammation.
  • the polynucleotides, genetic constructs, pharmaceutical compositions, and methods of the invention are useful in the treatment of human or non- human animal subjects suffering from, or at risk of developing, disorders associated with inflammation including, but not limited to, airway diseases, viral infections, and cancers.
  • a subject with the relevant condition or disease involving aberrant inflammation is identified, or a subject at risk for the condition or disease is identified.
  • a subject may be someone who has not been diagnosed with the disease or condition (diagnosis, prognosis, and/or staging) or someone diagnosed with the disease or condition (diagnosis, prognosis, monitoring, and/or staging), including someone treated for the disease or condition (prognosis, staging, and/or monitoring).
  • the subject may not have been diagnosed with the disease or condition but suspected of having the disease or condition based either on patient history or family history, or the exhibition or observation of characteristic symptoms.
  • an "effective amount" of a NPRA inhibitor e.g., isatin or another oxindole, an siRNA, an antisense nucleotide sequence or strand, and/or a ribozyme
  • an "effective amount" of a NPRA inhibitor is that amount effective to bring about the physiological changes desired in the cells to which the polynucleotide is administered in vitro (e.g., ex vivo) or in vivo.
  • NPRA inhibitor e.g., isatin or other oxindole, an siRNA, an antisense oligonucleotide, and/or a ribozyme
  • NPRA inhibitor e.g., isatin or other oxindole, an siRNA, an antisense oligonucleotide, and/or a ribozyme
  • a NPRA inhibitor can be administered to a subject in combination with other agents effective for alleviating or preventing the symptoms of inflammation, such as the gene expression vaccines (Mohapatra et al 2004).
  • RNAi methodology a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing a siRNA of the invention into a cell or organism.
  • a "suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits.
  • a "suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.
  • RNA Interference RNAi is an efficient process whereby double-stranded RNA (dsRNA, also referred to herein as siRNAs or ds siRNAs, for double-stranded small interfering RNAs) induces the sequence-specific degradation of targeted mRNA in animal and plant cells (Hutvagner and Zamore, Curr. Opin. Genet. Dev., 12:225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001). In mammalian cells, RNAi can be triggered by 21 -nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al, MoI.
  • siRNA small interfering RNA
  • RNA polymerase III promoters Zeng et al, MoI Cell 9:1327-1333 (2002); Paddison et al, Genes Dev. 16:948-958 (2002); Lee et al, Nature Biotechnol.
  • the invention includes such molecules that are targeted to NPRA mRNAs encoding at least a portion of one or more of NPRA-Iike receptors.
  • the nucleic acid molecules or constructs of the invention include dsRNA molecules comprising 16-30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA of the RSV mRNA, and the other strand is identical or substantially identical to the first strand.
  • dsRNA molecules comprising 16-30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or
  • the dsRNA molecules of the invention can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from, e.g., shRNA.
  • the dsRNA molecules can be designed using any method known in the art, for instance, by using the following protocol:
  • the invention includes polynucleotides having 35-55% G/C content.
  • the strands of the siRNA can be paired in such a way as to have a 3' overhang of 1 to 4, e.g., 2, nucleotides.
  • the polynucleotides can have a 3' overhang of 2 nucleotides.
  • the overhanging nucleotides can be either RNA or DNA.
  • BLAST BLAST sequence homology search
  • siRNA Select one or more sequences that meet your criteria for evaluation. Further general information regarding the design and use of siRNA can be found in "The siRNA User Guide,” available at the web site of the laboratory of Dr. Thomas Tuschl at Rockefeller University.
  • Negative control siRNAs preferably have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.
  • the polynucleotides of the invention can include both unmodified siRNAs and modified siRNAs as known in the art. Thus, the invention includes siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked.
  • a 3' OH terminus of one of the strands can be modified, or the two strands can be crosslinked and modified at the 3' OH terminus.
  • the siRNA derivative can contain a single crosslink (e.g., a psoralen crosslink).
  • the siRNA derivative has at its 3' terminus a biotin molecule (e.g., a photocleavable biotin), a peptide (e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organic compounds (e.g., a dye such as a fluorescent dye), or dendrimer.
  • Modifying siRNA derivatives in this way can improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.
  • the nucleic acid compositions of the invention can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability, and/or half-life.
  • the conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al, Drug Deliv. Rev. 47(1): 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al, J.
  • the nucleic acid molecules of the present invention can also be labeled using any method known in the art; for instance, the nucleic acid compositions can be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine.
  • the labeling can be carried out using a kit, e.g., the SILENCER siRNA labeling kit (AMBION).
  • the siRNA can be radiolabeled, e.g., using 3 H, 32 P, or other appropriate isotope.
  • the dsRNA molecules of the present invention can comprise the following sequences as one of their strands, and the corresponding sequences of allelic variants thereof: SEQ ID NO: 1 or SEQ ID NO:2.
  • RNAi is believed to progress via at least one single-stranded RNA intermediate
  • ss-siRNAs ⁇ e.g., the antisense strand of a ds-siRNA
  • ss-siRNAs can also be designed as described herein and utilized according to the claimed methodologies.
  • Synthetic siRNAs can be delivered into cells by methods known in the art, including cationic liposome transfection and electroporation. However, these exogenous siRNA generally show short-term persistence of the silencing effect (4 to 5 days in cultured cells), which may be beneficial in certain embodiments. To obtain longer term suppression of RSV gene expression and to facilitate delivery under certain circumstances, one or more siRNA duplexes, e.g., RSV ds siRNA, can be expressed within cells from recombinant DNA constructs.
  • Such systems for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer-term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., Hl or U6/snRNA promoter systems (Tuschl (2002), supra) capable of expressing functional double-stranded siRNAs; (Bagella et al, J. Cell. Physiol. 177:206-213 (1998); Lee et al. (2002), supra; Miyagishi et al. (2002), supra; Paul et al. (2002), supra; Yu et al. (2002), supra; Sui et al. (2002), supra).
  • mammalian Pol III promoter systems e.g., Hl or U6/snRNA promoter systems (Tuschl (2002), supra) capable of expressing functional double-stranded siRNAs; (Bagella et al, J. Cell. Physiol. 177:206-213 (
  • RNA Pol III Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence.
  • the siRNA is complementary to the sequence of the target gene in 5 '-3' and 3 '-5' orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs.
  • Hairpin siRNAs, driven by an Hl or U6 snRNA promoter can be expressed in cells, and can inhibit target gene expression (Bagella et al. (1998), supra; Lee et al (2002), supra; Miyagishi et al, (2002), supra; Paul et al (2002), supra; Yu et al. (2002), supra; Sui et al.
  • Constructs containing siRNA sequence(s) under the control of a T7 promoter also make functional siRNAs when co-transfected into the cells with a vector expressing T7 RNA polymerase (Jacque (2002), supra).
  • a single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the RSV NS 1 mRNA and/or other RSV genes, and can be driven, for example, by separate PoIIII promoter sites.
  • miRNAs animal cells express a range of non-coding RNAs of approximately 22 nucleotides termed micro RNA (miRNAs) that can regulate gene expression at the post transcriptional or translational level during animal development.
  • miRNAs are all excised from an approximately 70 nucleotide precursor RNA stem-loop, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof.
  • a vector construct that expresses the novel miRNA can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng (2002), supra).
  • micro-RNA designed hairpins can silence gene expression (McManus (2002), supra).
  • Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al. (2002), supra). Infection of HeLa cells by these recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of the recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression.
  • siRNA In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into post- implantation mouse embryos (Calegari et al, Proc. Natl, Acad. ScL USA 99(22): 14236- 40 (2002)). In adult mice, efficient delivery of siRNA can be accomplished by the "high- pressure" delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA-containing solution into animal via the tail vein (Liu (1999), supra; McCaffrey (2002), supra; Lewis, Nature Genetics 32:107-108 (2002)). Nanoparticles and liposomes can also be used to deliver siRNA into animals. Uses of Engineered RNA Precursors to Induce RNAi
  • RNA precursors introduced into cells or whole organisms as described herein, will lead to • the production of a desired siRNA molecule.
  • Such an siRNA molecule will then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage and destruction.
  • the mRNA to be targeted by the siRNA generated from the engineered RNA precursor will be depleted from the cell or organism, leading to a decrease in the concentration of the RSV protein (such as RSV NSl protein) encoded by that mRNA in the cell or organism.
  • the RNA precursors are typically nucleic acid molecules that individually encode either one strand of a dsRNA or encode the entire nucleotide sequence of an RNA hairpin loop structure.
  • an “antisense” nucleic acid sequence can include a nucleotide sequence that is complementary to a "sense" nucleic acid sequence encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to at least a portion of an RSV gene.
  • the antisense nucleic acid sequence can be complementary to an entire coding strand of a target sequence, or to only a portion thereof (for example, the RSV NSl gene and/or RSV NS2 gene, or a portion of either or both).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence within the RSV gene.
  • An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • An antisense nucleic acid sequence can be designed such that it is complementary to the entire RSV gene, but can also be an oligonucleotide that is antisense to only a portion of the RSV gene.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.
  • An antisense oligonucleotide sequence can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • an antisense nucleic acid sequence of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. , phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid sequence also can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid sequence will be of an antisense orientation to a target nucleic acid sequence of interest, described further in the following subsection).
  • antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., systemically or locally by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to RSV mRNA to thereby inhibit expression of the viral protein.
  • antisense nucleic acid molecules can be modified to target selected cells (such as respiratory epithelial cells, dendritic cells, and/or monocytes) and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter can be used.
  • the antisense oligonucleotide of the invention is an alpha-anomeric nucleic acid molecule.
  • An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al, Nucleic Acids. Res. 15:6625-6641 (1987)).
  • the antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. Nucleic Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al.
  • Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene to form triple helical structures that prevent expression of the gene in target cells. See generally, Helene, C. Anticancer Drug
  • Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA 5 ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme encoding nucleotide sequences can be introduced into cells through gene-delivery mechanisms known in the art.
  • a ribozyme having specificity for RSV RNA can include one or more sequences complementary to the nucleotide sequence of at least a portion of one or more RSV mRNA (e.g.
  • RNA RNA sequence having known catalytic sequence responsible for mRNA cleavage
  • a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the RSV mRNA, such as RSV NSl mRNA (see, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al U.S. Patent No. 5,1 16,742).
  • RSV mRNA encoding an RSV protein can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel, D. and Szostak, J. W. Science 261 :1411-1418 (1993)). Nucleic Acid Targets
  • the nucleic acid targets of the polynucleotides of the invention may be ANP receptor gene, or a portion thereof, such as NPRA, NPRB or NPRC or portion of any of the foregoing.
  • the nucleic acid target is the NPRA gene, or a portion thereof.
  • the nucleic acid target may be any location within the NPRA or transcript.
  • the nucleic acid target is located at a site selected from the group consisting of the 5' untranslated region (UTR), transcription start site, translation start site, and the 3 ' UTR.
  • UTR 5' untranslated region
  • the nucleic acid target may be located within a NPRA gene of any human or mammal.
  • the nucleic acid target is at least a portion of a non-structural NPRA gene. More preferably, the nucleic acid target is at least a portion of an NPRAgene encoding a protein.
  • the nucleic acid target is located within an NPRA that normally down-regulates host inflammation.
  • the nucleic acid target is located within the human NPRA or mammalian NPRA, selected from the group consisting of the 5' untranslated region (UTR), transcription start site, translation start site, and the 3' UTR.
  • UTR 5' untranslated region
  • the nucleic acid target may be located within a human NPRA gene (NCBI accession no. AF 190631, which is incorporated herein by reference in its entirety) or an ortholog thereof, such as a non-human, mammalian NPRA gene.
  • the polynucleotide selected for administration to the subject is preferably one targeted to a NPRA gene.
  • the nucleic acid target is preferably located within a human NPRA gene, or the nucleic acid target has sufficient homology with the human NPRA gene, so as to reduce expression of the human NPRA gene.
  • substantially identical refers to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity.
  • amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as substantially identical.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the length of the reference sequence.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity” is equivalent to amino acid or nucleic acid "homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. MoI. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at the official Accelrys web site), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at the official Accelrys web site), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • One set of parameters are a Blossurn 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other orthologs, e.g., family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. MoI. Biol. 215:403-10 (1990).
  • Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • Orthologs can also be identified using any other routine method known in the art, such as screening a cDNA library, e.g., using a probe designed to identify sequences that are substantially identical to a reference sequence.
  • compositions typically include the polynucleotide and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Supplementary active compounds can also be incorporated into the compositions.
  • Formulations (compositions) are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington 's Pharmaceutical Sciences (Martin E.W., Easton Pennsylvania, Mack Publishing Company, 19 th ed., 1995) describes formulations which can be used in connection with the subject invention.
  • a pharmaceutical composition is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g. , intravenous, intradermal, subcutaneous, oral (e.g., inhalation), nasal, topical, transdermal, transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediarninetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • Isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate or gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g. , a polynucleotide of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g. , a polynucleotide of the invention
  • dispersions are prepared by incorporating the polynucleotide into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • suitable methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as macrocrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as macrocrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the NPRA inhibitors can be delivered in the form of drops or an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays, drops, or suppositories.
  • the active compound e.g., polynucleotides of the invention
  • the active compound are formulated into ointments, salves, gels, or creams, as generally known in the art.
  • compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • the NPRA inhibitor is a polynucleotide
  • the polynucleotides can be administered by transfection or infection using methods known in the art, including but not limited to, the methods described in McCaffrey et al, Nature 418(6893):38-39 (2002) (hydrodynamic transfection)- Xia et al, Nature Biotechnol. 20(10):1006-10 (2002) (viral-mediated delivery); or Putnam, Am. J. Health Syst. Pharm. 53(2):15M60 (1996), erratum at Am. J. Health Syst. Pharm. 53(3):325 (1996).
  • the polynucleotides can also be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine.
  • methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Patent No. 6,168,587. Additionally, intranasal delivery is possible, as described in Hamajima et al, Clin. Immunol. Immunopathol 88(2):205-10 (1998).
  • Liposomes e.g., as described in U.S. Patent No. 6,472,375
  • microencapsulation can also be used.
  • Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Patent No. 6,471,996).
  • the polynucleotides of the invention are administered to the subject such that an effective amount are delivered to the respiratory epithelial cells, DC, and/or monocytes within the subject's airway, resulting in an effective amount of reduction in NPRA gene expression.
  • the polynucleotides are prepared with carriers that will protect the polynucleotide against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
  • Such formulations can be prepared using standard techniques.
  • Liposomal suspensions (including liposomes targeted to antigen-presenting cells with monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • the NPRA inhibitors of the subject invention are administered locally or systemically such that they are delivered to target cells, such as cells of the airway, e.g., airway epithelial cells, which line the nose as well as the large and small airways.
  • target cells such as cells of the airway, e.g., airway epithelial cells, which line the nose as well as the large and small airways.
  • the NPRA inhibitors of the invention be delivered to dendritic cells and/or monocytes.
  • Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compositions which exhibit high therapeutic indices can be used. While compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the dosage of such compositions generally lies within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • compositions of the invention can be administered on any appropriate schedule, e.g. , from one or more times per day to one or more times per week; including once every other day, for any number of days or weeks, e.g. , 1 day, 1 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months, 6 months, or more, or any variation thereon.
  • treatment of a subject with a therapeutically effective amount of a NPRA inhibitor can include a single treatment or can include a series of treatments.
  • Mammalian species that benefit from the disclosed methods include, but are not limited to, primates, such as apes, chimpanzees, orangutans, humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets; domesticated farm animals such as cows, buffalo, bison, horses, donkey, swine, sheep, and goats; exotic animals typically found in zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sea lions, elephant seals, otters, porpoises,
  • the terms "subject”, “host”, and “patient” are used interchangeably and intended to include such human and non-human mammalian species.
  • in vitro methods of the present invention can be carried out on cells of such mammalian species.
  • Host cells comprising exogenous polynucleotides of the invention may be administered to the subject, and may, for example, be autogenic (use of one's own cells), allogenic (from one person to another), or transgenic or xenogenic (from one species to another), relative to the subject.
  • the polynucleotides of the invention can be inserted into genetic constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et ai, (2002), supra.
  • Genetic constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g. , Chen et ai, Proc. Natl. Acad. Sci. USA 91 :3054-3057 (1994)).
  • the pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the polynucleotide delivery system.
  • the polynucleotides of the invention can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site.
  • shRNAs small hairpin RNAs
  • poly III polymerase III
  • shRNAs Upon expression, shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21 nucleotides (Brummelkamp et al, Science 296:550-553 (2002); Lee et al, (2002), supra; Miyagishi and Taira, Nature Biotechnol. 20:497-500 (2002); Paddison et al. (2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al (2002), supra.
  • SiRNAs of the invention may be fused to other nucleotide molecules, or to polypeptides, in order to direct their delivery or to accomplish other functions.
  • fusion proteins comprising a siRNA oligonucleotide that is capable of specifically interfering with expression of NPRA gene may comprise affinity tag polypeptide sequences, which refers to polypeptides or peptides that facilitate detection and isolation of the polypeptide via a specific affinity interaction with a ligand.
  • the ligand may be any molecule, receptor, counter-receptor, antibody or the like with which the affinity tag may interact through a specific binding interaction as provided herein.
  • Such peptides include, for example, poly-His or "FLAG” or the like, e.g., the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., (Bio/Technology 6:1204, 1988), or the XPRESS epitope tag (INVITROGEN, Carlsbad, Calif.).
  • the affinity sequence may be a hexa-histidine tag as supplied, for example, by a pBAD/His (INVITROGEN) or a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the affinity sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g., COS-7 cells, is used.
  • the HA tag corresponds to an antibody defined epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984 Cell 21:761).
  • the present invention also relates to vectors and to constructs that include or encode polynucleotides of the present invention (e.g., siRNA), and in particular to "recombinant nucleic acid constructs" that include any nucleic acid such as a DNA polynucleotide segment that may be transcribed to yield NPRAmRNA-specific siRNA polynucleotides according to the invention as provided above; to host cells which are genetically engineered with vectors and/or constructs of the invention and to the production of siRNA polynucleotides, polypeptides, and/or fusion proteins of the invention, or fragments or variants thereof, by recombinant techniques.
  • siRNA polynucleotides of the present invention
  • RNA polynucleotides may be engineered to produce corresponding DNA sequences using well-established methodologies such as those described herein.
  • a DNA polynucleotide may be generated from any siRNA sequence described herein, such that the present siRNA sequences will be recognized as also providing corresponding DNA polynucleotides (and their complements).
  • These DNA polynucleotides are therefore encompassed within the contemplated invention, and can, for example, be incorporated into the subject invention recombinant nucleic acid constructs from which siRNA may be transcribed.
  • a vector may comprise a recombinant nucleic acid construct containing one or more promoters for transcription of an RNA molecule, for example, the human U6 snRNA promoter (see, e.g., Miyagishi et al, Nat. Biotechnol. 20:497-500 (2002); Lee et al, Nat. Biotechnol. 20:500-505 (2002); Paul et al, Nat. Biotechnol. 20:505-508 (2002); Grabarek et al, BioTechniques 34:73544 (2003); see also Sui et al, Proc. Natl. Acad. Sci. USA 99:5515-20 (2002)).
  • the human U6 snRNA promoter see, e.g., Miyagishi et al, Nat. Biotechnol. 20:497-500 (2002); Lee et al, Nat. Biotechnol. 20:500-505 (2002); Paul et al, Nat. Bio
  • Each strand of a siRNA polynucleotide may be transcribed separately each under the direction of a separate promoter and then may hybridize within the cell to form the siRNA polynucleotide duplex.
  • Each strand may also be transcribed from separate vectors (see Lee et al, supra).
  • the sense and antisense sequences specific for an RSV sequence may be transcribed under the control of a single promoter such that the siRNA polynucleotide forms a hairpin molecule (Paul et al, supra).
  • the complementary strands of the siRNA specific sequences are separated by a spacer that comprises at least four nucleotides, but may comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 94 18 nucleotides or more nucleotides as described herein.
  • siRNAs transcribed under the control of a V6 promoter that form a hairpin may have a stretch of about four uridines at the 3' end that act as the transcription termination signal (Miyagishi et al, supra; Paul et al, supra).
  • the siRNA hairpin polynucleotide (beginning at the 5' end) has a 19-nucleotide sense sequence followed by a spacer (which as two uridine nucleotides adjacent to the 3' end of the 19-nucleotide sense sequence), and the spacer is linked to a 19 nucleotide antisense sequence followed by a 4-uridine terminator sequence, which results in an overhang.
  • siRNA polynucleotides with such overhangs effectively interfere with expression of the target polypeptide.
  • a recombinant construct may also be prepared using another RNA polymerase III promoter, the Hl RNA promoter, that may be operatively linked to siRNA polynucleotide specific sequences, which may be used for transcription of hairpin structures comprising the siRNA specific sequences or separate transcription of each strand of a siRNA duplex polynucleotide (see, e.g., Brummelkamp et ai, Science 296:550-53 (2002); Paddison et al, supra).
  • DNA vectors useful for insertion of sequences for transcription of an siRNA polynucleotide include pSUPER vector (see, e.g., Brummelkamp et al, supra ⁇ ; pAV vectors derived from pC WRSVN (see, e.g., Paul et al, supra); and pIND (see, e.g. , Lee et al. , supra), or the like.
  • Polynucleotides of the invention can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters, providing ready systems for evaluation of NPRA polynucleotides that are capable of interfering with expression of NPRA gene, as provided herein.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, by Sambrook, et al, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N. Y., (2001).
  • the appropriate DNA sequence(s) may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.
  • Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art.
  • a number of standard techniques are described, for example, in Ausubel et al. (1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al. (2001 Molecular Cloning, Third Ed., Cold Spring Harbor Laboratory, Plainview, N. Y.); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.); and elsewhere.
  • the DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis.
  • appropriate expression control sequences include LTR or SV40 promoter, the E. coli lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter, or regulated promoter, operably linked to a polynucleotide of the invention is described herein.
  • the vector may be a viral vector such as a mammalian viral vector (e.g., retrovirus, adenovirus, adeno-associated virus, lentivirus).
  • retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the viral vector includes one or more promoters.
  • Suitable promoters include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and beta-actin promoters).
  • CMV cytomegalovirus
  • viral promoters include, but are not limited to, adenovirus promoters, adeno-associated virus promoters, thymidine kinase (TK) promoters, and Bl 9 parvovirus promoters.
  • a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be from among either regulated promoters (e.g., tissue-specific or inducible promoters) or promoters as described above.
  • tissue-specific promoter allows preferential expression of the polynucleotide in a given target tissue (such as tissue of the respiratory tract), thereby avoiding expression in other tissues.
  • cardiac-specific regulatory elements can be used.
  • An example of a cardiac-specific promoter is the ventricular form of MLC-2v promoter (see, Zhu et al., Mol Cell Biol. 13:4432-4444, 1993; Navankasattusas et al, MoI. Cell Biol. 12:1469- 1479, 1992) or a variant thereof such as a 281 bp fragment of the native MLC-2v promoter (nucleotides -264 to +17, Genebank Accession No. U26708).
  • Examples of other cardiac-specific promoters include alpha myosin heavy chain (Minamino et al, Circ. Res.
  • Endothelial cell gene promoters include endoglin and ICAM-2. See Velasco et al., Gene Ther. 8:897-904, 2001.
  • Liver-specific promoters include the human phenylalanine hydroxylase (PAH) gene promoters (Bristeau et al, Gene 274:283-291, 2001), hBlF (Zhang et al, Gene 273:239-249, 2001), and the human C-reactive protein (CRP) gene promoter (Ruther et al, Oncogene 8:87-93, 1993).
  • PAH human phenylalanine hydroxylase
  • CRP C-reactive protein
  • Promoters that are kidney-specific include CLCN5 (Tanaka et al, Genomics 58:281-292, 1999), renin (Sinn et al, Physical Genomics 3:25-31, 2000), androgen-regulated protein, sodium-phosphate cotransporter, renal cytochrome P-450, parathyroid hormone receptor and kidney-specific cadherin. See Am. J. Physiol. Renal Physiol. 279:F383-392, 2000.
  • An example of a pancreas-specific promoter is the pancreas duodenum homeobox 1 (PDX-I) promoter (Samara et al, MoI. Cell Biol. 22:4702-4713, 2002).
  • a number of brain-specific promoters may be useful in the invention and include the thy-1 antigen and gamma- enolase promoters (Vibert et al, Eur. J. Biochem. 181:33-39, 1989), the glial-specific glial fibrillary acidic protein (GFAP) gene promoter (Cortez et al, J. Neurosci. Res. 59:39-46, 2000), and the human FGFl gene promoter (Chiu et al, Oncogene 19:6229- 6239, 2000).
  • the GATA family of transcription factors have promoters directing neuronal and thymocyte-specific expression (see Asnagli et al, J. Immunol. 168:4268- 4271, 2002).
  • the present invention relates to host cells containing the above described recombinant constructs.
  • Host cells are genetically engineered/modified (transduced, transformed or transfected) with the vectors and/or expression constructs of this invention that may be, for example, a cloning vector, a shuttle vector, or an expression construct.
  • the vector or construct may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying particular genes such as genes encoding siRNA polynucleotides or fusion proteins thereof.
  • the culture conditions for particular host cells selected for expression such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • appropriate host cells include, but need not be limited to, bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo.
  • a mammalian cell culture systems can also be employed to produce polynucleotides of the invention from recombinant nucleic acid constructs of the present invention.
  • the invention is therefore directed in part to a method of producing a polynucleotide, such as a siRNA, by culturing a host cell comprising a recombinant nucleic acid construct that comprises at least one promoter operably linked to a polynucleotide of the invention that is specific for NPRA gene.
  • the promoter may be a regulated promoter as provided herein, for example a tetracycline- repressible promoter.
  • the recombinant expression construct is a recombinant viral expression construct as provided herein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa, HEK, and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences, for example as described herein regarding the preparation of recombinant polynucleotide constructs.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • constructs into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, liposomes including cationic liposomes, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et ah, 1986 Basic Methods in Molecular Biology), or other suitable technique.
  • the expressed polynucleotides may be useful in intact host cells; in intact organelles such as cell membranes, intracellular vesicles or other cellular organelles; or in disrupted cell preparations including but not limited to cell homogenates or lysates, microsomes, uni- and multilamellar membrane vesicles or other preparations.
  • expressed polynucleotides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
  • HPLC high performance liquid chromatography
  • administer As used herein, the terms “administer”, “apply”, “treat”, “transplant”, “implant”, “deliver”, and grammatical variations thereof, are used interchangeably to provide NPRA inhibitors of the subject invention ⁇ e.g., vectors containing or encoding polynucleotides of the subject invention) to target cells in vitro or in vivo, or provide genetically modified (engineered) cells of the subject invention to a subject ex vivo.
  • co-administration refers to the administration of two or more agents simultaneously (in one or more preparations), or consecutively.
  • one or more types of NPRA inhibitors of the invention e.g., vectors containing or encoding polynucleotides of the subject invention
  • a polynucleotide includes more than one such polynucleotide.
  • a reference to “a nucleic acid sequence” includes more than one such sequence.
  • a reference to “a cell” includes more than one such cell.
  • the terms “comprising”, “consisting of and “consisting essentially of are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.
  • Example 1 ANP overexpression in the lung augments inflammation and cytokine production in splenocytes.
  • ANP has been suspected to play a role in decreasing inflammation, as it was shown to play a role in decreasing TNF- ⁇ production from macrophages and slightly decreased NFkB activation (Mohapatra et al. JACI, 2004). Also, NPRA deficient mice did not exhibit inflammation. Since excess ANP expression activates the clearance receptor, it was hypothesized that ANP actually increases inflammation. To test this, na ⁇ ve mice were administered intranasally a plasmid pVAX expressing the ANP peptide. The results show that ANP overexpression actually increases inflammation. Materials and Methods
  • ANP expression vector Total RNA was isolated from murine heart using Trizol reagent (LIFE TECHNOLOGY, Gaithersburg, MD) following the manufacturer's protocol. The cDNA sequence for the ANP, residues 99-126 of pro ANP was amplified by RT-PCR. A translation initiation codon was inserted in the forward primers, so that the recombinant peptides had an additional amino acid, methionine, as the first amino acid apart from its known content. The PCR product was cloned in pVAX vector (INVITROGEN, Carlsbad, CA) at HindIII and Xhol sites. The cloned ANPsequence was verified by DNA sequencing and its expression was checked in A549 human epithelial cells.
  • PMA 50 ng/ml
  • ionomycin 500 ng/ml
  • GOLGISTOP PHARMINGEN, San Diego, CA
  • CD8 or CD4 mAb BD BIOSCIENCES, San Diego, CA
  • mice were removed after 24 hours of intranasal pANP administration, fixed, and sections stained with H&E. Results. Normal BALB/c mice were given i.n. nanoparticles carrying pANP or pVAX and their lungs were examined 3 days after by staining the sections (H&E), showing goblet cell hyperplasia. These results directly demonstrate that in normal mice over expression of ANP results in bronchial inflammation. To demonstrate that ANP over expression also stimulates immune system, BALB/c mice were given i.p. OVA (with alum) and then challenged i.n. OVA.
  • mice were sacrificed, the spleens aseptically removed and the cells were cultured for 48 hours in the presence of OVA (Sigma) and recombinant IL-2.
  • Cells were removed from culture and stained for surface markers CD4 and CD3 and intracellular cytokines IL-4, IL-IO and IFN-g (BD Pharmingen). The results show that in normal mice in absence of any antigen sensitization, ANP overexpression increases expression of boANP in general augments inflammation by activating both innate and adaptive immunity.
  • Example 2 Inhibitory effect of transfected siRNA plasmids on NPRA expression.
  • siRNAs To determine whether siRNAs can be produced that will effectively decrease NPRA expression, 11 different siRNA oligos were designed and cloned in a pU6 vector. Cells transfected with each of the construct was examined for NPRA protein expression by western blotting.
  • Plasmid constructs The nucleotide sequence for each siRNA is described previously (SEQ ID #1-1 1). Each pair of oligos was inserted into pU6 plasmid at appropriate sites respectively, to generate the corresponding siRNA for siNPRA.
  • NPRA polyclonal antibody SANTA CRUZ BIOTECH, Santa Cruz, CA
  • immunoblot signals were developed by SUPER SIGNAL ULTRA chemiluminescent reagent (PIERCE 3 Rockford, IL).
  • HEKGCA cells grown in 6-well plates were transfected with psiNPRA (2ug), as indicated and forty eight hours later total protein were extracted western blotted using an antibody to NPRA (Figure 3C). Untransfected cells and cells transfected with U6 vector plasmid without any siNPRA were used as control. Also, filters were stripped and reprobed with antibody to beta-actin. The experiments were repeated. The results showed that 3 of 11 siNPRA constructs consistently decreased NPRA protein expression in the HEKGCA cells.
  • Example 3 Demonstration that oral siNPRA treatment decreases inflammation, eosinophilia and Th2 cytokines in BALB/c mice.
  • Thoracic lymph node cells (A) and spleen cells (B) were removed and cells cultured for 48 hours in the presence of OVA (Sigma Grade V) and recombinant mouse IL-2. Na ⁇ ve mice received no treatment. Cells were treated with GolgiStop (BD Pharmingen) and stained for surface and intracellular cytokines (Antibodies obtained from BD Pharmingen). Percent cytokine secreting cells were quantified by intracellular cytokine staining using flow cytometry, as described.
  • lung histology i.e., lung sections stained by H &E revealed that compared to untreated Ovalbumin-sensitized and mice treated with scrambled si-NPRA group, treated mice showed a significant reduction in lung inflammation.
  • the lung histology was very similar to the na ⁇ ve mice. There was significant reduction in epithelial goblet cell hyperplasia and a significant reduction in peribroncial, perivascular and interstitial infiltration of the inflammatory cells to the lung ( Figures 6A-6C).
  • Example 4 Demonstration that transdermal siNPRA treatment decreases inflammation, eosinophilia and Th2 cytokines in BALB/c mice. Patients are more compliant when the drug is delivered by transdermal route.
  • siNPRA8 delivered by transdermal route was attempted to determine whether such siRNA therapy would decrease pulmonary inflammation in this ovalbumin-induced mouse model of asthma.
  • mice were sensitized (i.p.) as in example #3 and challenged (i. n.) with 50 ⁇ g of OVA.
  • Example 5 Demonstration that transfection of A549 cells with psiNPRA9 decreases the number of respiratory syncytial virus (RS ⁇ O infection infected cells.
  • Respiratory syncytial virus infection also causes bronchiolitis in newborns and in elderly causing pneumonitis, which is characterized severe acute lung inflammation.
  • RSV infection typically requires certain host cell proteins and transcription factors for its replication and subsequent infection of others cells. Since siNPRA treatment decreases pulmonary inflammation, the effect of siNPRA9 transfection on RSV infection was examined in pulmonary type-II epithelial cells was examined.
  • RNA extraction RNA fragment was amplified by RT-PCR using NPRA specific primers (F:5'-GCA AAG GCC GAG TTA TCT ACA TC-3', R:5'-AAC GTA GTC CTC CCC ACA CAA -3) and analyzed in 1% agarose gel.
  • mice infected with RSV and intranasally treated with pU6 control plasmid given with chitosan nanoparticles showed NPRA expression in the lung cells.
  • mice infected with RSV and intranasally given psiNPRA9 showed an amplification product that was reduced in band intensity compared to cells from mice given pU6 plasmid.
  • the lung cells from NPRA knock-out mice showed the band as well but it was reduced in intensity.
  • results showed whether prophylactic approach or therapeutic approach the results showed a 20% reduction in rgRSV infected cells in cells treated with siNPRA ⁇ and/or siNPRA9 compared to siU6 control plasmid.
  • siNPRA treatment can decrease RSV infection in addition to inflammation as seen in other studies.
  • Example 6 Demonstration that siNPRA treatment decreases melanoma tumor formation in Bl 6 mouse model.
  • siNPRA molecules are deliverable by transdermal route and treatment with siNPRA decreases local and systemic inflammation, which has been recently attributed toward the origin of certain cancers, the effect of siNPRA on melanoma was tested.
  • the neoplastic transformation of the melanocyte involves differential ability of the melanoma cell versus the melanocyte to cope with oxidative stress.
  • Melanocytes produce reactive radicals and have a low level of anti-oxidant enzymes, responding to UV with a large but transient increase in superoxide anion whereas keratinocytes and fibroblasts do not.
  • Bl 6 melanoma cells (1.3x105) were injected subcutaneously into twelve-week old female C57BL/6 mice or NPRA-deficient mice produced in B6 background. These mice were then treated with 33 ⁇ g of siNPRA-oligos, siNPRA9 plasmid, or scrambled oligos. All of these were mixed with Chitosan at ratio of 1:2.5. Mixed chitosan and plasmid or oligos were mixed again with cream before application to the injection area. The control group was given cream only. These treatments were given twice a week. Mice were sacrificed on day twenty second, tumors were removed and weighed. Results.
  • NPRA-deficient mice may have other abnormalities which might make it resistant
  • the WT mice were injected with 3 X 10 5 B16F10.9 cells and were then treated with a cream containing siNPRA 9 given twice a week at the location of tumor cell injection.
  • both treated and control mice treated with cream alone without siNPRA were compared for their tumor burden.
  • Figure 13C shows a comparison of both groups of mice. Excision of these tumors revealed that siNPRA, but not siNPRA scrambled, showed significant reductions in tumor burden. These results together show that siNPRA can be used to treat melanomas.
  • Example 7 Demonstration that siNPRA treatment decreases melanoma tumor formation in Lewis Lung Carcinoma B 16 mouse model.
  • LLCl cells grown in DMEM were washed with phosphate buffered saline (PBS) and resuspended in PBS at 2x10 7 cells per ml.
  • PBS phosphate buffered saline
  • Example 8 Demonstration that siNPRA treatment decreases melanoma tumor formation in ID8 ovarian cancer mouse model.
  • mice were washed with PBS and resuspended in PBS at 2x10 7 cells per ml.
  • Tumor sizes were measured at days 10, 13, 15 and 17 after injection. All animals were sacrificed on day 17 and the tumors were removed and weighed.
  • mice and NPRA-deficient C57BL/6 mice were injected with 2 x 10 6 ID8 mouse ovarian cancer cells at day 1 and mice were monitored at weekly intervals for tumor growth. By week 8 after cancer cell inoculation, all mice from the
  • WT group developed solid tumors but no tumors were found in NPRA-deficient mice.

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Abstract

This invention pertains to inhibitors of atrial natriuretic peptide receptor A (NPRA) function, such as small interfering RNA (siRNA), useful for reducing the inflammation associated with many human diseases, such as asthma, respiratory syncytial virus (RSV) infection, and cancers (such as melanoma, lung cancer, and/or ovarian cancer) by interfering with NPRA gene expression or otherwise reducing NPRA function within a subject; and methods for treating a subject suffering from, or at risk of developing, an inflammatory disease, respiratory allergy (such as allergic rhinitis and asthma), viral infection, and/or cancer by administering such NPRA inhibitors to the subject.

Description

DESCRIPTION
MATERIALS AND METHODS FOR REDUCING INFLAMMATION BY INHIBITION OF THE ATRIAL NATRIURETIC PEPTIDE RECEPTOR
CROSS-REFERENCE TO RELATED APPLICATION
The present application claims the benefit of U.S. Provisional Application Serial o. 60/796,278, filed April 28, 2006, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, and drawings.
BACKGROUND OF THE INVENTION
An atrial peptide with natriuretic and diuretic properties was first reported from rat atrial muscle in 1981. Since then a family of natriuretic hormone peptides (NP) with broad physiologic effects including vasodilation and inhibition of aldosterone secretion has been described. Atrial natriuretic factor (ANF), a 126 amino acid prohormone gives rise to four peptides: long acting natriuretic peptide (LANP, amino acids 1-30), vessel dilator (VD, residues 31-67), kaliuretic peptide (KP, residues 79-98) and atrial natriuretic peptide (ANP, residues 99-126, also referred to here as NP99-126) (Vesely, DL Cardiovasc Res, 2001 51 :647-58). In addition, renal tubular cells produce urodilatin, a 32 amino acid peptide (residues 95-126 of ANF), which is realeased to circulation following differential processing of ANF (Forssmann et al. Cardiovassc Res, 2001,51 :450-62.). There is also a pro-brain natriuretic peptide (BNP) first discovered in porcine brain, which is analogous to ANP is found in circulation. The third type of natriuretic hormone, the C-type (CNP) comprises two peptides, 53 and 22 amino acids in length, which are produced by many cell types (Levin, ER et al. N Eng J Med, 1998, 339321-8). Of these peptides, the C-terminal pro-ANF, ANP, has been studied most extensively.
In keeping with the diversity of these NPs, there are three NP receptors (Misono, KS MoI Cell Biochem, 2002, 230(l-2):49-60; Tremblay, J et al. MoI Cell Biochem, 2002, 230(l-2):31-47). NPRa and NPRb, which are coupled to guanylyl cyclase, and the cGMP-independent receptor NPRc. ANP and BNP signal primarily through NPRa, which increases cGMP and activates cGMP-dependent protein kinase (PKG). PKG activation turns on the ion transport mechanism and activates specific transcription factors, which together affect a range of cellular activities including, cell growth and proliferation, apoptosis and inflammation. NPRC functions as a clearance receptor but also appears to signal phospholipase C activation and a decrease in adenylyl cyclase activity (Silberbach, M et al Cell Signal, 2001 13:221-31). Numerous tissues of various organ systems including the lung express these receptors in diverse cells.
The NPs are produced in various tissues of the mucosa (lung, gastrointestinal and genitourinary systems), cental nervous system and cardiovascular systems and released into the circulation. The signaling mechanisms underlying ANP' s growth inhibitory effects are poorly understood, although a number of reports suggest that ANP affects signaling via activation of mitogen-activated protein kinases (Silberbach, M et al. Cell Signal, 2001 13:221-31). The potential effects may include inhibition of ERK activation of epidermal growth factor, PKG-induced uncoupling of Ras/Rafl interaction, or induction of MKP- 1 , a MAPK phosphatase that inactivates signaling through a number of growth factors such as endothelin, EGF and FGF (Clark, AR J Endocrinol, 2003, 178: 5- 12). ANP has been shown to mediate anti-inflammatory (Kiemer, AK and Vollmar J Biol Chem, 1998 273:134444-51) and cytoprotective (Kiemer, AK et al, J Immunol, 2000, 165:175-81; Sprenger, H et al, Immunobiology, 1991, 183:94-101) effects. It has been shown to decrease cytokine and stress stimulated activation of NFKB in various cell types, leading to a decrease in pro-inflammatory cytokine production (Kiemer, AK and Vollmar J 5/ø/ Chem, 1998 273:134444-51; Kiemer, AK et al., J Immunol, 2000, 165:175-81; Morita. R et al, J Immunol, 2003:170:5869-75). ANP can reduce tumor necrosis factor-α (TNF-α)-stimulated production of adhesion molecules in endothelium.(Kiemer, AK and Vollmar J Biol Chem, 1998 273:134444-51). It has also been shown to attenuate TNF-α- induced actin polymerization, through activation of MAPK phoshatase-1 (MKP-I) and inhibition of p38 activity, leading to decreased permeability (Clark, AR J Endocrinol, 2003, 178(1):5-12).
ANP stimulates migration of human neutrophils (Izumi, T et al. J Clin Invest, 2001, 108(2):203-21345), and inhibits nitric oxide (NO) and TNF-a production by murine macrophages (Vesely, DL et al Chest, 1990, 97(6): 1295- 1298, Vesely, DL Am J Obstet Gynecol, 1991, 165(3):567-573). Human peripheral blood monocytes, however, do not express ANP receptors nor do they respond to ANP (Sprenger, H et al. Jmmunobiology, 1991, 183(l-2):94-101). The NP system, acting via cells of the innate immune system, modulates the immune response to antigens. Evidence to date suggests that it may augment allergic inflammation by acting on a number of cells in the lung (Kurihara, M et al. Biochem Biophys Res Commun, 1987, 149(3):1132-1140). The primary evidence supporting this notion is the finding that ANP acts via its receptor on dendritic cells to polarize these cells toward a Th2 phenotype, which is the hallmark of allergic immune response (Morita R et al. J Immunol, 2003, 170(12):5869-5875). In asthma, the production of inflammatory mediators secreted from resident epithelial cells and recruited immune cells results in airway hyperreactivity, which characterizes the late-phase airway response. Without intervention, this event leads to non-reversible airway remodeling (including sub-basement-membrane collagen deposition, smooth muscle hyperplasia and hypertrophy, and goblet cell hyperplasia), with subsequent airway narrowing and progression of the asthma. 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 J5 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. Patent No. 6,506,559; Fire et al, Nature 391:806-11 (1998); Sharp, Genes Dev. 13: 139-41 (1999); Elbashir et al Nature 41 1. -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 investigated (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 Na/. 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. ScL 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 in 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. Patent No. 5,168,053; U.S. Patent No. 5,190,931; U.S. Patent No. 5,135,917; U.S. Patent 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. Patent No. 5,272,262; U.S. Patent No. 5,144,019; and U.S. Patent 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. Patent 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 anti sense 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 in vitro and as a potential strategy for in vivo target validation and therapeutic product development (Novina, CD. and Sharp, P.A., Nature, 2004, 430:161-164; Lieberman, J. et al Trends MoI. Med., 2003, 9(9):397-403). In vivo gene silencing with RNAi has been reported using viral vector delivery, liposomal delivery, and high- pressure, high-volume intravenous (i.v.) injection of synthetic iRNAs (Haider, J. et al Clin. Cancer Res., 2006, 12( 16) :4916-4924; Landen, CN. et al, Cancer Biol. Ther., 2006, 5(12):1708-1713; 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, SJ. et al. MoI. 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; Tolentino, MJ. et al. Retina, 2004, 24:132-138). Silencing of endogenous genes by systemic administration of siRNAs has also been demonstrated (Zimmerman, T.S. et al, Nature, 2006, 441(7089):l l 1-114; Soutschek, J. et al Nature, 2004, 432:173-178).
The present inventors have demonstrated that, in contrast to prior knowledge that ANP decreases inflammatory mechanisms in the macrophages, ANP actually increases lung inflammation and this is caused by ANP-NPRA signaling. This signaling can be blocked by utilizing a small interference RNA (siRNA) approa ch, in which specific siRNAs targeted to NPRA can significantly decrease the inflammation. This results in amelioration of inflammation in allergic disease which may be caused by allergens and exacerbated by respiratory viral infections, pollutants, and smoke. Also, this may be beneficial in the amelioration of inflammation and tumorigenesis in cancers.
BRIEF SUMMARY OF THE INVENTION
The present invention pertains to a method for treating inflammatory diseases, respiratory allergies, such as allergic rhinitis and asthma, viral infections, and cancers using a polynucleotide (e.g., an siRNA, antisense nucleotide sequence, and/or ribozyme) or other agent that reduces expression of the atrial natriuretic peptide receptor A, NPRA, or otherwise reduces activity of the receptor (referred to herein as an NPRA inhibitor).
In one embodiment, the method of the present invention comprises administering a therapeutically effective amount of an NPRA inhibitor to a subject in need of such treatment. In one embodiment, the NPRA inhibitor is an interfering RNA molecule, such as siRNA, specifically targeted to NPRA. As used herein, NHP refers to atrial natriuretic factor (ANF) hormone, or a biologically active fragment or homolog thereof. Specifically exemplified siRNAs comprise an oligonucleotide sequence deduced from selected nucleotide sequence from the NPRA gene. Preferably, the siRNA is targeted to a sequence within the mRNA sequence encoded by SEQ ID NO:4.
In another embodiment, the method of the present invention comprises administering an effective amount of at least one nucleic acid molecule encoding an siRNA specifically targeted to NPRA (siNPRA) to a patient in need of such treatment. The present inventor has determined that introduction of a nucleic acid molecule encoding siNPRA is capable of inhibiting NPRA expression when introduced via a plasmid vector or a virus, in association with a desirable carrier molecule such as a lipid or polymer-based system. The siRNA delivery method of the present invention permits long-term expression of siNPRA encoding nucleic acid sequences in vivo, thereby conferring bronchoprotective effect and/or anti-inflammatory effect against respiratory allergies, such as asthma. Preferably, the siNPRA is targeted to a sequence within the mRNA sequence encoded by SEQ ID NO:4.
In one embodiment, a therapeutically effective amount of at least one nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or biologically active homologs of any of the foregoing, are administered to the subject. The nucleic acid molecule(s) can be administered with other inhibitors of NPRA and/or other agents having therapeutic efficacy in treatment of the disease.
In another aspect, the present invention concerns synthetic oligonucleotide having the sequence that acts as the interfering RNA (SEQ ID NOs: 1-3) or a biologically active homolog of the foregoing. In another aspect, the present invention concerns a pharmaceutical composition comprising a nucleic acid sequence encoding an siNPRA and a pharmaceutically acceptable carrier, which can be administered by an accepted route. BRIEF DESCRIPTION OF THE DRAWINGS
For a fuller understanding of the nature and objects of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:
Figures IA and IB show a diagram depicting that overexpression of ANP in the lung augments inflammation and cytokine production in splenocytes. A) Normal BALB/c mice were given i.n. nanoparticles carrying pANP or pVAX and their lungs were examined 3 days after by staining the sections (H&E), showing goblet cell hyperplasia. B) Female BALB/c mice were given i.p. OVA (with alum) and then challenged i.n. OVA. Mice were sacrificed, the spleens aseptically removed and the cells were cultured for 48 hours in the presence of OVA (Sigma) and recombinant IL-2. Cells were removed from culture and stained for surface markers CD4 and CD3 and intracellular cytokines IL-4, IL-IO and IFN-g (BD Pharmingen). Figure 2 shows cloning of siNPRA sequences in the pU6 vector. The siNPRA sequences were designed as shown in Sequence IDs and cloned in pSilencer 1(U6) vector using standard procedures. The transformants were tested by digestion with Apa I and EcoR I to release the siRNA inserts. Lanel, lOObp ladder; lane 2:pSilencerl(U6), Lane3- 6, siNPRAδ, Lane7-10, siNPRA9 are shown for illustration. Figures 3 A-3C show the inhibitor y effect of transfected siRNA plasmids on
NPRA expression. HEKGCA cells grown in 6-well plates were transfected with psiNPRA (2ug). Forty eight hours later, total protein was extracted and Western blotted using an antibody to NPRA. Plasmids encoding ANP, Kp73-102 and VD were used as controls, since they have been shown to downregulate NPRA expression. In the third experiment, HEKGCA cells grown in 6-well plates were transfected with psiNPRA (2ug), as indicated and forty eight hours later total protein were extracted western blotted using an antibody to NPRA (Figure 3C). Untransfected cells and cells transfected with U6 vector plasmid without any siNPRA were used as control. Also, filters were stripped and reprobed with antibody to beta-actin. Figures 4 A and 4B show inhibitory effect of siRNA in vitro and in vivo.
HEKGCA cells grown in 6-well plates were transfected with psiNPRA (2ug). Forty eight hours later, cells were subjected to flow cytometry to detect NPRA positive cells using an antibody to NPRA. U6 plasmid without any siRNA and plasmid encoding Kp73-102 were used as controls, since the latter has been shown to downregulate NPRA expression. Results are shown in Figure 4A. Mice (n=4) were intranasally administered with 25ug siRNA plasmids complexed with 125ul of chitosan nanoparticles. BAL was done 72 hours later. Cells were stained by NPRA Ab. NPRA expression cells were counted.
Figures 5 A, 5B-1, and 5B-2 show that SiNPRA treatment appears to reduce cytokine production in BALB/c mice. 4-6 week old BALB/c mice (n=3) were sensitized and challenged with OVA (50μg). All mice were sensitized intra-peritoneally (i.p.) and then challenged intranasally (i.n.). Mice were given two Si NPRA treatments by gavage and challenged 24 hours later. Thoracic lymph node cells (Figure 5A) and spleen cells (Figures 5B-1 and 5B-2) were removed and cells cultured for 48 hours in the presence of OVA (Sigma Grade V) and recombinant mouse IL-2. Naϊve mice received no treatment. Cells were treated with GolgiStop (BD Pharmingen) and stained for surface and intracellular cytokines (Antibodies obtained from BD Pharmingen). Percent cytokine secreting cells were quantified by intracellular cytokine staining using flow cytometry.
Figures 6A and 6B show that administration of siNPRA decreases inflammation of the lung in BALB/c mice. 4-6 week old BALB/c mice (n=3) were sensitized and challenged with OVA (50μg). All mice were sensitized intra-peritoneally (i.p.) and then challenged intranasally (i.n.). Mice were given two Si NPRA treatments by gavage and challenged 24 hours later. Lungs were obtained 24 hours after challenge, fixed in formalin, sectioned and stained with hematoxylin and eosin.
Figures 7A-7C show that administration of siNPRAδ by the transdermal route decreases NPRA expression, eosinophilia of the lung and BAL IL-4 cytokine. BALB/c mice (n=5 each group) were sensitized (i.p.) and challenged (i.n.) with 50 μg of OVA. Mice were given siNPRAδ oligonucleotide treatments by transdermal route and challenged 4 hours later. Following 24 hours of challenge two mice were sacrificed to obtain lungs and which were fixed sectioned and immunostained for NPRA expression (Figure 7A). Mice (n=3) were sacrificed and lavaged and the percentage of eosinophils (Figure 7B) and IL-4 concentration (Figure 7C) in the lavage fluid was determined. Figures 8 A and 8B show that administration of siNPRA decreases inflammation of the lung in BALB/c mice. BALB/c mice (n=5 each group) were sensitized (i.p.) and challenged (i. n.) with 50 μg of OVA. All mice were sensitized intra-peritoneally (i.p) and then challenged intranasally (i.n.) Mice were given siNPRAδ oligonucleotide treatments transdermally (si8) and challenged 4 hours later. Lungs were obtained 24 hours after challenge, fixed in formalin, sectioned and stained with hematoxylin and eosin.
Figure 9 shows that administration of siNPRA inhibits NPRA expression in the respiratory syncytial virus (RSV) infected lung. RT-PCR analysis of NPRA expression in the lung of mice treated with siRNA. psiNPRA9 was encapsulated with chitosan nanoparticles and intranasally delivered to mice. Twenty-four hours later, mice were infected with RSV (5x106 pfu/mouse). Four days later, mice were sacrificed and lung were collected for RNA extraction. NPRA fragment were amplified by RT-PCR and analyzed in 1% agarose gel.
Figures 1OA and 1OB show that administration of siNPRA inhibits the Respiratory syncytial virus infection of A549 cells. A549 cells were grown in 6 well plate, transfected by siNPRAδ, siNPRA9 or control U6 plasmid (2.0ug) and 2 hours after infected by rgRSV (MOI=0.2). Cells were checked for infection 48 hours later, FACS was done. Results are shown in Figure 1OA. A549 cells were grown in 6 well plate infected by rgRSV (MOI=0.2) and 24 hours after infection they were transfected by siNPRA8, SΪNPRA9 or control U6 plasmid (2.0 μg) and further 24 hours later, flow cytometry was performed to estimate percentage of infected cells. Results are shown in Figure 1OB. Figure 11 shows that NPRA-deficient mice are resistant to melanoma tumor formation and metastasis in the B 16 mouse model. B 16 melanoma cells (1.3x105) were injected subcutaneously into twelve-week-old female C57BL/6 mice and NPRA-deficient mice. Mice were observed for tumor formation for one month, then sacrificed on day-22. Tumors were then removed and weighed. Figures 12A-12E show that siNPRA treatment decreases melanoma tumor formation in bl6 mouse model. Bl 6 melanoma cells (1.3x105) were injected subcutaneously into twelve-week old female C57BL/6 mice. These mice were then treated with 33 μg of siNPRA-oligos, siNPRA plasmid, or scrambled oligos. All of these were mixed with chitosan at a ratio of 1 :2.5. Mixed chitosan and plasmid or oligos were mixed again with cream before application to the injection area. The control group was given cream only. These treatments were given twice a week. Mice were sacrificed on day-22, and tumors were removed and weighed. Figures 13A-13C show the effect of NPRA deficiency on melanoma. To test of the anti-melanoma activity of decreased NPRA levels, NPRA'A mice (n = 12) and wild type (n=12) were injected s.c. with B16 melanoma cells. The tumor size (Figure 13A) over several days post injection and tumor burden (Figure 13B) at day 18 were measured. Figure 13C shows that siNPRA treatment decreases melanoma tumor formation in the B16 mouse model. B16 melanoma cells (1.3xlO5) were injected subcutaneously into twelve-week old female C57BL/6 mice. These mice were then treated with 33 μg of siNPRA-oligos, siNPRA plasmid, or scrambled oligos. All of these were mixed with chitosan at a ratio of 1:2.5. Mixed chitosan and plasmid or oligos were mixed again with cream before application to the injection area. The control group was given cream only. These treatments were given twice a week. Mice were sacrificed on day-22, and tumors were removed and weighed.
Figures 14A and 14B show that siNPRA treatment decreases lewis lung carcinoma. Groups of wild type and NPRA'A mice (n = 8 per group) were injected s.c. with 2 xlO6 LLCl cells. Tumor sizes were measured on day 10, 13, 15 and 17 (Figure 14A) and tumor weights at day 17 (Figure 14B) were compared.
Figure 15 shows that siNPRA treatment decreases ovarian cancer. Groups of wild type and NPRA'A mice (n = 8) were injected s.c. with 2 xlO6 mouse ovarian cancer ID-8 cells and tumor sizes were measured every week after ID8 injection.
BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 is the nucleotide sequence of an siRNA for NPRA (siNPRAlV. (targeting position 33): 5'-CAT ATG ggg ccc GGG CGC TGC TGC TGC TAC Cct cga aat GGT AGC AGC AGC AGC GCC CTT gaa ttc CCA TGG-3'.
SEQ ID NO:2 is the nucleotide sequence of an siRNA for NPRA (siNPRA2) (targeting position 72): 5'-CAT ATG ggg ccc GCG GCC ACG CGA GCG ACC Tct cga aat AGG TCG CTC GCG TGG CCG CTTgaa ttc CCA TGG-3'.
SEQ ID NO:3 is the nucleotide sequence of an siRNA for NPRA (siNPRA3): (targeting position 33)siNPRA187top (silO): 5'-CAT ATG ggg ccc GGC TCG GCC GGA CTT GCT Get cga aat CAG CAA GTC CGG CCG AGC CTT gaa ttc CCA TGG-3'. SEQ ID NO:4 is the nucleotide sequence encoding human NPRA (NCBI Accession # AF190631:
1 ggatcccaaa ccagcacacc tttccctctt cccccgagga gaccaggtag gaggcgaggg 61 aaaaggtggg gcgcaagtgg gccccggttg cttccacaca caccctccgt tcagccgtcc
121 tttccatccc ggcgagggcg caccttcaga gggtcctgtc ctccaaagag gtaggcgtgg 181 ggcggccgag accggggaag atggtccacg gggaagcgcg cgggctgggc ggcggggagg 241 aaggagtcta tgatcctgga ttggctcttc tgtcactgag tctgggaggg gaagcggctg 301 ggagggaggg ttcggagctt ggctcgggtc ctccacggtt ccctccggat agccggagac 361 ttgggccggc cggacgcccc ttctggcaca ctccctgggg caggcgctca cgcacgctac
421 aaacacacac tcctctttcc tccctcgcgc gccctctctc atccttcttc acgaagcgct, 481 cactcgcacc ctttctctct ctctctctct ctctaacacg cacgcacact cccagttgtt 541 cacactcggg tcctctccag cccgacgttc tcctggcacc cacctgctcc gcggcgccct 601 gcacgccccc ctcggtcgcg ccccttgcgc tctcggccca gaccgtcgca gctacagggg 661 gcctcgagcc ccggggtgag cgtccccgtc ccgctcctgc tccttcccat agggacgcgc
721 ctgatgcctg ggaccggccg ctgagcccaa ggggaccgag gaggccatgg taggagcgct 781 cgcctgctgc ggtgcccgct gaggccatgc cggggccccg gcgccccgct ggctcccgcc 841 tgcgcctgct cctgctcctg ctgctgccgc cgctgctgct gctgctccgg ggcagccacg 901 cgggcaacct gacggtagcc gtggtactgc cgctggccaa tacctcgtac ccctggtcgt 961 gggcgcgcgt gggacccgcc gtggagctgg ccctggccca ggtgaaggcg cgccccgact
1021 tgctgccggg ctggacggtc cgcacggtgc tgggcagcag cgaaaacgcg ctgggcgtct 1081 gctccgacac cgcagcgccc ctggccgcgg tggacctcaa gtgggagcac aaccccgctg 1141 tgttcctggg ccccggctgc gtgtacgccg ccgccccagt ggggcgcttc accgcgcact 1201 ggcgggtccc gctgctgacc gccggcgccc cggcgctggg cttcggtgtc aaggacgagt 1261 atgcgctgac cacccgcgcg gggcccagct acgccaagct gggggacttc gtggcggcgc
1321 tgcaccgacg gctgggctgg gagcgccaag cgctcatgct ctacgcctac cggccgggtg 1381 acgaagagca ctgcttcttc ctcgtggagg ggctgttcat gcgggtccgc gaccgcctca 1441 atattacggt ggaccacctg gagttcgccg aggacgacct cagccactac accaggctgc 1501 tgcggaccat gccgcgcaaa ggccgaggtg agacgctggc acaccccgtc ccgccgctta 1561 gccgcagggc ctcccctctg acctgccgga ggcatcggga ctttctctct catctggggg
1621 cactcttctt tctcctcgcc gttcttcatt ctactttcag ctccctggcc ctttctacag 1681 ctgagtttct atttccctct cttcttccgc cacccccacc acgtctctat cctctcatct 1741 ccccgacccc cactcattcc ctcccaccct agcacagctc ggttccggtc cctttttccc 1801 tcccacattt tctctcttcc ctatagcctt ctcccttctt tcatcctctc ctctcatggc 1861 gcctcatccc ctctcttctc cccctccctc tccctcctct ctccctcctg gccccatcct 1921 tctccacctt cagctccact atccccctct ccctacccgt tccttcctcc cttccgcctc 1981 ccccttcctc ctcccgccca ccgccccgca cccgcccgtt ccacccttcg actttctcct
2041 gctgtggcct aggctgagcc gggagttacc acttaactct cactgggtct ctcctgcacc 2101 ctatctctaa acttcctccc ttgggtgccc cagctttcct actcctgtct ctcccgcagt 2161 acctaggctt ctctctctga ctctccgtct ttctccagtt atctacatct gcagctcccc 2221 tgatgccttc agaaccctca tgctcctggc cctggaagct ggcttgtgtg gggaggacta 2281 cgttttcttc cacctggata tctttgggca aagcctgcaa ggtggacagg gccctgctcc
2341 ccgcaggccc tgggagagag gggatgggca ggatgtcagt gcccgccagg cctttcaggt 2401 gagtacctag gtttgaagcc caggctgtct cagcttgtgg cacatcattt ctgggcactg 2461 tgtccctcag catctgaaag aattccagaa aagaggtttt tgtctgtttg tttctttatg 2521 cactcctggt aactcacaga acagaaaaga ggttggtgat gctcactggg aattaggcaa 2581 tgaagggcag gggactgccc aggggcgctt cgccaccagc aggctaaaaa gataagaaaa
2641 tgggcttgag gcgggaggag gataaagtcc cacagcctgg acaggacttg gagaaggcat 2701 cccattggat cccctgcttt ggaatgggca tcacttcatg cagggcatag ggtccagttt 2761 gaccttgagc taagcagaga cgcagctctg ggaggtgggc tcccaactgt tggggcccca 2821 cagtactagg gaatagtcag ctcccaactc tctgctctcc actgacccct ttctcaggct 2881 gccaaaatca ttacatataa agacccagat aatcccgagt acttggaatt cctgaagcag
2941 ttaaaacacc tggcctatga gcagttcaac ttcaccatgg aggatggcct ggtaagaagg 3001 ggtcccggga ccctccagcg tggacctcca gcccccactc catgaccctc tgccagcctc 3061 catccttccc tattcccagt tctccccttc cttccctccc ttcccattgt tccatgtttc 3121 tcgtgatgat ggaggaggac actggcaagt tcagcctctg aaactcaggt catcatcagt 3181 aatatggaga cgatacatcc tgccctgtct acctagtagg attcaggaag tgatgctaat
3241 ccaaaggcat cgtttaaata gtaaaatctc cctgtgatat aggggtgtta ttttctccca 3301 tcctcttcca aaatcccagt gcctcttgtt cccttcccca cagctcccac ctccatgccc 3361 ttcatatgcc caccccagcc gacctctgtt tgcccctaca ggtgaacacc atcccagcat 3421 ccttccacga cgggctcctg ctctatatcc aggcagtgac ggagactctg gcacatgggg 3481 gaactgttac tgatggggag aacatcactc agcggatgtg gaaccgaagc tttcaaggtc
3541 agggcctgga ggtggctgga atgggctgcc ttgggggatg aatcccaggt gcccagtgtc 3601 aagccatgag aagcctattg tcctgcagca gttacctatg cacaccagcc ttttcctcca 3661 cagctttttt caggcccatc cctcagaagt cctacaaagt gtccaatctc aatcatccct 3721 gctgggcact gagttctttt acctttcttt ttcttttttc tttttttttt gagatggagt 3781 ctcgctctgt ccccaagact ggagtgtggt ggtgcaatct cggctcactt caacctccgc 3841 ctcccaggtt caagcaattc tcctgcctca gcctcctgag tagctgggat tacaggtgcc 3901 ctccaccaac acttggctaa ttttttgtat tttttttagt agagacaggg tttcaccacg
3961 ttggtcaggc tggtcttgaa ctcctgacgt caggtgatct gcccgcctca gcctcccaaa 4021 gtgctgggat tacaagcatg agccacagtg cccggccgtt ttaccattta ctatcattct 4081 gtatacatgt atgtttggaa ggcaaggcaa aaaagattag aggatgaaga gatgaagtgg 4141 ggcacccctg aacttctatt ctctcaaaca tagtcatctt cccccatgtc ctcaggtgtg 4201 acaggatacc tgaaaattga tagcagtggc gatcgggaaa cagacttctc cctctgggat
4261 atggatcccg agaatggtgc cttcagggta agtttgtgca cccagaagac agtgccaatt 4321 ccaaatgaca tctcaccctc ctacttcccc cccacagccc tgccagggca cctgtttatc 4381 ctgtagccat tccaccatgc ctggacactt acaagagccc tggataaaac agacccagct 4441 ccagtctggg gaagccacca gaatgatagg gactcacagg catcacactt ggggagcccc 4501 atgcctgagg agggagcaca agcctgccct cggggagctc cgaagggagg caggcaggac
4561 cgcctcccag cagagacagg gctgtgaaag atgcacatta cacagctctg caagcgagca 4621 gggacaggaa ggcgctgagg ccaatggcca caagggacag gtcatccaga gaaggcctcc 4681 tggaagacgg gcacatggac tgggcctgcg aatgtaggct aaggtgaaca ttaccttctc 4741 ctgttttcta ccaagaaaat aagtagagaa aaatcaatgc ttggttggta cttcaaccaa 4801 gattataaac tccctgagtg tagagatcgg gttctaaatg gagttttctt tataaacccc
4861 ttgatagttt tcaggtgttt ccacttgagt actatgtgtg tggtatgagg tcctgtgtcc 4921 agttgcagtg gggacttggt aagcaggtga caacccagat atatatgtag gctctagaag 4981 cagagctggg gtaggtggga ggtgagactg ctgcactcac agcatgcctt ccccgcaggc 5041 cctggcctag ccaccactcc tgctctccct taggttgtac tgaactacaa tgggacttcc 5101 caagagctgg tggctgtgtc ggggcgcaaa ctgaactggc ccctggggta ccctcctcct
5161 gacatcccca aatgtggctt tgacaacgaa gacccagcat gcaaccaagg tgactgcccc 5221 ttgccttcca ggcctccatc ccagagatgc tgcatccttc ccctaagcac agtcgagtag 5281 gtgctcctgt cccatgctga gggctttctg gagaatgact cctgcctttt tcttcccttc 5341 atccatcatc ccagttcact gatggactat tagaaagttc ttcctcctgc tgtctaaccc 5401 aaatctctct tgctgcaata tggactctct cctgcagatc acctttccac cctggaggtg
5461 ctggctttgg tgggcagcct ctccttgctc ggcattctga ttgtctcctt cttcatatac 5521 aggtgagctg tgatgtgggg ggttgagtga ggctggggga cccggagaac caagagcaga 5581 ggaggcggtg gggacccaga gggaagaggg caggggtgaa ggggcagcag gggaaaacca 5641 agggagatga ggaagaaagg aggcttaaaa gccagaggag aaagaaagag aagggaatgg 5701 cagggcgagg ggaggagaca aggataggaa tggccaagga gagtcagaaa gatccaagaa 5761 gcagagaagt tgatgggtga catcataggg gcgtggactg gttttccttg ctactcttgc 5821 aggccagata ggaagcaact ttctgaacct ttgcaatcat gcccatgtta gctgaggagg
5881 gtgagccctg gtgtgtgcca ggtgcccaac ctagaatgga gaagggagct gaatgagcct 5941 tgttcctgcc gtccagtgga ggctaaaatg aagtacagga ggagttaatg atatacaaaa 6001 gcaaggaggg aggggagaaa aatcactgct ggttgagcat ataatgtgtg ccaggcactt 6061 ccacgtacac tatttctttc tttctttttt tttttttttt tttttttttg agacggagtc 6121 tcgctctgtt gccagactgg agtgcagtgg catgatctag gctcactgca acctccgcct
6181 cccagtttca agcaattctc ctgcctcagc ctcccatgta gctgggacta caggcacatg 6241 ccaccacgct cagctaattt ttgtattttt agtagagaca gggtttcacc atgttggcca 6301 ggatggtctc gatctcttga cctcatgatc cacccacctt ggcctcccaa agtgctggga 6361 ttacaggcat gagccactgt gcctggcctc atgttcacta tttcttttca ttcttataat 6421 agttaagaat gaaatagata ttgcggcctc attcccaagt aaggacattg aggtgattcc
6481 cccaaggtcc ccagtaaggc agaatttccc ccagccatcc tgattctcag tccagaggat 6541 agaattcccc ctccatctct gagtgcatgg tgtggtccca cggctctgag gaggggctgc 6601 tgagcaccct gccctgggtc agcggctcag ccacaggctc agatgcagcc ttcgtatccc 6661 aggaagatgc agctggagaa ggaactggcc tcggagctgt ggcgggtgcg ctgggaggac 6721 gttgagccca gtagccttga gaggcacctg cggagtgcag gcagccggct gaccctgagc
6781 ggggtaagaa cgctggtgtt tgtgttgggg ggcaataaag gagaggtggg tacaaggggc 6841 agtgcctgag ggataggtaa gggagcagga ttctagtccc agctctgctt tcacttgctg 6901 tgtgaccttg agcgactcat agtccctctc cgagactgtc tcagatgatg attacagcag 6961 cagagcctcc ctcacagggc tcttttaaag gtcagaggag atagtacctg tgaaaacact 7021 ttaaaaaaaa aaaaagtaaa tgaggaggaa attttatgat gtggaacata aagcagggtg
7081 ggccaggcac agtggctcac atctgcaatc ccagcacttt gggagaccga ggcaggagga 7141 ttgcttgtgc ctgggagttc aagaccagcc tgggcaacag agcaagacat cgtctctaca 7201 aagaatacaa agattagcag ggcatggtgg cgcatacctg tagtcccagc tactctggag 7261 gctgaggtga aaggatcatc tgagcccagg agtctgaggc ggcagtgacc taggatagca 7321 ccactgcact ccagcctgga tgacacaatg atactacatc tcaaaaaaaa acccaacaac
7381 aaaaaggaag ggtgacacaa agataaggca ggataaggca gggaaataaa gaccagagca 7441 caagcaatca ggatgcagac tgggcccacc ggctgaccat tcctcctgct ctccctcctt 7501 tcagagaggc tccaattacg gctccctgct aaccacagag ggccagttcc aagtctttgc 7561 caagacagca tattataagg tgggcctggg gaaagatcac tgggccttgg gactggggca 7621 ggagtgtact ctgatggagg actggtgggg ggttctgagg gaaggagtaa gctggtgggg 7681 agcagcagat gggggccctg ggggtgggct attgggaaca agtgagggtc ctgagggcag 7741 ggatgggctg tcgggagcag ctggaattcc caggacatgg gaccatgctc ttcacagtga
7801 cagtctccat tccatgccca gggcaacctc gtggctgtga aacgtgtgaa ccgtaaacgc 7861 attgagctga cacgaaaagt cctgtttgaa ctgaagcatg taatgtgggg agtgaggcag 7921 tggcatggag aaggggccct cggggacgca agggagactg gccaacagaa ctagttatgg 7981 agggacctca gggtacccca agaaaggggc agggactgga gccctggatg accttcatct 8041 tgtggtggag tgggggtatc ctaagtagga gaagagacca ctgagataac ctggaggaat
8101 cttgaggggc catatgtgat gtccctgggg gagagagggc ttaggatgcc agagggagta 8161 ggagcagatt ctggggaggg tgggctaaag gacatgggtg ggaatcacca gggaagatct 8221 tagtgatggt tgcagaaagt gaataaggag ttaagaagag tgagggtccc tgaagctagt 8281 gagcagcttg gtgaggagcg aggtctctgt caagctcctg atgctggtcc cacttgcaga 8341 tgcgggatgt gcagaatgaa cacctgacca ggtttgtggg agcctgcacc gaccccccca
8401 atatctgcat cctcacagag tactgtcccc gtgggagcct gcaggtgagg gggacaaggg 8461 gtgtcaagaa acctgggttc tagccctggc tctgcccctg actggccata agaccccagg 8521 catgcctcgc cctctttctg acctttctgg ccccatctgt aaaaatggga gttggggaag 8581 ggcagtggca ctagagtcaa tccaaagttt tgtcctgttc taccagttca catcagtagg 8641 accctgcacc ctcctccaac tcccaggggg atctgcaggg gattggtctt gactcttatt
8701 gccccagcag gacattctgg agaatgagag catcaccctg gactggatgt tccggtactc 8761 actcaccaat gacatcgtca aggtatgccc ctaagcacct attggatgtg tagagcaggg 8821 gccaggcatg cttctcctgg ccacgggtgt aggtcccact cctggccaat acctctgccc 8881 actcacattt ccagggcatg ctgtttctac acaatggggc tatctgttcc catgggaacc 8941 tcaagtcatc caactgcgtg gtagatgggc gctttgtgct caagatcacc gactatgggc
9001 tggagagctt cagggacctg gacccagagc aaggacacac cgtttatgcc agtgagcctt 9061 gactcttgaa cctaacacct gcccccagca ccacccagta gggagactga tgcaaggcct 9121 ctgatgggct tgggcatgct tgtcctgact ccagcctcaa ttcattcacc catgaaaaag 9181 ggaaggccag acgaagtggt ttctaaggcc tcctctagct ctaacactct gtgatgcatc 9241 cagatcagtt tcggccacac ccttgtttcc ccctcacccc ttagctttgg gctccctcac
9301 tcggtgacta ccgacctctg acccacagaa aagctgtgga cggcccctga gctcctgcga 9361 atggcttcac cccctgtgcg gggctcccag gctggtgacg tatacagctt tgggatcatc ■ 9421 cttcaggaga ttgccctgag gagtggggtc ttccacgtgg aaggtttgga cctgagcccc 9481 aaaggtgaga ggagcacacc ttccttaaac ccagccacag tctcaacgaa ccccagcccc 9541 agggagaggg tcccctggca gcaccaccac accttccttc tgtaatgggg ttcagtcacc 9601 accctttgac ccattgctgc cagtgaccag tcccccgccc ccatgccttg gtcttggact 9661 tcccctgcca tctcagctgg ttgccccagt ctctcactag gcccttggcc agccccaccc
9721 ctcagctcct ctacccccca atacagagat catcgagcgg gtgactcggg gtgagcagcc 9781 ccccttccgg ccctccctgg ccctgcagag tcacctggag gagttggggc tgctcatgca 9841 gcggtgctgg gctgaggacc cacaggagag gccaccattc cagcagatcc gcctgacgtt 9901 gcgcaaattt aacaggtccc tggtgtttgt catggatccc ccaggccctt cctccacagc 9961 caccatttac ctaatgcttc tggctctggc ttatcccagc agtggcagag ggagaccact
10021 cacctcctcc ctgtacatag tcagctccag ctcagcacag cctcatgacc ctcttcgcaa 10081 gtacagcatg actcagctgt ccccacagtc ccctgccatt catgcccctt ccctccacca 10141 tcgacacccc acacccttcc tgcccactcg ccttgctggc ctctagactt ctcagcagtg 10201 tgtaggatag atgggcctcc cgcctcctgc cctgtaggct cttggccctc cacgggagct 10261 cctgccccac cccttgattt cccttcccca gcgtgcccac caggcccagt tcctccagac
10321 acacccttct gtggacatca ctttgtccgc aattgaccct tgtcattctc cacctccttt 10381 acctccttct aactcactgg gttcaacaaa gatgaacaaa atgtccatat gtctgaagct 10441 tcatacttga ccttggggtc tcagaaaaga attgaacttt cttccttctg ttttcccctg 10501 ctccccggta tcctgctatg ccctcaaccc tgagcgtctc tagagacctc actgcagtct 10561 ggagggggaa gtgcctaggg gcgggcgctc acgtaggctg tgctgctcct ctcttaccac
10621 ccccaccgcc accctctgcc cccagggaga acagcagcaa catcctggac aacctgctgt 10681 cccgcatgga gcagtacgcg aacaatctgg aggaactggt ggaggagcgg acccaggcat 10741 acctggagga gaagcgcaag gctgaggccc tgctctacca gatcctgcct cagtgagtgc 10801 ctgagtctgg ggaccccccc caacacaaag cccctgtccc gacccccaac tctgatcctg 10861 cacctgccct gaccccttag ctcagtggct gagcagctga agcgtgggga gacggtgcag
10921 gccgaagcct ttgacagtgt taccatctac ttcagtgaca ttgtgggttt cacagcgctg 10981 tcggcggaga gcacacccat gcaggtaggc cagggttcag ccacaggtgc caggcaagct 11041 cagcatctgg atcccaccag acctgccttc tggttctgct ttacccacct gaccccaggt 11101 ggggtcccct acttcctgtc tctcttagct tctcttccct tccaggtggt gaccctgctc 11161 aatgacctgt acacttgctt tgatgctgtc atagacaact ttgatgtgta caaggtgagg
11221 gtgggagtgg ggatgggaag ggacagacag acatggacaa ggtcagaaaa agatgagggg 11281 taggcagaat gatgtggagt cttaagagag gagatcgggg acacgggcag agacagtgac 11341 acagggagac ccgggaacag gcagagaacc catgtgggat gggggatgag caaagacaga 11401 tgagggtaca gaatgacaga cgctgcaccc ggtgtgacgg tgtggccggc cgcacagttg 11461 cagccgtcaa gtcctgcacc ccctcgccac tcccacaggt ggagacaatt ggcgatgcct 11521 acatggtggt gtcagggctc cctgtgcgga acgggcggct acacgcctgc gaggtagccc 11581 gcatggccct ggcactgctg gatgctgtgc gctccttccg aatccgccac cggccccagg
11641 agcagctgcg cttgcgcatt ggcatccaca caggtaaggc cactgaaggt gcaggcgggc 11701 atccagaggc caaggctttg caagggaaac ttgtcccctg gcccagcccc tcgccctttc 11761 atctctctct ctctctctct ctctctctct ctctctctct gtctctctct ctctctctct 11821 ctctctctct ctcacacaca cacacacaca cacacacaga gctgggacct cagatcctgc 11881 ctcctgcctg tcttggattg tccacctacc tcccttaaca cccctccctc cctcactcgc
11941 tgatgggctc tgctccttcc cttgctcctc ccaggacctg tgtgtgctgg agtggtggga 12001 ctgaagatgc cccgttactg tctctttggg gatacagtca acacagcctc aagaatggag 12061 tctaatgggg aaggtacagt gccccctcct agagggaatg gggagggcag ggtggctgag 12121 ggaaatgcca tcctggggca gcctgtgcct gcacagcccg tttcagctcc tagccctttc 12181 gcctcccaag ttccccttct cataatatta agagttcaac ctgggctcat caacttgact
12241 gtaaccagag actcaggttc ctgctgcccc tcttgtcaaa cgatgtaaaa gtatttccgg 12301 gccagtgctg gagagttccc agcaggaatc tgattttaag accctctgtg ggccgggcgt 12361 ggtgactcac acctgtgatc ccagcacttt gggaagctga ggcaggcgga tcacctgagg 12421 tcgggggttt cgagaccagc ctgaccaaca tgatgaaatc ccgtctctac taaaaataca 12481 aaaaactagc caggtgtgat ggcaggctcc tgtaatccca gctacttggg aggcttgagg
12541 cagaagaatt gcttgaaccc gggaggcaga ggttgcgatg agccaagatt acaccacgca 12601 ccccagcttg ggcaataaga gttaaactct gtctcaaaaa aaaaaaaaaa aaaaaaaaaa 12661 agggccctct gctccacctt tgatgtggta aagatggctt cagagccagc ataagtgagg 12721 ctgtgaatct cagctccaca gctggctgtg tgtcagtttg ctatacctct ctgagccatg 12781 gttttcctca tctgtaaaaa gagggaaaaa atctatctca caggaattat gtgagaaacc
12841 cattaaaaat gtctaccaca taattgtcat ttaacttttc caagccttag cggattatct 12901 gtaaaatgat gtctatctca ggattgcaag aagcctagca caaaccctgg tacccagcag 12961 gcacctaata aattcttact cctacccgcc ccttgctctt gcctcctgtt tatcttctat 13021 ccttctgctg tattcgacac aattcaatgc agtaaacatt tattgagtga ctactgagtg 13081 ccaggccctg ggatagtaac atggcccaga tccagagtta gctgagaaat tcatgtggac
13141 cccatctaaa ccttatggtg aaagaaaggc tgcttgggag ccagtcctgg gagcccagag 13201 ggatctagtt cggcaaatat tccctgggca ctatttgggg gctgcagagt cagcccttgt 13261 tgagggtcca gtcctcaagg agcacattcc cagaaatgtt cacattctgg cgctggggtg 13321 ctgtaatccc agcactttgg gaggccgagg tgggcagatc acttgaggcc aggagtggag 13381 actagcctgg ccaacatggt gacctcctgt ctctactaaa aatacaaaaa attagctggg 13441 cgtggtggca cgtgcccgta atcccagcta ctcaggaggc ttgagacatg aaaatcactt 13501 gaacccagga ggtggatgtt gcagtgagcc gagactgcac ccctgggcaa cagagcgaga
13561 ctctgtctca aaaaaaaaaa agagagaaag aaagaaaaga aaagaaagaa actgttaaac 13621 acaacaaggc cactgtgatt gatgcaaacc ccagaagtag ggacatgagt tcagacagtg 13681 gtcaaagaga gggtgtggca atattgggcc ccactccatc actgacctcc tcagccactt 13741 gggcagatca ccctgggcct cagttcctcg gccacaaaat gagggtatag catgaaatca 13801 tgaaagcaac aatttacata gtgcttccta ggtagcacat tccgtttgaa tactttatgg
13861 atgttaaatt taatcctcac aacaaggttt tgagatgggt actgacacta tcagcatttt 13921 acagattagg aaaatgaagc agagagaatt tattttacat acctaagcaa gtatccaagc 13981 tgaggttcat actgaggcag tgcaggatcc aaagtgccag ctcctaacca ccatgctgtg 14041 tagagccggg tgacactcca gagagtgctg tccaacagga tgttccatag tcatgaaaat 14101 gttctgtatt ctgtgctgtc caatacagta gcctctaggc acatatggct acttatcact
14161 ggaaatgtga cgggtgcaac tgaggccctg attttttttt tttttttgga gacagagttt 14221 cgctctgtcg cccagcctgg atggagtgca gtggtgcaat ctcggctcac tgcaacctcc 14281 gcctcccagg ttcaagcgat tctcctgcct cagcctccca agtagctgga attacaggtg 14341 agtgccacca cacacagcta atttttgtat ttttagtaga gacggggttt cgccatattg 14401 gccaggatgg tctcgaactc ctggcctcaa gtgatcctcc tgcctcagcc tcccaaagtg
14461 ctgggattac aggtgtgagc cacagcaccc agcctgaatt tttaactgta tttagtttaa 14521 attaatttaa gttgaaacag gcacatgtga ttagtggcta ctgtattgga ttacacagct 14581 ccagagttct aaatgagagg ctaatgtggt cacgcactac attcaggggg tggggcccct 14641 ctgagctaga gggcttcctg gcccaaaaga gggagagagg gtacctgtcc acctgtccac 14701 ccccacagtc cctggtctct tttgcctcta ctttcctgct ctcctctctc acattgctca
14761 ccttcccttc tcccctgtec tacccagccc tgaagatcca cttgtcttct gagaccaagg 14821 ctgtcctgga ggagtttggt ggtttcgagc tggagcttcg aggggatgta gaaatgaagg 14881 tagagcgaga agcctctgcc ctccccacct tttggggtcc tagagggagt tacccttctc 14941 aagcagccga tgccactccc atccctaagg ctctcatctg actggggaaa gggcatgtgc 15001 cactccccag cccatcctct tttttccctc cagggcaaag gcaaggttcg gacctactgg
15061 ctccttgggg agagggggag tagcacccga ggctgacctg cctcctctcc tatccctcca 15121 cacctcccct accctgtgcc agaagcaaca gaggtgccag gcctcagcct cacccacagc 15181 agccccatcg ccaaaggatg gaagtaattt gaatagctca ggtgtgctga ccccagtgaa 15241 gacaccagat aggacctctg agaggggact ggcatggggg gatctcagag cttacaggct 15301 gagccaagcc cacggccatg cacagggaca ctcacacagg cacacgcacc tgctctccac 15361 ctggactcag gccgggctgg gctgtggatt cctgatcccc tcccctcccc atgctctcct 15421 ccctcagcct tgctaccctg tgacttactg ggaggagaaa gagtcacctg aaggggaaca
15481 tgaaaagaga ctaggtgaag agagggcagg ggagcccaca tctggggctg gcccacaata 15541 cctgctcccc cgaccccctc cacccagcag tagacacagt gcacagggga gaagaggggt 15601 ggcgcagaag ggttgggggc ctgtatgcct tgcttctacc atgagcagag acaattaaaa 15661 tctttattcc agtgacagtg tctcttcttg agggagagag ggttgccaga aaacagtcag 15721 ttctccactc tctacttcaa ataagactca cttcttgttc tacaagggtc tagaaggaaa
15781 agtaaaaaaa aaagactctc gattcttaac
DETAILED DISCLOSURE OF THE INVENTION
The present invention provides a method for reducing atrial natriuretic peptide receptor A (NPRA) gene expression and/or function within a subject by administering an effective amount of an NPRA inhibitor to the subject. In one embodiment, the NPRA inhibitor is a polynucleotide that is specific for one or more target NPRA genes such that the polynucleotide decreases NPRA gene expression within the subject. In another embodiment, the NPRA inhibitor is a chemical compound, such as an oxindole (e.g., isatin). The method of the invention is useful for treating inflammatory diseases in human subjects and non-human subjects suffering from, or at risk for developing, inflammatory reactions.
The present invention includes, but is not limited to, the following embodiments: Embodiment 1: an isolated polynucleotide targeted to a target nucleic acid sequence within a natriuretic peptide receptor A (NPRA) gene or NPRA transcript, wherein said polynucleotide inhibits expression of said NPRA gene or transcript.
Embodiment 2: the polynucleotide of embodiment 1, wherein the NPRA is human NPRA (e.g., encoded by SEQ ID NO:4).
Embodiment 3: the polynucleotide of embodiment 1, wherein the target nucleic acid sequence is at least a portion of the human NPRA gene or transcript.
Embodiment 4: the polynucleotide of any of embodiments 1 to 3, wherein the target nucleic acid sequence is located in a region selected from the group consisting of the 5' untranslated region GJTR), transcription start site, translation start site, and 3' UTR.
Embodiment 5: the polynucleotide of any of embodiments 1 to 4, wherein the polynucleotide is a small interfering RNA (siRNA). Embodiment 6: the polynucleotide of any of embodiments 1 to 4, wherein the polynucleotide is an antisense molecule.
Embodiment 7: the polynucleotide of any of embodiments 1 to 4, wherein the polynucleotide is a ribozyme.
Embodiment 8: the polynucleotide of embodiment 1, wherein the polynucleotide comprises SEQ ID NO: 1 , or SEQ ID NO:2, or SEQ ID No:3.
Embodiment 9: the polynucleotide of embodiment 1 , wherein the NPRA gene or NPRA transcript is at least a portion of the mammal gene or transcript.
Embodiment 10: a method for reducing NPRA function in a subject, comprising administering an NPRA inhibitor to the subject, such as the polynucleotide of any of embodiments 1 to 9, wherein the polynucleotide is administered in an effective amount to reduce expression of the NPRA gene or transcript.
Embodiment 11 : the method of embodiment 10, wherein the subject is suffering from an inflammatory disease, respiratory allergy, viral infection (such as respiratory virus infection), or cancer (such as melanoma, lung cancer, or ovarian cancer). Embodiment 12: the method of embodiment 10, wherein the subject is not suffering from an inflammatory disease, respiratory allergy, viral infection, or cancer.
Embodiment 13: the method of any one of embodiments 10 to 12, wherein the subject is human.
Embodiment 14: the method of any one of embodiments 10 to 12, wherein the subject is a non-human mammal.
Embodiment 15: the method of any one of embodiments 10 to 14, wherein the NPRA inhibitor is delivered to cells within the subject selected from the group consisting of respiratory epithelial cells, dendritic cells, and monocytes.
Embodiment 16: the method of any one of embodiments 10 to 15, wherein the NPRA inhibitor is administered to the subject intranasally. Embodiment 17: the method of any one of embodiments 10 to 16, wherein the NPRA inhibitor is administered intranasally as drops or as an aerosol, or orally or transdermally.
Embodiment 18: the method of any one of embodiments 10 to 17, wherein step of administering comprises administering a combination of NPRA inhibitors that reduce the function of NPRA within the subject (such as a combination of polynucleotide, e.g., an siRNA pool).
Embodiment 19: the method of any one of embodiments 10 to 18, wherein the NPRA inhibitor is a siRNA and wherein the siRNA reduces expression of NPRA within the subject
Embodiment 20: the method of any one of embodiments 10 to 18, wherein the NPRA inhibitor is an oxindole, such as 5-hydroxyoxindole or isatin, or a pharmaceutically acceptable salt thereof (Cane, A. et al. Biochem. Biophy. Res Comm, 2000, 276:379-384; Vine, K.L. et al. Bioorg Med Chem, 2007, 15(2):931-938; Abadi, A.H. et al. Eur J Med Chem, 2006, 41(3):296-305; Igosheva, N. et al. Neurochem Int, 2005, 47(3):216-224; Liu, Y. et al. Chem Biol, 2003, 10(9):837-846; Levy, J.A. et al. Virology, 1976, 74(2):426-431; Popp, F.O. J Med Chem, 1969, 12(1):182-184). Isatin (also known as lH-indole-293-dione) is an indole derivative (Sumpter, W.C. Chem Rev, 34(3):393-434; Ogata, A. et al J Neurol Sci, 2003, 206(l):79-83; Glover, V. et al. J Neurochem, 1988, 51(2):656-659; Filomeni, G. et al J Biol Chem, 2007, 282(16):12010- 12021).
As used herein, the term "polypeptide" refers to any polymer comprising any number of amino acids, and is interchangeable with "protein", "gene product", and "peptide". As used herein, the term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. The term "nucleotide" refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3' carbon atoms. As used herein, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" refers generally to a polymer of ribonucleotides. The term "DNA" or "DNA molecule" or deoxyribonucleic acid molecule" refers generally to a polymer of deoxyribonucleotides. DNA and RNA molecules can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA molecules can be post- transcriptionally modified. DNA and RNA molecules can also be chemically synthesized. DNA and RNA molecules can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). Based on the nature of the invention, however, the term "RNA" or "RNA molecule" or "ribonucleic acid molecule" can also refer to a polymer comprising primarily (i.e., greater than 80% or, preferably greater than 90%) ribonucleotides but optionally including at least one non-ribonucleotide molecule, for example, at least one deoxyribonucleotide and/or at least one nucleotide analog.
As used herein, the term "nucleotide analog", also referred to herein as an "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non- naturally occurring ribonucleotides or deoxyribonucleotides. Preferred nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function.
As used herein, the term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. Exemplary RNA analogues include sugar- and/or backbone- modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate (mediates) RNA interference or otherwise reduce target gene expression.
As used herein, the term "operably-linked" or "operatively-linked" refers to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function. Thus, a flanking sequence operably-linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably-linked to a promoter when the promoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably-linked" to the coding sequence. Each nucleotide sequence coding for a siRNA will typically have its own operably-linked promoter sequence.
The term "vector" or "vehicle" is used to refer to any molecule (e.g. , nucleic acid, plasmid, or virus) used to transfer coding information (e.g., a polynucleotide of the invention) to a host cell. The term "expression vector" refers to a vector that is suitable for use in a host cell (e.g., a subject's cell) and contains nucleic acid sequences which direct and/or control the expression of exogenous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present. The vectors of the present invention can be conjugated with chitosan or chitosan derivatives. Such chitosan conjugates can be administered to hosts according to the methods of the present invention. For example, polynucleotide chitosan nanospheres can be generated, as described by Roy, K. et αl. (Nat Med, 1999, 5:387). Chitosan allows increased bioavailability of the nucleic acid sequences because of protection from degradation by serum nucleases in the matrix and thus has great potential as a mucosal gene delivery system. Chitosan also has many beneficial effects, including anticoagulant activity, wound-healing properties, and immunostimulatory activity, and is capable of modulating immunity of the mucosa and bronchus-associated lymphoid tissue. In one embodiment of the present invention, the vectors are conjugated with chitosan-derived nanoparticles.
As used herein, the term "RNA interference" ("RNAi") refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.
As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNAs") refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.
As used herein, a siRNA having a "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the siRNA has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process. RSV "mRNA", "messenger RNA", and "transcript" each refer to single-stranded RNA that specifies the amino acid sequence of one or more RSV polypeptides. This information is translated during protein synthesis when ribo somes bind to the mRNA.
As used herein, the term "cleavage site" refers to the residues, e.g. , nucleotides, at which RISC* cleaves the target RNA, e.g., near the center of the complementary portion of the target RNA, e.g., about 8-12 nucleotides from the 5' end of the complementary portion of the target RNA.
As used herein, the term "mismatch" refers to a basepair consisting of non- complementary bases, e.g., not normal complementary G:C, A:T or A:U base pairs. As used herein, the term "isolated" molecule (e.g., isolated nucleic acid molecule) refers to molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, the NPRA inhibitors of the invention are administered in an isolated form. As used herein, the term "in vitro" has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells in an organism, e.g., immortalized cells, primary cells, and/or cell lines in an organism.
A gene "involved in" or "associated with" a disorder includes a gene, the normal or aberrant expression or function of which affects or causes a disease or disorder or at least one symptom of the disease or disorder. For example, NPRA protein has been found to have a significant role in pulmonary inflammation and immune modulation. Without being bound by theory, it has been found that signaling through the NPRA protein results in increased cGMP production and activation of protein kinase G3 leading to regulation of transcription of many genes involved in the cell cycle, apoptosis, and inflammation. The polynucleotides, genetic constructs, pharmaceutical compositions, and methods of the invention are useful in decreasing expression of NPRA gene, in vitro or in vivo, consequently causing decreased production of the NPRA protein and decreased inflammation. Thus, the polynucleotides, genetic constructs, pharmaceutical compositions, and methods of the invention are useful in the treatment of human or non- human animal subjects suffering from, or at risk of developing, disorders associated with inflammation including, but not limited to, airway diseases, viral infections, and cancers.
The methods of the invention may include further steps. In some embodiments, a subject with the relevant condition or disease involving aberrant inflammation (e.g., asthma, RSV infection, cancers) is identified, or a subject at risk for the condition or disease is identified. A subject may be someone who has not been diagnosed with the disease or condition (diagnosis, prognosis, and/or staging) or someone diagnosed with the disease or condition (diagnosis, prognosis, monitoring, and/or staging), including someone treated for the disease or condition (prognosis, staging, and/or monitoring). Alternatively, the subject may not have been diagnosed with the disease or condition but suspected of having the disease or condition based either on patient history or family history, or the exhibition or observation of characteristic symptoms.
As used herein, an "effective amount" of a NPRA inhibitor (e.g., isatin or another oxindole, an siRNA, an antisense nucleotide sequence or strand, and/or a ribozyme), which selectively interferes with expression of the NPRA gene and/or function of the receptor, is that amount effective to bring about the physiological changes desired in the cells to which the polynucleotide is administered in vitro (e.g., ex vivo) or in vivo. The term "therapeutically effective amount" as used herein, means that amount of NPRA inhibitor (e.g., isatin or other oxindole, an siRNA, an antisense oligonucleotide, and/or a ribozyme), which selectively reduces expression of the NPRA gene(s) and/or function of the receptor, alone or in combination with another agent according to the particular aspect of the invention, that elicits the biological or medicinal response in cells (e.g., tissue(s)) that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation and/or prevention of the symptoms of the disease or disorder being treated. For example, a NPRA inhibitor can be administered to a subject in combination with other agents effective for alleviating or preventing the symptoms of inflammation, such as the gene expression vaccines (Mohapatra et al 2004).
Various methods of the present invention can include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control", referred to interchangeably herein as an "appropriate control". A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing a siRNA of the invention into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.
RNA Interference RNAi is an efficient process whereby double-stranded RNA (dsRNA, also referred to herein as siRNAs or ds siRNAs, for double-stranded small interfering RNAs) induces the sequence-specific degradation of targeted mRNA in animal and plant cells (Hutvagner and Zamore, Curr. Opin. Genet. Dev., 12:225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001). In mammalian cells, RNAi can be triggered by 21 -nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al, MoI. Cell, 10:549-561 (2002); Elbashir et al, Nature 411 :494-498 (2001), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which can be expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al, MoI Cell 9:1327-1333 (2002); Paddison et al, Genes Dev. 16:948-958 (2002); Lee et al, Nature Biotechnol. 20:500-505 (2002); Paul et al, Nature Biotechnol 20:505-508 (2002); Tuschl, T., Nature Biotechnol 20:440-448 (2002); Yu et al, Proc. Natl. Acad. ScI USA 99(9):6047-6052 (2002); McManus et al, RNA 8:842-850 (2002); Sui et al, Proc. Natl Acad. Set. USA 99(6):5515-5520 (2002).
Accordingly, the invention includes such molecules that are targeted to NPRA mRNAs encoding at least a portion of one or more of NPRA-Iike receptors.
siRNA Molecules
The nucleic acid molecules or constructs of the invention include dsRNA molecules comprising 16-30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA of the RSV mRNA, and the other strand is identical or substantially identical to the first strand. The dsRNA molecules of the invention can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNA molecules can be designed using any method known in the art, for instance, by using the following protocol:
1. Beginning with the AUG start codon, look for AA dinucleotide sequences; each AA and the 3' adjacent 16 or more nucleotides are potential siRNA targets. Further, siRNAs with lower G/C content (35-55%) may be more active than those with G/C content higher than 55%. Thus, in one embodiment, the invention includes polynucleotides having 35-55% G/C content. In addition, the strands of the siRNA can be paired in such a way as to have a 3' overhang of 1 to 4, e.g., 2, nucleotides. Thus, in another embodiment, the polynucleotides can have a 3' overhang of 2 nucleotides. The overhanging nucleotides can be either RNA or DNA. 2. Using any method known in the art, compare the potential targets to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences for which reduced expression is not desired. One such method for such sequence homology searches is known as BLAST, which is available at the National Center for Biotechnology Information web site of the National Institutes of Health.
3. Select one or more sequences that meet your criteria for evaluation. Further general information regarding the design and use of siRNA can be found in "The siRNA User Guide," available at the web site of the laboratory of Dr. Thomas Tuschl at Rockefeller University.
4. Negative control siRNAs preferably have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. The polynucleotides of the invention can include both unmodified siRNAs and modified siRNAs as known in the art. Thus, the invention includes siRNA derivatives that include siRNA having two complementary strands of nucleic acid, such that the two strands are crosslinked. For example, a 3' OH terminus of one of the strands can be modified, or the two strands can be crosslinked and modified at the 3' OH terminus. The siRNA derivative can contain a single crosslink (e.g., a psoralen crosslink). In some embodiments, the siRNA derivative has at its 3' terminus a biotin molecule (e.g., a photocleavable biotin), a peptide (e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organic compounds (e.g., a dye such as a fluorescent dye), or dendrimer. Modifying siRNA derivatives in this way can improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.
The nucleic acid compositions of the invention can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability, and/or half-life. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al, Drug Deliv. Rev. 47(1): 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al, J. Control Release 53(l-3):137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al, Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al, Eur. J, Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).
The nucleic acid molecules of the present invention can also be labeled using any method known in the art; for instance, the nucleic acid compositions can be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine. The labeling can be carried out using a kit, e.g., the SILENCER siRNA labeling kit (AMBION). Additionally, the siRNA can be radiolabeled, e.g., using 3H, 32P, or other appropriate isotope.
The dsRNA molecules of the present invention can comprise the following sequences as one of their strands, and the corresponding sequences of allelic variants thereof: SEQ ID NO: 1 or SEQ ID NO:2.
Moreover, because RNAi is believed to progress via at least one single-stranded RNA intermediate, the skilled artisan will appreciate that ss-siRNAs {e.g., the antisense strand of a ds-siRNA) can also be designed as described herein and utilized according to the claimed methodologies.
siRNA Delivery for Longer-Term Expression
Synthetic siRNAs can be delivered into cells by methods known in the art, including cationic liposome transfection and electroporation. However, these exogenous siRNA generally show short-term persistence of the silencing effect (4 to 5 days in cultured cells), which may be beneficial in certain embodiments. To obtain longer term suppression of RSV gene expression and to facilitate delivery under certain circumstances, one or more siRNA duplexes, e.g., RSV ds siRNA, can be expressed within cells from recombinant DNA constructs. Such systems for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer-term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., Hl or U6/snRNA promoter systems (Tuschl (2002), supra) capable of expressing functional double-stranded siRNAs; (Bagella et al, J. Cell. Physiol. 177:206-213 (1998); Lee et al. (2002), supra; Miyagishi et al. (2002), supra; Paul et al. (2002), supra; Yu et al. (2002), supra; Sui et al. (2002), supra). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5 '-3' and 3 '-5' orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by an Hl or U6 snRNA promoter can be expressed in cells, and can inhibit target gene expression (Bagella et al. (1998), supra; Lee et al (2002), supra; Miyagishi et al, (2002), supra; Paul et al (2002), supra; Yu et al. (2002), supra; Sui et al. (2002) supra). Constructs containing siRNA sequence(s) under the control of a T7 promoter also make functional siRNAs when co-transfected into the cells with a vector expressing T7 RNA polymerase (Jacque (2002), supra). A single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the RSV NS 1 mRNA and/or other RSV genes, and can be driven, for example, by separate PoIIII promoter sites.
Animal cells express a range of non-coding RNAs of approximately 22 nucleotides termed micro RNA (miRNAs) that can regulate gene expression at the post transcriptional or translational level during animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with miRNA sequence complementary to the target mRNA, a vector construct that expresses the novel miRNA can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng (2002), supra). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression (McManus (2002), supra). Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al. (2002), supra). Infection of HeLa cells by these recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of the recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into post- implantation mouse embryos (Calegari et al, Proc. Natl, Acad. ScL USA 99(22): 14236- 40 (2002)). In adult mice, efficient delivery of siRNA can be accomplished by the "high- pressure" delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA-containing solution into animal via the tail vein (Liu (1999), supra; McCaffrey (2002), supra; Lewis, Nature Genetics 32:107-108 (2002)). Nanoparticles and liposomes can also be used to deliver siRNA into animals. Uses of Engineered RNA Precursors to Induce RNAi
Engineered RNA precursors, introduced into cells or whole organisms as described herein, will lead to • the production of a desired siRNA molecule. Such an siRNA molecule will then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage and destruction. In this fashion, the mRNA to be targeted by the siRNA generated from the engineered RNA precursor will be depleted from the cell or organism, leading to a decrease in the concentration of the RSV protein (such as RSV NSl protein) encoded by that mRNA in the cell or organism. The RNA precursors are typically nucleic acid molecules that individually encode either one strand of a dsRNA or encode the entire nucleotide sequence of an RNA hairpin loop structure.
Antisense
An "antisense" nucleic acid sequence (antisense oligonucleotide) can include a nucleotide sequence that is complementary to a "sense" nucleic acid sequence encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to at least a portion of an RSV gene. The antisense nucleic acid sequence can be complementary to an entire coding strand of a target sequence, or to only a portion thereof (for example, the RSV NSl gene and/or RSV NS2 gene, or a portion of either or both). In another embodiment, the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence within the RSV gene. An antisense oligonucleotide can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
An antisense nucleic acid sequence can be designed such that it is complementary to the entire RSV gene, but can also be an oligonucleotide that is antisense to only a portion of the RSV gene. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of the target mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest. An antisense oligonucleotide sequence can be, for example, about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length. An antisense nucleic acid sequence of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. , phosphorothioate derivatives and acridine substituted nucleotides can be used. The antisense nucleic acid sequence also can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid sequence will be of an antisense orientation to a target nucleic acid sequence of interest, described further in the following subsection).
The antisense nucleic acid molecules of the invention are typically administered to a subject (e.g., systemically or locally by direct injection at a tissue site), or generated in situ such that they hybridize with or bind to RSV mRNA to thereby inhibit expression of the viral protein. Alternatively, antisense nucleic acid molecules can be modified to target selected cells (such as respiratory epithelial cells, dendritic cells, and/or monocytes) and then administered systemically. For systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter can be used.
In yet another embodiment, the antisense oligonucleotide of the invention is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual beta-units, the strands run parallel to each other (Gaultier et al, Nucleic Acids. Res. 15:6625-6641 (1987)). The antisense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. Nucleic Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al. FEBS Lett., 215:327-330 (1987)). Gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene to form triple helical structures that prevent expression of the gene in target cells. See generally, Helene, C. Anticancer Drug
Des. 6:569-84 (1991); Helene, C. Ann. N.Y. Acad. Sci. 660:27-36 (1992); and Maher, Bioassays 14:807-15 (1992). The potential sequences that can be targeted for triple helix formation can be increased by creating a so-called "switchback" nucleic acid molecule.
Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
Ribozymes
Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. By cleaving the target RNA5 ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the art. Alternatively, ribozyme encoding nucleotide sequences can be introduced into cells through gene-delivery mechanisms known in the art. A ribozyme having specificity for RSV RNA can include one or more sequences complementary to the nucleotide sequence of at least a portion of one or more RSV mRNA (e.g. , RSV NS 1 mRNA), and a sequence having known catalytic sequence responsible for mRNA cleavage (see U.S. Patent No. 5,093,246 or Haselhoff and Gerlach Nature 334:585-591 (1988)). For example, a derivative of a Tetrahymena L- 19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in the RSV mRNA, such as RSV NSl mRNA (see, e.g., Cech et al. U.S. Patent No. 4,987,071; and Cech et al U.S. Patent No. 5,1 16,742). Alternatively, RSV mRNA encoding an RSV protein can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel, D. and Szostak, J. W. Science 261 :1411-1418 (1993)). Nucleic Acid Targets
The nucleic acid targets of the polynucleotides of the invention (e.g., antisense, RNAi, and ribozymes) may be ANP receptor gene, or a portion thereof, such as NPRA, NPRB or NPRC or portion of any of the foregoing. In some embodiments, the nucleic acid target is the NPRA gene, or a portion thereof. The nucleic acid target may be any location within the NPRA or transcript. Preferably, the nucleic acid target is located at a site selected from the group consisting of the 5' untranslated region (UTR), transcription start site, translation start site, and the 3 ' UTR.
The nucleic acid target may be located within a NPRA gene of any human or mammal. Preferably, the nucleic acid target is at least a portion of a non-structural NPRA gene. More preferably, the nucleic acid target is at least a portion of an NPRAgene encoding a protein. In a particularly preferred embodiment, the nucleic acid target is located within an NPRA that normally down-regulates host inflammation. In another preferred embodiment, the nucleic acid target is located within the human NPRA or mammalian NPRA, selected from the group consisting of the 5' untranslated region (UTR), transcription start site, translation start site, and the 3' UTR.
The nucleic acid target may be located within a human NPRA gene (NCBI accession no. AF 190631, which is incorporated herein by reference in its entirety) or an ortholog thereof, such as a non-human, mammalian NPRA gene. For treating and/or preventing inflammation within a particular subject, the polynucleotide selected for administration to the subject is preferably one targeted to a NPRA gene. For example, for treating and/or preventing inflammation within a human subject, the nucleic acid target is preferably located within a human NPRA gene, or the nucleic acid target has sufficient homology with the human NPRA gene, so as to reduce expression of the human NPRA gene. The term "substantially identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as substantially identical.
Calculations of homology or sequence identity between sequences (the terms are used interchangeably herein) are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. MoI. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at the official Accelrys web site), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at the official Accelrys web site), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. One set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a Blossurn 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other orthologs, e.g., family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. MoI. Biol. 215:403-10 (1990). BLAST nucleotide searches can be performed with the NBLAST program, score=100, word length=12, to obtain nucleotide sequences homologous to known RSV DNA and RNA sequences. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to known RSV polypeptide products. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see the National Center for Biotechnology Information web site of the National Institutes of Health).
Orthologs can also be identified using any other routine method known in the art, such as screening a cDNA library, e.g., using a probe designed to identify sequences that are substantially identical to a reference sequence.
Pharmaceutical Compositions and Methods of Administration
The NPRA inhibitors of the subject invention (e.g., isatin or other oxindols, siRNA molecules, antisense molecules, and ribozymes) can be incorporated into pharmaceutical compositions. Such compositions typically include the polynucleotide and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions. Formulations (compositions) are described in a number of sources that are well known and readily available to those skilled in the art. For example, Remington 's Pharmaceutical Sciences (Martin E.W., Easton Pennsylvania, Mack Publishing Company, 19th ed., 1995) describes formulations which can be used in connection with the subject invention.
A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g. , intravenous, intradermal, subcutaneous, oral (e.g., inhalation), nasal, topical, transdermal, transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediarninetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride can also be included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate or gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g. , a polynucleotide of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the polynucleotide into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, suitable methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as macrocrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the NPRA inhibitors can be delivered in the form of drops or an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays, drops, or suppositories. For transdermal administration, the active compound (e.g., polynucleotides of the invention) are formulated into ointments, salves, gels, or creams, as generally known in the art.
The pharmaceutical compositions can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. In embodiments in which the NPRA inhibitor is a polynucleotide, the polynucleotides can be administered by transfection or infection using methods known in the art, including but not limited to, the methods described in McCaffrey et al, Nature 418(6893):38-39 (2002) (hydrodynamic transfection)- Xia et al, Nature Biotechnol. 20(10):1006-10 (2002) (viral-mediated delivery); or Putnam, Am. J. Health Syst. Pharm. 53(2):15M60 (1996), erratum at Am. J. Health Syst. Pharm. 53(3):325 (1996).
The polynucleotides can also be administered by any method suitable for administration of nucleic acid agents, such as a DNA vaccine. These methods include gene guns, bio injectors, and skin patches as well as needle-free methods such as the micro-particle DNA vaccine technology disclosed in U.S. Patent No. 6,194,389, and the mammalian transdermal needle-free vaccination with powder-form vaccine as disclosed in U.S. Patent No. 6,168,587. Additionally, intranasal delivery is possible, as described in Hamajima et al, Clin. Immunol. Immunopathol 88(2):205-10 (1998). Liposomes (e.g., as described in U.S. Patent No. 6,472,375) and microencapsulation can also be used. Biodegradable targetable microparticle delivery systems can also be used (e.g., as described in U.S. Patent No. 6,471,996). Preferably, the polynucleotides of the invention are administered to the subject such that an effective amount are delivered to the respiratory epithelial cells, DC, and/or monocytes within the subject's airway, resulting in an effective amount of reduction in NPRA gene expression.
In one embodiment, the polynucleotides are prepared with carriers that will protect the polynucleotide against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using standard techniques. Liposomal suspensions (including liposomes targeted to antigen-presenting cells with monoclonal antibodies) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
Preferably, the NPRA inhibitors of the subject invention (e.g., compositions containing them) are administered locally or systemically such that they are delivered to target cells, such as cells of the airway, e.g., airway epithelial cells, which line the nose as well as the large and small airways. For some disorder, it is preferred that the NPRA inhibitors of the invention be delivered to dendritic cells and/or monocytes.
Toxicity and therapeutic efficacy of compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compositions which exhibit high therapeutic indices can be used. While compositions that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
Data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compositions generally lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test composition which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography. The compositions of the invention can be administered on any appropriate schedule, e.g. , from one or more times per day to one or more times per week; including once every other day, for any number of days or weeks, e.g. , 1 day, 1 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 3 months, 6 months, or more, or any variation thereon. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a NPRA inhibitor can include a single treatment or can include a series of treatments.
Mammalian species that benefit from the disclosed methods include, but are not limited to, primates, such as apes, chimpanzees, orangutans, humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets; domesticated farm animals such as cows, buffalo, bison, horses, donkey, swine, sheep, and goats; exotic animals typically found in zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sea lions, elephant seals, otters, porpoises, dolphins, and whales. As used herein, the terms "subject", "host", and "patient" are used interchangeably and intended to include such human and non-human mammalian species. Likewise, in vitro methods of the present invention can be carried out on cells of such mammalian species. Host cells comprising exogenous polynucleotides of the invention may be administered to the subject, and may, for example, be autogenic (use of one's own cells), allogenic (from one person to another), or transgenic or xenogenic (from one species to another), relative to the subject.
The polynucleotides of the invention can be inserted into genetic constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et ai, (2002), supra. Genetic constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Patent No. 5,328,470) or by stereotactic injection (see, e.g. , Chen et ai, Proc. Natl. Acad. Sci. USA 91 :3054-3057 (1994)). The pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the polynucleotide delivery system.
The polynucleotides of the invention can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21 nucleotides (Brummelkamp et al, Science 296:550-553 (2002); Lee et al, (2002), supra; Miyagishi and Taira, Nature Biotechnol. 20:497-500 (2002); Paddison et al. (2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al (2002), supra.
SiRNAs of the invention may be fused to other nucleotide molecules, or to polypeptides, in order to direct their delivery or to accomplish other functions. Thus, for example, fusion proteins comprising a siRNA oligonucleotide that is capable of specifically interfering with expression of NPRA gene may comprise affinity tag polypeptide sequences, which refers to polypeptides or peptides that facilitate detection and isolation of the polypeptide via a specific affinity interaction with a ligand. The ligand may be any molecule, receptor, counter-receptor, antibody or the like with which the affinity tag may interact through a specific binding interaction as provided herein. Such peptides include, for example, poly-His or "FLAG" or the like, e.g., the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., (Bio/Technology 6:1204, 1988), or the XPRESS epitope tag (INVITROGEN, Carlsbad, Calif.). The affinity sequence may be a hexa-histidine tag as supplied, for example, by a pBAD/His (INVITROGEN) or a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the affinity sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g., COS-7 cells, is used. The HA tag corresponds to an antibody defined epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984 Cell 21:761). The present invention also relates to vectors and to constructs that include or encode polynucleotides of the present invention (e.g., siRNA), and in particular to "recombinant nucleic acid constructs" that include any nucleic acid such as a DNA polynucleotide segment that may be transcribed to yield NPRAmRNA-specific siRNA polynucleotides according to the invention as provided above; to host cells which are genetically engineered with vectors and/or constructs of the invention and to the production of siRNA polynucleotides, polypeptides, and/or fusion proteins of the invention, or fragments or variants thereof, by recombinant techniques. siRNA sequences disclosed herein as RNA polynucleotides may be engineered to produce corresponding DNA sequences using well-established methodologies such as those described herein. Thus, for example, a DNA polynucleotide may be generated from any siRNA sequence described herein, such that the present siRNA sequences will be recognized as also providing corresponding DNA polynucleotides (and their complements). These DNA polynucleotides are therefore encompassed within the contemplated invention, and can, for example, be incorporated into the subject invention recombinant nucleic acid constructs from which siRNA may be transcribed.
According to the present invention, a vector may comprise a recombinant nucleic acid construct containing one or more promoters for transcription of an RNA molecule, for example, the human U6 snRNA promoter (see, e.g., Miyagishi et al, Nat. Biotechnol. 20:497-500 (2002); Lee et al, Nat. Biotechnol. 20:500-505 (2002); Paul et al, Nat. Biotechnol. 20:505-508 (2002); Grabarek et al, BioTechniques 34:73544 (2003); see also Sui et al, Proc. Natl. Acad. Sci. USA 99:5515-20 (2002)). Each strand of a siRNA polynucleotide may be transcribed separately each under the direction of a separate promoter and then may hybridize within the cell to form the siRNA polynucleotide duplex. Each strand may also be transcribed from separate vectors (see Lee et al, supra). Alternatively, the sense and antisense sequences specific for an RSV sequence may be transcribed under the control of a single promoter such that the siRNA polynucleotide forms a hairpin molecule (Paul et al, supra). In such an instance, the complementary strands of the siRNA specific sequences are separated by a spacer that comprises at least four nucleotides, but may comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 94 18 nucleotides or more nucleotides as described herein. In addition, siRNAs transcribed under the control of a V6 promoter that form a hairpin may have a stretch of about four uridines at the 3' end that act as the transcription termination signal (Miyagishi et al, supra; Paul et al, supra). By way of illustration, if the target sequence is 19 nucleotides, the siRNA hairpin polynucleotide (beginning at the 5' end) has a 19-nucleotide sense sequence followed by a spacer (which as two uridine nucleotides adjacent to the 3' end of the 19-nucleotide sense sequence), and the spacer is linked to a 19 nucleotide antisense sequence followed by a 4-uridine terminator sequence, which results in an overhang. siRNA polynucleotides with such overhangs effectively interfere with expression of the target polypeptide. A recombinant construct may also be prepared using another RNA polymerase III promoter, the Hl RNA promoter, that may be operatively linked to siRNA polynucleotide specific sequences, which may be used for transcription of hairpin structures comprising the siRNA specific sequences or separate transcription of each strand of a siRNA duplex polynucleotide (see, e.g., Brummelkamp et ai, Science 296:550-53 (2002); Paddison et al, supra). DNA vectors useful for insertion of sequences for transcription of an siRNA polynucleotide include pSUPER vector (see, e.g., Brummelkamp et al, supra}; pAV vectors derived from pC WRSVN (see, e.g., Paul et al, supra); and pIND (see, e.g. , Lee et al. , supra), or the like.
Polynucleotides of the invention can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters, providing ready systems for evaluation of NPRA polynucleotides that are capable of interfering with expression of NPRA gene, as provided herein. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, by Sambrook, et al, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N. Y., (2001).
The appropriate DNA sequence(s) may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described, for example, in Ausubel et al. (1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., Boston, Mass.); Sambrook et al. (2001 Molecular Cloning, Third Ed., Cold Spring Harbor Laboratory, Plainview, N. Y.); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.); and elsewhere.
The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequences (e.g., a promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include LTR or SV40 promoter, the E. coli lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter, or regulated promoter, operably linked to a polynucleotide of the invention is described herein.
As noted above, in certain embodiments the vector may be a viral vector such as a mammalian viral vector (e.g., retrovirus, adenovirus, adeno-associated virus, lentivirus). For example, retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
The viral vector includes one or more promoters. Suitable promoters that may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques 7:980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and beta-actin promoters). Other viral promoters that may be employed include, but are not limited to, adenovirus promoters, adeno-associated virus promoters, thymidine kinase (TK) promoters, and Bl 9 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be from among either regulated promoters (e.g., tissue-specific or inducible promoters) or promoters as described above. A tissue-specific promoter allows preferential expression of the polynucleotide in a given target tissue (such as tissue of the respiratory tract), thereby avoiding expression in other tissues. For example, to express genes specifically in the heart, a number of cardiac-specific regulatory elements can be used. An example of a cardiac-specific promoter is the ventricular form of MLC-2v promoter (see, Zhu et al., Mol Cell Biol. 13:4432-4444, 1993; Navankasattusas et al, MoI. Cell Biol. 12:1469- 1479, 1992) or a variant thereof such as a 281 bp fragment of the native MLC-2v promoter (nucleotides -264 to +17, Genebank Accession No. U26708). Examples of other cardiac-specific promoters include alpha myosin heavy chain (Minamino et al, Circ. Res. 88:587-592, 2001) and myosin light chain-2 (Franz et al, Circ. Res. 73:629- 638, 1993). Endothelial cell gene promoters include endoglin and ICAM-2. See Velasco et al., Gene Ther. 8:897-904, 2001. Liver-specific promoters include the human phenylalanine hydroxylase (PAH) gene promoters (Bristeau et al, Gene 274:283-291, 2001), hBlF (Zhang et al, Gene 273:239-249, 2001), and the human C-reactive protein (CRP) gene promoter (Ruther et al, Oncogene 8:87-93, 1993). Promoters that are kidney-specific include CLCN5 (Tanaka et al, Genomics 58:281-292, 1999), renin (Sinn et al, Physical Genomics 3:25-31, 2000), androgen-regulated protein, sodium-phosphate cotransporter, renal cytochrome P-450, parathyroid hormone receptor and kidney-specific cadherin. See Am. J. Physiol. Renal Physiol. 279:F383-392, 2000. An example of a pancreas-specific promoter is the pancreas duodenum homeobox 1 (PDX-I) promoter (Samara et al, MoI. Cell Biol. 22:4702-4713, 2002). A number of brain-specific promoters may be useful in the invention and include the thy-1 antigen and gamma- enolase promoters (Vibert et al, Eur. J. Biochem. 181:33-39, 1989), the glial-specific glial fibrillary acidic protein (GFAP) gene promoter (Cortez et al, J. Neurosci. Res. 59:39-46, 2000), and the human FGFl gene promoter (Chiu et al, Oncogene 19:6229- 6239, 2000). The GATA family of transcription factors have promoters directing neuronal and thymocyte-specific expression (see Asnagli et al, J. Immunol. 168:4268- 4271, 2002).
In another aspect, the present invention relates to host cells containing the above described recombinant constructs. Host cells are genetically engineered/modified (transduced, transformed or transfected) with the vectors and/or expression constructs of this invention that may be, for example, a cloning vector, a shuttle vector, or an expression construct. The vector or construct may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying particular genes such as genes encoding siRNA polynucleotides or fusion proteins thereof. The culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan.
The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Representative examples of appropriate host cells according to the present invention include, but need not be limited to, bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells, such as Drosophila S2 and Spodoptera Sf9; animal cells, such as CHO, COS or 293 cells; adenoviruses; plant cells, or any suitable cell already adapted to in vitro propagation or so established de novo.
Various mammalian cell culture systems can also be employed to produce polynucleotides of the invention from recombinant nucleic acid constructs of the present invention. The invention is therefore directed in part to a method of producing a polynucleotide, such as a siRNA, by culturing a host cell comprising a recombinant nucleic acid construct that comprises at least one promoter operably linked to a polynucleotide of the invention that is specific for NPRA gene. In certain embodiments, the promoter may be a regulated promoter as provided herein, for example a tetracycline- repressible promoter. In certain embodiments, the recombinant expression construct is a recombinant viral expression construct as provided herein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa, HEK, and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences, for example as described herein regarding the preparation of recombinant polynucleotide constructs. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, for example, liposomes including cationic liposomes, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et ah, 1986 Basic Methods in Molecular Biology), or other suitable technique.
The expressed polynucleotides may be useful in intact host cells; in intact organelles such as cell membranes, intracellular vesicles or other cellular organelles; or in disrupted cell preparations including but not limited to cell homogenates or lysates, microsomes, uni- and multilamellar membrane vesicles or other preparations. Alternatively, expressed polynucleotides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
As used herein, the terms "administer", "apply", "treat", "transplant", "implant", "deliver", and grammatical variations thereof, are used interchangeably to provide NPRA inhibitors of the subject invention {e.g., vectors containing or encoding polynucleotides of the subject invention) to target cells in vitro or in vivo, or provide genetically modified (engineered) cells of the subject invention to a subject ex vivo.
As used herein, the term "co-administration" and variations thereof refers to the administration of two or more agents simultaneously (in one or more preparations), or consecutively. For example, one or more types of NPRA inhibitors of the invention (e.g., vectors containing or encoding polynucleotides of the subject invention) can be coadministered with other agents.
As used in this specification, including the appended claims, the singular "a", "an", and "the" include plural reference unless the contact dictates otherwise. Thus, for example, a reference to "a polynucleotide" includes more than one such polynucleotide. A reference to "a nucleic acid sequence" includes more than one such sequence. A reference to "a cell" includes more than one such cell. The terms "comprising", "consisting of and "consisting essentially of are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.
Example 1 — ANP overexpression in the lung augments inflammation and cytokine production in splenocytes.
ANP has been suspected to play a role in decreasing inflammation, as it was shown to play a role in decreasing TNF-α production from macrophages and slightly decreased NFkB activation (Mohapatra et al. JACI, 2004). Also, NPRA deficient mice did not exhibit inflammation. Since excess ANP expression activates the clearance receptor, it was hypothesized that ANP actually increases inflammation. To test this, naϊve mice were administered intranasally a plasmid pVAX expressing the ANP peptide. The results show that ANP overexpression actually increases inflammation. Materials and Methods
Animals. Six-week old female BALB/c mice from Jackson laboratory (Bar Harbor, ME) were maintained in pathogen free conditions in accordance with animal research committee regulations.
Construction of ANP expression vector. Total RNA was isolated from murine heart using Trizol reagent (LIFE TECHNOLOGY, Gaithersburg, MD) following the manufacturer's protocol. The cDNA sequence for the ANP, residues 99-126 of pro ANP was amplified by RT-PCR. A translation initiation codon was inserted in the forward primers, so that the recombinant peptides had an additional amino acid, methionine, as the first amino acid apart from its known content. The PCR product was cloned in pVAX vector (INVITROGEN, Carlsbad, CA) at HindIII and Xhol sites. The cloned ANPsequence was verified by DNA sequencing and its expression was checked in A549 human epithelial cells.
Analysis of intracellular cytokine production in T cells. Mouse spleen T cells purified using mouse T-cell enrichment column kit (R & D Systems, Minneapolis, MN) were cultured in 6-well plates for 4 days. Finally, cells were stimulated with PMA (50 ng/ml) and ionomycin (500 ng/ml) (SIGMA, Saint Louis, Missouri) for 6 hours in the presence of GOLGISTOP (PHARMINGEN, San Diego, CA) and then fixed and stained using CD8 or CD4 mAb (BD BIOSCIENCES, San Diego, CA) for flow cytometry analysis.
Histological analysis. Mouse lungs were removed after 24 hours of intranasal pANP administration, fixed, and sections stained with H&E. Results. Normal BALB/c mice were given i.n. nanoparticles carrying pANP or pVAX and their lungs were examined 3 days after by staining the sections (H&E), showing goblet cell hyperplasia. These results directly demonstrate that in normal mice over expression of ANP results in bronchial inflammation. To demonstrate that ANP over expression also stimulates immune system, BALB/c mice were given i.p. OVA (with alum) and then challenged i.n. OVA. Mice were sacrificed, the spleens aseptically removed and the cells were cultured for 48 hours in the presence of OVA (Sigma) and recombinant IL-2. Cells were removed from culture and stained for surface markers CD4 and CD3 and intracellular cytokines IL-4, IL-IO and IFN-g (BD Pharmingen). The results show that in normal mice in absence of any antigen sensitization, ANP overexpression increases expression of boANP in general augments inflammation by activating both innate and adaptive immunity.
Example 2 — Inhibitory effect of transfected siRNA plasmids on NPRA expression.
To determine whether siRNAs can be produced that will effectively decrease NPRA expression, 11 different siRNA oligos were designed and cloned in a pU6 vector. Cells transfected with each of the construct was examined for NPRA protein expression by western blotting.
Materials and Methods
Plasmid constructs. The nucleotide sequence for each siRNA is described previously (SEQ ID #1-1 1). Each pair of oligos was inserted into pU6 plasmid at appropriate sites respectively, to generate the corresponding siRNA for siNPRA.
DNA transfection. Cells were transfected with siNPRA or controls (siU6) using LIPOFECTAMINE 2000 reagent (INVITROGEN, Carlsbad, CA). pEGFP plasmid (STRATAGENE, La Jolla, CA) was used for measurement of transfection efficiency. Protein expression analysis by Western blotting. Transfected cells were used to prepare whole cell lysates, which were electrophoresed on 12% polyacrylamide gels and the proteins were transferred to PVDF membranes (BIO-RAD, Hercules, CA). The blot was incubated separately with NPRA polyclonal antibody (SANTA CRUZ BIOTECH, Santa Cruz, CA), immunoblot signals were developed by SUPER SIGNAL ULTRA chemiluminescent reagent (PIERCE3 Rockford, IL).
Results. Eleven different siRNA oligos were designed specifically targeting NPRA gene. The siRNA oligos were cloned in pU6 vector. Fig 2 shows results the inserts being present in the plasmids. The inserts were sequenced to confirm the presence of siRNA inserts in them. Cells in 6-well plates were transfected with psiNPRA (2ug). Forty eight hours later total protein were extracted western blotted using an antibody to NPRA. Results from two different experiments are shown in Figures 3A-3C. Plasmids encoding ANP, Kp73-102 and VD were used as control, since they have been shown to downregulate NPRA expression. In the third experiment, HEKGCA cells grown in 6-well plates were transfected with psiNPRA (2ug), as indicated and forty eight hours later total protein were extracted western blotted using an antibody to NPRA (Figure 3C). Untransfected cells and cells transfected with U6 vector plasmid without any siNPRA were used as control. Also, filters were stripped and reprobed with antibody to beta-actin. The experiments were repeated. The results showed that 3 of 11 siNPRA constructs consistently decreased NPRA protein expression in the HEKGCA cells.
To confirm these results, inhibitory effect of siRNA in vitro was examined using HEKGCA cells. Cells grown in 6-well plates were transfected with psiNPRA (2ug). Forty eight hours later, cells were subjected to flow cytometry to detect NPRA positive cells using an antibody to NPRA (Figure 4A). U6 plasmid without any siRNA and Plasmid encoding Kp73-102 was used as controls, since the latter has been shown to downregulate NPRA expression. B) Mice (n=4) were intranasally administered with 25ug siRNA plasmids complexed with 125ul of chitosan nanoparticles. BAL was done 72 hours later. Cells were stained by NPRA Ab. NPRA expression cells were counted (Figure 4B).
Together the results show that SΪNPRA8, siNPRA9 and siNR 10 were the most effective siRNAs that significantly reduced NPRA expression. Example 3 — Demonstration that oral siNPRA treatment decreases inflammation, eosinophilia and Th2 cytokines in BALB/c mice.
To determine whether decreased expression of NPRA by siNPRA treatment will reduce inflammation in asthma, the effect of intranasal siNPRA9 was tested in ovalbumin-induced mouse model of asthma.
Materials and Methods. Six to eight week-old BALB/c mice (n=6) were sensitized by i.p. injection of ovalbumin (50ug in 2mg of alum/mouse) and challenged intranasally with OVA (50μg). Mice were given two SΪNPRA9 treatments by gavage and challenged 24 hours later. After a further 24 hours of challenge, mice were sacrificed and their lungs removed for histology in a subgroup (n=3) of mice. The remainder of the group were lavaged and a cell differential was performed as described, especially to enumerate the eosinophil numbers in the BAL fluid. Thoracic lymph node cells (A) and spleen cells (B) were removed and cells cultured for 48 hours in the presence of OVA (Sigma Grade V) and recombinant mouse IL-2. Naϊve mice received no treatment. Cells were treated with GolgiStop (BD Pharmingen) and stained for surface and intracellular cytokines (Antibodies obtained from BD Pharmingen). Percent cytokine secreting cells were quantified by intracellular cytokine staining using flow cytometry, as described.
Results. The results of lung histology, i.e., lung sections stained by H &E revealed that compared to untreated Ovalbumin-sensitized and mice treated with scrambled si-NPRA group, treated mice showed a significant reduction in lung inflammation. The lung histology was very similar to the naϊve mice. There was significant reduction in epithelial goblet cell hyperplasia and a significant reduction in peribroncial, perivascular and interstitial infiltration of the inflammatory cells to the lung (Figures 6A-6C). There was also a significant reduction in the number of eosinophils in BAL fluid (Figure 5A) and reduction in Th2 cytokines in thoracic lymph nodes as determined by intracellular cytokine staining (Figure 5B- 1 and 5B-2).
Example 4 — Demonstration that transdermal siNPRA treatment decreases inflammation, eosinophilia and Th2 cytokines in BALB/c mice. Patients are more compliant when the drug is delivered by transdermal route.
Therefore, siNPRA8 delivered by transdermal route was attempted to determine whether such siRNA therapy would decrease pulmonary inflammation in this ovalbumin-induced mouse model of asthma.
Materials and Methods. BALB/c mice (n=5 each group) were sensitized (i.p.) as in example #3 and challenged (i. n.) with 50 μg of OVA. Mice were given siNPRA (oligonucleotide) treatments by transdermal route (siNPRA8) and challenged 4 hours later. Following 24 hours of challenge two mice were sacrificed to obtain lungs and which were fixed sectioned and immunostained for NPRA expression(A). Mice (n=3) were sacrificed and lavaged and the percentage of eosinophils (B) and IL-4 concentration (C) in the lavage fluid was determined. Results. Since intradermal delivery of siRNA has not been shown previously, the lung sections were first checked for the expression of NPRA and whether siRNA delivered by transdermal route decreases NPRA expression. The results are shown in Figure 7A and indicate that lungs of ova-sensitized mice and mice treated with scrambled si-NPRA8 show higher number of cells expressing NPRA. siNPRA treatment decreased the expression level significantly. Typically, epithelial cells did not express NPRA and although not verified it is the dendritic cells appear to be involved in NPRA expression. The siNPRAδ treated mice also showed a significant reduction in eosinophil numbers (Figure 7B) and levels of IL-4 (Figure 7C) in the BAL. The results of H & E staining of lung sections showed that compared to untreated Ovalbumin-sensitized and mice treated with scrambled si-NPRA8 group, treated mice showed a significant reduction in lung inflammation (Figures 8A and 8B). There was a significant reduction in epithelial goblet cell hyperplasia and a significant reduction in peribroncial, perivascular and interstitial infiltration of the inflammatory cells to the lung. Together these results show that transdermal delivery of siNPRA8 decreases NPRA expression and inflammation of the lung and reduction of IL-4 and eosinophils in the lung.
Example 5 — Demonstration that transfection of A549 cells with psiNPRA9 decreases the number of respiratory syncytial virus (RS\O infection infected cells.
Respiratory syncytial virus infection also causes bronchiolitis in newborns and in elderly causing pneumonitis, which is characterized severe acute lung inflammation.
RSV infection typically requires certain host cell proteins and transcription factors for its replication and subsequent infection of others cells. Since siNPRA treatment decreases pulmonary inflammation, the effect of siNPRA9 transfection on RSV infection was examined in pulmonary type-II epithelial cells was examined.
Materials and Methods. RT-PCR analysis of NPRA expression in the lung of mice treated with siRNA. psiNPRA9 was encapsulated with chitosan nanoparticles and intranasally delivered to mice. Twenty-four hours later, mice were infected with RSV(5xl06 pfu/mouse). Four days later, mice were sacrificed and lung cells were collected for RNA extraction. NPRA fragment were amplified by RT-PCR using NPRA specific primers (F:5'-GCA AAG GCC GAG TTA TCT ACA TC-3', R:5'-AAC GTA GTC CTC CCC ACA CAA -3) and analyzed in 1% agarose gel. To determine the effect of siNPRA9 on RSBV infection of epithelial cells, A549 cells were grown in 6 well plate, transfected by siNPRA8, siNPRA9 or control U6 plasmid (2.0ug) and 2 hours after infected by rgRSV (MOI=0.2). Cells were checked for infection 48 hours later, FACS was done. Also, A549 cells were grown in 6 well plate infected by rgRSV (MOI=O.2) and 24 hours after infection they were transfected by siNPRAδ, siNPRA9 or control U6 plasmid (2.0ug) and further 24hr later, Flow cytometry was performed to estimate percentage of infected cells.
Results. The RT-PCR analysis showed that both RSV infected mice and mice infected with RSV and intranasally treated with pU6 control plasmid given with chitosan nanoparticles showed NPRA expression in the lung cells. However, mice infected with RSV and intranasally given psiNPRA9 showed an amplification product that was reduced in band intensity compared to cells from mice given pU6 plasmid. The lung cells from NPRA knock-out mice showed the band as well but it was reduced in intensity.
To determine the effect of siNPRA9 on rgRSV infection of A549 cells, either cells were grown in 6 well plate, transfected by siNPRA8, siNPRA9 or control U6 plasmid (2.0ug) and 2 hours after infected by rgRSV (MOI=0.2) (prophylactic approach), or A549 cells were grown in 6 well plate infected by rgRSV (MOI=O.2) and 24 hours after infection they were transfected by siNPRA8, siNPRA9 or control U6 plasmid (2.0ug) (therapeutic approach) and further 24hr later, flow cytometry was performed to estimate percentage of infected cells. The results showed whether prophylactic approach or therapeutic approach the results showed a 20% reduction in rgRSV infected cells in cells treated with siNPRAδ and/or siNPRA9 compared to siU6 control plasmid. Thus these results show that siNPRA treatment can decrease RSV infection in addition to inflammation as seen in other studies.
Example 6 — Demonstration that siNPRA treatment decreases melanoma tumor formation in Bl 6 mouse model.
Because siNPRA molecules are deliverable by transdermal route and treatment with siNPRA decreases local and systemic inflammation, which has been recently attributed toward the origin of certain cancers, the effect of siNPRA on melanoma was tested. The neoplastic transformation of the melanocyte involves differential ability of the melanoma cell versus the melanocyte to cope with oxidative stress. Melanocytes produce reactive radicals and have a low level of anti-oxidant enzymes, responding to UV with a large but transient increase in superoxide anion whereas keratinocytes and fibroblasts do not. Also, the comparative resting levels of the subunits forming the transcription factor NFkB are altered between melanocytes and melanoma cells both under resting and UVB stimulated conditions (Chin, L et al Genes Dev, 1998, 12(22) .3467-348126). Thus, the effect of the role of NPRA in melanoma was investigated.
Materials and Methods. Bl 6 melanoma cells (1.3x105) were injected subcutaneously into twelve-week old female C57BL/6 mice or NPRA-deficient mice produced in B6 background. These mice were then treated with 33μg of siNPRA-oligos, siNPRA9 plasmid, or scrambled oligos. All of these were mixed with Chitosan at ratio of 1:2.5. Mixed chitosan and plasmid or oligos were mixed again with cream before application to the injection area. The control group was given cream only. These treatments were given twice a week. Mice were sacrificed on day twenty second, tumors were removed and weighed. Results. To determine the role of NPRA in melanoma, groups of wild-type (WT) and NPRA'Λ mice (n=8) were given subcutaneously 3 X 105 B16F10.9 cells and the tumor progression was observed after 14 days. The WT mice produced tumors whereas NPRA-/- mice did not have any tumors ANP pathway is a major pathway promoting melanoma tumors in C57BL/6-B16F10.9 model (Figures 12A- 12E). To quantify the results, the tumor size and burden were measured in WT and NPRA"7' mice injected s.c. with B 16 melanoma cells. A significant reduction (P<0.01) in mean tumor volume measured over 18 days after B16 cell injection and a significant decrease in tumor weight at day 18 was found in NPRA"7' mice (n = 12) compared to WT (Figures 13A and 13B).
Since, NPRA-deficient mice may have other abnormalities which might make it resistant, the WT mice were injected with 3 X 105 B16F10.9 cells and were then treated with a cream containing siNPRA 9 given twice a week at the location of tumor cell injection. Three weeks later, both treated and control mice treated with cream alone without siNPRA were compared for their tumor burden. Figure 13C shows a comparison of both groups of mice. Excision of these tumors revealed that siNPRA, but not siNPRA scrambled, showed significant reductions in tumor burden. These results together show that siNPRA can be used to treat melanomas.
Example 7 — Demonstration that siNPRA treatment decreases melanoma tumor formation in Lewis Lung Carcinoma B 16 mouse model.
Methods: For challenge with Lewis lung cancer cells, LLCl cells grown in DMEM were washed with phosphate buffered saline (PBS) and resuspended in PBS at 2x107 cells per ml. Two groups of mice (n = 8 per group) were tested: WT C57BL/6 mice and C57BL/6 NPRA-deficient mice. Animals were injected subcutaneously with 2x106 LLCl cells (100 μl) in the right flank. Tumor sizes were measured at days 10, 13, 15 and 17 after injection. All animals were sacrificed on day 17 and the tumors were removed and weighed.
Results: Using the Lewis lung carcinoma model, C57BL/6 WT mice and NPRA gene knockout (NPRA''') mice (n = 8 for each group) were injected s.c. with 2 xlO6 cells LLCl cells in the right flank. Tumors appeared within one week after injection and tumor size was measured with a digital caliper beginning on day 10. The tumors in WT mice grew rapidly after day 10, but tumors in NPRA7' mice gradually shrank. On day 17, all mice were sacrificed, and tumor sizes and weights were measured. In one of the NPRA"'" mice, there were no visible tumors at all. Significant differences (P<0.001) in tumor size and weight were observed between the two groups Example 8 — Demonstration that siNPRA treatment decreases melanoma tumor formation in ID8 ovarian cancer mouse model.
Methods: For challenge with ovarian cancer cells, ID-8 ovarian cancer cells grown in
DMEM were washed with PBS and resuspended in PBS at 2x107 cells per ml. Two groups of mice (n = 8 per group) were tested: WT C57BL/6 mice and C57BL/6 NPRA- deficient mice. Animals were injected subcutaneously with 2xlO6 ID8 cells (100 μl).
Tumor sizes were measured at days 10, 13, 15 and 17 after injection. All animals were sacrificed on day 17 and the tumors were removed and weighed.
Results: Groups (n = 8) of WT mice and NPRA-deficient C57BL/6 mice were injected with 2 x 106 ID8 mouse ovarian cancer cells at day 1 and mice were monitored at weekly intervals for tumor growth. By week 8 after cancer cell inoculation, all mice from the
WT group developed solid tumors but no tumors were found in NPRA-deficient mice.
The results indicate that NPRA deficiency significantly protects mice from ovarian cancer. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.

Claims

CLAIMSWe claim:
1. An isolated polynucleotide targeted to a nucleic acid sequence within a natriuretic peptide receptor A (NPRA) gene or NPRA transcript, wherein said polynucleotide inhibits expression of said NPRA gene or transcript.
2. The polynucleotide of claim 1, wherein the NPRA is human NPRA.
3. The polynucleotide of claim 1, wherein the target nucleic acid sequence is at least a portion of the human NPRA gene or transcript.
4. The polynucleotide of any of claims 1 to 3, wherein the target nucleic acid sequence is located in a region selected from the group consisting of the 5' untranslated region (UTR), transcription start site, translation start site, and 3' UTR.
5. The polynucleotide of any of claims 1 to 45 wherein the polynucleotide is a small interfering RNA (siRNA).
6. The polynucleotide of any of claims 1 to 4, wherein the polynucleotide is an antisense molecule.
7. The polynucleotide of any of claims 1 to 4, wherein the polynucleotide is a ribozyme.
8. The polynucleotide of claim 1, wherein the polynucleotide comprises SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3.
9. The polynucleotide of claim 1, wherein the NPRA gene or NPRA transcript is at least a portion of the mammal gene or transcript.
10. A composition comprising the polynucleotide of any of claims 1-9; and a pharmaceutically acceptable carrier.
11. A vector comprising the polynucleotide of any of claims 1-9; and an operably linked promoter.
12. A method for reducing the function of the atrial natriuretic peptide receptor A (NPRA) in a subject, comprising administering an NPRA inhibitor to the subject, wherein the NPRA inhibitor is administered in an effective amount to reduce NPRA function.
13. The method of claim 12, wherein the subject is suffering from an inflammatory disease, respiratory allergy, viral infection, and/or cancer.
14. The method of claim 12, wherein the subject is not suffering from an inflammatory disease, respiratory allergy, viral infection, and/or cancer.
15. The method of any one of claims 12 to 14, wherein the subject is human.
16. The method of any one of claims 12 to 14, wherein the subject is a non-human mammal.
17. The method of any one of 12 to 16, wherein the NPRA inhibitor is delivered to cells within the subject selected from the group consisting of respiratory epithelial cells, dendritic cells, and monocytes.
18. The method of any one of claims 12 to 16, wherein the NPRA inhibitor is administered to the subject intranasally.
19. The method of any one of claims 12 to 165 wherein the NPRA inhibitor is administered intranasally as drops or as an aerosol, or orally or transdermally.
20. The method of any one of claims 12 to 19, wherein step of administering comprises administering a combination of NPRA inhibitors to the subject.
21. The method of any one of claims 12 to 20, wherein the NPRA inhibitor is a polynucleotide of any one of claims 1 to 9 wherein the polynucleotide reduces expression of the NPRA gene or transcript.
22. The method of claim 21, wherein the polynucleotide is an siRNA and wherein the siRNA reduces expression of NPRA within the subject.
23. The method of any one of claims 12 to 20, wherein the NPRA inhibitor is an oxindole.
24. The method of claim 23, wherein the NPRA inhibitor is isatin.
25. The method of any of claims 12 to 24, wherein the subject is suffering from respiratory virus infection, melanoma, lung cancer, or ovarian cancer.
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