ALDOSTERONISM

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the following U.S. Provisional Application No.: 62/688,792, filed June 22, 2018, the entire contents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

This invention was made with government support under grant No. 1R01DK100653A awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Primary aldosteronism (PA) (a.k.a primary hyperaldosteronism or Conn’s syndrome) is the most common form of secondary (low renin) hypertension, affecting -10% of all hypertensive patients, estimated at 100 million people world- wide. PA results from autonomous production of aldosterone from the zona glomerulosa (zG) of the adrenal gland. In addition to the adverse cardiovascular outcomes associated with hypertension, excess aldosterone is an independent risk factor for cardiovascular disease and renal damage. Thus, prompt treatment of PA is important to minimize the resulting morbidity and mortality.

SUMMARY OF THE INVENTION

The invention provides compositions for ameliorating the symptoms of PA and methods for treating PA.

PA is the most common form of secondary hypertension and is often associated with hypokalemia and low plasma renin activity. Delays in diagnosis and treatment result in a higher risk of stroke, cardiac failure, and renal damage. Thus, understanding the mechanisms underlying the pathogenesis of PA is crucial to facilitate early diagnosis and to develop alternative treatment strategies. The present inventors generated a mouse model with zG-specific beta-catenin gain-of- function ( CatGOF), which develops the hallmarks of PA, including zG hyperplasia, hyperaldosteronism, and high blood pressure. Using RNA sequencing, the present inventors have identified 790 transcripts that are differentially expressed in CatGOF adrenals, including increased levels of Fibroblast Growth Factor Receptor 2 (Fgfr2). Results presented herein show that Fgfr2 deletion in the adult adrenal results in disrupted zG morphology and impaired aldosterone production, suggesting an important role for FGFR2 in regulating zG function. These findings suggested that FGFR2 is an important mediator of PA progression driven by WNT/beta-catenin activation, and that its inhibition will effectively block the progression of PA. To test these hypotheses, the present inventors have investigated the mechanism by which FGFR2 regulates aldosterone production and the impact of FGFR2 inhibition on PA progression in CatGOF mice, using genetic ablation and pharmacological inhibition strategies. Results presented herein provide the first molecular insights into how WNT/beta-catenin signaling drives PA progression in vivo. Accordingly, results presented herein provide critical proof-of-principle evidence in support of a novel non-invasive therapeutic strategy for the treatment of hypertension resulting from PA.

In an aspect, a method of inhibiting cell proliferation in the zona granulosa is presented, the method comprising contacting a cell of the zona granulosa with an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

In another aspect, a method of reducing aldosterone secretion in a subject is presented, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

In a further aspect, a method for treating primary aldosteronism (PA) in a subject in need thereof is presented, the method comprising administering to the subject a

therapeutically effective amount of an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

In a still further aspect, a method for treating primary aldosteronism (PA) in a subject in need thereof is presented, the method comprising treating a subject selected for having elevated aldosterone levels and administering to the subject a therapeutically effective amount of an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

In an embodiment of methods described herein, the agent is a small molecule, protein, or polynucleotide. In a particular embodiment, the agent binds to FGFR2 and inhibits ligand binding. In a further particular embodiment, the agent is a tyrosine kinase inhibitor. In another particular embodiment, the agent is a small molecule inhibitor, an antibody specific for fgfr2, or an antigen-binding fragment of an antibody specific for FGFR2. In particular embodiments thereof, the small molecule inhibitor is selected from the group consisting of AZD4547, BGJ398 (infigratinib), Alofanib (RPT835), and SSR128129E; and the protein is Bemarituzumab (FPA144).

In another particular embodiment, the agent inhibits FGFR2 expression. In a more particular embodiment, the agent is a polynucleotide. In a still more particular embodiment, the agent is an inhibitory nucleic acid molecule. Exemplary inhibitory nucleic acid molecules are oligonucleotides comprising: single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes an FGFR2 polypeptide (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers). In another particular embodiment, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA or a vector encoding an inhibitory nucleic acid molecule.

In an embodiment of methods described herein, the subject is a mammal. In a more particular embodiment, the mammal is a human.

In another embodiment of methods described herein, the agent or composition comprising same is administered intravenously, subcutaneously, intraperitoneally, orally, via inhalation, or locally. In a particular embodiment, the administering involves direct administration into and around the adrenal gland. In a more particular embodiment, the direct administration into and around the adrenal gland is achieved via injection or implantation of a drug-delivery device.

In an embodiment of methods described herein, the subject has at least one of expansion of a zona glomerulosa (zG) expansion in an adrenal gland, hyperaldosteronism, and hypertension.

In a particular embodiment, the subject has resistant hypertension. Resistant hypertension may be used to refer to high blood pressure that requires several medications to control.

In an embodiment of methods described herein, the method further comprises determining at least one of the subject’s blood pressure, aldosterone levels, and serum renin levels. In a particular embodiment, the aldosterone levels are determined in at least one of the subject’s serum and urine.

In another embodiment of methods described herein, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent. In a particular embodiment, the at least one additional therapeutic agent is an aldosterone blocking drug. In a more particular embodiment, the aldosterone-blocking drug is a mineralocorticoid receptor antagonist.

Also encompassed herein is an inhibitor of fibroblast growth factor receptor 2 (FGFR2) for use in a method of treating primary aldosteronism (PA), the method comprising administering the inhibitor of fgfr2 in an amount effective to reduce PA symptoms.

In another aspect, use of an inhibitor of fibroblast growth factor receptor 2 (fgfr2) for the treatment of primary aldosteronism (PA) is presented.

In another aspect, use of an inhibitor of fibroblast growth factor receptor 2 (fgfr2) for the manufacture of a medicament for the treatment of primary aldosteronism (PA) is presented.

In a further aspect, a composition is presented comprising a therapeutically effective amount of at least one inhibitor of fibroblast growth factor receptor 2 (fgfr2) and a therapeutically effective amount of at least one inhibitor of WNT/beta-catenin signaling pathway and a pharmaceutically acceptable carrier.

Accordingly the invention provides compositions for ameliorating the symptoms of PA and methods for treating PA. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "agent" is meant any small molecule chemical compound, antibody or functional fragment thereof, nucleic acid molecule, peptide, or polypeptide.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, more particularly a 25% change, more particularly a 40% change, and most particularly a 50% or greater change in expression levels.

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include PA, both familial forms and non-familial forms of the disease. In that non-familial forms of PA are most commonly caused by unilateral aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia (BAH), it is envisioned that therapeutic methods described herein may be used to advantage to treat APA and/or BAH. In a particular embodiment thereof, a patient with PA does not have a detectable cancer.

By "effective amount" is meant the amount required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject.

Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

In a particular embodiment, the dosage regimen calls for twice daily, once daily, twice per week, or once per week. The duration of treatment may extend from 7-28 days. The duration of treatment may be extended for longer periods of time for the purposes of chronic treatment of, for example, hypertension.

In a particular embodiment, a dosage regimen is intermittent, wherein a period during which time an effective amount of active compound(s) is administered is followed by a period of time wherein no active compound is administered. Such an intermittent regimen may be used when the active compound(s) is associated with deleterious side effects. As is understood by skilled practitioners, subjects/patients may respond differently to various active compound(s) and thus, sensitivities may also vary in a subject/patient dependent manner. A skilled practitioner may employ an intermittent dosage regimen under circumstances wherein a subject/patient exhibits side effects to an extent unacceptable to the subject/patient.

The invention provides a number of targets that are useful for the development of highly specific drugs to treat a disease or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high-volume throughput, high sensitivity, and low complexity.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. "Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By“marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

By“reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%.

By“reference” is meant a standard or control condition.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate,

1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.

Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,

BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;

aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e ³ and e ¹⁰⁰ indicating a closely related sequence.

By "subject" is meant a mammal, including, but not limited to, a human or non human mammal, such as a rodent (e.g., a mouse) bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 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, or 50.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

By“Fibroblast Growth Factor Receptor 2 (FGFR2) polypeptide” is meant a cell surface receptor for fibroblast growth factor or a fragment thereof having tyrosine kinase activity and having at least about 85% or greater amino acid sequence identity to Uniprot Identifier P21802-1. An exemplary FGFR2 amino acid sequence is provided below.

>sp | P21802 | FGFR2_HUMAN Fibroblast growth factor receptor 2 OS=Homo sapiens OX=9606 GN=FGFR2 ^_ PE=1 SV=1

MVSWGRFICLVWTMATLSLARPS SLVEDTTLEPEEPPTKYQISQPEVYVAAPGESLEV RCLLKDAAVI SWTKDGVHLGPNNRTVLIGEYLQIKGATPRDSGLYACTASRTVDSETWYF MV VTDAI SSGDDEDDTDGAEDFVSENSNNKRAPYWTNTEKMEKRLHAVPAANTVKFRCP AGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESWPSDKGNYTCWENEYGSI NHTYHLDWERSPHRPILQAGLPANASTWGGDVEFVCKVYSDAQPHIQWIKHVEKNGSK YGPDGLPYLKVLKAAGV TTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFHSAWLTVL PAPGREKEITASPDYLEIAIYCIGVFLIACMWTVILCRMKNTTKKPDFSSQPAVHKLTK RIPLRRQVTVSAESSSSMNSNTPLVRITTRLSSTADTPMIAGVSEYELPEDPKWEFPRDK LTLGKPLGEGCFGQWMAEAVGIDKDKPKEAVTVAVKMLKDDATEKDLSDLVSEMEMMKM IGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLRARRPPGMEYSYDINRVPEEQMTF KDLVSCTYQLARGMEYIASQKCIHRDIAARNVLVTENNVMKIADFGLARDINNIDYYKKT TNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLMWEIFTLGGSPYPGI PVEELFKLLKEGH RMDKPANCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRILTLTTNEEYLDLSQPLEQYS

PSYPDTRSSCSSGDDSVFSPDPMPYEPCLPQYPHINGSVKT

By“Fibroblast Growth Factor Receptor 2 (FGFR2) polynucleotide” is meant a nucleic acid sequence encoding an FGFR2 polypeptide or fragment thereof. An exemplary FGFR2 nucleic acid sequence is provided at NM_000l4l.4, which is reproduced below.

1 ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg

61 ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgeggegta

121 cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg

181 ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg

241 tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggeagee

301 cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gegtgaagee egggaggett

361 ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg

421 ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtegeggag

481 gagcgttgcc attcaagtga ctgcagcagc ageggeageg cctcggttcc tgagcccacc

541 gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa

601 cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg

661 gtcgtttcat ctgcctggtc gtggtcaeca tggcaacctt gtccctggcc cggccctcct

721 tcagtttagt tgaggatacc acattagagc cagaagagcc accaaccaaa taccaaatct

781 ctcaaccaga agtgtacgtg gctgcgccag gggagteget agaggtgcgc tgcctgttga

841 aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc aacaatagga

901 cagtgcttat tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct

961 atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg gtgaatgtca

1021 cagatgccat ctcatccgga gatgatgagg atgacaccga tggtgcggaa gattttgtca

1081 gtgagaacag taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc

1141 ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc ggggggaacc

1201 caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt taageaggag catcgcattg

1261 gaggctacaa ggtacgaaac cagcactgga gcctcattat ggaaagtgtg gtcccatctg

1321 acaagggaaa ttatacctgt gtagtggaga atgaataegg gtccatcaat cacacgtacc

1381 acctggatgt tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa

1441 atgcctccac agtggtcgga ggagaegtag agtttgtctg caaggtttac agtgatgccc

1501 agccccacat ccagtggatc aagcacgtgg aaaagaacgg cagtaaatac gggcccgacg

1561 ggctgcccta cctcaaggtt ctcaaggccg ccggtgttaa caccacggac aaagagattg

1621 aggttctcta tattcggaat gtaacttttg aggacgctgg ggaatatacg tgcttggcgg

1681 gtaattctat tgggatatcc tttcactctg catggttgac agttctgcca gcgcctggaa

1741 gagaaaagga gattacagct tccccagact acctggagat agccatttac tgcatagggg

1801 tcttcttaat cgcctgtatg gtggtaacag tcatcctgtg ccgaatgaag aacacgacca

1861 agaagccaga cttcagcagc cagccggctg tgcacaagct gaccaaacgt atccccctgc

1921 ggagacaggt aacagtttcg gctgagtcca gctcctccat gaactccaac accccgctgg

1981 tgaggataac aacacgcctc tcttcaacgg cagacacccc catgctggca ggggtctccg

2041 agtatgaact tccagaggac ccaaaatggg agtttccaag agataagetg acactgggca

2101 agcccctggg agaaggttgc tttgggcaag tggtcatggc ggaagcagtg ggaattgaca

2161 aagacaagcc caaggaggcg gtcaccgtgg ccgtgaagat gttgaaagat gatgccacag

2221 agaaagacct ttctgatctg gtgtcagaga tggagatgat gaagatgatt gggaaacaca

2281 agaatatcat aaatcttctt ggagcctgca cacaggatgg gcctctctat gtcatagttg

2341 agtatgcctc taaaggcaac ctccgagaat acctccgagc ccggaggcca cccgggatgg

2401 agtactccta tgacattaac cgtgttcctg aggageagat gaccttcaag gacttggtgt

2461 catgcaccta ccagctggcc agaggeatgg agtacttggc ttcccaaaaa tgtattcatc

2521 gagatttagc agccagaaat gttttggtaa cagaaaacaa tgtgatgaaa atagcagact

2581 ttggactcgc cagagatatc aacaatatag actattacaa aaagaccacc aatgggcggc

2641 ttccagtcaa gtggatggct ccagaagccc tgtttgatag agtatacact catcagagtg

2701 atgtctggtc cttcggggtg ttaatgtggg agatcttcac tttagggggc tcgccctacc

2761 cagggattcc cgtggaggaa ctttttaagc tgctgaagga aggacacaga atggataage

2821 cagccaactg caccaacgaa ctgtacatga tgatgaggga ctgttggcat gcagtgccct 2881 cccagagacc aacgttcaag cagttggtag aagacttgga tcgaattctc actctcacaa

2941 ccaatgagga atacttggac ctcagccaac ctctcgaaca gtattcacct agttaccctg

3001 acacaagaag ttcttgttct tcaggagatg attctgtttt ttctccagac cccatgcctt

3061 acgaaccatg ccttcctcag tatccacaca taaacggcag tgttaaaaca tgaatgactg

3121 tgtctgcctg tccccaaaca ggacagcact gggaacctag ctacactgag cagggagacc

3181 atgcctccca gagcttgttg tctccacttg tatatatgga tcagaggagt aaataattgg

3241 aaaagtaatc agcatatgtg taaagattta tacagttgaa aacttgtaat cttccccagg

3301 aggagaagaa ggtttctgga gcagtggact gccacaagcc accatgtaac ccctctcacc

3361 tgccgtgcgt actggctgtg gaccagtagg actcaaggtg gacgtgcgtt ctgccttcct

3421 tgttaatttt gtaataattg gagaagattt atgtcagcac acacttacag agcacaaatg

3481 cagtatatag gtgctggatg tatgtaaata tattcaaatt atgtataaat atatattata

3541 tatttacaag gagttatttt ttgtattgat tttaaatgga tgtcccaatg cacctagaaa

3601 attggtctct ctttttttaa tagctatttg ctaaatgctg ttcttacaca taatttctta

3661 attttcaccg agcagaggtg gaaaaatact tttgctttca gggaaaatgg tataacgtta

3721 atttattaat aaattggtaa tatacaaaac aattaatcat ttatagtttt ttttgtaatt

3781 taagtggcat ttctatgcag gcagcacagc agactagtta atctattgct tggacttaac

3841 tagttatcag atcctttgaa aagagaatat ttacaatata tgactaattt ggggaaaatg

3901 aagttttgat ttatttgtgt ttaaatgctg ctgtcagacg attgttctta gacctcctaa

3961 atgccccata ttaaaagaac tcattcatag gaaggtgttt cattttggtg tgcaaccctg

4021 tcattacgtc aacgcaacgt ctaactggac ttcccaagat aaatggtacc agcgtcctct

4081 taaaagatgc cttaatccat tccttgagga cagaccttag ttgaaatgat agcagaatgt

4141 gcttctctct ggcagctggc cttctgcttc tgagttgcac attaatcaga ttagcctgta

4201 ttctcttcag tgaattttga taatggcttc cagactcttt ggcgttggag acgcctgtta

4261 ggatcttcaa gtcccatcat agaaaattga aacacagagt tgttctgctg atagttttgg

4321 ggatacgtcc atctttttaa gggattgctt tcatctaatt ctggcaggac ctcaccaaaa

4381 gatccagcct catacctaca tcagacaaaa tatcgccgtt gttccttctg tactaaagta

4441 ttgtgttttg ctttggaaac acccactcac tttgcaatag ccgtgcaaga tgaatgcaga

4501 ttacactgat cttatgtgtt acaaaattgg agaaagtatt taataaaacc tgttaatttt

4561 tatactgaca ataaaaatgt ttctacagat attaatgtta acaagacaaa ataaatgtca

4621 cgcaacttat ttttttaata aaaaaaaaaa aaaa

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1A-1F depicts (A) a schematic of the conditional deletion of Ctnnbl exon 3 following Cre-mediated recombination; (B) a histogram depicting results comparing b wildtype (b WT) and b gain of function (b GOF); and (C)-(F) immunohistochemistry results comparing b WT and b GOF adrenals.

Fig. 2A-2F depicts (A) immunohistochemistry results comparing b WT and b GOF adrenal staining patterns; (B) a schematic of functional lineage-tracing experiments using the R26R ^mTmG reporter allele; (C)-(F) immunohistochemistry results comparing b WT and b GOF adrenals.

Fig. 3A-3H depicts (A) - (D) histograms depicting results comparing b WT and b GOF biological read outs; (E) immunohistochemistry results comparing b WT and b GOF adrenals; (F) histograms depicting results comparing b WT and b GOF adult and aged mice; (G) a plot depicting plasma aldosterone levels in b WT and b GOF mice; and (H) histogram plots demonstrating that b-catenin gain-of-function (Bcat-GOF) increases the adrenal NADPH/NADP+ ratio. Fig. 4A-4F depicts (A) a schematic depicting the generation of Aff ^re/Cre :: Ctnnb l ^FloxedEx3 (AS(KO)-Pcat-GOF) mice; (B) a cartoon illustrating RAAS activation; (C) immunohistochemistry results comparing b WT and b GOF adrenals; (D) a histogram depicting results comparing b WT and b GOF biological parameters; (E) immunohistochemistry results comparing b WT and b GOF adrenals; and (F) a histogram depicting results comparing b WT and b GOF.

Fig. 5A-5F depicts (A) a cartoon illustrating the aldosterone/Renin/Angll pathway;

(B) a cartoon illustrating the experimental design for evaluating the impact of spironolactone on b WT and b GOF mice; (C) immunohistochemistry results comparing b WT and b GOF adrenals; (D) a histogram depicting results comparing b WT and b GOF biological parameters; (E) immunohistochemistry results comparing b WT and b GOF adrenals; and (F) a histogram depicting results comparing b WT and b GOF.

Fig. 6A-6D depicts (A) a histogram depicting results comparing b WT and b GOF in young adult, adult, and aged mice; (B) - (C) immunohistochemistry results comparing b WT and b GOF adrenals; and (D) ) a histogram depicting results comparing b WT and b GOF adrenals.

Fig. 7A-7D depicts (A) and (C) immunohistochemistry results comparing b WT and b GOF adrenals; and (B) and (D) histograms depicting results comparing b WT and b GOF adrenals.

Fig. 8A-8E depicts (A) and (B) histograms depicting results comparing b WT and b GOF mice biological parameters; and (C) - (E) immunohistochemistry results comparing b WT and b GOF adrenals.

Fig. 9A-9E depicts (A) and (B) histograms depicting results comparing b WT and b GOF mice adrenal glands; and (C) - (E) immunohistochemistry results comparing b WT and b GOF adrenals.

Fig. 10A-10E depicts (A), (C), and (E) immunohistochemistry results comparing b WT and b GOF adrenals; and (B) and (D) histograms depicting results comparing b WT and b GOF mice biological parameters and adrenal gland weight.

Fig. 11A-11C depicts transcriptome analysis of b Cat-GOF adrenals. (A) Heat map of differentially expressed genes in b Cat-GOF adrenals compared to controls (N = 6,6), with fold change > 1.2, adjusted p-value <0.05, mean expression > 100 RPKM. (B) Functional annotation enrichment analysis using DAVID (v6.8). (C) RPKM fold change of Fgfr2 and FGF target genes, *, ***, ****, adjusted p-value < 0.05, 0.001, 0.0001. Error bar, SEM. Fig. 12 depicts a schematic of Fgfr2 cKO mouse model. Cre mediates deletion of exon 8-10, resulting in loss of function of Fgfr2.

Fig. 13 depicts zG morphology is disrupted in Fgfr2 cKO adrenals. Immunostaining of laminin bl (Lambl), outlining glomerulus structures in control and Fgfr2 cKO adrenals. Bar, 50pm.

Fig. 14A- 14B Fgfr2 deletion causes impaired aldosterone production. (A) Cypl lb2 expression by qRT-PCR (N = 6,6). (B) aldosterone renin ratio (N = 6,9). *, **, p < 0.05, 0.01, t-test. Error bars, SEM.

Fig. 15 presents histogram plots depicting results comparing aldosterone production and aldosterone to renin ratios in fgfr2 WT and fgfr2 knock out (KO) mice which show that FGFR2 is required for normal aldosterone production.

Fig. 16 presents photographs of AS-/- Control and AS-/- cKO mice and a histogram plot depicting results comparing weight gain in AS-/- Control and AS-/- cKO mice.

Fig. 17 presents a schematic of the conditional mouse alleles used to generate b Cat- GOF and fgfr2 loss of function (LOF)/KO mice.

Fig. 18 presents immunohistochemistry results comparing b GOF and b GOF :: fgfr2c LOF mice adrenals which shows that fgfr2 deletion rescues the b catenin GOF phenotype.

Fig. 19 presents immunohistochemistry results comparing b GOF adrenals with and without treatment with the indicated FGFR2 inhibitor and histogram plots depicting these results which show that FGFR2 inhibitor treatment impairs zG proliferation, a hallmark of the b catenin GOF phenotype.

FIGs. 20A and 20B illustrate that FGFR inhibitors impair zG function. FIG. 20 A is a plot showing urinary aldosterone levels over time in adult mice treated with AZD4547. The arrow denotes the start of treatments. denotes p < 0.01 and“*” denotes p < 0.05. FIG. 20B, is a graph showing the fold change in Cypl lb2 and Cyclin Dl transcript levels in adrenals from untreated mice and mice treated with BGJ398 for 7 days. “*” denotes p < 0.05. Error bars, denote the mean ± SEM.

FIG. 21 provides a series of images illustratrating that Fgfr2 deletion prevents zG hyperplasia driven by b-catenin gain-of-function. Representative images of GFP and Dab2 co-immunostaining of control (ASCre/+; mTmG), bCat GOF (ASCre/+; mTmG; b-catenin fl(ex3)/+), and bCat GOF; Fgfr2 cKO (ASCre/+; mTmG; b-catenin fl(ex3)/+; Fgfr2 fl/fl) adult mice adrenals. Bars denote 50 pm DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions for ameliorating the symptoms of PA and methods for treating PA.

Despite being a major cause of morbidity affecting -10% of patients with severe hypertension, PA is markedly underdiagnosed and managed with limited medical and surgical options. Apart from familial forms, PA is most commonly caused by unilateral aldosterone producing adenoma (APA) or bilateral adrenal hyperplasia (BAH). The WNT/b- catenin signaling pathway is a well-known regulator of adrenal homeostasis and is found activated in -70% of aldosterone-producing Conn’s adenomas. The present inventors have generated a mouse model with b-catenin gain-of-function ^Cat-GOF) in the adult zG, which develops the hallmarks of PA, including zG expansion, increased plasma aldosterone/renin ratio, and high blood pressure. Hence, this model offers a unique opportunity to study the downstream cellular and molecular mechanisms by which WNT^-catenin signaling leads to PA, and to develop targeted therapies for its treatment.

Using an unbiased RNA-seq approach, the present inventors identified increased expression of Fibroblast growth factor receptor 2 (Fgfr2) in bCat-GOF adrenals. Fgfr2 has previously been shown to regulate adrenal development and in the adult its expression is restricted to the zG. Genetic deletion of Fgfr2 in the adult adrenal results in disrupted zG morphology and impaired aldosterone production. In addition, treatment of mice with pharmacological antagonists of FGFR signaling results in a marked decrease in zG proliferation.

In accordance with the present findings, therefore, the present inventors have identified a novel aldosterone regulator, FGFR2. FGFR2 is a highly targetable receptor tyrosine kinase. Results from studies presented herein provide important insights into the mechanisms of PA pathogenesis and guidance regarding the development of novel, non- invasive therapeutic strategies for the treatment of PA using FGF inhibitors, including those currently under development for the treatment of cancer.

The invention features compositions and methods that are useful for treating PA and ameliorating symptoms thereof, including, without limitation, hypertension, zG hyperplasia, and hyperaldosteronism. The present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound of the formulae herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a PA or a symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof or a composition comprising same, under conditions such that the disease or disorder is treated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms“prevent,”“preventing,”“prevention,”“prop hylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

The therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of the compounds described herein, such as, for example a small molecule inhibitor of Fgfr2 to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

Inhibitors of Fgfr2 activity are known in the art and include small molecule inhibitors and monoclonal antibodies specific for Fgfr2. Inhibitors of Fgfr2 activity include, without limitation, AZD4547, a tyrosine kinase inhibitor which targets FGFR1-3 (available from AstraZeneca); BGJ398 (infigratinib), a Pan-FGF receptor kinase inhibitor (available from Novartis); Bemarituzumab (FPA144), a monoclonal antibody that binds to FGFR2b preventing binding of certain FGFs (available from Five Prime); Alofanib (RPT835), a novel first-in-class allosteric small-molecular inhibitor of FGFR2 (available from Ruspharmtech); and SSR128129E, an allosteric inhibitor of FGF receptor signaling (available from Sanofi Aventis).

Dosing parameters have been established for AZD4547 when used to treat cancer as follows: AZD4547 may be dosed at l2.5mg/kg once daily, 24d (Gavine et al, 2012, Cancer Res. 72:2045-2056); or in accordance with the following clinical trials: PHASE 1/2, 40, 60, 80mg, 2lday, lowest does resulted in dose limiting toxicity (NCT01824901); PHASE 2, 80mg, 2wk on lwk off (NCT01457846); or phase 1/2, 40, 80mg, tablet twice daily

(NCT01202591); the entire content of each of which is incorporated herein by reference.

Dosing parameters have been established for BGJ398 when used to treat cancer as follows: BGJ398 may be dosed at 30mg/kg/day (Guagnano et al., 2011, J Med Chem

54:7066-7083); or in accordance with the following clinical trial: phase 2, l25mg flat, 3wk on lwk off (NCT02160041) ); the entire content of each of which is incorporated herein by reference.

Dosing parameters have been established for FPAl44/bemarituzumab when used to treat cancer as follows: FPAl44/bemarituzumab may be dosed at 5mg/kg bidaily (Gemo et al. 2014); or in accordance with the following clinical trials: phase 1 (NCT03343301); the entire content of each of which is incorporated herein by reference.

Dosing parameters have been established for RPT835/Alofanib when used to treat cancer as follows: RPT835/Alofanib may be dosed at 50mg/kg oral daily, l-3wk.

Dosing parameters have been established for RPT835/Alofanib when used to treat cancer as follows: RPT835/Alofanib may be dosed at 50mg/kg oral daily, l-3wk.

Accordingly, in one embodiment, a therapeutically effective amount of at least one inhibitor of Fgfr2 activity or expression, such as those described herein, is administered to a subject in need thereof. In yet another embodiment, a therapeutically effective amount of at least one inhibitor of Fgfr2 activity is administered to a subject in need thereof in conjunction with a therapeutically effective amount of at least one inhibitor of WNT/beta-catenin signaling. Such a combination may be administered concomitantly or in succession. In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with PA (e.g., hypertension), in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject’s disease status. In particular embodiments, a second level of Marker in the subject is determined at a time point later than the

determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In particular embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

Pharmaceutical Compositions

For therapeutic use, a compound or agent (e.g., small molecule inhibitor of Fgfr2) or a pharmaceutically acceptable salt thereof is formulated with a carrier that is pharmaceutically acceptable and is appropriate for delivering the compound or agent by the chosen route of administration. Suitable pharmaceutically acceptable carriers are those used conventionally with small molecules, such as diluents, excipients and the like. See, for example, "Remington s Pharmaceutical Sciences", l7th Ed., Mack Publishing Company, Easton, Pa., 1995, for guidance on drug formulations. In one embodiment, the compounds are formulated for administration by infusion or by injection, either sub-cutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered to a slightly acidic or physiological pH. Thus, the compounds/agents may be administered in distilled water, saline, buffered saline or 5% dextrose solution. Water solubility of compositions comprising a compound or agent may be enhanced by

incorporating a solubility enhancer, such as acetic acid.

Methods of Delivery Compounds/agents and compositions comprising same may be administered via a variety of methods. Such methods include, without limitation, intravesicular, intralesional (in and around an adrenal gland), oral, intravenous (iv), subcutaneous (sc or sq), intraperitoneal, intramuscular intradermal, rectal, nasal, or topical administration, or inhalation via nebulizer or inhaler, to a subject (e.g., a mammal) in need thereof.

Therapeutic Dosing and Regimen

The therapeutic dosing and regimen best suited for treatment of a subject (e.g., a human patient) vary with the disorder or condition to be treated, and according to the patient's weight and other parameters. A dose of at least one compound/agent described herein may, for example, be administered at about 2.5 mg/kg, administered twice daily over 10 days. Smaller doses, e.g., in the pg/kg range, and shorter or longer duration or frequency of treatment, are also envisioned to produce therapeutically useful results, i.e., a statistically significant decrease in hypertension. It is, moreover, envisioned that localized administration to, e.g., at least one adrenal gland, may be optimized based on the response of adrenal cells therein.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject, including the size, age, and general condition of the patient, the particular disorder being treated, the severity of the disorder, and the presence of other drugs in the patient. Trial dosages may be chosen after consideration of the results of animal studies and the clinical literature.

A typical human dose of a compound/agent may be from about 10 pg/kg body weight/day to about 10 mg/kg/day, more particularly from about 50 pg/kg/day to about 5 mg/kg/day, and even more particularly about 100 pg/kg/day to 1 mg/kg/day.

Therapeutic efficacy of a compound/agent and/or compositions comprising same may be determined by evaluating and comparing patient symptoms and quality of life pre- and post-administration. Such methods apply irrespective of the mode of administration. In a particular embodiment, pre-administration refers to evaluating patient symptoms and quality of life prior to onset of therapy and post-administration refers to evaluating patient symptoms and quality of life at least 2-8 weeks after onset of therapy. In a particular embodiment, the post-administration evaluating is performed about 2-8, 2-6, 4-6, or 4 weeks after onset of therapy. In a particular embodiment, patient symptoms (e.g., hypertension) and quality of life pre- and post-administration are evaluated via questionnaire assessment.

In some embodiments, the formulation comprising a compound/agent comprises one or more additional components, wherein the additional component is at least one of an osmolar component that provides an isotonic, or near isotonic solution compatible with human cells or blood, and a preservative.

In some embodiments, the osmolar component is a salt, such as sodium chloride, or a sugar or a combination of two or more of these components. In some embodiments, the sugar may be a monosaccharide such as dextrose, a disaccharide such as sucrose or lactose, a polysaccharide such as dextran 40, dextran 60, or starch, or a sugar alcohol such as mannitol. The osmolar component is readily selected by those skilled in the art.

In some embodiments, the preservative is at least one of parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.

In some embodiments, the formulation comprising a compound/agent is in the form of a sustained release formulation and further comprises one or more additional components, wherein the additional component is at least one of an anti-inflammatory agent; and a preservative.

In some embodiments, the sustained release formulation is administered as a suppository.

In some embodiments, the sustained release formulation is administered in an implant designed for subcutaneous (sc or sq) implantation. Exemplary sc implants are known to those of skill in the art and may involve a port or catheter or the like. In a particular embodiment, the port or catheter is implanted in or near an adrenal gland.

Recombinant DNA/Polypeptides

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,

“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);

“Methods in Enzymology”“Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Inhibitory Nucleic Acids

Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of an FGFR2 polypeptide. Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes an FGFR2 polypeptide (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).

siRNA

Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al, Cell 101 : 25-33; Elbashir et al., Nature 411: 494-498, 2001, hereby incorporated by reference). The therapeutic effectiveness of an siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38- 39.2002).

Given the sequence of a target gene, siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat FSHD. The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.

In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047- 6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.

Small hairpin RNAs (shRNAs) comprise an RNA sequence having a stem-loop structure. A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The term "hairpin" is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches. The multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecule, or some combination thereof.

As used herein, the term "small hairpin RNA" includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA" also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that are about 22 -nucleotides long and generally expressed in a highly tissue- or developmental- stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function analogously to small interfering RNAs (siRNAs). Thus, one can design and express artificial miRNAs based on the features of existing miRNA genes.

shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type. In some embodiments, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations. Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14c, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. A producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.

Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid molecule encoding an FGFR2 polypeptide). The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al, Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 Al, each of which is incorporated by reference.

Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Example of hairpin motifs are described by Hampel et al, "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al, Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

Essentially any method for introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct. A viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA. Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.

For expression within cells, DNA vectors, for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed. Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al, 2005, Nat. Genet. 39: 914-921). In some embodiments, expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters. Examples of useful promoters in the context of the invention are tetracycline- inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline trans activator (rtTA) systems. Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types. A certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application

PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et al, 2006, Drug Discovery Today 11 : 975-982, for a description of inducible shRNA.

Delivery of Polynucleotides

Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

Oligonucleotides and other Nucleobase Oligomers

At least two types of oligonucleotides induce the cleavage of RNA by RNase H: polydeoxynucleotides with phosphodi ester (PO) or phosphorothioate (PS) linkages. Although 2'-OMe-RNA sequences exhibit a high affinity for RNA targets, these sequences are not substrates for RNase H. A desirable oligonucleotide is one based on 2'-modified oligonucleotides containing oligodeoxynucleotide gaps with some or all intemucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of

methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies that may be developed, including covalently-closed multiple antisense (CMAS) oligonucleotides (Moon et al, Biochem J. 346:295-303, 2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS) oligonucleotides (Moon et al, J. Biol. Chem. 275:4647-4653, 2000; PCT Publication No.

WO 00/61595), and large circular antisense oligonucleotides (U.S. Patent Application Publication No. US 2002/0168631 Al).

As is known in the art, a nucleoside is a nucleobase-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.

For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure; open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Specific examples of preferred nucleobase oligomers useful in this invention include oligonucleotides containing modified backbones or non-natural intemucleoside linkages. As defined in this specification, nucleobase oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone are also considered to be nucleobase oligomers. Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,

thionoalkylphosphonates, thionoalkylphosphotriest- ers, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.

Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos.

5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

In other nucleobase oligomers, both the sugar and the intemucleoside linkage, i.e., the backbone, are replaced with novel groups. The nucleobase units are maintained for hybridization with a gene encoding an FGFR2 polypeptide. One such nucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an

aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in "Peptide Nucleic Acids: Protocols and Applications" Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.

In particular embodiments of the invention, the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular - CH2-NH-O-CH2-, -CH2-N(CH3)-0-CH2- (known as a methylene (methylimino) or MMI backbone), -CH ₂ -0-N(CH ₃ )-CH ₂ -, -CH ₂ -N(CH ₃ )-N(CH ₃ )-CH ₂ -, and -0-N(CH ₃ )-CH ₂ -CH ₂ -. In other embodiments, the oligonucleotides have morpholino backbone structures described in U.S. Pat. No. 5,034,506.

Nucleobase oligomers may also contain one or more substituted sugar moieties. Nucleobase oligomers comprise one of the following at the 2' position: OH; F; O-, S-, or N- alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted Ci to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are 0[(CH2)n0] _n CH ₃ , 0(CH2) nOCH ₃ , 0(CH2) 11NH2, 0(CH2) nCH ₃ , 0(CH2) 11ONH2, and 0(CH2) nON[(CH2) nCH ₃ )]2, where n and m are from 1 to about 10. Other preferred nucleobase oligomers include one of the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O- aralkyl, SH, SOU, OCN, Cl, Br, CN, CF ₃ , OCF ₃ , SOCTU, S0 ₂ CH ₃ , ONO2, NO2, NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the

pharmacokinetic properties of a nucleobase oligomer, or a group for improving the pharmacodynamic properties of an nucleobase oligomer, and other substituents having similar properties. Preferred modifications are 2'-0-methyl and 2'-methoxyethoxy (2'-0- CH2CH20CH ₃ , also known as 2'-0-(2-methoxyethyl) or 2'-MOE). Another desirable modification is 2'-dimethylaminooxyethoxy (i.e., 0(CH2) 20N(CH ₃ ) 2), also known as 2'- DMAOE. Other modifications include, 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'- fluoro (2'-F). Similar modifications may also be made at other positions on an

oligonucleotide or other nucleobase oligomer, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.

Nucleobase oligomers may also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me- C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5- propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4- thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7- deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2. degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are desirable base substitutions, even more particularly when combined with 2'-0-methoxy ethyl or 2'-0-methyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include U.S. Pat. Nos.

4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is herein incorporated by reference.

Another modification of a nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan et al, Bioorg. Med. Chem. Let, 4: 1053-1060, 1994), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770, 1993), a thiocholesterol (Oberhauser et al, Nucl. Acids Res., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO I, 10: 1111- 1118, 1991; Kabanov et al, FEBS Lett., 259:327-330, 1990; Svinarchuk et al, Biochimie, 75:49-54, 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di- O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al, Tetrahedron Lett., 36:3651- 3654, 1995; Shea et al, Nucl. Acids Res., 18:3777-3783, 1990), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al, J. Pharmacol. Exp. Ther., 277:923-937, 1996. Representative United States patents that teach the preparation of such nucleobase oligomer conjugates include U.S. Pat. Nos. 4,587,044;

4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045; 5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077; 5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,608,046; and 5,688,941, each of which is herein incorporated by reference.

The present invention also includes nucleobase oligomers that are chimeric compounds. "Chimeric" nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide. These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate deoxy oligonucleotides hybridizing to the same target region.

Chimeric nucleobase oligomers of the invention may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

The nucleobase oligomers used in accordance with this invention may be

conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The nucleobase oligomers of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

EXAMPLES

Aberrant activation of WNT/beta-catenin signaling has been found in -70% of aldosterone-producing adenomas, a major cause of PA. How this pathway leads to PA remains largely unclear. To address this, the present inventors generated a mouse model with zG-specific beta-catenin gain-of-function ( CatGOF), which develops the hallmarks of PA, including zG hyperplasia, hyperaldosteronism, and high blood pressure. Using RNA sequencing, the present inventors have identified 790 transcripts that are differentially expressed in CatGOF adrenals, including increased levels of Fibroblast Growth Factor Receptor 2 (Fgfr2). See, for example, Figure 11. Results presented herein show that Fgfr2 deletion in the adult adrenal results in disrupted zG morphology and impaired aldosterone production, suggesting an important role for FGFR2 in regulating zG function. These findings suggested that FGFR2 is an important mediator of PA progression driven by WNT/beta-catenin activation, and that its inhibition will effectively block the progression of PA. To test these hypotheses, the present inventors have investigated the mechanism by which FGFR2 regulates aldosterone production and the impact of FGFR2 inhibition on PA progression in CatGOF mice, using genetic ablation and pharmacological inhibition strategies. Results presented herein provide the first molecular insights into how WNT/beta- catenin signaling drives PA progression in vivo. Accordingly, results presented herein provide critical proof-of-principle evidence in support of a novel non-invasive therapeutic strategy for the treatment of hypertension resulting from PA. Example 1: Stabilization of b-catenin in zG cells results in ectopic accumulation of the zG.

To investigate the mechanisms by which activation of cWnt signaling leads to hyperaldosteronism, the present inventors generated mice with zG-specific stabilization of b- catenin [Cypl lb2(AS) ^Cre/+ :: Ctnnb l ^FloxedEx3 ) (pcat-GOF] (Figure 1A}. In this model, conditional deletion of Ctnnbl exon 3 following Cre-mediated recombination results in stabilization of b- catenin and constitutive activation of the canonical (c)WNT pathway) specifically within zG cells (Figure 1A). Mice heterozygous for the AS ^Cre allele or wild type mice were used as controls (Pcat- WT). Increased expression of Axin2 and Lefl, well-established targets of cWNT signaling, confirmed activation of this pathway in Pcat-GOF adrenals (Figure IB).

To begin to understand the impact of Pcat-GOF on adrenal homeostasis, the present inventors analyzed the b-catenin expression domain in young adult, adult and aged mice. As expected, in Pcat-WT adrenals b-catenin expression was restricted to a thin layer of cells beneath the capsule at all ages (Figure 1C). In contrast, Pcat-GOF adrenals showed progressive expansion of b-catenin expressing cells extending into the orthotopic zF (Figure 1C; Figure 6A). Male and female mice displayed a similar pattern and pace of zG expansion (Figure 6B), thus, unless otherwise stated, the following studies were performed with female mice.

To determine the identity of the ectopic b-catenin-expressing cells, the present inventors performed co-immunostaining for b-catenin and Dab2, an established zG marker. In both Pcat-WT and Pcat-GOF adrenals, Dab2 expression co-localized with b-catenin (Figure ID), suggesting that the ectopic b-catenin-expressing cells maintain a zG-like identity. Co- immunostaining for Gocq, the alpha subunit of the heterotrimeric G protein activated by Angiotensin II (Angll), which revealed co-localization with both b-catenin and Dab2 (Figure IE, Figure 6C). To determine whether this domain demonstrated features of zF cells, the present inventors examined the expression pattern of Aldose reductase-related protein 1 (Akrlb7), a zF cell marker, which revealed mutually exclusive expression of b-catenin and Akrlb7 in both Pcat- WT and Pcat-GOF adrenals (Figure IF). Finally, since the histomorphometry of the orthotopic zG and zF are notably distinct, the present inventors analyzed the cellular density of the ectopic domain by assessing the number of DAPI-positive nuclei per unit area, which was

indistinguishable from the eutopic zG (Figure 6D). In agreement with this result, analysis of the laminin subunit beta-1 (LAMB1) expression pattern, which is localized specifically in the zG in Pcat-WT sections, is also expanded in Pcat-GOF adrenals, in parallel to the ectopic b-catenin- positive domain. Upon homeostatic conditions, LAMB1 immunoreactivity was detected surrounding glomerular-like structures of 4 to 7 cells each. Notably, in Pcat-GOF sections, LAMB1 was expressed around centripetally stretched glomerular figures, containing more cells than normal, which extend in the orthotopic zF region. Together, these data indicate that stabilization of b-catenin within zG cells leads to a progressive expansion of the zG.

Example 2: Stabilization of b-catenin in zG cells blocks zG-to-zF cell transdifferentiation.

To understand the cellular mechanisms underlying the ectopic accumulation of zG cells in Pcat-GOF mice, the present inventors first tested the hypothesis that accumulation of zG cells resulted from increased proliferation of zG cells in response to the oncogenic activity of stabilized b-catenin. To address this, the present inventors determined the proliferation index in bq3R\UT and bq8uOOR adrenals by scoring the number of Ki67-positive cells per zG area, defined by the b-catenin expression domain. Surprisingly, the proliferation index in bq8uOOR adrenals was reduced compared to bq8RnnT controls (Figure 2A), ruling out increased cell division as the explanation for the expanded zG. Since an increase in cell survival could also explain the increase in zG cells, the present inventors next assessed the impact of bq8uOOR on apoptosis by staining for activated caspase-3 or TUNEL in bqBίAUT and bq8uOOR adrenals. Despite the overall low level of apoptosis, no difference was observed between groups (Figure

Under homeostatic conditions, zG cells are converted to zF cells through a process of transdifferentiation. To determine whether cat-GOF impacts zG-to-zF transdifferentiation, the present inventors performed functional lineage-tracing experiments using the R26R ^mTmG reporter allele (Figure 2B). As expected, in bq3u\UT adrenals GFP expression marked both zG cells (defined by b-catenin, Dab2 or Gocq co-expression) as well as zF cells (defined by the presence of Akrlb7 or the absence of b-catenin, Dab2 or Gocq co-expression) (Figure 2 C-F). In contrast, in cat-GOF adrenals GFP expression was largely confined to zG cells, whereas nearly all zF cells failed to express GFP (Figure 2 C-F). In fact, the few GFP+ cells that did

transdifferentiate into zF cells did not express b-catenin, establishing that these cells escaped recombination of the Ctnnbl ^FloxedEx3 allele (Figure 2C, arrowheads). Together, these data indicate that stabilization of b-catenin within zG cells blocks zG-to-zF transdifferentiation, leading to a progressive accumulation of zG cells within the orthotopic zF.

Example 3: Stabilization of b-catenin in zG cells results in increased aldosterone and high blood pressure.

Since cWnt signaling is active exclusively in the zG, the site of aldosterone production, and activation of this pathway has been shown to arise in up to 70% of patients with primary aldosteronism, the present inventors assessed aldosterone levels in pcat-GOF mice. Compared with age-matched Pcat-WT controls, Pcat-GOF adult and aged mice showed higher plasma aldosterone levels (Figure 3A). Since overproduction of aldosterone is associated with increased blood pressure, the present inventors next assessed systolic, diastolic, and mean arterial blood pressure, which revealed higher overall levels in Pcat-GOF mice, compared with age-matched Pcat-WT controls (Figure 3B; Figure 8A and 8B). Together, these data indicate that p-catenin stabilization in the zG is sufficient to increase plasma aldosterone levels, which in turn leads to elevated blood pressure, suggesting Pcat-GOF mice serve as a pre-clinical model of

hyperaldosteronism.

Example 4: Stabilization of b-catenin in zG cells does not lead to autonomous aldosterone production.

Primary aldosteronism results from autonomous production of aldosterone and a decreased ability to respond to volume expansion, owing to an impaired feedback response. To assess whether the higher level of aldosterone in Pcat-GOF mice is the result of autonomous production from zG cells, Pcat-WT and Pcat-GOF mice were subjected to volume expansion by feeding a high-salt diet for a period of 7 days. Analysis of plasma aldosterone levels on the high- salt diet, compared with matched levels obtained on a normal-salt diet, revealed aldosterone suppression in both groups (Figure 3C). These results indicate that the higher level of aldosterone in Pcat-GOF mice does not arise from autonomous aldosterone production and that aldosterone-producing cells in these mice maintain normal feedback regulation.

Example 5: Stabilization of b-catenin in zG cells is not associated with increased CYP11B2 expression.

Because Pcat-GOF mice demonstrate ectopic expansion of the zG and increased expression of zG markers (Dab2 and Gocq), the present inventors next asked whether higher aldosterone levels in these mice might be explained by increased Cypllb2 (aldosterone synthase) expression. Quantitiatve RT-PCR of whole adrenals, however, showed no difference in the overall level of Cypllb2 expression (Figure 3D). The present inventors next assessed the number and distribution of CYPllB2-expressing cells by immunostaining, which revealed isolated patches of CYPllB2-expressing cells in the orthotopic zG in the subcapsular region, as previously described (Figure 3E, Figure 8C). Moreover, consistent with the gene expression analysis, no difference in the overall number of CYPllB2-expressing cells was found between Pcat-WT and Pcat-GOF adrenals (Figure 3F). Together, these data indicate that the higher level of plasma aldosterone in Pcat-GOF mice does not result from an increase in CYP11B2- expressing cells.

Example 6: Stabilization of b-catenin does not change zG responsiveness to Angll.

The present inventors next hypothesized that Pcat-GOF might lead to an increase in Angll responsiveness of zG cells leading to an increase in aldosterone production on a per cell basis. To investigate this, Pcat-WT and Pcat-GOF mice were subjected to intravascular volume depletion (using either a low salt diet or acute water restriction) to activate the renin- angiotensin-aldosterone system (RAAS). Immunostaining for CYPllB2-expressing cells revealed the expected overall increase in the number of cells in response to volume depletion, including in the expanded zG domain, but no difference in the absolute number of CYP11B2- expressing cells between Pcat-WT and Pcat-GOF mice (Figure 8D-E). These data suggest a similar level of Angll responsiveness of zG cells in Pcat-GOF and Pcat-WT mice and confirm that cells in the expanded zG domain can express CYP11B2 in response to physiological demand.

To further assess the responsiveness of zG cells to Angll, an Angll stimulation test was performed in Pcat-WT and Pcat-GOF mice. Plasma aldosterone levels were assayed from individual mice at baseline, 30, 60 or 90 minutes after a single dose of Angll (1.2 nM) administered i.p. Despite an overall increased level of aldosterone in Pcat-GOF mice, there was no difference in the responsiveness to Angll between Pcat-WT and Pcat-GOF mice (Figure 3G). Immunostaining for AS following Angll stimulation revealed similar results to treatment with either a low salt diet or acute water restriction (Figure 8F).

Example 7: Stabilization of b-catenin in zG cells leads to increased NADPH and

steroidogenesis

To begin to understand the molecular basis for higher aldosterone levels in Pcat-GOF mice the present inventors assessed the expression of genes involved in aldosterone biosynthesis. As determined by qPCR and RNAseq, expression of the following genes was unchanged: Nr4al, Nr4a2, Star, (Cypllb2), Fdxl, Psatl, and AngRla. The expression of AngRlb, Me3, Gpdl, Assl, and Acsslwas changed as determined by qPCR and RNAseq. See Figure 3H. Taken together, these results demonstrate that Pcat-GOF leads to increased steroidogenesis of zG cells to Angll, which in turn leads to increased levels of aldosterone.

Example 8: Chronic activation of RAAS in ficat-GOF mice leads to accelerated zG

hyperplasia. Despite b-catenin’s well-established role as an oncogene, its ability to block zG-to-zF transdifferentiation suggests it also functions as a pro-differentiation agent, at least under baseline physiological conditions. The impact of Pcat-GOF was assessed in response to the increased trophic drive associated with chronic activation of RAAS using genetic, dietary and pharmacologic models. To achieve genetic activation of RAAS, the present inventors employed AS ^Cre / ^Cre mice (AS(KO)), a model of CYP11B2 deficiency that results in a ~3 fold increase in plasma renin activity and generated A^f ^re/Cre : : ( 'innh 1 ' AS ( K 0 ) - b ca t- G 0 F) mice (Figure

4A). Gross analysis of adrenals from AS(K0)^cat-G0F mice showed a progressive increase in adrenal weight, culminating in an ~90-fold increase in aged animals, compared with adrenals from AS(KO), bqB^OR and bq3ΐ-\UT mice (Figure 9A and B). Immunostaining was performed for Gocq in young mice to define the initial impact on the zG of combined boh^OR and chronic RAAS activation. While the zG was notably expanded in AS(KO) mice, compared with bohRqOR and bq8ίAUT controls, in AS(K0)^cat-G0F adrenals the zG occupied the entire cortex (Figure 4C and D). Analysis of the zG proliferation index revealed that the zG in young adult bohRqOR animals contained as many proliferating cells as bΰ8ίAUT zG, while AS(KO) and AS(K0)^cat- GOF adrenals exhibited a significant increase in Ki67-positive cells within the b-catenin-positive domain (Figure 4, E and F; Figure 9C and 9D). The impact of RAAS activation on

transdifferentiation was assessed using lineage-tracing. Consistent with the present inventors’ prior findings, bq8ΐ-OOR also blocked zG-to-zF transdifferentiation in AS(K0)^cat-G0F adrenals (Figure 9E). Thus, the combination of increased proliferation and decreased transdifferentiation explains the dramatic increase in zG size in AS(K0)^cat-G0F adrenals.

To validate the combined impact of chronic RAAS activation and bq8ί-OOR on the zG, the present inventors next sought to activate RAAS using spironolactone (a mineralocorticoid receptor antagonist) or a low-salt diet, two treatments commonly prescribed for patients with hyperaldosteronism. bcat-WT and bq8ί-OOR mice were treated with spironolactone for 50 days (Figure 5, A, B). At the end of the treatment period, Cypllb2 transcript levels and the overall number of CYPllB2-expressing cells were increased in both bcat-WT and bq8ί-OOR mice, compared to non-treated controls (Figure 10A and 10B). Remarkably, spironolactone-treated bq8ί-OOR mice demonstrated increased adrenal mass compared with age-matched

spironolactone-treated bcat-WT mice (Figure 10C). Immunostaining for Gocq also revealed that spironolactone treatment led to a marked expansion of the zG in bohRqOR adrenals compared to non-treated controls, while treatment of bcat-WT mice did not increase the zG domain (Figure 5, C and D). To assess the role of proliferation in spironolactone-treated mice, the present inventors quantified Ki67-positive cells in bq8ΐ^T and bq8ΐ-OOR mice. While spironolactone had no impact on the proliferation index in Pcat-WT adrenals, it restored the levels of proliferation in Pcat-GOF adrenals to control levels (Figure 5, E and F; Figure 10 D and 10E). Together, these data establish chronic RAAS activation as a critical mediator for enhanced zG expansion in the setting of activated cWnt signaling.

Example 9: Transcriptome analysis of^Cat-GOF adrenals revealed upregulation of FGF Signaling.

To begin to elucidate the mechanisms by which WNT/p-catenin activation leads to PA, the present inventors performed RNA sequencing analysis on whole adrenal mRNA from the present inventors pCat-GOF and control animals. Differential expression analysis revealed 790 unique genes significantly changed in pCat-GOF adrenals compared to controls (Figure 11 A). The present inventors further analyzed the data for enrichment of Gene Ontology and KEGG pathway annotations and found significant enrichment in genes associated with pathways such as WNT signaling, MAPK signaling, as well as biological processes such as cell adhesion, migration, and morphogenesis (Figure 11B). Interestingly, the present inventors noticed that Fibroblast growth factor receptor 2 (Fgfr2) expression is significantly upregulated in pCat-GOF adrenals (Figure 11C), while the expression of other FGFRs and FGF ligands are unchanged. Further to this point, FGF-activated ETV transcription factors, Etv4 and Etv5, were both significantly upregulated in pCat-GOF adrenals, along with other FGF-regulated target

genes such as Duspl, Dusp , and Spry 4. suggesting FGF signaling is activated in pCat-GOF adrenals (Figure 11C).

Example 10: FGFR2 regulates aldosterone production in adult mice.

To understand the significance of FGFR2 signaling in mediating PA progression in pCat-GOF mice, the present inventors first sought to determine its function in wild type animals. The function of FGFR2 in adult adrenals was evaluated by generating a zG-specific knockout mouse model (ASCre/+ : : Fgfr2flox/flox mice, hereafter referred to as Fgfr2 cKO). In this model, the ligand binding and transmembrane domains of FGFR2 are deleted postnatally in zG cells (Figure 12). Preliminary analyses revealed Irf2 cKO adrenals have severely disrupted zG morphology, as shown by the decreased size and amorphous shape of glomerulus structures outlined by Laminin bl, a zG-specific extracellular matrix protein (Figure 13). Furthermore, it was found that Cypllb2 transcript (encoding aldosterone synthase) was significantly downregulated in Fgfr2 cKO adrenals (Figure 14A), suggesting a potential failure in zG differentiation. Consistent with this result, physiological analysis of Fgfr2 cKO mice revealed compensated hypoaldosteronism, as demonstrated by a suppressed aldosterone/renin ratio (Figure 14B). These data indicate that FGFR2 signaling plays an important role in regulating zG physiological function.

Further to the above, the present inventors demonstrated using Fgfr2 cKOs that FGFR2 is required for normal aldosterone production (Figure 15). Moreover, Fgfr2 cKOs cells cannot respond to Angll stimulation (Figure 16).

Example 11: Evaluation of the effects of inhibiting FGFR2 signaling on PA progression in Cat-GOF mice.

Results presented herein indicate that activation of Wnt/p-catenin signaling in zG cells, which leads to PA in mice, may result from activation of FGFR2 and its downstream signaling. Targeting the FGFR2 signaling pathway, therefore, represents a robust and non- invasive therapeutic strategy for the treatment of patients with PA. The present inventors investigated this therapeutic avenue by experimentally testing whether inhibition of FGFR2 signaling prevents and/or reverses the progression of PA in pCat-GOF animals.

Experimental Design : The present inventors employed two complementary strategies to inhibit FGFR2 signaling in pCat-GOF mice: (1) genetic ablation using th c Fgfr2flox conditional allele and (2) pharmacological inhibition using selective FGFR small molecule inhibitors. Turning to the first strategy of using genetic ablation, the present inventors assessed whether concomitant Fgfr2 deletion in pCat-GOF animals can prevent the onset of PA including zG expansion, hyperaldosteronism, and high blood pressure. The conditional mouse alleles and crossing to achieve this plan experimentally are shown in schematic in Figure 17. To genetically delete Fgfr2 from bCat-GOF mice, the present inventors combined the Fgfr2 cKO and pCat-GOF mouse models. Male ririCre/Cre :: Fgfr2+/flox mice were bred with female Fgfr2flox/flox : : fi-catenin(Ex3)flox/flox mice to generate ASCre/+ ::

Fgfr2flox/flox : : ficatenin (Ex3)flox/+ double mutant mice (pCat-FKO) and ASCre/+ ::

Fgfr2+/flox :: fi-catenin(Ex3)flox/+ single mutant mice (pCat-GOF). Results presented in Figure 18 show that Fgfr2 deletion in pCat-GOF rescues the pcatenin GOF phenotype. Turning to the second strategy of pharmacological inhibition using selective FGFR small molecule inhibitors, the present inventors used pCat-GOF and ASCre/+ (pCat-Ctrl) mice. For pharmacological inhibition studies, since FGFR inhibitors are widely studied chemotherapeutic agents, the present inventors selected two commercially available small molecules, AZD4547 (AstraZeneca) and BGJ398 (Novartis). Both small molecule agents are currently in phase II clinical trials for the treatment of various malignancies and are second generation FGFR inhibitors that target FGFR1/2/3 with high selectivity and minimum off- target effects. Based on published data in tumor xenograft models, both compounds demonstrate strong antitumor effects at a dose of lOmg/kg/day with minimum toxicity for a maximum period of 28 days. Age and sex-matched adult pCat-Ctrl mice were treated with vehicle or lOmg/kg inhibitor (AZD4547 or BGJ398) daily by oral gavage for 12 days. Figure 19 depicts the results of experiments wherein the effects of AZD4547 and BGJ398 were evaluated in the context of pCat-Ctrl adrenals. As shown in Figure 19, each of AZD4547 and BGJ398 impaired zG proliferation in pCat-Ctrl adrenals. To determine the effect FGFR inhibitors have on zG function, mice were treated with 30 mg/kg/day per os (p.o.) of AZD4547 (N=6) or with vehicle only (N=6) starting on day 4. Aldosterone levels for the treatment mice and control mice were compared using two-way ANOVA analysis, followed by Bonferroni’s multiple comparison correction test. Decreased aldosterone levels were observed in treated mice compared to untreated mice (FIG. 20A). An additional cohort of mice were treated with 30 mg/kg/day p. o. of BGJ398 for 7 days. qRT-PCR analysis was performed to determine Cypl lb2 and Cyclin Dl transcript levels in adrenals from treated and control mice. The results were analyzed using two sample t-test. Both Cypl lb2 and Cyclin Dl transcript levels were reduced in treated mice compared to untreated controls (FIG. 20B). Taken together, these results indicate that FGFR inhibitors impair zG function and that therapeutic intervention via treatment with FGFR inhibitors will provide clinical benefit to subjects afflicted by PA.

The impact of Fgfr2 deletion on zG hyperplasia driven by b-catenin gain-of-function was determined. Adult mouse adrenals were isolated and imaged for GFP, DAPI, and Dab2 co-immunostaining. The adult mice belonged to one of the following cohorts: control (ASCre/+; mTmG), Cat GOF (ASCre/+; mTmG; b-catenin fl(ex3)/+), and bCat GOF; Fgfr2 cKO (ASCre/+; mTmG; b-catenin fl(ex3)/+; Fgfr2 fl/fl). Referring to FIG. 21, the representative images demonstrate that Fgfr2 deletion prevents zG hyperplasia driven by b- catenin gain-of-function.

To understand further the physiological impact of pharmacological inhibition of FGFR2 activity in the context of pCat-GOF animals, plasma aldosterone levels and blood pressure in pCat-GOF animals can be determined before and after administration of at least one FGFR inhibitor. For these studies, both female and male mice are studied at 12-15 weeks of age. Adrenal collection and statistical analyses is performed as described herein above. For hormone analyses, blood is collected retro-orbitally from conscious mice using EDTA- containing capillary tubes and centrifuged to obtain plasma. Aldosterone levels are determined using a radioimmunoassay (RIA; IBL International). Renin activity is determined by RIA (IBL International) for Angiotensin I generated by incubating plasma with excess angiotensinogen.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.