Title:

Materials and methods for treatment of inflammatory and cell proliferation disorders

Document Type and Number:

United States Patent Application 20050272650

Kind Code:

Abstract:

The present invention pertains to methods for treatment of inflammatory and cell proliferation disorders, such as cancer, by administering an agent that reduces atrial natriuretic peptide receptor-A (NPR-A) activity. In one aspect, the invention concerns a method for treatment of inflammatory and cell proliferation disorders, such as cancer, by administration of an effective amount of natriuretic hormone peptide (NP), or a polynucleotide encoding NP and an operably-linked promoter sequence. In another aspect, the present invention includes a pharmaceutical composition comprising an agent that reduces the activity of atrial natriuretic peptide receptor-A (NPR-A), and an anti-cancer agent. In another aspect, the present invention further concerns a method for identifying an agent useful for treating an inflammatory or cell proliferation disorder, comprising determining whether the agent reduces the activity of atrial natriuretic peptide receptor-A (NPR-A).

Representative Image:

Materials and methods for treatment of inflammatory and cell proliferation disorders

Inventors:

Mohapatra, Shyam S. (Tampa, FL, US)

Plaque It!

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. Provisional Application Ser. No. 60/521,072, filed Feb. 17, 2004, which is hereby incorporated by reference herein in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, or drawings.

BACKGROUND OF THE INVENTION

The vast majority of cancers of the lung, breast and colon are adenocarcinomas, which arise from pre-existing adenomatous polyps that develop in the normal colonic mucosa. This adenoma-carcinoma sequence is a well-characterized clinical and histopathologic series of events with which discrete molecular genetic alterations have been associated. Lung tumor development and metastasis are complex processes that include transformation, proliferation, resistance to apoptosis, neovascularization, and metastatic spread. A number of gene products have been identified that play critical roles in these processes. It has been suggested that the development of epithelial-derived tumors, the most common class of cancers, involves mutations of tumor suppressors and proto-oncogenes or epigenetic alterations of signaling pathways affecting cell proliferation and/or survival, which in turn may be caused by inflammation induced by infections and reactive oxygen species (ROS) (Ernst, P. Aliment Pharmacol Ther., 1999, 13(1):13-18).

A group of four peptide hormones, originating from the 126-amino acid atrial natriuretic factor (ANF) prohormone, have become known for their vasodilator activity. These four peptide hormones, consisting of amino acids 1-30, 31-67, 79-98, and 99-126 of this prohormone, have been named long acting natriuretic peptide (LANP), vessel dilator (VD), kaliuretic peptide (KP), and atrial natriuretic peptide (ANP), respectively, for their most prominent effects (Angus R. M. et al., Clin Exp Allergy 1994, 24:784-788). The ANP sequence, particularly the C-terminal portion, is highly conserved among species (Seidman et al., Science, 1984, 226:1206-1209). ANP has been proposed to be useful for treatment of various cardiovascular, respiratory, and renal diseases (Vesely, D. L. Cardiovascular, 2001, 51:647-658), but also causes inflammation. The family of natriuretic hormone peptides has been shown to have broad physiologic effects, including vasodilation and inhibition of aldosterone secretion and cardiovascular homeostasis.

As indicated above, ANF, the 126 amino acid prohormone, gives rise to four peptides: LANP (amino acids 1-30), VD (amino acids 31-67), KP (amino acids 79-98) and ANP (amino acids 99-126, also referred to herein as NP _99-126) (Angus R. M. et al, Clin Exp Allergy, 1994, 24:784-788). The ANP sequence particularly the C-terminal portion is highly conserved among species (Seidman et al., Science, 1984, 226: 1206-1209). The natriuretic peptide receptors (NPRs), NPR-A and NPR-B, are expressed in many different tissues of various organs systems, and are coupled to guanylyl cyclase. ANP and BNP are thought to signal primarily through NPR-A by increasing cGMP and activating cGMP-dependent protein kinase (PKG). NPR-A is the primary receptor for ANP while NPR-B seems to bind CNP most effectively. PKG activation in turn activates ion transporters and transcription factors, which together affect cell growth and proliferation, apoptosis and inflammation. NPR-C is a clearance receptor for ANP removal, but also appears to signal phospholipase C activation and a decrease in adenylyl cyclase activity through a cGMP-independent pathway (Abbey and Potter, Endocrinology, 2003, 144: 240-246; Silberbach and Roberts, Cell Signal, 2001, 13:221-231). The signaling mechanisms underlying ANP's growth regulatory effects are poorly understood, although a number of reports suggest that ANP acts through mitogen-activated protein kinases (Silberbach and Roberts, Cell Signal, 2001, 13:221-231).

Most cells of the mucosal immune system have ANP receptors (NPRs) and there is evidence that natriuretic peptides regulate the immune response and inflammation (Kurihara et al., Biochem Biophys Res Commun 1987, 149:1132-1140). ANP stimulates migration of human neutrophils (Izumi et al., J Clin Invest 2001, 108:203-213), and inhibit nitric oxide and TNF-α production by murine macrophages (Kiemer and Vollmar, J Biol Chem 1998, 273:13444-13451; Kiemer et al., J Immunol 2000, 165:175-81). It has been suggested that the ANP system may be a critical component of the immune response through its actions on both immune and non-immune cells. In patients with lung tumors, the immune response plays a large part in the progression of the disease and, consequently, the NPR system may potentially be involved. The alveolar macrophages in lung cancer patients secrete more pro-inflammatory cytokines, such as IL-6 and IL-1β, after LPS stimulation than in persons with non-malignant disease (Matanic et al., Scand J Immunol 2003, 57: 173-178). Increased IL-6 in lung cancer patients enhances the acute phase response, and is correlated with poor nutritional status and lowered survival (Martin et al., Cytokine 1999, 11; 267-273). The cells of the immune system, such as natural killer (NK) cells, Vα24 NKT, which are necessary for cancer surveillance may also be reduced in lung tumor patients (Motohashi et al., Int J Cancer 2002, 102:159-165). The most common clinical paraneoplastic syndrome in patients with small-cell lung cancer (SCLC) is hyponatremia, which is believed to be caused by tumor secretion of vasopressin. Tumor biopsies from patients with SCLC and hyponatremia expressed the gene for ANP (Shimizu et al., Cancer 1991, 68: 2284-2288; Bliss et al., J Natl Can Inst, 1990, 82: 305-310). Thus, the reduced sodium levels seen in SCLC patients may be attributed to the secretion of ANP (Bliss et al., J Natl Can Inst, 1990, 82: 305-310). Human SCLC cell lines express functional ANP receptors (Ohsaki et al., Cancer Res 1993, 53: 3165-3171). A majority of SCLC cell lines produce ANP and some produce BNP as well (Ohsaki et al., Oncology 1999, 56: 155-159). In contrast, in NSCLC cell lines, which are derived mostly from adenocarcinomas that comprise about two-thirds of all lung cancers, little is known about their growth regulation in response to ANP cascade.

The present inventor has found that the N-terminal natriuretic peptides, such as pNP73-102, are capable of inhibiting NFkB activation (Mohapatra, international application WO 2004/022003, published Mar. 18, 2004, which is incorporated herein by reference in its entirety), and that the ANP cascade plays a critical role in cell proliferation and inflammation. NFkB, a transcription factor and a key player in inflammatory processes, has been implicated in the development of cancer in liver and mammary tissues (Greten F. R. et al. Cell, 2004, 118: 285-296; Pikarsky E. et al. Nature, 2004, 431: 461-466). Activation of the NF-κB pathway enhances tumor development and may act primarily in the late stages of tumorigenesis. Inhibition of NF-κB signaling uniformly suppressed tumor development; however, depending upon the model studied, this salutary effect was attributed to an increase in tumor cell apoptosis, reduced expression of tumor cell growth factors supplied by surrounding stromal cells, or abrogation of a tumor cell dedifferentiation program that is critical for tumor invasion/metastasis.

BRIEF SUMMARY OF THE INVENTION

In another aspect, the present invention concerns methods for identifying agents useful for treating inflammatory and cell proliferation disorders by determining whether the candidate agent reduces activity of the natriuretic peptide receptor-A (also known in the art as NPRA, NPR-A, and guanylate cyclase A) in vitro and/or in vivo (also referred to herein as the diagnostic method or assay of the invention).

In another aspect, the method of the present invention may be used for reducing the growth of cancer cells in vitro or in vivo. In one aspect, the method is useful for treating cancers, such as adenocarcinomas of lung, breast, ovary and melanomas, which may be caused by cell proliferation and inflammation induced by the atrial natriuretic peptide (ANP) cascade.

In one embodiment, the method of the present invention comprises administering a therapeutically effective amount of an agent that reduces NPR-A activity. In another embodiment, the method of the present invention comprises administering a therapeutically effective amount of an N-terminal natriuretic peptide (referred to herein as NP or NP peptide), or a polynucleotide encoding NP and an operably-linked promoter sequence, to a patient in need of such treatment. As used herein, NP refers to peptides derived from atrial natriuretic factor (ANF) hormone, or a biologically active fragment, homolog, or variant thereof. In another embodiment, the method of the present invention comprises administering an effective amount of NP, or a polynucleotide encoding NP and an operably-linked promoter, to one or more cancer cells, wherein the NP is capable of reducing cell proliferation and/or tumor growth. The effect of the NP or a biologically active fragment, homolog, or variant thereof, is capable of reducing cancer cell growth in vitro or in vivo.

Specifically exemplified NPs comprise an amino acid sequence selected from the group consisting of amino acids 1-30 of ANF (also known as “long acting natriuretic peptide” and referred to herein as NP _1-30or SEQ ID NO:1), amino acids 31-67 of ANF (also known as “vessel dilator” and referred to herein as NP _31-67or SEQ ID NO:2), and amino acids 79-98 of ANF (also known as “kaliuretic peptide” and referred to herein as NP _79-98or SEQ ID NO:3), or biologically active fragments or homologs of any of the foregoing. Other exemplified NPs comprise amino acids 73-102 of proANF (referred to herein as NP _73-102or SEQ ID NO:5), or SEQ ID NO:6, or biologically active fragment(s) or homolog(s) of the foregoing. In one embodiment, the NP administered to the patient does not consist of NP _99-126(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 NP to a patient in need of such treatment. The present inventor has determined that introduction of a nucleic acid molecule encoding NP is capable of inhibiting tumor growth and tumor metastasis. The gene delivery method of the present invention permits long-term expression of NP-encoding nucleic acid sequences in vivo, thereby conferring anti-cancer effects. In one embodiment, a therapeutically effective amount of at least one nucleic acid molecule encoding a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5 or biologically active fragments or homologs of any of the foregoing, are administered to the patient.

In another aspect, the present invention concerns an isolated peptide comprising the amino acid sequence NP _73-102(SEQ ID NO:5) or SEQ ID NO:6, or a biologically active fragment or homolog of the foregoing. In another aspect, the present invention concerns an isolated nucleic acid molecule encoding the amino acid sequence of NP _73-102(SEQ ID NO:5) or encoding the amino acid sequence of SEQ ID NO:6, or a biologically active fragment or homolog thereof.

In another aspect, the present invention concerns an expression vector comprising a nucleic acid sequence encoding an NP, and a promoter sequence that is operably linked to the NP-encoding nucleic acid sequence. In one embodiment, the expression vector is a DNA plasmid or virus. In another aspect, the present invention concerns a pharmaceutical composition comprising a nucleic acid sequence encoding an NP, and a pharmaceutically acceptable carrier.

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:

FIG. 1 shows pNP 73-102 inhibits NPRA expression. Pregnant (12 days) mice were injected i.p. with pVAX (vector), or pNP73-102. After 1 day, mice were sacrificed, thymi removed from the embryo, and homogenized. Cells were centrifuged and erythrocytes were lysed and incubated with anti-NPR-Ab or anti-NPR-C for 1 hour, washed, and incubated with PE-conjugated secondary antibodies. Levels of NPRA and NPRC were determined by flow cytometry.

FIGS. 2A-2D show NPRA deficiency decreases pulmonary inflammation. Groups (n=3) of wild type DBA/2 (wt) (FIG. 2A) and NPR-C deficient (NPRC ^−/−) (FIG. 2B) mice and wild type C57/BL6 (wt) (FIG. 2C) and NPR-A (NPRA ^−/−) (FIG. 2D) were sensitized with OVA (20 mg/mouse) and after 2 weeks challenged i.n. with OVA (20 mg/mouse). One day later mice were sacrificed and lung sections were stained with H & E to examine inflammation.

FIGS. 3A-3D demonstrate that A549 cells transfected with pNP _73-102show a significantly higher level of apoptosis compared to control and pANP or pVAX (FIGS. 3A-3C). Cells were transfected with pNP73-102, pANP and pVAX (as control) and cells were stained with PI and annexin and quantified by flow cytometry (FIG. 3D). The proteins were isolated and an equal amount of the cell lysates were western-blotted using an antibody to poly-ADP ribose polymerase (PARP). The results demonstrate that pNP73-102 shows a higher accumulation of apoptotic cells compared to cells transfected with pANP and pVAX controls.

FIG. 4 shows that pNP73-102 decreases tumorigenesis in a colony formation assay by A549. Six centimeter tissue culture plates were covered with 4 ml of 0.5% soft agar. A549 cells were transfected with pANP, pNP _73-102and pVAX plasmid DNA (V) or nothing (C). After 40 h of transfection, cells were suspended in 2 ml of 0.3% soft agar and added to each plate. Cells were plated in duplicate at a density of 2×10 ⁴cells/dish and incubated for two weeks. Plates were photographed under a microscope. Cell colonies were counted and plotted. The results of one representative experiment of two is shown.

FIGS. 5A-5E show expression of NP _73-102-FLAG in the BAL cells after i.n. administration of chitosan encapsulated plasmid pNP _73-102-FLAG construct. BAL was performed in mice (n=3) after 24 hours and BAL cells were stained with either the second antibody control or anti-FLAG antibody (SIGMA) and then with DAPI. A representative staining is shown (FIGS. 5A-5C). FIG. 5D shows lungs removed from mice treated with chitosan nanoparticles carrying pNP _73-102(CPNP73-102) (Rx) or empty plasmid pVAX (control). The lungs of control mice showed several lung nodules in contrast to mice treated with CPNP73-102, which showed very few tumors. Intranasal CPNP73-102 administration abrogated tumor formation in A549 injected nude mice. Nude mice were given 5×10 ⁶cells intravenously (tail vein) and weekly injections of nanoparticle carrying either empty plasmid (control) or pNP73-102 (Rx). Three weeks later, mice were sacrificed and lung sections were stained with H & E to examine the lung nodules (FIG. 5D). Control shows nodules and tumor cell mass, whereas the treated group had no tumors. Sections were also stained with antibodies to cyclinB and to phospho-Bad (FIG. 5E). The results show that mice treated with CPNP73-102 had no tumors in the lung and did not show any staining for pro-mitotic Cyclin-B and anti-apoptotic marker phospho-Bad.

FIGS. 6A-6D demonstrate that treatment with chitosan nanoparticles carrying pNP _73-102(CPNP73-102) decreases the tumor burden in a spontaneous tumorigenesis model of immunocompetent BALB/c mice. Two groups of mice (n=4) were administered with the Line-1 tumor cells (100,000 cells/mouse) at the flanks. One group was administered with CPNP73-102 the same day, whereas another group was administered with vehicle alone (nanoparticle carrying a plasmid without NP73-102) and the third group was given the saline. Treatment was continued with CPNP73-102 or control at weekly intervals for 5 weeks. The tumors were dissected out from the mice of each group (FIGS. 6A-6C) and the tumor burden was calculated by weighing them on a balance and expressed as tumor mass per g lung weight. Results are shown in FIG. 6D.

FIG. 7 shows that CPNP73-102 induces apoptosis in chemoresistant ovarian cancer cells. C-13 and OV2008 ovarian cancer cells were transfected with pNP73-102. Forty-eight hours later, cells were processed for TUNEL assay to examine apoptosis. The results of one of two representative experiments are shown.

FIG. 8 shows breast cancer MCF-7 cell counts. The cells were transfected with pVAX, pANP, and pANP _73-102and counted at 24 and 48 hours after transfection. 30 ml of Trypan Blue was mixed with 30 ml for measuring the cell viability. The results of one of two representative experiments are shown.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the amino acid sequence of human “long acting natriuretic peptide” or NP _1-30: ¹NPMYN AVSNADLMDF KNLLDHLEEK MPLED ³⁰(SEQ ID NO:1).

SEQ ID NO:2 is the amino acid sequence of human “vessel dilator” or NP _31-67: ³¹EVVPP QVLSEPNEEA GAALSPLPEV PPWTGEVSPA QR ⁶⁷(SEQ ID NO:2).

SEQ ID NO:3 is the amino acid sequence of human “kaliuretic peptide” or NP _79-98: ⁷⁹SSDRSAL LKSKLRALLT APR ⁹⁸(SEQ ID NO:3).

SEQ ID NO:4 is the amino acid sequence of human “atrial natriuretic peptide” (ANP) or NP _99-126: ⁹⁹SLRRSSC FGGRMDRIGA QSGLGCNSFR Y ¹²⁶(SEQ ID NO:4).

SEQ ID NO:5 is the amino acid sequence of cloned mouse pNP _73-102: ⁷³GSPWDPSDRS ALLKSKLRAL LAGPRSLRR ¹⁰²(SEQ ID NO:5).

SEQ ID NO:6 is the amino acid sequence of cloned mouse NP fragment: VSNTDLMDFK NLLDHLEEKM PVEDEVMPPQ ALSEQTE (SEQ ID NO:6).

SEQ ID NO:7 is the amino acid sequence for the human preproANP (NCBI ACCESSION # NM _—006172) wherein the underlined amino acids represent the signal sequence which is cleaved off to form the mature peptide:


¹ MSSFSTTTVS FLLLLAFQLL GQTRA NPMYN AVSNADLMDF KNLLDHLEEK	(SEQ ID NO:7)

MPLEDEVVPP QVLSEPNEEA GAALSPLPEV PPWTGEVSPA QRDGGALGRG

PWDSSDRSAL LKSKLRALLT APRSLRRSSC FGGRMDRIGA QSGLGCNSFR Y ¹⁵¹.

SEQ ID NO:8 is a forward primer for the cDNA sequence encoding mouse prepro ANF protein:


5′-gac ggc aag ctt act atg ggc agc	(SEQ ID NO:8)

ccc tgg gac cc-3′.

SEQ ID NO:9 is a reverse primer for the cDNA sequence encoding mouse pre-proANF protein:


(SEQ ID NO:9)
5′-acc ccc ctc gag tta tta tct tcg tag gct ccg-3′.

SEQ ID NO:10 is a forward primer for the cDNA sequence encoding mouse NP fragment:


(SEQ ID NO:10)
5′-aat cct aag ctt agt atg gtg tcc aac aca gat-3′.

SEQ ID NO:11 is a reverse primer for the cDNA sequence encoding mouse NP fragment:


5′-tgc gaa ctc gag tta ctc agt ctg	(SEQ ID NO:11)

ctc act cag ggc ctg cg-3′.

SEQ ID NO:12 is the nucleotide sequence encoding cloned mouse pNP _73-102:


atg ggc agc ccc tgg gac ccc tcc gat	(SEQ ID NO:12)

aga tct gcc ctc ttg aaa agc aaa ctg

agg gct ctg ctc gct ggc cct cgg agc

cta cga aga taa.

SEQ ID NO:13 is the nucleotide sequence encoding cloned mouse pNP fragment:


atg gtg tcc aac aca gat ctg atg gat	(SEQ ID NO:13)

ttc aag aac ctg cta gac cac ctg gag

gag aag atg ccg gta gaa gat gag gtc

atg ccc ccg cag gcc ctg agt gag cag

act gag taa.

SEQ ID NO:14 is the mRNA nucleotide sequence encoding human ANP (NCBI Accession # NM _—006172:


1	tggcgaggga cagacgtagg ccaagagagg ggaaccagag aggaaccaga ggggagagac	(SEQ ID NO:14)

61	agagcagcaa gcagtggatt gctccttgac gacgccagca tgagctcctt ctccaccacc

121	accgtgagct tcctcctttt actggcattc cagctcctag gtcagaccag agctaatccc

181	atgtacaatg ccgtgtccaa cgcagacctg atggatttca agaatttgct ggaccatttg

241	gaagaaaaga tgcctttaga agatgaggtc gtgcccccac aagtgctcag tgagccgaat

301	gaagaagcgg gggctgctct cagccccctc cctgaggtgc ctccctggac cggggaagtc

361	agcccagccc agagagatgg aggtgccctc gggcggggcc cctgggactc ctctgatcga

421	tctgccctcc taaaaagcaa gctgagggcg ctgctcactg cccctcggag cctgcggaga

481	tccagctgct tcgggggcag gatggacagg attggagccc agagcggact gggctgtaac

541	agcttccggt actgaagata acagccaggg aggacaagca gggctgggcc tagggacaga

601	ctgcaagagg ctcctgtccc ctggggtctc tgctgcattt gtgtcatctt gttgccatgg

661	agttgtgatc atcccatcta agctgcagct tcctgtcaac acttctcaca tcttatgcta

721	actgtagata aagtggtttg atggtgactt cctcgcctct cccaccccat gcattaaatt

781	ttaaggtaga acctcacctg ttactgaaag tggtttgaaa gtgaataaac ttcagcacca

841	tggac.

SEQ ID NO:15 is the human gene for atrial natriuretic factor propeptide (coding sequence includes—join (570 . . . 692, 815 . . . 1141, 2235 . . . 2240); sig. peptide=570 . . . 644; mat. peptide=join (645 . . . 692, 815 . . . 1141, 2235 . . . 2237), (NCBI ACCESSION NO: X01471; Greenberg, B. D. et al., Nature, 1984, 312(5995):656-658):


1	ggatccattt gtctcgggct gctggctgcc tgccatttcc tcctctccac ccttatttgg	(SEQ ID NO:15)

61	aggccctgac agctgagcca caaacaaacc aggggagctg ggcaccagca agcgtcaccc

121	tctgtttccc cgcacggtac cagcgtcgag gagaaagaat cctgaggcac ggcggtgaga

181	taaccaagga ctctttttta ctcttctcac acctttgaag tgggagcctc ttgagtcaaa

241	tcagtaagaa tgcggctctt gcagctgagg gtctgggggg ctgttggggc tgcccaaggc

301	agagaggggc tgtgacaagc cctgcggatg ataactttaa aagggcatct cctgctggct

361	tctcacttgg cagctttatc actgcaagtg acagaatggg gagggttctg tctctcctgc

421	gtgcttggag agctgggggg ctataaaaag aggcggcact gggcagctgg gagacaggga

481	cagacgtagg ccaagagagg ggaaccagag aggaaccaga ggggagagac agagcagcaa

541	gcagtggatt gctccttgac gacgccagca tgagctcctt ctccaccacc accgtgagct

601	tcctcctttt actggcattc cagctcctag gtcagaccag agctaatccc atgtacaatg

661	ccgtgtccaa cgcagacctg atggatttca aggtagggcc aggaaagcgg gtgcagtctg

721	gggccagggg gctttctgat gctgtgctca ctcctcttga tttcctccaa gtcagtgagg

781	tttatccctt tccctgtatt ttccttttct aaagaatttg ctggaccatt tggaagaaaa

841	gatgccttta gaagatgagg tcgtgccccc acaagtgctc agtgagccga atgaagaagc

901	gggggctgct ctcagccccc tccctgaggt gcctccctgg accggggaag tcagcccagc

961	ccagagagat ggaggtgccc tcgggcgggg cccctgggac tcctctgatc gatctgccct

1021	cctaaaaagc aagctgaggg cgctgctcac tgcccctcgg agcctgcgga gatccagctg

1081	cttcgggggc aggatggaca ggattggagc ccagagcgga ctgggctgta acagcttccg

1141	ggtaagagga actggggatg gaaatgggat gggatggaca ctactgggag acaccttcag

1201	caggaaaggg accaatgcag aagctcattc cctctcaagt ttctgcccca acacccagag

1261	tgccccatgg gtgtcaggac atgccatcta ttgtccttag ctagtctgct gagaaaatgc

1321	ttaaaaaaaa aagggggggg gctgggcacg gtcgtcacgc ctgtaatccc agcactttgg

1381	gaggccaggc agcggatcat gaggtcaaga gatcaagact atcctggcca acatggtgaa

1441	accccagctc tactaaaaat acaaaaatta gctgggtgtg tggcgggcac ctgtactctc

1501	agctacttgg gaggctgagg caggagaatc acttgaaccc aggaggcaga ggttgcagtg

1561	agcagagatc acgccactgc agtccagcct aggtgataga gcgagactgt ctcaaaaaaa

1621	aaaaaaaaag gccaggcgcg gtggctcacg cctgtaatcc cagcgctttg ggaggccaag

1681	gcgggtggat cacgaggtca ggagatggag accatcctgg ctaacacggt gaaaccccgt

1741	ctctactaaa aatacaaaaa attagccagg cgtggtggca ggcgcctgta agtcctagct

1801	actccggagg ctgaggcagg agaatggcgt gaacccggga ggcggagctt gcagtgagca

1861	gagatggcac cactgcactc cagcctgggc gacagagcaa gactccgtct caaaaaaaaa

1921	aaaaaaaaaa gcaactgcca ctagcactgg gaaattaaaa tattcataga gccaagttat

1981	ctttgcatgg ctgattagca gttcatattc ctccccagaa ttgcaagatc ctgaagggct

2041	taagtgaaat ttactctgat gagtaacttg cttatcaatt catgaagctc agagggtcat

2101	caggctgggg tgggggccgg tgggaagcag gtggtcagta atcaagttca gaggatgggc

2161	acactcatac atgaagctga cttttccagg acagccaggt caccaagcca gatatgtctg

2221	tgttctcttt gcagtactga agataacagc cagggaggac aagcagggct gggcctaggg

2281	acagactgca agaggctcct gtcccctggg gtctctgctg catttgtgtc atcttgttgc

2341	catggagttg tgatcatccc atctaagctg cagcttcctg tcaacacttc tcacatctta

2401	tgctaactgt agataaagtg gtttgatggt gacttcctcg cctctcccac cccatgcatt

2461	aaattttaag gtagaacctc acctgttact gaaagtggtt tgaaagtgaa taaacttcag

2521	caccatggac agaagacaaa tgcctgcgtt ggtgtgcttt ctttcttctt gggaagagaa

2581	ttc.

SEQ ID NO:16 is the amino acid sequence for the mouse preproANP peptide:


MGSFSITLGF FLVLAFWLPG HIGANPVYSA VSNTDLMDFK NLLDHLEEKM	(SEQ ID NO:16)

PVEDEVMPPQ ALSEQTEEAG AALSSLPEVP PWTGEVNPPL RDGSALGRSP

WDPSDRSALL KSKLRALLAG PRSLRRSSCF GGRIDRIGAQ SGLGCNSFRY RR.

SEQ ID NO:17 is the genetic sequence for the mouse preproANP peptide wherein the coding sequence starts at nucleic acid molecule position 81 and ends at nucleic acid molecule position 539:


1	caaaagctga gagagagaga gaaagaaacc agagtgggca gagacagcaa acatcagatc	(SEQ ID NO:17)

61	gtgccccgac ccacgccagc atgggctcct tctccatcac cctgggcttc ttcctcgtct

121	tggccttttg gcttccaggc catattggag caaatcctgt gtacagtgcg gtgtccaaca

181	cagatctgat ggatttcaag aacctgctag accacctgga ggagaagatg ccggtagaag

241	atgaggtcat gcccccgcag gccctgagtg agcagactga ggaagcaggg gccgcactta

301	gctccctccc cgaggtgcct ccctggactg gggaggtcaa cccacctctg agagacggca

361	gtgctctagg gcgcagcccc tgggacccct ccgatagatc tgccctcttg aaaagcaaac

421	tgagggctct gctcgctggc cctcggagcc tacgaagatc cagctgcttc gggggtagga

481	ttgacaggat tggagcccag agtggactag gctgcaacag cttccggtac cgaagataac

541	agccaaggag gaaaaggcag tcgattctgc ttgagcagat cgcaaaagat cctaagccct

601	tgtggtgtgt cacgcagctt ggtcacattg ccactgtggc gtggtgaaca ccctcctgga

661	gctgcggctt cctgccttca tctatcacga tcgatgttaa atgtagatga gtggtctagt

721	ggggtcttgc ctctcccact ctgcatatta aggtagatcc tcaccctttt cagaaagcag

781	ttggaaaaaa aaaaaaagaa taaacttcag caccaaggac agacgccgag gccctgatgt

841	gcttctttgg cttctgccct cagttctttg ctctcccc.

SEQ ID NO:18 is amino acid sequence of human natriuretic peptide receptor-A (NPR-A):


MPGPRRPAGSRLRLLLLLLLPPLLLLLRGSHAGNLT	(SEQ ID NO:18)

VAVVLPLANTSYPWSWARVGPAVELALAQVKARPDL

LPGWTVRTVLGSSENALGVCSDTAAPLAAVDLKWEH

NPAVFLGPGCVYAAAPVGRFTAHWRVPLLTAGAPAL

GFGVKDEYALTTRAGPSYAKLGDFVAALHRRLGWER

QALMLYAYRPGDEEHCFFLVEGLFMRVRDRLNITVD

HLEFAEDDLSHYTRLLRTMPRKGRVIYICSSPDAFR

TLMLLALEAGLCGEDYVFFHLDIFGQSLQGGQGPAP

RRPWERGDGQDVSARQAFQAAKIITYKDPDNPEYLE

FLKQLKHLAYEQFNFTMEDVLVNTIPASFHDGLLLY

IQAVTETLAHGGTVTDGENITQRMWNRSFQGVTGYL

KIDSSGDRETDFSLWDMDPENGAFRVVLNYNGTSQE

LVAVSGRKLNWPLGYPPPDIPKCGFDNEDPACNQDH

LSTLEVLALVGSLSLLGILIVSFFIYRKMQLEKELA

SELWRVRWEDVEPSSLERHLRSAGSRLTLSGRGSNY

GSLLTTEGQFQVFAKTAYYKGNLVAVKRVNRKRIEL

TRKVLFELKHMRDVQNEHLTRFVGACTDPPNICILT

EYCPRGSLQDILENESITLDWMFRYSLTNDIVKGML

FLHNGAICSHGNLKSSNCVVDGRFVLKITDYGLESF

RDLDPEQGHTVYAKKLWTAPELLRMASPPVRGSQAG

DVYSFGIILQEIALRSGVFHVEGLDLSPKEIIERVT

RGEQPPFRPSLALQSHLEELGLLMQRCWAEDPQERP

PFQQIRLTLRKFNRENSSNILDNLLSRMEQYANNLE

ELVEERTQAYLEEKRKAEALLYQILPHSVAEQLKRG

ETVQAEAFDSVTIYFSDIVGFTALSAESTPMQVVTL

LNDLYTCFDAVIDNFDVYKVETIGDAYMVVSGLPVR

NGRLHACEVARMALALLDAVRSFRIRHRPQEQLRLR

IGIHTGPVCAGVVGLKMPRYCLFGDTVNTASRMESN

GEALKIHLSSETKAVLEEFGGFELELRGDVEMKGKG

KVRTYWLLGERGSSTRG.

(NCBI ACCESSION NO. NM _—000906; Airhart N. et al., J. Biol. Chem., 2003, 278(40):38693-38698; Pitzalis M. V. et al., J. Hypertens., 2003, 21(8):1491-1496; Mokentin J. D. J. Clin. Invest., 2003, 111(9):1275-1277; De L. et al., J. Biol. Chem., 2003, 278(13):11159-11166; Knowles J. W. et al., Hum. Genet., 2003, 12(1):62-70; Pandy K. N. et al., J. Biol. Chem., 2002, 277(7):4618-4627).

SEQ ID NO:19 is the nucleotide coding sequence for human natriuretic peptide receptor-A (NPR-A):


	ggttccctcc ggatagccgg agacttgggc cggccggacg ccccttctgg cacactccct	(SEQ ID NO:19)

61	ggggcaggcg ctcacgcacg ctacaaacac acactcctct ttcctccctc gcgcgccctc

121	tctcatcctt cttcacgaag cgctcactcg caccctttct ctctctctct ctctctctaa

181	cacgcacgca cactcccagt tgttcacact cgggtcctct ccagcccgac gttctcctgg

241	cacccacctg ctccgcggcg ccctgcgcgc ccccctcggt cgcgcccctt gcgctctcgg

301	cccagaccgt cgcagctaca gggggcctcg agccccgggg tgagcgtccc cgtcccgctc

361	ctgctccttc ccatagggac gcgcctgatg cctgggaccg gccgctgagc ccaaggggac

421	cgaggaggcc atggtaggag cgctcgcctg ctgcggtgcc cgctgaggcc atgccggggc

481	cccggcgccc cgctggctcc cgcctgcgcc tgctcctgct cctgctgctg ccgccgctgc

541	tgctgctgct ccggggcagc cacgcgggca acctgacggt agccgtggta ctgccgctgg

601	ccaatacctc gtacccctgg tcgtgggcgc gcgtgggacc cgccgtggag ctggccctgg

661	cccaggtgaa ggcgcgcccc gacttgctgc cgggctggac ggtccgcacg gtgctgggca

721	gcagcgaaaa cgcgctgggc gtctgctccg acaccgcagc gcccctggcc gcggtggacc

781	tcaagtggga gcacaacccc gctgtgttcc tgggccccgg ctgcgtgtac gccgccgccc

841	cagtggggcg cttcaccgcg cactggcggg tcccgctgct gaccgccggc gccccggcgc

901	tgggcttcgg tgtcaaggac gagtatgcgc tgaccacccg cgcggggccc agctacgcca

961	agctggggga cttcgtggcg gcgctgcacc gacggctggg ctgggagcgc caagcgctca

1021	tgctctacgc ctaccggccg ggtgacgaag agcactgctt cttcctcgtg gaggggctgt

1081	tcatgcgggt ccgcgaccgc ctcaatatta cggtggacca cctggagttc gccgaggacg

1141	acctcagcca ctacaccagg ctgctgcgga ccatgccgcg caaaggccga gttatctaca

1201	tctgcagctc ccctgatgcc ttcagaaccc tcatgctcct ggccctggaa gctggcttgt

1261	gtggggagga ctacgttttc ttccacctgg atatctttgg gcaaagcctg caaggtggac

1321	agggccctgc tccccgcagg ccctgggaga gaggggatgg gcaggatgtc agtgcccgcc

1381	aggcctttca ggctgccaaa atcattacat ataaagaccc agataatccc gagtacttgg

1441	aattcctgaa gcagttaaaa cacctggcct atgagcagtt caacttcacc atggaggatg

1501	tcctggtgaa caccatccca gcatccttcc acgacgggct cctgctctat atccaggcag

1561	tgacggagac tctggcacat gggggaactg ttactgatgg ggagaacatc actcagcgga

1621	tgtggaaccg aagctttcaa ggtgtgacag gatacctgaa aattgatagc agtggcgatc

1681	gggaaacaga cttctccctc tgggatatgg atcccgagaa tggtgccttc agggttgtac

1741	tgaactacaa tgggacttcc caagagctgg tggctgtgtc ggggcgcaaa ctgaactggc

1801	ccctggggta ccctcctcct gacatcccca aatgtggctt tgacaacgaa gacccagcat

1861	gcaaccaaga tcacctttcc accctggagg tgctggcttt ggtgggcagc ctctccttgc

1921	tcggcattct gattgtctcc ttcttcatat acaggaagat gcagctggag aaggaactgg

1981	cctcggagct gtggcgggtg cgctgggagg acgttgagcc cagtagcctt gagaggcacc

2041	tgcggagtgc aggcagccgg ctgaccctga gcgggagagg ctccaattac ggctccctgc

2101	taaccacaga gggccagttc caagtctttg ccaagacagc atattataag ggcaacctcg

2161	tggctgtgaa acgtgtgaac cgtaaacgca ttgagctgac acgaaaagtc ctgtttgaac

2221	tgaagcatat gcgggatgtg cagaatgaac acctgaccag gtttgtggga gcctgcaccg

2281	acccccccaa tatctgcatc ctcacagagt actgtccccg tgggagcctg caggacattc

2341	tggagaatga gagcatcacc ctggactgga tgttccggta ctcactcacc aatgacatcg

2401	tcaagggcat gctgtttcta cacaatgggg ctatctgttc ccatgggaac ctcaagtcat

2461	ccaactgcgt ggtagatggg cgctttgtgc tcaagatcac cgactatggg ctggagagct

2521	tcagggacct ggacccagag caaggacaca ccgtttatgc caaaaagctg tggacggccc

2581	ctgagctcct gcgaatggct tcaccccctg tgcggggctc ccaggctggt gacgtataca

2641	gctttgggat catccttcag gagattgccc tgaggagtgg ggtcttccac gtggaaggtt

2701	tggacctgag ccccaaagag atcatcgagc gggtgactcg gggtgagcag ccccccttcc

2761	ggccctccct ggccctgcag agtcacctgg aggagttggg gctgctcatg cagcggtgct

2821	gggctgagga cccacaggag aggccaccat tccagcagat ccgcctgacg ttgcgcaaat

2881	ttaacaggga gaacagcagc aacatcctgg acaacctgct gtcccgcatg gagcagtacg

2941	cgaacaatct ggaggaactg gtggaggagc ggacccaggc atacctggag gagaagcgca

3001	aggctgaggc cctgctctac cagatcctgc ctcactcagt ggctgagcag ctgaagcgtg

3061	gggagacggt gcaggccgaa gcctttgaca gtgttaccat ctacttcagt gacattgtgg

3121	gtttcacagc gctgtcggcg gagagcacgc ccatgcaggt ggtgaccctg ctcaatgacc

3181	tgtacacttg ctttgatgct gtcatagaca actttgatgt gtacaaggtg gagacaattg

3241	gcgatgccta catggtggtg tcagggctcc ctgtgcggaa cgggcggcta cacgcctgcg

3301	aggtagcccg catggccctg gcactgctgg atgctgtgcg ctccttccga atccgccacc

3361	ggccccagga gcagctgcgc ttgcgcattg gcatccacac aggacctgtg tgtgctggag

3421	tggtgggact gaagatgccc cgttactgtc tctttgggga tacagtcaac acagcctcaa

3481	gaatggagtc taatggggaa gccctgaaga tccacttgtc ttctgagacc aaggctgtcc

3541	tggaggagtt tggtggtttc gagctggagc ttcgagggga tgtagaaatg aagggcaaag

3601	gcaaggttcg gacctactgg ctccttgggg agagggggag tagcacccga ggctgacctg

3661	cctcctctcc tatccctcca cacctcccct accctgtgcc agaagcaaca gaggtgccag

3721	gcctcagcct cacccacagc agccccatcg ccaaaggatg gaagtaattt gaatagctca

3781	ggtgtgctta ccccagtgaa gacaccagat aggacctctg agaggggact ggcatggggg

3841	gatctcagag cttacaggct gagccaagcc cacggccatg cacagggaca ctcacacagg

3901	cacacgcacc tgctctccac ctggactcag gccgggctgg gctgtggatt cctgatcccc

3961	tcccctcccc atgctctcct ccctcagcct tgctaccctg tgacttactg ggaggagaaa

4021	gagtcacctg aaggggaaca tgaaaagaga ctaggtgaag agagggcagg ggagcccaca

4081	tctggggctg gcccacaata cctgctcccc cgaccccctc cacccagcag tagacacagt

4141	gcacagggga gaagaggggt ggcgcagaag ggttgggggc ctgtatgcct tgcttctacc

4201	atgagcagag acaattaaaa tctttattcc aaaaaaaaaa aaaaaa

(NCBI ACCESSION NO. NM _—000906; Airhart N. et al., J. Biol. Chem., 2003, 278(40):38693-38698; Pitzalis M. V. et al., J Hypertens., 2003, 21(8):1491-1496; Mokentin J. D. J. Clin. Invest., 2003, 111(9):1275-1277; De L. et al., J. Biol. Chem., 2003, 278(13):11159-11166; Knowles J. W. et al., Hum. Genet., 2003, 12(1):62-70; Pandy K. N. et al., J. Biol. Chem., 2002, 277(7):4618-4627).

SEQ ID NO:20 is amino acid sequence of the human atrial natriuretic peptide clearance receptor precursor (ANP-C; also referred to as NPR-C, NPRC, and atrial natriuretic peptide C-type receptor):


MPSLLVLTFS PCVLLGWALL AGGTGGGGVG GGGGGAGIGG GRQEREALPP	(SEQ ID NO:20)

QKIEVLVLLP QDDSYLFSLT RVRPAIEYAL RSVEGNGTGR RLLPPGTRFQ

VAYEDSDCGN RALFSLVDRV AAARGAKPDL ILGPVCEYAA APVARLASHW

DLPMLSAGAL AAGFQHKDSE YSHLTRVAPA YAKMGEMMLA LFRHHHWSRA

ALVYSDDKLE RNCYFTLEGV HEVFQEEGLH TSIYSFDETK DLDLEDIVRN

IQASERVVIM CASSDTIRSI MLVAHRHGMT SGDYAFFNIE LFNSSSYGDG

SWKRGDKHDF EAKQAYSSLQ TVTLLRTVKP EFEKFSMEVK SSVEKQGLNM

EDYVNMFVEG FHDAILLYVL ALHEVLRAGY SKKDGGKIIQ QTWNRTFEGI

AGQVSIDANG DRYGDFSVIA MTDVEAGTQE VIGDYFGKEG RFEMRPNVKY

PWGPLKLRID ENRIVEHTNS SPCKSSGGLE ESAVTGIVVG ALLGAGLLMA

FYFFRKKYRI TIERRTQQEE SNLGKHRELR EDSIRSHFSV A

(NCBI ACCESSION NO. P17342; Lowe D. G. et al., Nucleic Acids Res., 1990, 18(11):3412; Porter J. G. et al., Biochem. Biophys. Res. Commun., 1990, 171(2):796-803; Stults J. T. et al., Biochemistry, 1994, 33(37):11372-11381).


	SEQ ID NO:21 is an siRNA specific for NPR-A
	(human).
	tat tac ggt gga cca cct gtt caa gag aca ggt ggt

	cca ccg taa tat ttttt

	SEQ ID NO:22 is an siRNA specific for NPR-A
	(human).
	aga att cca gaa acg cag ctt caa gag agc tgc gtt

	tct gga att ctt ttttt

DETAILED DISCLOSURE

The present invention pertains to methods for reducing natriuretic peptide receptor-A (also known in the art as NPRA, NPR-A, and guanylate cyclase A) activity in vitro or in vivo. In one aspect, the method of the invention may be used for treating inflammatory and cell proliferation disorders, such as cancer. In another aspect, the present invention concerns methods for identifying agents useful for treating inflammatory and cell proliferation disorders by determining whether the candidate agent reduces activity of the natriuretic peptide receptor-A (also known in the art as NPRA, NPR-A, and guanylate cyclase A) in vitro and/or in vivo (also referred to herein as the diagnostic method or assay of the invention).

As used herein, an “inflammatory disorder” includes those conditions characterized by an aberrant increase in one or more of the following: IL-6, IL-1 beta, TNF-alpha, IL-8, eosinophil production, neutrophil production, release of histamines, proliferants, hyperplasia, and cell adhesion molecule expression. As used herein, a “cell proliferation disorder” is characterized by one or more of the following: uncontrolled proliferation, a high mitogenic index, over-expression of cyclin D1, cyclin B1, expression of an oncogene such as c-jun and/or c-fos, aberrant activation of NFkB and/or ERK (extracellular receptor kinase), and matrix metalloproteinase expression (such as MMP-2 and/or MMP-9).

In one embodiment, the inflammatory disorder and cell proliferation disorder is not one that is amenable to effective treatment by administration of a vasodilator. In one embodiment, the inflammatory disorder and cell proliferation disorder is not a cardiovascular disorder (such as hypertension or stroke). In another embodiment, the inflammatory disorder and cell proliferation disorder is not a disorder of the central nervous system (such as Alzheimer's disease or other dementia). In another embodiment, the inflammatory disorder and cell proliferation disorder is not kidney failure or other kidney disorder.

The agent used to reduce NPR-A activity in vitro or in vivo can be virtually any substance and can encompass numerous chemical classes, including organic compounds or inorganic compounds. Preferably, an effective amount of the agent is administered to the cells with a pharmaceutically acceptable carrier. The agent may be a substance such as genetic material, protein, lipid, carbohydrate, small molecules, a combination of any of two or more of foregoing, or other compositions. The agent may be naturally occurring or synthetic, and may be a single substance or a mixture. The agent can be obtained from a wide variety of sources including libraries of compounds. The agent can be or include, for example, a polypeptide, peptidomimetic, amino acid(s), amino acid analog(s), function-blocking antibody, polynucleotide(s), polynucleotide analog(s), nucleotide(s), nucleotide analog(s), or other small molecule(s). A polynucleotide may encode a polypeptide that potentially reduces NPR-A activity within the cell, or the polynucleotide may be a short interfering RNA (siRNA), a hairpin RNA (shRNA), antisense oligonucleotide, ribozyme, or other polynucleotide that targets an endogenous or exogenous gene for silencing of gene expression and potentially NPR-A activity within the cell.

In one embodiment, the agent used to reduce NPR-A activity is an interfering RNA specific for NPR-A mRNA, preferably human NPR-A mRNA. Interfering RNA is capable of hybridizing with the mRNA of a target gene and reduce and/or eliminate translation through the mechanism of RNA interference. Examples of such interfering RNA include SEQ ID NO:21 and SEQ ID NO:22, which were determined to have a relatively high probably of reducing NPR-A activity using an siRNA Target Finder program (AMBION) and in accordance with published guidelines (Tuschl T., Nature Biotechnol., 2002, 20:446448). 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) that is capable of directing or mediating RNA interference. In one embodiment, the siRNA is between about 10-50 nucleotides (or nucleotide analogs). Optionally, a polynucleotide (e.g., DNA) encoding the siRNA may be administered to cells in vitro or in vivo, such as in a vector, wherein the DNA is transcribed.

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. “mRNA” or “messenger RNA” or “transcript” is single-stranded RNA that specifies the amino acid sequence of one or more polypeptides. This information is translated during protein synthesis when ribosomes bind to the mRNA.

The scientific literature is replete with reports of endogenous and exogenous gene expression silencing using siRNA, highlighting their therapeutic potential (Gupta, S. et al. PNAS, 2004, 101:1927-1932; Takaku, H. Antivir Chem. Chemother, 2004, 15:57-65; Pardridge, W. M. Expert Opin. Biol. Ther., 2004, 4:1103-1113; Zheng, B. J. Antivir. Ther., 2004, 9:365-374; Shen, W. G. Chin. Med. J . ( Engl ), 2004, 117:1084-1091; Fuchs, U. et al. Curr. Mol. Med., 2004, 4:507-517; Wadhwa, R. et al. Mutat. Res., 2004, 567:71-84; Ichim, T. E. et al. Am. J. Transplant, 2004, 4:1227-1236; Jana, S. et al. Appl. Microbiol. Biotechnol., 2004, 65:649-657; Ryther, R. C. et al. Gene Ther., 2005, 12:5-11; Chae, S-S. et al., J. Clin. Invest., 2004, 114:1082-1089; Fougerolles, A. et al., Methods Enzymol., 2005, 392:278-296), each of which is incorporated herein by reference in its entirety. Therapeutic silencing of endogenous genes by systemic administration of siRNAs has been described in the literature (Kim B. et al., American Journal of Pathology, 2004, 165:2177-2185; Soutschek J. et al., Nature, 2004, 432:173-178; Pardridge W. M., Expert Opin. Biol. Ther., 2004, July, 4(7): 1103-1113), each of which is incorporated herein by reference in its entirety.

In another embodiment, the decrease in NPR-A activity (e.g., a reduction in NPR-A expression) may be achieved by administering an analogue of ANP (e.g., ANP4-23) or non-peptide antagonists (e.g., HS-142-1; Rutherford et al., Br. J. Pharmacol., 1994, 113:931-939; El-Ayoubi et al., Br. J. Pharmacol., 2005, Feb. 7, Epub ahead of print; Delport C. et al., Eur. J. Pharmacol., 1992, 224(2-3):183-188; Ohyama Y. et al., Biochem. Biophys. Res. Commun., 1992, 189(1):336-342). In another embodiment, the agent is an anti-human NPR-A function-blocking antibody (preferably, humanized), or soluble NPR-A or NPR-C (as a receptor decoy). Other examples of agents include NPR-A antagonists that specifically inhibit cGMP-dependent protein kinase (PKG) such as A71915 and KT5823 (Pandey K. N. et al., Biochemical and Biophysical Research Communications, 2000, 271:374-379).

The methods of the invention may include further steps. In some embodiments, a subject with the relevant inflammatory disorder and/or cell proliferation disorder is identified or a patient at risk for the disorder is identified. A patient may be someone who has not been diagnosed with the disease or condition (diagnosis, prognosis, and/or staging) or someone diagnosed with disease or condition (diagnosis, prognosis, monitoring, and/or staging), including someone treated for the disease or condition (prognosis, staging, and/or monitoring). Alternatively, the person 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.

In one aspect, the therapeutic method of the invention involves administering a natriuretic hormone peptide (NP), or a fragment, homolog or variant thereof, or a nucleic acid sequence encoding an NP, or a fragment, homolog, or variant thereof, to a patient. The present inventor has demonstrated that a prolonged, substantial reduction of tumor burden in lungs can be achieved by intranasal delivery of pDNA-encoding a peptide comprising amino acid residues 73 to 102 (NP73-102). Without being bound by theory, the NP decreased viability due to the induction of apoptosis in a lung adenocarcinoma cell line A549 cell, and can reduce tumorigenesis and metastasis in a number of cancers.

In specific embodiments, the peptides used in the subject invention comprise at least one amino acid sequence selected from the group consisting of NP _1-30, NP _31-67, NP _79-98, and NP _73-102, (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:5, respectively), SEQ ID NO:6, or a biologically active fragment or homolog thereof. In some embodiments, a combination of NP or NP-encoding nucleic acid sequences is utilized. In one embodiment, the peptide utilized does not consist of the amino acid sequence of NP _99-126(SEQ ID NO: 4).

In another aspect, the therapeutic method of the invention involves administering an agent that reduces activity of the natriuretic peptide receptor-A (also known in the art as NPRA, NPR-A, and guanylate cyclase A) to a patient, wherein the agent is administered in an amount effective to reduce receptor (NPR-A) activity. NPR-A activity can be determined, for example, by one or more of the following biological parameters: production/accumulation of cGMP, expression of the NPR-A (transcription or translation), and/or cellular internalization of the NPR-A.

According to the gene therapy method of the present invention, the NP-encoding nucleic acid sequence is administered locally at the target site (e.g., at the site of cancer or pre-cancer), or systemically to the patient. In order to treat cancer of the lung, for example, the NP-encoding nucleic acid sequence is preferably administered to the airways of the patient, e.g., nose, sinus, throat and lung, for example, as nose drops, by nebulization, vaporization, or other methods known in the art. More preferably, the nucleic acid sequence encoding NP is administered to the patient orally or intranasally, or otherwise intratracheally. For example, the nucleic acid sequence can be inhaled by the patient through the oral or intranasal routes, or injected directly into tracheal or bronchial tissue.

In specific embodiments, the nucleic acid sequences used in the subject invention encode at least one amino acid sequence selected from the group consisting of NP _1-30, NP _31-67, NP _79-98, NP _99-126, and NP _73-102, (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively), SEQ ID NO:6, or a biologically active fragment or homolog of any of the foregoing.

Preferably, the nucleic acid sequence encoding the NP is administered with a nucleic acid sequence that is operatively linked with the NP-encoding nucleic acid sequence and operates as a regulatory sequence. For example, the regulatory sequence can be a promoter sequence that controls transcription and drives expression of the NP-encoding nucleic acid sequence at the desired site, such as at, or adjacent to, the patient's respiratory epithelial cells. The promoter can be a constitutive or inducible promoter to allow selective transcription. The promoter can be a vertebrate or viral promoter. Optionally, enhancers may be used to obtain desired transcription levels. An enhancer is generally any non-translated nucleic acid sequence that works contiguously with the coding sequence (in cis) to change the basal transcription level dictated by the promoter.

The NP-encoding nucleic acid sequences used in the methods, expression vectors, and pharmaceutical compositions of the present invention are preferably isolated. According to the present invention, an isolated nucleic acid molecule or nucleic acid sequence, is a nucleic acid molecule or sequence that has been removed from its natural milieu. As such, “isolated” does not necessarily reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule or sequence useful in the present composition can include DNA, RNA, or any derivatives of either DNA or RNA. An isolated nucleic acid molecule or sequence can be double stranded (i.e., containing both a coding strand and a complementary strand) or single stranded.

A nucleic acid molecule can be isolated from a natural source, or it can be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid molecules can be generated or modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site-directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules, and combinations thereof.

Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases are used interchangeably herein. As used herein, a “coding” nucleic acid sequence refers to a nucleic acid sequence that encodes at least a portion of a peptide or protein (e.g., a portion of an open reading frame), and can more particularly refer to a nucleic acid sequence encoding a peptide or protein which, when operatively linked to a transcription control sequence (e.g., a promoter sequence), can express the peptide or protein.

The nucleotide sequences encoding NP used in the subject invention include “homologous” or “modified” nucleotide sequences. Modified nucleic acid sequences will be understood to mean any nucleotide sequence obtained by mutagenesis according to techniques well known to persons skilled in the art, and exhibiting modifications in relation to the normal sequences. For example, mutations in the regulatory and/or promoter sequences for the expression of a polypeptide that result in a modification of the level of expression of a polypeptide according to the invention provide for a “modified nucleotide sequence”. Likewise, substitutions, deletions, or additions of nucleic acids to the polynucleotides of the invention provide for “homologous” or “modified” nucleotide sequences. In various embodiments, “homologous” or “modified” nucleic acid sequences have substantially the same biological or serological activity as the native (naturally occurring) natriuretic peptide. A “homologous” or “modified” nucleotide sequence will also be understood to mean a splice variant of the polynucleotides of the instant invention or any nucleotide sequence encoding a “modified polypeptide” as defined below.

A homologous nucleotide sequence, for the purposes of the present invention, encompasses a nucleotide sequence having a percentage identity with the bases of the nucleotide sequences of between at least (or at least about) 20.00% to 99.99% (inclusive). The aforementioned range of percent identity is to be taken as including, and providing written description and support for, any fractional percentage, in intervals of 0.01%, between 20.00% and 99.99%. These percentages are purely statistical and differences between two nucleic acid sequences can be distributed randomly and over the entire sequence length.

In various embodiments, homologous sequences exhibiting a percentage identity with the bases of the nucleotide sequences of the present invention can have 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity with the polynucleotide sequences of the instant invention. Homologous nucleotide and amino acid sequences include mammalian homologs of the human NP sequences.

The NP homologs include peptides containing, as a primary amino acid sequence, all or part of an exemplified NP polypeptide sequence. The NP homologs thus include NP polypeptides having conservative substitutions, i.e., altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a peptide which is biologically active. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. In one aspect of the present invention, conservative substitutions for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 1). Conservative substitutions also include substitutions by amino acids having chemically modified side chains which do not eliminate the biological activity of the resulting NP homolog.

	TABLE 1


	Class of Amino Acid	Examples of Amino Acids

	Nonpolar	Ala, Val, Leu, Ile, Pro, Met, Phe, Trp
	Uncharged Polar	Gly, Ser, Thr, Cys, Tyr, Asn, Gln
	Acidic	Asp, Glu
	Basic	Lys, Arg, His

Both protein and nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman Proc. Natl. Acad. Sci. USA, 1988, 85(8):2444-2448; Altschul et al. J. Mol. Biol., 1990, 215(3):403-410; Thompson et al. Nucleic Acids Res., 1994, 22(2):4673-4680; Higgins et al. Methods Enzymol., 1996, 266:383-402; Altschul et al. J. Mol. Biol., 1990, 215(3):403-410; Altschul et al. Nature Genetics, 1993, 3:266-272).

Identity and similarity of related nucleic acid molecules and polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; York (1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; York (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; York (1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

The methods, pharmaceutical compositions, and vectors of the present invention can utilize biologically active fragments of nucleic acid sequences encoding the 126-amino acid atrial natriuretic factor (ANF) prohormone, such as nucleic acid sequences encoding NP _1-30, NP _31-67, NP _79-98, NP _99-126, and NP _73-102, (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively), SEQ ID NO:6, and including biologically active fragments of the nucleic acid sequences encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

Representative fragments of the nucleotide sequences according to the invention will be understood to mean any polynucleotide fragment having at least 8 or 9 consecutive nucleotides, preferably at least 12 consecutive nucleotides, and still more preferably at least 15 or at least 20 consecutive nucleotides of the sequence from which it is derived, with retention of biological activity as described herein. The upper limit for such fragments is one nucleotide less than the total number of nucleotides found in the full-length sequence (or, in certain embodiments, of the full length open reading frame (ORF) identified herein).

In other embodiments, fragments can comprise consecutive nucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, and up to one nucleotide less than the full length ANF prohormone. In some embodiments, fragments comprise biologically active fragments of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

It is also well known in the art that restriction enzymes can be used to obtain biologically active fragments of the nucleic acid sequences, such as those encoding SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. For example, Bal31 exonuclease can be conveniently used for time-controlled limited digestion of DNA (commonly referred to as “erase-a-base” procedures). See, for example, Maniatis et al. [1982 ] Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, New York; Wei et al., J. Biol. Chem., 1983, 258:13006-13512.

The methods and pharmaceutical compositions of the present invention can utilize amino acid sequences that are biologically active fragments of the 126-amino acid atrial natriuretic factor (ANF) prohormone, such as NP _1-30, NP _31-67, NP _79-98, NP _99-126, and NP _73-102(SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively), SEQ ID NO:6, and including biologically active fragments of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

Representative fragments of the polypeptides according to the invention will be understood to mean any polypeptide fragment having at least 8 or 9 consecutive amino acids, preferably at least 12 amino acids, and still more preferably at least 15 or at least 20 consecutive amino acids of the polypeptide sequence from which it is derived, with retention of biological activity as described herein. The upper limit for such fragments is one amino acid less than the total number of amino acids found in the full-length sequence.

In other embodiments, fragments of the polypeptides can comprise consecutive amino acids of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and up to one amino acid less than the full-length ANF prohormone. Fragments of polypeptides can be any portion of the full-length ANF prohormone amino acid sequence (including human or non-human mammalian homologs of the ANF prohormone) that exhibit biological activity as described herein, e.g., a C-terminally or N-terminally truncated version of the ANF prohormone, or an intervening portion of the ANF prohormone. In some embodiments, fragments comprise biologically active fragments of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

The present invention can be practiced using other biologically equivalent forms of ANF fragments or homologs thereof as can be appreciated by the sequence comparison below. Sequence similarities between mouse and human forms of ANP are shown where areas of conservation are clearly seen.

NCBI BLAST Comparison of Mouse (Query) to Human (Sbjct) ANP a.a. Sequences.


Query:	1	MGSFSIT-

		LGFFLVLAFWLPGHIGANPVYSAVSNTDLMDFKNLLDHLEEKMPVEDEVMPP

		M SFS T + F L + LAF L G ANP + Y + AVSN DLMDFKNLLDHLEEKMP + EDEV + PP

Sbjct:	1	MSSFSTTTVSFLLLLAFQLLGQTRANPMYNAVSNADLMDFKNLLDHLEEKMPLEDE VVPP

Query:	60	QALSEQTEEAGAALSSLPEVPPWTGEVNPPLRDGSALGRSPWDPSDXXXXXXXXXX XXXX

		Q LSE EEAGAALS LPEVPPWTGEV + P RDG ALGR PWD SD

Sbjct:	61	QVLSEPNEEAGAALSPLPEVPPWTGEVSPAQRDGGALGRGPWDSSDRSALLKSKLR ALLT

Query:	120	GPRSLRRSSCFGGRIDRIGAQSGLGCNSFRY	150

		PRSLRRSSCFGGR + DRIGAQSGLGCNSFRY

Sbjct:	121	APRSLRRSSCFGGRMDRIGAQSGLGCNSFRY	151

The NP utilized in the subject invention can be peptide derivatives, such as those disclosed in U.S. Patent Publication No. 2004/0266673 (Bakis et al.), which is incorporated herein by reference in its entirety. These NP derivates include an NP and a reactive entity coupled to the NP peptide. The reactive entity is able to covalently bond with a functionality on a blood component. Such NP derivatives are reported to have an extended half-life in vivo. The NP utilized in the subject invention can be a modified NP, such as those described in U.S. Patent Publication No. 2004/0002458 (Seilhamer et al.) and U.S. Patent Publication No. 2003/0204063 (Gravel et al.), which are incorporated herein by reference in their entirety.

The NP utilized may be a fusion polypeptide comprising an NP, or fragment or homolog thereof, and one or more additional polypeptides, such as another NP or a carrier protein, including those described in U.S. Patent Publication No. 2004/0138134 (Golembo et al.), which is incorporated herein by reference in its entirety. The NP utilized may be a chimeric polypeptide, such as those described in U.S. Patent Publication No. 2003/0069186 (Burnett et al.), which is incorporated herein by reference in its entirety. The fusion polypeptide or chimeric polypeptide may be administered to cells in vitro or in vivo directly (i.e., as a polypeptide), or the fusion polypeptide may be administered as a polynucleotide encoding the fusion polypeptide with an operably linked promoter sequence. See, for example, Wang W. et al., “Albubnp, a Recombinant B-type Natriuretic Peptide and Human Serum Albumin Fusion Hormone, as a Long-Term Therapy of Congestive Heart Failure”, Pharmaceutical Research, Springer Science and Business Media B.V., Formerly Kluwer Academic Publishers B.V., ISSN:0724-8741, volume 21, Number 11, November, 2004, pages 2105-2111.

The NP includes all hydrates and salts of natriuretic peptides that can be prepared by those of skill in the art. Under conditions where the compounds of the present invention are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids that form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts of NP may be obtained using standard procedures well known in the art, for example, by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

The NP of the invention can be prepared by well-known synthetic procedures. For example, the polypeptides can be prepared by the well-known Merrifield solid support method. See Merrifield, J. Amer. Chem. Soc., 1963, 85:2149-2154 and Merrifield (1965) Science 150:178-185. This procedure, using many of the same chemical reactions and blocking groups of classical peptide synthesis, provides a growing peptide chain anchored by its carboxyl terminus to a solid support, usually cross-linked polystyrene or styrenedivinylbenzene copolymer. This method conveniently simplifies the number of procedural manipulations since removal of the excess reagents at each step is effected simply by washing of the polymer.

Alternatively, these peptides can be prepared by use of well-known molecular biology procedures. Polynucleotides, such as DNA sequences, encoding the NP of the invention can be readily synthesized. Such polynucleotides are a further aspect of the present invention. These polynucleotides can be used to genetically engineer eukaryotic or prokaryotic cells, for example, bacteria cells, insect cells, algae cells, plant cells, mammalian cells, yeast cells or fungi cells for synthesis of the peptides of the invention.

For purposes of the present invention, the biological activity attributable to the homologs and fragments of NP and NP-encoding nucleic acid sequences means the capability to prevent or alleviate symptoms associated with inflammatory and/or cell proliferation disorders such as cancer. This biological activity can be mediated by one or more of the following mechanisms: increased production of intracellular Ca ⁺⁺ concentration (e.g., in epithelial cells), increased production of nitric oxide (NO), and decreased activation of transcription factors such as NFkB, ERK1, 2 and/or AP1.

The methods of the subject invention also contemplate the administration of cells that have been genetically modified to produce NP, or biologically active fragments, variants, or homologs thereof. Such genetically modified cells can be administered alone or in combinations with different types of cells. Thus, genetically modified cells of the invention can be co-administered with other cells, which can include genetically modified cells or non-genetically modified cells. Genetically modified cells may serve to support the survival and function of the co-administered cells, for example.

The term “genetic modification” as used herein refers to the stable or transient alteration of the genotype of a cell of the subject invention by intentional introduction of exogenous nucleic acids by any means known in the art (including for example, direct transmission of a polynucleotide sequence from a cell or virus particle, transmission of infective virus particles, and transmission by any known polynucleotide-bearing substance) resulting in a permanent or temporary alteration of genotype. The nucleic acids may be synthetic, or naturally derived, and may contain genes, portions of genes, or other useful polynucleotides in addition to those encoding NP. A translation initiation codon can be inserted as necessary, making methionine the first amino acid in the sequence. The term “genetic modification” is not intended to include naturally occurring alterations such as that which occurs through natural viral activity, natural genetic recombination, or the like. The genetic modification may confer the ability to produce NP, wherein the cell did not previously have the capability, or the modification may increase the amount of NP endogenously produced by the cell, e.g., through increased expression.

Exogenous nucleic acids and/or vectors encoding NP can be introduced into a cell by viral vectors (retrovirus, modified herpes virus, herpes virus, adenovirus, adeno-associated virus, lentivirus, and the like) or direct DNA transfection (lipofection, chitosan-nanoparticle mediated transfection, calcium phosphate transfection, DEAE-dextran, electroporation, and the like), microinjection, cationic lipid-mediated transfection, transduction, scrape loading, ballistic introduction and infection (see, for example, Sambrook et al. [1989 ] Molecular Cloning: A Laboratory Manual, 2 ^ndEd., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Preferably, the exogenous polynucleotide encoding the NP is operably linked to a promoter sequence that permits expression of the polynucleotide in a desired tissue within the patient. The promoters can be inducible, tissue-specific, or event-specific, as necessary.

The genetically modified cell may be chosen from eukaryotic or prokaryotic systems, for example, bacterial cells (Gram negative or Gram positive), yeast cells, animal cells, plant cells, and/or insect cells using baculovirus vectors, for example. In some embodiments, the genetically modified cell for expression of the nucleic acid sequences encoding NP, are human or non-human mammal cells.

According to the methods of the present invention, NP or polynucleotides encoding NP can be administered to a patient in order to alleviate (e.g., reduce or eliminate) a variety of symptoms associated with cancers, in various stages of pathological development. Treatment with NP or nucleic acid sequences encoding NP is intended to include prophylactic intervention to prevent or reduce cancer cell growth (e.g., tumor growth) and onset of the symptoms associated with cancer cell growth (e.g., tumor growth), such as pain. The nucleic acid sequences and pharmaceutical compositions of the invention can be co-administered (concurrently or consecutively) to a patient with other therapeutic agents useful for treating cancers of the lung, ovarian, breast, as well as melanomas.

Suitable expression vectors for NP include any that are known in the art or yet to be identified that will cause expression of NP-encoding nucleic acid sequences in mammalian cells. Suitable promoters and other regulatory sequences can be selected as is desirable for a particular application. The promoters can be inducible, tissue-specific, or event-specific, as necessary. For example, the cytomegalovirus (CMV) promoter (Boshart et al., Cell, 1985, 41:521-530) and SV40 promoter (Subramani et al., Mol. Cell. Biol., 1981, 1:854-864) have been found to be suitable, but others can be used as well. Optionally, the NP-encoding nucleic acid sequences used in the subject invention include a sequence encoding a signal peptide upstream of the NP-encoding sequence, thereby permitting secretion of the NP from a host cell. Also, various promoters may be used to limit the expression of the peptide in specific cells or tissues, such as lung cells.

A tissue-specific and/or event-specific promoter or transcription element that responds to the target microenviroment and physiology can also be utilized for increased transgene expression at the desired site. There has been an immense amount of research activity directed at strategies for enhancing the transcriptional activity of weak tissue-specific promoters or otherwise increasing transgene expression with viral vectors. It is possible for such strategies to provide enhancement of gene expression equal to one or two orders of magnitude, for example (see Nettelbeck et al., Gene Ther., 1998, 5(12):1656-1664 and Qin et al., Hum. Gene Ther., 1997, 8(17):2019-2019, the abstracts of which are submitted herewith for the Examiner's convenience). Examples of cardiac-specific promoters are the ventricular form of MLC-2v promoter (see, Zhu et al., Mol. Cell Biol., 1993, 13:4432-4444, Navankasattusas et al., Mol. Cell Biol., 1992, 12:1469-1479, 1992) and myosin light chain-2 promoter (Franz et al., Circ. Res., 1993, 73:629-638). The E-cadherin promoter directs expression specific to epithelial cells (Behrens et al., PNAS, 1991, 88:11495-11499), while the estrogen receptor (ER) 3 gene promoter directs expression specifically to the breast epithelium (Hopp et al., J. Mammary Gland Biol . Neoplasia, 1998, 3:73-83). The human C-reactive protein (CRP) gene promoter (Ruther et al., Oncogene 8:87-93, 1993) is a liver-specific promoter. An example of a muscle-specific gene promoter is human enolase (ENO3) (Peshavaria et al., Biochem. J., 1993, 292(Pt 3):701-704). A number of brain-specific promoters are available such as the thy-1 antigen and gamma-enolase promoters (Vibert et al., Eur. J. Biochem. 181:33-39, 1989). The prostate-specific antigen promoter provides prostate tissue specificity (Pang et al., Gene Ther., 1995, 6(11):1417-1426; Lee et al., Anticancer Res., 1996, 16(4A): 1805-1811). The surfactant protein B promoter provides lung specificity (Strayer et al., Am. J. Respir. Cell Mol. Biol., 1998, 18(1):1-11). Any of the aforementioned promoters may be selected for targeted or regulated expression of the NP-encoding polynucleotide.

Various viral or non-viral vectors may be used to deliver polynucleotides encoding NP to cells in vitro or in vivo, resulting in expression and production of NP. Tissue-specific promoters or event-specific promoters may be utilized with polynucleotides encoding NP to further optimize and localize expression at target sites, such as within diseased tissues (e.g., cancer cells or tissues containing cancer cells). Robson et al. review various methodologies and vectors available for delivering and expressing a polynucleotide in vivo for the purpose of treating cancer (Robson, T. Hirst, D. G., J. Biomed. and Biotechnol., 2003, 2003(2): 110-137). Among the various targeting techniques available, transcriptional targeting using tissue-specific and event-specific transcriptional control elements is discussed. For example, Table 1 at page 112 of the Robson et al. publication lists several tissue-specific promoters useful in cancer therapy. Tables 2-4 of the Robson et al. publication list tumor-specific promoters, tumor environment-specific promoters, and exogenously controlled inducible promoters, many of which were available at the time the patent application was filed. The successful delivery and expression of the p53 tumor suppressor gene in vivo has been documented (Horowitz, J. Curr. Opin. Mol. Ther., 1999, 1(4):500-509; Von Gruenigen, V. E. et al. Int. J. Gynecol. Cancer, 1999, 9(5):365-372; Fujiwara, T. et al., Mol. Urol., 2000, 4(2):51-54, respectively).

Many techniques for delivery of drugs and proteins are available in the art to reduce the effects of enzymatic degradation, to facilitate cell uptake, and to reduce any potential toxicity to normal (undiseased) cells, etc. Such methods and reagents can be utilized for administration of NP to cells in vitro or in vivo. For example, peptides known as “cell penetrating peptides” (CPP) or “protein transduction domains” (PTD) have an ability to cross the cell membrane and enter the cell. PTDs can be linked to a cargo moiety such as a drug, peptide, or full-length protein, and can transport the moiety across the cell membrane. One well characterized PTD is the human immunodeficient virus (HIV)-1 Tat peptide (see, for example, Frankel et al., U.S. Pat. Nos. 5,804,604; 5,747,641; 6,674,980; 5,670,617; and 5,652,122; Fawell, S. et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91:664-668). Peptides such as the homeodomain of Drosophila antennapedia (ANTp) and arginine-rich peptides display similar properties (Derossi, D. et al., J. Biol. Chem., 1994, 269:10444-10450; Derossi, D. et al., Trends Cell Biol., 1998, 8:84-87; Rojas, M. et al., Nat. Biotechnol., 1998, 16:370-375; Futaki, S. et al., J. Biol. Chem., 2001, 276:5836-5840). VP22, a tegument protein from Herpes simplex virus type 1 (HSV-1), also has the ability to transport proteins across a cell membrane (Elliot et al., Cell, 1997, 88:223-233; Schwarze S. R. et al., Trends Pharmacol. Sci., 2000, 21:45-48). A common feature of these carriers is that they are highly basic and hydrophilic (Schwarze S. R. et al., Trends Cell Biol., 2000, 10:290-295). Coupling of these carriers to marker proteins such as beta-galactosidase has been shown to confer efficient internalization of the marker protein into cells. More recently, chimeric, in-frame fusion proteins containing these carriers have been used to deliver proteins to a wide spectrum of cell types both in vitro and in vivo. For example, VP22-p53 chimeric protein retained its ability to spread between cells and its pro-apoptotic activity, and had a widespread cytotoxic effect in p53 negative human osteosarcoma cells in vitro (Phelan, A. et al., Nature Biotechnol., 1998, 16:440-443). Intraperitoneal injection of the beta-galactosidase protein fused to the HIV-1 Tat peptide resulted in delivery of the biologically active fusion protein to all tissues in mice, including the brain (Schwarze S. R. et al., Science, 1999, 285:1569-1572).

Liposomes of various compositions can also be used for site-specific delivery of proteins and drugs (Witschi, C. et al., Pharm. Res., 1999, 16:382-390; Yeh, M. K. et al., Pharm. Res., 1996, 1693-1698). The interaction between the liposomes and the protein cargo usually relies on hydrophobic interactions or charge attractions, particularly in the case of cationic lipid delivery systems (Zelphati, O. et al., J. Biol. Chem., 2001, 276:35103-35110). Tat peptide-bearing liposomes have also been constructed and used to deliver cargo directly into the cytoplasm, bypassing the endocytotic pathway (Torchilin V. P. et al., Biochim. Biophys. Acta—Biomembranes, 2001, 1511:397-411; Torchilin V. P. et al., Proc. Natl. Acad. Sci. USA, 2001, 98:8786-8791). When encapsulated in sugar-grafted liposomes, pentamidine isethionate and a derivative have been found to be more potent in comparison to normal liposome-encapsulated drug or to the free drug (Banerjee, G. et al., J. Antimicrob. Chemother., 1996, 38(1):145-150). A thermo-sensitive liposomal taxol formulation (heat-mediated targeted drug delivery) has been administered in vivo to tumor-bearing mice in combination with local hyperthermia, and a significant reduction in tumor volume and an increase in survival time was observed compared to the equivalent dose of free taxol with or without hyperthermia (Sharma, D. et al., Melanoma Res., 1998, 8(3):240-244). Topical application of liposome preparations for delivery of insulin, IFN-alpha, IFN-gamma, and prostaglandin E1 have met with some success (Cevc G. et al., Biochim. Biophys, Acta, 1998, 1368:201-215; Foldvari M. et al., J. Liposome Res., 1997, 7:115-126; Short S. M. et al., Pharm. Res., 1996, 13:1020-1027; Foldvari M. et al., Urology, 1998, 52(5):838-843; U.S. Pat. No. 5,853,755).

Antibodies represent another targeting device that may make liposome uptake tissue-specific or cell-specific (Mastrobattista, E. et al., Biochim. Biophys. Acta, 1999, 1419(2):353-363; Mastrobattista, E. et al., Adv. Drug Deliv. Rev., 1999, 40(1-2):103-127). The liposome approach offers several advantages, including the ability to slowly release encapsulated drugs and proteins, the capability of evading the immune system and proteolytic enzymes, and the ability to target tumors and cause preferentially accumulation in tumor tissues and their metastases by extravasation through their leaky neovasculature. Other carriers have also been used to deliver anti-cancer drugs to neoplastic cells, such as polyvinylpyrrolidone nanoparticles and maleylated bovine serum albumin (Sharma, D. et al., Oncol. Res., 1996, 8(7-8):281-286; Mukhopadhyay, A. et al., FEBS Lett., 1995, 376(1-2):95-98). Thus, using targeting and encapsulation technologies, which are very versatile and amenable to rational design and modification, delivery of NP to desired cells can be facilitataed. Furthermore, because many liposome compositions are also viable delivery vehicles for genetic material, many of the advantages of liposomes are equally applicable to polynucleotides encoding NP.

As indicated above, the pharmaceutical composition of the present invention can include a liposome component. According to the present invention, a liposome comprises a lipid composition that is capable of fusing with the plasma membrane of a cell, thereby allowing the liposome to deliver a nucleic acid molecule and/or a protein composition into a cell. Some preferred liposomes include those liposomes commonly used in gene delivery methods known to those of skill in the art. Some preferred liposome delivery vehicles comprise multilamellar vesicle (MLV) lipids and extruded lipids, although the invention is not limited to such liposomes. Methods for preparation of MLVs are well known in the art. “Extruded lipids” are also contemplated. Extruded lipids are lipids that are prepared similarly to MLV lipids, but which are subsequently extruded through filters of decreasing size, as described in Templeton et al., Nature Biotech., 1997, 15:647-652, which is incorporated herein by reference in its entirety. Small unilamellar vesicle (SUV) lipids can also be used in the compositions and methods of the present invention. Other preferred liposome delivery vehicles comprise liposomes having a polycationic lipid composition (i.e., cationic liposomes). For example, cationic liposome compositions include, but are not limited to, any cationic liposome complexed with cholesterol, and without limitation, include DOTMA and cholesterol, DOTAP and cholesterol, DOTIM and cholesterol, and DDAB and cholesterol. Liposomes utilized in the present invention can be any size, including from about 10 to 1000 nanometers (nm), or any size in between.

A liposome delivery vehicle can be modified to target a particular site in a mammal, thereby targeting and making use of an NP-encoding nucleic acid molecule of the present invention at that site. Suitable modifications include manipulating the chemical formula of the lipid portion of the delivery vehicle. Manipulating the chemical formula of the lipid portion of the delivery vehicle can elicit the extracellular or intracellular targeting of the delivery vehicle. For example, a chemical can be added to the lipid formula of a liposome that alters the charge of the lipid bilayer of the liposome so that the liposome fuses with particular cells having particular charge characteristics. In one embodiment, other targeting mechanisms, such as targeting by addition of exogenous targeting molecules to a liposome (i.e., antibodies) may not be a necessary component of the liposome of the present invention, since effective immune activation at immunologically active organs can already be provided by the composition when the route of delivery is intravenous or intraperitoneal, without the aid of additional targeting mechanisms. However, in some embodiments, a liposome can be directed to a particular target cell or tissue by using a targeting agent, such as an antibody, soluble receptor or ligand, incorporated with the liposome, to target a particular cell or tissue to which the targeting molecule can bind. Targeting liposomes are described, for example, in Ho et al., Biochemistry, 1986, 25: 5500-6; Ho et al., J Biol Chem, 1987a, 262: 13979-84; Ho et al., J Biol Chem, 1987b, 262: 13973-8; and U.S. Pat. No. 4,957,735 to Huang et al., each of which is incorporated herein by reference in its entirety). In one embodiment, if avoidance of the efficient uptake of injected liposomes by reticuloendothelial system cells due to opsonization of liposomes by plasma proteins or other factors is desired, hydrophilic lipids, such as gangliosides (Allen et al., FEBS Lett, 1987, 223: 42-6) or polyethylene glycol (PEG)-derived lipids (Klibanov et al., FEBS Lett, 1990, 268: 235-7), can be incorporated into the bilayer of a conventional liposome to form the so-called sterically-stabilized or “stealth” liposomes (Woodle et al., Biochim Biophys Acta, 1992, 1113: 171-99). Variations of such liposomes are described, for example, in U.S. Pat. No. 5,705,187 to Unger et al., U.S. Pat. No. 5,820,873 to Choi et al., U.S. Pat. No. 5,817,856 to Tirosh et al.; U.S. Pat. No. 5,686,101 to Tagawa et al.; U.S. Pat. No. 5,043,164 to Huang et al., and U.S. Pat. No. 5,013,556 to Woodle et al., all of which are incorporated herein by reference in their entireties).

The NP-encoding nucleic acid sequences of the present invention can be conjugated with chitosan. For example, DNA chitosan nanospheres can be generated, as described by Roy, K. et al. (1999 , Nat Med 5:387). Chitosan allows increased bioavailability of the NP-encoding 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.

Mammalian species which benefit from the disclosed methods of treatment include, and are not limited to, 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. The terms “patient” and “subject” are used interchangeably herein are intended to include such human and non-human mammalian species. According to the method of the present invention, human or non-human mammalian NP (or nucleic acid sequences encoding human or non-human mammalian NP) can be administered to the patient. The NP may be naturally occurring within the patient's species or a different mammalian species. The expression vectors used in the subject invention can comprise nucleic acid sequences encoding any human or non-human mammalian NP. In instances where genetically modified cells are administered to a patient, the cells may be autogenic, allogeneic, or xenogeneic, for example.

In another aspect, the present invention concerns pharmaceutical compositions containing a therapeutically effective amount of agent that reduces NPR-A activity, such as an NP, or polynucleotides encoding NP, and a pharmaceutically acceptable carrier. Preferably, if the agent is a polynucleotide, such as an NP-encoding nucleic acid sequence, the polynucleotide is contained within an expression vector, such as plasmid DNA or a virus. Pharmaceutical compositions including a therapeutically effective amount of an agent that reduces NPR-A activity such as NP, or nucleic acid sequences encoding NP, and a pharmaceutically acceptable carrier, can be administered to a patient by any effective route, including local or systemic delivery. Administration can be continuous or at distinct intervals as can be determined by a person skilled in the art.

The agent that reduces NPR-A activity, such as NP or polynucleotides encoding NP (and pharmaceutical compositions containing them), can be administered to a patient by any route that results in prevention (or reduction of onset) or alleviation of symptoms associated with cancer, such as pain. For example, the agent (e.g., NP or NP-encoding nucleic acid molecule) can be administered parenterally, intravenously (I.V.), intramuscularly (I.M.), subcutaneously (S.C.), intradermally (I.D.), topically, transdermally, orally, intranasally, etc.

If desired, the pharmaceutical composition can be adapted for administration to the airways of the patient, e.g., nose, sinus, throat and lung, for example, as nose drops, as nasal drops, by nebulization as an inhalant, vaporization, or other methods known in the art. Examples of intranasal administration can be by means of a spray, drops, powder or gel and also described in U.S. Pat. No. 6,489,306, which is incorporated herein by reference in its entirety. One embodiment of the present invention is the administration of the invention as a nasal spray. Alternate embodiments include administration through any oral or mucosal routes, sublingual administration and even eye drops. However, other means of drug administrations are well within the scope of the present invention.

The pharmaceutical compositions of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Furthermore, as used herein, the phrase “pharmaceutically acceptable carrier” includes any of the standard pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can include diluents, adjuvants, and vehicles, as well as implant carriers, and inert, non-toxic solid or liquid fillers, diluents, or encapsulating material that does not react with the active ingredients of the invention. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water, and emulsions, such as oil/water emulsions. The carrier can be