US 20130203827A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0203827 A1 Sucharov et al. (43) Pub. Date: Aug. 8, 2013

(54) POLYMORPHISMS IN THE PDE3A GENE Related U.S. Application Data (75) Inventors: Carmen Sucharov, Superior, CO (US); (60) Pygal application No. 61/241,730, filed on Sep. Michael Bristow, Englewood, CO (US); Matthew Taylor, Denver, CO (US); Dobromir Slavov, Aurora, CO (US) Publication Classification (73) Assignee: The Regents of the University of ( 51) Int. Cl. Colorado. a Body, Denver, CO (US) CI2O I/68 (2006.01) (52) U.S. Cl. (21) Appl. No.: 13/395,311 CPC ...... CI2O I/6883 (2013.01) USPC ...... 514/392:435/6.11:506/9 (22) PCT Filed: Sep. 13, 2010 (57) ABSTRACT (86). PCT No.: PCT/US10/48629 Embodiments of the invention are directed to identifying or S371 (c)(1), treating a patient that would benefit from phosphodiesterase (2), (4) Date: Nov. 5, 2012 inhibitor therapy. Patent Application Publication Aug. 8, 2013 Sheet 1 of 8 US 2013/0203827 A1

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POLYMORPHSMS IN THE PDE3A GENE PDE3A levels and reduced response to treatment, commonly 0001. This application claims benefit of priority to U.S. known as pharmacological tolerance. Provisional Application Ser. No. 61/241,730, filed Sep. 11, 0007 Certain embodiments are directed to methods for 2009, the entire contents of which are hereby incorporated by treating a patient with heart failure comprising treating the reference. patient with an effective amount of a PDE3A inhibitor after the patient is determined to be homozygous for an insertional BACKGROUND OF THE INVENTION polymorphism comprising an Evi-1 binding site in the pro moter of the phosphodiesterase type 3A (PDE3A) gene. The 0002 I. Field of the Invention Evi-1 insertion polymorphism refers to a nucleic acid 0003 Embodiments of this invention are directed gener sequence inserted in the promoter of PDE3A comprising a ally to biology, molecular genetics, and medicine. Certain nucleic acid sequence that is 85,90, 95, 98, to 100% identical embodiments are directed to identifying a patient that would to the nucleic acid sequence of SEQ ID NO:2. In certain benefit from phosphodiesterase inhibitor therapy. aspects the insertional polymorphism comprises an ectotro 0004 II. Background pic viral integration site-1 protein (Evi-1) binding site. In a 0005 Cardiac hypertrophy in response to an increased further aspects the Evi-1 binding site comprises a 5, 10, 15 to workload imposed on the heart is a fundamental adaptive 10, 15, 25, 29 nucleotides or more, including all values and mechanism. It is a specialized process reflecting a quantita ranges there between of the nucleotide sequence of SEQID tive increase in cell size and mass (rather than cell number) as NO:2. the result of any, or a combination of neural, endocrine or 0008. In certain aspects a PDE3A inhibitor is aminone, mechanical stimuli. When heart failure occurs, the left ven cilostazol, milrinone, quaZinone, SiguaZodan, trequinsin, or tricle usually is hypertrophied and dilated and indices of enoximone. In a further aspect the PDE3A inhibitor is enoxi systolic function, such as ejection fraction, are reduced. mone. The PDE3A inhibitor can be administered at a dose of Clearly, the cardiac hypertrophic response is a complex Syn 1, 5, 10, 15.20, 25, 30,35, 40, 45, 50 mg to 40, 50, 60, 70,80, drome and the elucidation of the pathways leading to cardiac 90, 100, or 150 mg, including all values and ranges there hypertrophy and systolic dysfunction will be beneficial in the between. In certain aspects the PDE3A inhibitor is adminis treatment of heart disease resulting from various stimuli. tered 1, 2, 3, 4, 5 or more times a day, a week, or a month, There remains a need for additional treatments for patients including all values and ranges there between. suffering from heart failure. 0009 Certain embodiments are directed to methods for evaluating a heart failure patient comprising analyzing a bio SUMMARY OF THE INVENTION logical sample from the patient for the presence of an ecto 0006 Chronic activation of the f-Adrenergic Receptor tropic viral integration site-1 protein (Evi-1) binding site (B-AR) can have deleterious effects on the heart, and animal deletion or insertional polymorphism in the promoter of one models over-expressing the B-AR develop heart failure. In the or more phosphodiesterase type 3A (PDE3A) genes of the classical B-AR pathway, activation of the receptor results in patient, wherein a patient determined to be homozygous for increased cyclic AMP (cAMP) levels. However, B-ARs are the insertional polymorphism is responsive to PDE3A inhibi desensitized in the failing heart and cAMP levels are tor therapy. decreased. Phosphodiesterase 3A (PDE3A) hydrolyzes 0010. In certain aspects the methods described herein can cAMP in certain Subcellular compartments in cardiac myo further comprise obtaining a biological sample from the cytes, regulating cAMP levels and Subsequent protein kinase patient. A biological sample can be a blood sample, a buccal A mediated cell signaling. Since cAMP is reduced in certain Smear, a tissue sample, or a primary culture of Somatic cells important cardiac myocyte Subcellular compartments such as from the patient. In certain aspects analyzing the sample the microdomain occupied by phospholamban and SR Ca2+ comprises performing nucleic acid sequencing, restriction ATPase, it has been postulated that PDE3A inhibition will digestion, allele-specific nucleic acid amplification, single benefit the heart failure clinical syndrome by restoring cAMP Stranded conformational polymorphism analysis, or allele levels in the phospholamban microdomain/suncellular com specific hybridization analysis. The methods described partment. However, a recently published large clinical trial herein can further comprising preparing a report containing showed benefits of a PDE3 inhibitor on only a subset of information regarding the genotype of one or more PDE3A patients. Here we show that the promoter region of PDE3A genes of the patient. In a further aspect the patient has symp contains a 29 nucleotide insertional polymorphism (SEQID toms of or has been diagnosed with heart failure. In still a NO:2) that renders the PDE3A promoter non-responsive to further aspect the patient is determined to be homozygous for increased cAMP levels. The inventors demonstrate that the insertional polymorphism in the PDE3A gene and is sub increased cAMP levels in the cardiac cell, mediated by iso sequently treated with a PDE3A inhibitor. proterenol, a cAMP analogue or PDE3A inhibition, results in 0011. In certain embodiments the patient is determined increased promoter activity when the promoter construct not to be homozygous for the insertional polymorphism in the lacks the 29 nucleotide sequence. However, promoteractivity PDE3A gene and is subsequently treated with a non-PDE3A does not go up when the sequence is present. Similarly, inhibitor treatment. mRNA levels for heart failure patients containing the inser 0012. Further embodiments are directed to methods for tion polymorphism were not up-regulated in response to evaluating a patient having or at risk of developing heart PDE3A inhibition treatment while mRNA levels for patients failure for responsiveness to phosphodiesterase inhibitor lacking this region were up-regulated 2-fold in response to therapy comprising obtaining a determination of the patients treatment. These results indicate that only patients who are genotype for an insertional polymorphism in a phosphodi homozygous for the insertion should be treated with a esterase type 3A (PDE3A) gene, wherein homozygosity for PDE3A inhibition. Inhibition of PDE3A in other patients will the insertional polymorphism is predictive of responsiveness create a positive feedback loop that will result in increased to phosphodiesterase inhibitor therapy for treating heart fail US 2013/0203827 A1 Aug. 8, 2013

ure in the patient. In certain aspects the nucleic acid sequence TGACTAGCTG). A second primer can comprise the nucleic insertion comprises an ectotropic viral integration site-1 pro acid sequence of SEQID NO:7 (GCCAAAAGGAGATCCT tein (Evi-1) binding site. In a further aspect the Evi-1 binding TGAGAT). site has a nucleic acid sequence that is 85,90, 95, 98, or 100% 0016 Certain embodiments are directed to a nucleic acid identical to the nucleic acid sequence of SEQID NO:2. The probe that specifically hybridizes to a phosphodiesterase type methods can include obtaining a patient history. In certain 3A nucleic acid comprising nucleotides 274 to 302 of SEQID aspects this includes determination of the patient’s genotype NO:3 or a nucleic acid that is 85,90, 95, 98, or 1005 identical for an insertional polymorphism in a phosphodiesterase type to SEQID NO:2. In certain aspects the nucleic acid probe is 3A (PDE3A) gene by nucleic acid sequencing, restriction labeled. Inafurther aspect the nucleic acid probe is detectable digestion, allele-specific nucleic acid amplification, single upon binding or hybridization to a PDE3A nucleic acid com Stranded conformational polymorphism analysis, or allele prising an Evi-1 insertion. specific hybridization analysis. The patient can have symp 0017. Another aspect is a kit for genotyping a phosphodi esterase type 3A gene comprising oligonucleotides of at least toms of or has been diagnosed with heart failure. In certain 10 contiguous nucleotides of SEQ ID NO:3 that amplify a aspects the methods further comprise treating a patient deter nucleic acid segment comprising an Evi-1 binding site inser mined to be homozygous for the insertional polymorphism in tional polymorphism or a nucleic acid probe that specifically the PDE3A gene with a PDE3A inhibitor, including but not hybridizes to a PDE3A gene comprising an Evi-1 binding site limited to, enoXimone. insertional polymorphism. 0013 Embodiments are directed to methods of determin 0018 To achieve these methods, a doctor, medical practi ing the efficacy of phosphodiesterase type 3A (PDE3A) inhi tioner, or their staff may obtain directly from the patient a bition therapy (including but not limited to enoXimone) com biological sample for evaluation. The sample may be ana prising detecting a nucleic acid insertion having a nucleotide lyzed by the practitioner or their staff, or it may be sent to an sequence that is 80, 85,90, 95, 98, or 100% identical to the outside or independent laboratory. The medical practitioner nucleic acid sequence of SEQID NO:2 in a promoter of one may be cognizant of whether the test is providing information or both phosphodiesterase type 3A (PDE3A) genes of a sub regarding the patient's PDE3A genes, or the medical practi ject. The nucleic acid insertion can be within the 2000 nucle tioner may be aware only that the test indicates directly or otides 5' of the transcription start site. In certain aspects indirectly that the genotype of the patient reflects the inser detection of the nucleic acid insertion uses at least one oligo tional polymorphism phenotype (“homozygous for inser nucleotide that anneals to SEQID NO:3, SEQID NO:4, or tional polymorphism’ sequence). SEQ ID NO:3 and SEQID NO:4. The detection method can 0019. Similarly, the medical practitioner may be cogni comprise at least one allele specific amplification primer or Zant of whether the test is providing information regarding the allele specific nucleic acid probe. The allele specific amplifi patient’s PDE3A genes or the medical practitioner may be cation primer orallele specific nucleic acid probe can specifi aware only that the test indicates directly or indirectly that the cally hybridizes with an Evi-1 insertion under high Stringency genotype of the patient reflects the homozygous wildtype conditions. In certain aspects, the probe or primer is labeled sequence (no insertion in either allele), the heterozygous with a detectable label. In a further aspect the primer or probe genotype, or the homozygous insertional genotype. is detectable after hybridization with a PDE3A nucleic acid 0020. In any of these circumstances, the medical practi comprising an Evi-1 insertion. The nucleic acid insertion can tioner"knows” the relevant information that will allow him or be detected using nucleic acid amplification, nucleic acid her to determine whether a PDE3A inhibitor is an appropriate hybridization, restriction fragment length polymorphism medicinal option. It is contemplated that, for example, a labo (RFLP) analysis, single stranded conformational polymor ratory conducts the test to determine that patient’s genotype phism (SSCP) analysis, nucleic acid sequencing, denaturing Such its personnel also know the appropriate information. high performance liquid chromatography, comparative They may report back to the practitioner with the specific genome hybridization, and/or Southern blotting. In certain result of the test performed or the laboratory may simply aspects nucleic acid amplification comprises polymerase report that a PDE3A inhibitor is an appropriate drug based on chain reaction amplification or ligase chain reaction amplifi the laboratory results. Depending on the result, the patient cation. In a further aspect nucleic acid hybridization detection may be subsequent treated with the PDE3A inhibitor. In method comprises an allele specific oligonucleotide probe or certain embodiments, a PDE3A inhibitor such as enoximone a microarray of nucleic acid probes. is administered to a patient after the patient has been geno typed as having the homozygous wildtype sequence (no 0014 Certain embodiments are directed to an isolated insertion in either allele), the heterozygous genotype, or the nucleic acid sequence comprising nucleotides 269 to nucle homozygous insertional genotype. The term 'genotyping is otide 307 of SEQID NO:3 or a nucleic acid having 85,90,95, refers to the physical manipulation and transformation of a 98, or 100% identity to SEQ ID NO:2, including all values biological sample to determine genotype information con and ranges there between. In certain aspects the nucleic acid tained in the sample. Obtaining genotype information refers sequence comprises 20, 30, 40, 50, 60, 70, 80,90, 100, 500, to obtaining some or all of the results of a genotyped sample. 1000 or more consecutive nucleotides of SEQ ID NO:3 or 0021 Certain embodiments are directed to a tangible, SEQID NO:4, including all values and ranges there between. computer-readable medium comprising a genotype of a Sub 0015. A further embodiment is directed to an amplifica ject, wherein the genotype exhibits the presence or absence of tion primer pair comprising two oligonucleotides that a Evi-1 insertion in the PDE3A gene. In certain aspects the amplify a nucleic acid segment comprising nucleotides 274 to medium comprising the genotype of the Subject exhibits the 302 of SEQIDNO:3 or a nucleic acid that is 85,90,95,98, or presence of a Evi-1 insertion in the promoter of one or more 100% identical to SEQID NO:2. A first primer can comprise PDE3A gene. Moreover, in further embodiments the medium the nucleic acid sequence of SEQID NO:6 (CCACTGCCAT or method involves an algorithm that determines the risk of a US 2013/0203827 A1 Aug. 8, 2013

phenotype based on the genotype information. In particular (0028. The term “phosphodiesterase inhibitor” or “PDE embodiments a method involves a computer specifically pro inhibitor” refers to a chemical compound or entity that is grammed to evaluate a genotype result and determine capable of blocking, either partially or completely, the activ whether a particular PDE3A inhibitor is an appropriate drug. ity of a phosphodiesterase enzyme. Some PDE inhibitors This determination may involve a probability analysis. A exhibit a degree of specificity for one PDE subtype (e.g., report may be subsequently prepared with the results of the phosphodiesterase type 3A (PDE3A)). The term “PDE3A genotyping and/or predicted phenotype. inhibitor” refers to chemical compounds that are selective for 0022. Moreover, in some methods a clinician may or may phosphodiesterase type 3A. The use of derivatives of known not order a genotyping test for a patient. In other embodi PDE3A inhibitors is encompassed by the methods of the ments, a clinician may retrieve a biological sample from a present invention. Indeed any compound, which functionally patient, which may or may not constitute a biopsy. In further behaves as a PDE3A inhibitor is encompassed by the methods embodiments, methods may involve receiving genotype of the present invention. information about a patient’s sample and Subsequently treat 0029. As used herein, the term “genotype” refers to the ing a patient with a PDE3A inhibitor based on the genotype actual genetic make-up of an organism, while “phenotype information. In further methods, an algorithm may be run to refers to physical traits displayed by an individual (respon evaluate possible phenotype based on genotyping informa siveness to PDE3A inhibitors for the treatment of heart fail tion. ure). 0023. As used herein, the term “heart failure' is broadly 0030. Other embodiments of the invention are discussed used to mean any condition that reduces the ability of the throughout this application. Any embodiment discussed with heart to pump blood. As a result, congestion and edema respect to one aspect of the invention applies to other aspects develop in the tissues. Most frequently, heart failure is caused of the invention as well and vice versa. The embodiments in by decreased contractility of the myocardium, resulting from the Example section are understood to be embodiments of the reduced coronary blood flow; however, many other factors invention that are applicable to all aspects of the invention. may result in heart failure, including damage to the heart 0031. The terms “inhibiting,” “reducing,” or “prevention.” valves, vitamin deficiency, and primary cardiac muscle dis or any variation of these terms, when used in the claims and/or ease. Though the precise physiological mechanisms of heart the specification includes any measurable decrease or com failure are not entirely understood, heart failure is generally plete inhibition to achieve a desired result. believed to involve disorders in several cardiac autonomic 0032. The use of the word “a” or “an when used in con properties, including sympathetic, parasympathetic, and junction with the term "comprising in the claims and/or the baroreceptor responses. The phrase “manifestations of heart specification may mean “one but it is also consistent with failure' is used broadly to encompass all of the sequelae the meaning of"one or more.” “at least one.” and “one or more associated with heart failure, such as shortness of breath, than one.” pitting edema, an enlarged tender liver, engorged neck veins, 0033. It is contemplated that any embodiment discussed pulmonary rates and the like including laboratory findings herein can be implemented with respect to any method or associated with heart failure. composition of the invention, and vice versa. Furthermore, 0024. The term “treatment” or equivalents encompasses compositions and kits of the invention can be used to achieve the improvement and/or reversal of the symptoms of heart methods of the invention. failure (i.e., the ability of the heart to pump blood). “Improve 0034. Throughout this application, the term “about is ment in the physiologic function' of the heart may be used to indicate that a value includes the standard deviation of assessed using any of the measurements described herein error for the device or method being employed to determine (e.g., measurement of ejection fraction, fractional shortening, the value. left ventricular internal dimension, heart rate, etc.), as well as 0035. The use of the teen “or” in the claims is used to mean any effect upon Subject's Survival. In certain embodiments, a “and/or unless explicitly indicated to refer to alternatives patient administers the drug to himself/herself. only or the alternatives are mutually exclusive, although the 0025. The term “dilated cardiomyopathy' refers to a type disclosure Supports a definition that refers to only alternatives of heart failure characterized by the presence of a symmetri and “and/or.” It is also contemplated that anything listed using cally dilated left ventricle with poor systolic contractile func the term “or” may also be specifically excluded. tion and, in addition, frequently involves the right ventricle. 0036. As used in this specification and claim(s), the words 0026. As used herein, the term “cardiac hypertrophy' “comprising (and any form of comprising, such as "com refers to the process in which adult cardiac myocytes respond prise' and "comprises”), “having (and any form of having, to stress through hypertrophic growth. Such growth is char such as “have and “has'), “including' (and any form of acterized by cell size increases without cell division, assem including, such as “includes and “include’) or “containing bling of additional sarcomeres within the cell to attempt to (and any foam of containing, such as "contains and “con increase force generation, and an activation of a fetal cardiac tain’) are inclusive or open-ended and do not exclude addi gene program that inherently reduces myocardial function. tional, unrecited elements or method steps. Cardiac hypertrophy is often associated with increased risk of 0037 Other objects, features and advantages of the morbidity and mortality, and thus studies aimed at under present invention will become apparent from the following standing the molecular mechanisms of cardiac hypertrophy detailed description. It should be understood, however, that could have a significant impact on human health. the detailed description and the specific examples, while indi 0027. As used herein, the terms “antagonist' and “inhibi cating specific embodiments of the invention, are given by tor” refer to molecules, compounds, or nucleic acids which way of illustration only, since various changes and modifica inhibit the action of a protein. Antagonists and inhibitors may tions within the spirit and scope of the invention will become include proteins, nucleic acids, carbohydrates, or any other apparent to those skilled in the art from this detailed descrip molecules which bind or interact with a protein of interest. tion. US 2013/0203827 A1 Aug. 8, 2013

DESCRIPTION OF THE DRAWINGS the phospholamban microdomain. However, providing infor mation to the clinician regarding the propensity of an indi 0038. The following drawings form part of the present vidual to experience adverse side effects, such as develop specification and are included to further demonstrate certain ment of tolerance to the inhibitor, is of value in selecting a aspects of the present invention. The invention may be better therapy regimen from which the individual will benefit most understood by reference to one or more of these drawings in and/or with which the patient will most likely comply with a combination with the detailed description of specific embodi prescribed regimen. Thus, it is of interest to provide a method ments presented herein. for assessing the level and propensity for developing toler 0039 FIG. 1. Scheme for identification of functionally ance for PDE3A inhibitors in selecting a PDE3A inhibitor important polymorphisms in the PDE3A gene promoter (no based therapy or prescribing an alternate therapy. NS SNPs in coding regions). 0049 Pharmacogenomics allows a clinician or physician 0040 FIG. 2. Ins/Del Polymorphism in the PDE3A pro to target prophylactic ortherapeutic treatments to individuals moter region. Twenty-nine nucleotide insertion/deletion who will most benefit from the treatment and to avoid treat within the 2 kb proximal promoter region. This region con ment of individuals who will experience symptomatic side tains the binding site for the Evi-1 (ectotropic viral integration effects. Transcriptional regulation and feedback downregula site-1) transcription factor, a strong repressor. Evi-1 is an tion of transcription can lead to therapeutic failure by altering inhibitory factor and its expression is increased in response to the quantity of enzymatic target in a tissue. Thus, a physician cAMP. Genotype frequencies: deletion homozygotes: 37%; or clinician may consider applying knowledge obtained in heterozygotes: 44%; insertion homozygotes: 19%. Allele fre relevant pharmacogenomics studies in determining whether quencies:—Del: 59%; Ins: 41%. to administer a PDE3A inhibitor as well as tailoring the 0041 FIG. 3. NRVMs transfected with PDE3A Del/Ins dosage, regimen, and/or therapeutically effective amounts to luciferase constructs: agents that cAMP increase promoter be administered so as to attain the effect desired by the treat activity in Del, not in Ins. ment. 0042 FIG. 4. Deletion of the CREB binding site (CRE) at 0050 Heart failure is one of the leading causes of morbid -1070 abolishes promoter response to enoximone and ity and mortality in the world. In the U.S. alone, estimates dbcAMP in NRVMS transfected with the Del construct. Note: indicate that 3 million people are currently living with cardi ~20 C/EBPbinding sites distributed throughout entire pro omyopathy and another 400,000 are diagnosed on a yearly moter region. basis. Dilated cardiomyopathy (DCM), also referred to as 0043 FIG. 5. In explanted LV samples from HF patients “congestive cardiomyopathy, is the most common form of treated with enoximone, mRNA expression of the Evi-1 tran the cardiomyopathies and has an estimated prevalence of Scription factor is increased (p values compared to non-fail nearly 40 per 100,000 individuals (Durand et al., 1995). ing, no enoXimone). Although there are other causes of DCM, familiar dilated 0044 FIG. 6. Predicted model of INS/DEL polymorphism cardiomyopathy has been indicated as representing approxi effects on PDE3A expression. mately 20% of "idiopathic' DCM. Approximately half of the 004.5 FIG. 7. PDE inhibition results in increased PDE3A DCM cases are idiopathic, with the remainder being associ mRNA expression in failing human hearts that are not ated with known disease processes. For example, serious homozygous for the INS polymorphism (Del carriers). myocardial damage can result from certain drugs used in Hypothesis: patients who are INS/INS will not develop tol cancer chemotherapy (e.g., doxorubicin and daunoribucin). erance to PDEIS. In DEL carriers CRE mediated feedback In addition, many DCM patients are chronic alcoholics. For loop up-regulation in PDE3A gene expression by cAMP tunately, for these patients, the progression of myocardial cancels effects of PDEIS. dysfunction may be stopped or reversed if alcohol consump 0046 FIG.8. PDE3A mRNA levels are up-regulated in the tion is reduced or stopped early in the course of disease. failing heart in patients that have the insertion polymorphism. Peripartum cardiomyopathy is another idiopathic of DCM, as Myocardial cAMP levels are decreased in HF, which is con is disease associated with infectious sequelae. In Sum, cardi sistent with increased mRNA levels in INS/INS hearts. Evi-1 omyopathies, including DCM, are significant public health binds to the INS and down-regulates expression of PDE3A. problems. Additional methods of improving the outcome of Failing hearts have lower cAMP levels, and therefore there is therapy for heart failure are needed. In certain embodiments less repression of PDE3A gene expression compared to non of the present application are directed to compositions and failing controls in individuals who are INS/INS. method for identifying a patient population responsive to PDE3A inhibitor treatment and methods of treating such a DETAILED DESCRIPTION OF THE INVENTION population are described. 0047. The present invention relates generally to assessing a PDE3A polymorphism in an individual and to assessing the I. PHOSPHODIESTERASE (PDE) individual regarding responsiveness to PDE3A inhibitor 0051 cAMP signaling plays important roles in both physi treatment. Some conditions associated with PDE3A treat ologic and pathologic regulation of cardiac performance ment are not advantageous to the individual, for example, (Wang and Dhalla, 2000). cAMP is one of the most well some patients develop a tolerance to PDE3A inhibitors. Spe characterized signaling molecules in B-AR signaling, but its cifically, the present invention relates to determining the contribution to Ang II signaling in cardiomyocytes is not fully genotype for an individual at the PDE3A gene (e.g., deter understood. Clinical and experimental studies indicate that mining if the Subject has an insertional polymorphism). acute stimulation of B-AR/cAMP signaling increases myo 0048. The methods of the invention can also be applied to cyte contractility, which is beneficial. In contrast, chronic facilitate the selection of a therapy for conditions in which stimulation of cAMP signaling promotes myocyte apoptosis, inhibition of PDE3A can be beneficial. The inhibition of which is harmful to the heart (Wang and Dhalla, 2000). The PDE3A can result in an increase in cAMP in the heart and in temporal and spatial features of cAMP production in the cell US 2013/0203827 A1 Aug. 8, 2013

are controlled by adenylyl cyclases that catalyze the synthesis tion of various genes. The Evi-1 protein binds to a consensus of cAMP, and phosphodiesterases (PDEs) that hydrolyze sequence of DGATADGADWAGATA (SEQ ID NO:5). cAMP. 0057 A. Nucleic Acids 0052 Phosphodiesterase as used herein refers to cyclic 0.058 Certain embodiments of the present invention con nucleotide phosphodiesterases. The cyclic nucleotide phos cern various nucleic acids, including amplification primers, phodiesterases (PDE) comprise a group of enzymes that oligonucleotide probes, and other nucleic acid elements degrade the phosphodiester bond in the second messenger involved in the analysis of genomic DNA. In certain aspects, molecules cAMP and coiMP. They regulate the localization, a nucleic acid comprises a wild-type, a mutant, or a polymor duration, and amplitude of cyclic nucleotide signaling within phic nucleic acid. subcellular domains. PDEs are therefore important regulators 0059. The terms “PDE3A polymorphism or “PDE3A of signal transduction mediated by these second messenger insertional polymorphism refer to polymorphisms in the molecules. promoter a PDE3A gene. The sequence of the PDE3A pro 0053 PDE enzymes are often targets for pharmacological moter segment containing an insertional polymorphism is inhibition due to their unique tissue distribution, structural provided as SEQ ID NO:3. The sequence of the PDE3A properties, and functional properties. Inhibitors of PDE can promoter segment lacking the insertional polymorphism is prolong or enhance the effects of physiological processes provided as SEQID NO:4. mediated by cAMP or cGMP by inhibition of their degrada 0060 For the purposes of identifying the location of a tion by PDE. For example, Sildenafil (Viagra) is an inhibitor polymorphism, the first nucleotide of a transcription start site of c(3MP specific phosphodiesterase type 5, which enhances is considered nucleotide "+1, i.e., the transcriptional start the vasodilatory effects of c(GMP in the corpus cavernosum designated in SEQID NO:4 is present at nucleotide 1406 of and is used to treat erectile dysfunction. Cilostazol (Pletal) SEQID NO:4. Nucleotide positions in the promoter are typi inhibits PDE3. This inhibition allows Red Blood Cells to be cally designated by negative number beginning with first more able to bend. This is useful in conditions such as inter nucleotide before the transcription start site as “-1 and mittent claudication, as the cells can maneuver through con decreasing in number as you progress along the PDE3A pro stricted veins and arteries more easily. moter sequence to the first nucleotide of the sequence repre 0054 PDE3 cyclic nucleotide phosphodiesterases bind sented in the sequence listing. Alternatively, the location of a cAMP and coMP with high affinity and hydrolyze both sub polymorphism can be designated based on the total number of strates in a mutually competitive manner (Shakur et al., nucleotides in the sequence starting as nucleotide 1 and pro 2000). Two PDE3 genes have been discovered: PDE3A is gressing in increments of one to the end of the sequence, i.e., expressed primarily in cardiac and vascular myocytes and nucleotide 1842 of SEQID NO:4. Given this convention, the platelets, whereas PDE3B is expressed primarily in adipo insertion in SEQ ID NO:3 begins at nucleotide 274 and the cytes, hepatocytes, and pancreatic cells (Reinhardt et al., last nucleotide of the insertion is at nucleotide 302 of SEQID 1995). Phosphodiesterase type 3 (PDE3) is an important NO:3. Thus, the insertion is defined as nucleotides 274-302 of regulator of cAMP-mediated responses within the cardiovas SEQID NO:3 (i.e., SEQID NO:2). Relative to SEQID NO:4 cular system. PDE3 inhibitors (e.g. milrinone and enoxi the insertion—an Evi-1 binding site—is inserted after nucle mone) have inotropic effects attributable to the elevation of otide 273 of SEQID NO:4. cAMP content in cardiac myocytes and vasodilatory effects 0061. In some embodiments, nucleic acids of the inven attributable to the elevation of cAMP and/or cGMP content in tion comprise or are complementary to all or 5, 6, 7, 8, 9, 10, vascular myocytes, and have been used to augment contrac 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, tility and reduce afterload in patients with dilated cardiomy 28, 29, 30, 31, 32,33, 34,35,36, 37,38, 39, 40, 41,42, 43,44, opathy (Movsesian, 1999). PDE3A is significantly down 45, 46,47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, 61, regulated in human failing hearts and in a mouse model of 62,63,64, 65,66, 67,68, 69,70, 71,72, 73,74, 75,76, 77,78, chronic pressure overload (Ding et al., 2005). Down-regula 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89,90,91, 92,93, 94, 95, tion of PDE3A expression/activity induced cardiomyocyte 96, 97,98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, apoptosis in cultured cardiomyocytes, due to PDE3A down 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, regulation-mediated induction of the proapoptotic transcrip 310,320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, tional repressor ICER (Ding et al., 2005). 430, 440, 450, 460, 470, 480,490, 500, 510,520, 530, 540, II. DETECTION OF POLYMORPHISMS 550,560,570, 580,590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 0055. The insertional polymorphism described herein is 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, present in the promoter of PDE3A genes and affect the tran 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, scrptional regulation the PDE3A gene. The presence of the 1165, 1200, 1300, 1400, 1500, 1840, 1870 or more contigu insertional polymorphism can be determined from the ous nucleotides, or any range derivable therein, of the human sequence of the PDE3A promoter or by using specific char PDE3A promoter segment provided in SEQID NO:3 or SEQ acteristics of the insertional polymorphism, e.g., amplicon ID NO:4. One of skill in the art knows how to design and use size. As a result, a variety of different methodologies can be primers and probes for hybridization and amplification of the employed for the purpose of detecting polymorphisms in the PDE3A promoter. promoter of PDE3A. 0062. These definitions generally refer to a single 0056. The insertional polymorphism comprises a binding stranded molecule, but in specific embodiments will also site for the transcription factor Evi-1. Evi-1 (Ecotropic viral encompass an additional Strand that is partially, Substantially integration site-1) is a human protein encoded by the Evi-1 or fully complementary to the single-stranded molecule. gene. The gene was first identified in AKXD murine myeloid Thus, a nucleic acid may encompass a double-stranded mol tumors. Evi-1 is a nuclear transcription factor involved in ecule or a triple-Stranded molecule that comprises one or many signaling pathways for both corepression and coactiva more complementary strand(s) or “complement(s) of a par US 2013/0203827 A1 Aug. 8, 2013 ticular sequence comprising a molecule. As used herein, a sequence. Thus, a “nucleic acid segment may comprise any single Stranded nucleic acid may be denoted by the prefix part of a gene sequence, including from about 2 nucleotides to “ss', a double stranded nucleic acid by the prefix “ds, and a the full length gene including promoter regions to the poly triple stranded nucleic acid by the prefix “ts.” adenylation signal and any length that includes all the coding 0063 1. Preparation of Nucleic Acids region. 0064. A nucleic acid may be made by any technique 0071 Various nucleic acid segments may be designed known to one of ordinary skill in the art, such as for example, based on a particular nucleic acid sequence, and may be of chemical synthesis, enzymatic production or biological pro any length. By assigning numeric values to a sequence, for duction. Non-limiting examples of a synthetic nucleic acid example, the first residue is 1, the second residue is 2, etc., an (e.g., a synthetic oligonucleotide), include a nucleic acid algorithm defining all nucleic acid segments can be created: made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and Solid phase in to n+y techniques such as described in European Patent 266.032, where n is an integer from 1 to the last number of the sequence incorporated herein by reference, or via deoxynucleoside and y is the length of the nucleic acid segment minus one, H-phosphonate intermediates as described by Froehler et al., where n+y does not exceed the last number of the sequence. 1986 and U.S. Pat. No. 5,705,629, each incorporated herein Thus, for a 10-mer, the nucleic acid segments correspond to by reference. In the methods of the present invention, one or bases 1 to 10, 2 to 11, 3 to 12... and so on. For a 15-mer, the more oligonucleotide may be used. In certain aspects ampli nucleic acid segments correspond to bases 1 to 15, 2 to 16, 3 fication oligonucleotides can be designed on either side or to 17 . . . and so on. For a 20-mer, the nucleic segments overlapping with the boundaries of the insertion site. In a correspond to bases 1 to 20, 2 to 21, 3 to 22. . . and so on. In further aspect an oligonucleotide specific for insertion as certain embodiments, the nucleic acid segment may be a compare to the PDE3A promoter laking the insertional poly probe or primer. As used herein, a “probe’ generally refers to morphism can be designed. These oligonucleotides can vary a nucleic acid used in a detection method or composition. As ing in length from 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, used herein, a “primer generally refers to a nucleic acid used 25, 26, 27, 28, 29, 30, 50, nucleotides or more, including all in an extension or amplification method or composition. values and ranges there between. Various different mecha (0072 4. Nucleic Acid Complements nisms of oligonucleotide synthesis have been disclosed in for 0073. The present invention also encompasses a nucleic example, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, acid that is complementary to a nucleic acid. A nucleic acid is 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, “complement(s) or is “complementary” to another nucleic 5,602.244, each of which is incorporated herein by reference. acid when it is capable of base-pairing with another nucleic 0065. A non-limiting example of an enzymatically pro acid according to the standard Watson-Crick, Hoogsteen or duced nucleic acid include one produced by enzymes in reverse Hoogsteen binding complementarity rules. As used amplification reactions such as PCRTM (see for example, U.S. herein “another nucleic acid may refer to a separate mol Pat. No. 4,683.202 and U.S. Pat. No. 4,682,195, each incor ecule or a spatial separated sequence of the same molecule. In porated herein by reference), or the synthesis of an oligo preferred embodiments, a complement is a hybridization nucleotide described in U.S. Pat. No. 5,645,897, incorporated probe or amplification primer for the detection of a nucleic herein by reference. A non-limiting example of a biologically acid polymorphism. produced nucleic acid includes a recombinant nucleic acid 0074 As used herein, the term “complementary' or produced (i.e., replicated) in a living cell. Such as a recombi “complement” also refers to a nucleic acid comprising a nant DNA vector replicated in bacteria (see for example, sequence of consecutive nucleobases or semiconsecutive Sambrook et al. 2001, incorporated herein by reference). nucleobases (e.g., one or more nucleobase moieties are not 0066 2. Purification of Nucleic Acids present in the molecule) capable of hybridizing to another 0067. A nucleic acid may be purified on polyacrylamide nucleic acid strand or duplex even if less than all the nucleo gels, cesium chloride centrifugation gradients, chromatogra bases do not base pair with a counterpart nucleobase. How phy columns or by any other means known to one of ordinary ever, in some diagnostic or detection embodiments, com skill in the art (see for example, Sambrook et al., 2001, incor pletely complementary nucleic acids are preferred. porated herein by reference). 0075 5. Nucleic Acid Detection and Evaluation 0068. In certain aspects, the present invention concerns a 0076 Genotyping can be performed using methods nucleic acid that is an isolated nucleic acid. As used herein, described in Small et al. (2002), which is incorporated herein the term "isolated nucleic acid refers to a nucleic acid mol by reference. It will be understood by the skilled artisan that ecule (e.g., an RNA or DNA molecule) that has been isolated other standard techniques are available for genotyping and free of, or is otherwise free of the bulk of the total genomic any technique may be used with the present invention. Gen and transcribed nucleic acids of one or more cells. In certain eral methods of nucleic acid detection methods are provided embodiments, "isolated nucleic acid' refers to a nucleic acid below. that has been isolated free of, or is otherwise free of, bulk of 0077 One genotyping method involves isolating from the cellular components or in vitro reaction components such as individual a nucleic acid mixture comprising the two copies for example, macromolecules Such as lipids or proteins, Small of the PDE3A gene, or a fragment thereof, that are present in biological molecules, and the like. the individual, and determining the nucleotide sequence or 0069. 3. Nucleic Acid Segments presence of a nucleotide insertion in the promoter of the 0070. In certain embodiments, the nucleic acid is a nucleic PDE3A gene. Other polymorphisms, such as single nucle acid segment. As used herein, the term “nucleic acid seg otide polymorphisms can be linked to and indicative of the ment, are fragments of a nucleic acid, Such as, for a non insertional polymorphism described herein. limiting example, those that encode only part of a PDE3A 0078 Those in the art will readily recognize that nucleic promoter sequence, or part of the PDE3A gene locus or gene acid molecules may be double-stranded molecules and that US 2013/0203827 A1 Aug. 8, 2013

reference to a particular site on one strand refers, as well, to used as differently labeled probe pairs, with one member of the corresponding site on a complementary Strand. Thus, in the pair showing a perfect match to one variant of a target defining a polymorphic site, reference to a sequence includ sequence and the other member showing a perfect match to a inganadenine, a thymine (uridine), a cytosine, or a guanine at different variant. In some embodiments, more than one poly a particular site on one strand of a nucleic acid molecule is morphic site may be detected at once using a set of allele also intended to include the thymine (uridine), adenine, gua specific oligonucleotides or oligonucleotide pairs. nine, or cytosine (respectively) at the corresponding site on a I0085) Hybridization of an allele-specific oligonucleotide complementary strand of a nucleic acid molecule. Thus, ref to a target polynucleotide may be performed with both enti erence may be made to either Strand and still comprise the ties in solution, or such hybridization may be performed when same polymorphic site and an oligonucleotide may be either the oligonucleotide or the target polynucleotide is designed to hybridize to either strand. Throughout the text, in covalently or noncovalently affixed to a solid Support. Attach identifying a polymorphic site, reference is made to SEQID ment may be mediated, for example, by antibody-antigen NO:3 or SEQID NO:4 for the purpose of convenience. interactions, poly-L-Lys, streptavidin or avidin-biotin, salt 007.9 Typically, the nucleic acid mixture is isolated from a bridges, hydrophobic interactions, chemical linkages, UV biological sample taken from the individual. Such as a blood cross-linking baking, etc. Allele-specific oligonucleotides sample or tissue sample using standard techniques such as may be synthesized directly on the solid support or attached to disclosed in Jones (1963) which is hereby incorporated by the Solid Support Subsequent to synthesis. Solid-supports Suit reference. Suitable tissue samples include whole blood, able for use in detection methods of the invention include semen, Saliva, tears, urine, fecal material, Sweat, buccal, skin, Substrates made of silicon, glass, plastic, paper and the like, and hair. The nucleic acid mixture may be comprised of which may be formed, for example, into wells (as in 96-well genomic DNA. plates), slides, sheets, membranes, fibers, chips, dishes, and 0080. The ability to predict a patient’s response to a PDE beads. The solid support may be treated, coated or derivatized inhibitor is useful for physicians in making decisions about to facilitate the immobilization of the allele-specific oligo how to treat a patient having heart failure. A patient whose nucleotide or target nucleic acid. genotype indicates the patient will probably respond well to I0086. The genotype for one or more polymorphic sites in the PDE inhibitor (i.e., a patient homozygous for a Evi-1 the PDE3A gene of an individual may also be determined by binding site insertional polymorphism) would be a better hybridization of one or both copies of the gene, or a fragment candidate for PDE inhibitor therapy than a patient who is thereof, to nucleic acid arrays and Subarrays such as described likely to exhibit tolerance to PDE inhibitor therapy, or an in PCT Appln. WO95/11995. The arrays would contain a intermediate response, or no response, and the physician battery of allele-specific oligonucleotides representing each would be able to determine with less trial and error which of the polymorphic sites to be included in the genotype or individuals should receive an alternative form of therapy. haplotype. 0081. In the genotyping methods used in the present I0087. The identity of polymorphisms may also be deter invention, the identity of a polymorphic site may be deter mined using a mismatch detection technique, including but mined by amplifying a target region containing the polymor not limited to the RNase protection method using riboprobes phic site directly from one or both copies of the PDE3A gene (Winter et al., 1985); Meyers et al., 1985) and proteins which present in the individual and the sequence of the amplified recognize nucleotide mismatches, such as the E. coli mutS region(s) determined by conventional methods or evaluated protein (Modrich, 1991). Alternatively, variant alleles can be directly. identified by single strand conformation polymorphism 0082. The target region(s) may be amplified using any (SSCP) analysis (Orita et al., 1989: Humphries et al., 1996) or oligonucleotide-directed amplification method, including but denaturing gradient gel electrophoresis (DGGE) (Wartell et not limited to polymerase chain reaction (PCR) (U.S. Pat. No. al., 1990; Sheffield et al., 1989). 4.965, 188), ligase chain reaction (LCR) (Barany et al., 1991; I0088 A polymerase-mediated primer extension method PCT Appln. WO90/01069), and oligonucleotide ligation may also be used to identify the polymorphism(s). Several assay (OLA) (Landegren et al., 1988). Oligonucleotides use such methods have been described in the patent and scientific ful as primers or probes in Such methods should specifically literature. Extended primers containing a polymorphism may hybridize to a region of the nucleic acid that contains or is be detected by mass spectrometry as described in U.S. Pat. adjacent to the polymorphic site. Typically, the oligonucle No. 5,605,798. An other primer extension method is allele otides are between 10 and 35 nucleotides in length and pref specific PCR (Ruano et al., 1989: Ruano et al., 1991; PCT erably, between 15 and 30 nucleotides in length. Most pref Applin. WO93/224.56: Turki et al., 1995). erably, the oligonucleotides are 20 to 25 nucleotides long. The I0089 Polymorphic variation in the promoter of the human exact length of the oligonucleotide will depend on many PDE3A gene can also be detected using differential digestion factors that are routinely considered and practiced by the of DNA by certain restriction enzymes (Small et al., 2002) or skilled artisan. by any other method that identifies the nucleotide insertion in 0083. Other known nucleic acid amplification procedures the PDE3A gene. may be used to amplify the target region including transcrip (0090 a. Hybridization tion-based amplification systems (U.S. Pat. No. 5,130.238; (0091. The use of a probe or primer of between 7, 8, 9, 10, EP 329,822; U.S. Pat. No. 5,169,766, PCT Appln. WO89/ 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 06700) and isothermal methods (Walker et al., 1992). 70, 80,90, or 100 consecutive nucleotides of SEQID NO:3 or 0084. A polymorphism in the target region may also be SEQID NO:4, preferably between 17 and 100 nucleotides in assayed before or after amplification using one of several length, or in Some aspects of the invention up to 1-2 kilobases hybridization-based methods known in the art. Typically, or more in length, allows the formation of a duplex molecule allele-specific oligonucleotides are utilized in performing that is both stable and selective. Molecules having comple Such methods. The allele-specific oligonucleotides may be mentary sequences over contiguous stretches greater than 20 US 2013/0203827 A1 Aug. 8, 2013

bases in length are generally preferred, to increase stability indicator Substrates are known that can be employed to pro and/or selectivity of the hybrid molecules obtained. One will vide a detection means that is visibly or spectrophotometri generally prefer to design nucleic acid molecules for hybrid cally detectable, to identify specific hybridization with ization having one or more complementary sequences of 20 to complementary nucleic acid containing samples. In other 30 nucleotides, or even longer where desired. Such fragments aspects, a particular nuclease cleavage site may be present may be readily prepared, for example, by directly synthesiz and detection of a particular nucleotide sequence can be ing the fragment by chemical means or by introducing determined by the presence or absence of nucleic acid cleav selected sequences into recombinant vectors for recombinant age. production. 0097. In general, it is envisioned that the probes or primers 0092. Accordingly, the nucleotide sequences of the inven described herein will be useful as reagents in solution hybrid tion may be used for their ability to selectively form duplex ization, as in PCR, for detection of expression or genotype of molecules with complementary stretches of DNAs and/or corresponding genes, as well as in embodiments employing a RNAs or to provide primers for amplification of DNA or RNA Solid phase. In embodiments involving a solid phase, the test from samples. Depending on the application envisioned, one DNA (or RNA) is adsorbed or otherwise affixed to a selected would desire to employ varying conditions of hybridization to matrix or Surface. This fixed, single-stranded nucleic acid is achieve varying degrees of selectivity of the probe or primers then subjected to hybridization with selected probes under for the target sequence. desired conditions. The conditions selected will depend on 0093. For applications requiring high selectivity, one will the particular circumstances (depending, for example, on the typically desire to employ relatively high Stringency condi G+C content, type of target nucleic acid, Source of nucleic tions to form the hybrids. For example, relatively low salt acid, size of hybridization probe, etc.). Optimization of and/or high temperature conditions, such as provided by hybridization conditions for the particular application of about 0.02 M to about 0.10 MNaCl attemperatures of about interest is well known to those of skill in the art. After washing 50° C. to about 70° C. Such high stringency conditions tol of the hybridized molecules to remove non-specifically erate little, if any, mismatch between the probe or primers and bound probe molecules, hybridization is detected, and/or the template or target Strand and would be particularly Suit quantified, by determining the amount of bound label. Rep able for isolating specific genes or for detecting a specific resentative solid phase hybridization methods are disclosed in polymorphism. It is generally appreciated that conditions can U.S. Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other be rendered more stringent by the addition of increasing methods of hybridization that may be used in the practice of amounts of formamide. For example, under highly stringent the present invention are disclosed in U.S. Pat. Nos. 5,849, conditions, hybridization to filter-bound DNA may be carried 481, 5,849,486 and 5,851,772. The relevant portions of these out in 0.5 M NaHPO, 7% sodium dodecyl sulfate (SDS), 1 and other references identified in this section of the Specifi mM EDTA at 65° C., and washing in 0.1xSSC/0.1% SDS at cation are incorporated herein by reference. 68°C. (Ausubel et al., 1989). (0098 b. Amplification of Nucleic Acids 0094 Conditions may be rendered less stringent by increasing salt concentration and/or decreasing temperature. 0099 Nucleic acids used as a template for amplification For example, a medium stringency condition could be pro may be isolated from cells, tissues or other samples according vided by about 0.1 to 0.25 MNaCl at temperatures of about to standard methodologies (Sambrook et al., 2001). In certain 37°C. to about 55°C., while a low stringency condition could embodiments, analysis is performed on whole cell or tissue be provided by about 0.15 M to about 0.9 M salt, attempera homogenates or biological fluid samples with or without Sub tures ranging from about 20° C. to about 55° C. Under low stantial purification of the template nucleic acid. The nucleic stringent conditions, such as moderately stringent conditions acid may be genomic DNA or fractionated or whole cell the washing may be carried out for example in 0.2xSSC/0.1% RNA. Where RNA is used, it may be desired to first convert SDS at 42°C. (Ausubel et al., 1989). Hybridization condi the RNA to a complementary DNA. tions can be readily manipulated depending on the desired 0100. The term “primer, as used herein, is meant to results. encompass any nucleic acid that is capable of priming the 0095. In other embodiments, hybridization may be synthesis of a nascent nucleic acid in a template-dependent achieved under conditions of for example, 50 mM Tris-HCl process. Typically, primers are oligonucleotides from ten to (pH 8.3), 75 mMKC1, 3 mM MgCl, 1.0 mM dithiothreitol, at twenty and/or thirty base pairs in length, but longer sequences temperatures between approximately 20° C. to about 37° C. can be employed. Primers may be provided in double Other hybridization conditions utilized could include Stranded and/or single-stranded form, although the single approximately 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 1.5 stranded form is preferred. mMMgCl2, attemperatures ranging from approximately 40° 0101 Pairs of primers designed to selectively hybridize to C. to about 72° C. nucleic acids corresponding to the PDE3A gene locus, or 0096. In certain embodiments, it will be advantageous to variants thereof, and fragments thereofare contacted with the employ nucleic acids of defined sequences of the present template nucleic acid under conditions that permit selective invention in combination with an appropriate means, such as hybridization. Depending upon the desired application, high a label, for determining hybridization. A wide variety of stringency hybridization conditions may be selected that will appropriate indicator means are known in the art, including only allow hybridization to sequences that are completely fluorescent, radioactive, enzymatic or other ligands, such as complementary to the primers. In other embodiments, avidin/biotin, which are capable of being detected. In pre hybridization may occur under reduced stringency to allow ferred embodiments, one may desire to employ a fluorescent for amplification of nucleic acids that contain one or more label oran enzyme tag such as urease, alkaline phosphatase or mismatches with the primer sequences. Once hybridized, the peroxidase, instead of radioactive or other environmentally template-primer complex is contacted with one or more undesirable reagents. In the case of enzyme tags, colorimetric enzymes that facilitate template-dependent nucleic acid syn US 2013/0203827 A1 Aug. 8, 2013 thesis. Multiple rounds of amplification, also referred to as scripts. Other amplification methods include “RACE and “cycles are conducted until a sufficient amount of amplifi “one-sided PCR" (Frohman, 1990; Ohara et al., 1989). cation product is produced. 0109 c. Detection of Nucleic Acids 0102 The amplification product may be detected, ana 0110. Following any amplification, it may be desirable to lyzed or quantified. In certain applications, the detection may separate the amplification product from the template and/or be performed by visual means. In certain applications, the the excess primer. In one embodiment, amplification products detection may involve indirect identification of the product are separated by agarose, agarose-acrylamide or polyacryla via chemiluminescence, radioactive Scintigraphy of incorpo mide gel electrophoresis using standard methods (Sambrook rated radiolabel or fluorescent label or even via a system using et al., 2001). Separated amplification products may be cut out electrical and/or thermal impulse signals (Affymax technol and eluted from the gel for further manipulation. Using low ogy: Bellus, 1994). melting point agarose gels, the separated band may be 0103) A number of template dependent processes are removed by heating the gel, followed by extraction of the available to amplify the oligonucleotide sequences present in nucleic acid. a given template sample. One of the best known amplification 0111 Separation of nucleic acids may also be effected by methods is the polymerase chain reaction (referred to as spin columns and/or chromatographic techniques known in PCRTM) which is described in detail in U.S. Pat. Nos. 4,683, art. There are many kinds of chromatography which may be 195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each used in the practice of the present invention, including of which is incorporated herein by reference in their entirety. adsorption, partition, ion-exchange, hydroxylapatite, 0104. Another method for amplification is ligase chain molecular sieve, reverse-phase, column, paper, thin-layer, reaction (“LCR), disclosed in European Applin. 320 308, and gas chromatography as well as HPLC. incorporated herein by reference in its entirety. U.S. Pat. No. 0112. In certain embodiments, the amplification products 4,883,750 describes a method similar to LCR for binding are visualized, with or without separation. A typical visual probe pairs to a target sequence. A method based on PCRTM ization method involves staining of a gel with ethidium bro mide and visualization of bands under UV light. Alterna and oligonucleotide ligase assay (OLA) (described in further tively, if the amplification products are integrally labeled with detail below), disclosed in U.S. Pat. No. 5,912,148, may also radio- or fluorometrically-labeled nucleotides, the separated be used. amplification products can be exposed to X-ray film or visu 0105. Alternative methods for amplification of target alized under the appropriate excitatory spectra. nucleic acid sequences that may be used in the practice of the 0113. In one embodiment, following separation of ampli present invention are disclosed in U.S. Pat. Nos. 5,843,650, fication products, a labeled nucleic acid probe is brought into 5,846,709, 5,846,783, 5,849,546, 5,849,497, 5,849,547, contact with the amplified marker sequence. The probe pref 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905, erably is conjugated to a chromophore but may be radiola 5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, beled. In another embodiment, the probe is conjugated to a Great Britain Applin. 2 202 328, and in PCT Application binding partner, Such as an antibody or biotin, or another PCT/US89/01025, each of which is incorporated herein by binding partner carrying a detectable moiety. reference in its entirety. Qbeta Replicase, described in PCT 0114. In particular embodiments, detection is by Southern Application PCT/US87/00880, may also be used as an ampli blotting and hybridization with a labeled probe. The tech fication method in the present invention. niques involved in Southern blotting are well known to those 010.6 An isothermal amplification method, in which of skill in the art (see Sambrook et al., 2001). One example of restriction endonucleases and ligases are used to achieve the the foregoing is described in U.S. Pat. No. 5.279,721, incor amplification of target molecules that contain nucleotide 5'- porated by reference herein, which discloses an apparatus and alpha-thio-triphosphates in one strand of a restriction site method for the automated electrophoresis and transfer of may also be useful in the amplification of nucleic acids in the nucleic acids. The apparatus permits electrophoresis and blot present invention (Walker et al., 1992). Strand Displacement ting without external manipulation of the gel and is ideally Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779, is Suited to carrying out methods according to the present inven another method of carrying out isothermal amplification of tion. nucleic acids which involves multiple rounds of strand dis 0115 Other methods of nucleic acid detection that may be placement and synthesis, i.e., nick translation. used in the practice of the instant invention are disclosed in 0107. Other nucleic acid amplification procedures include U.S. Pat. Nos. 5,840,873, 5,843,640, 5,843,651, 5,846,708, transcription-based amplification systems (TAS), including 5,846,717, 5,846,726, 5,846,729, 5,849,487, 5,853,990, nucleic acid sequence based amplification (NASBA) and 3SR 5,853,992, 5,853,993, 5,856,092, 5,861,244, 5,863,732, (Kwoh et al., 1989; PCT Applin. WO 88/10315, incorporated 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124, herein by reference in their entirety). European Applin. 329 5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 822 disclose a nucleic acid amplification process involving 5,929,227, 5,932,413 and 5,935,791, each of which is incor cyclically synthesizing single-stranded RNA (“ssRNA), porated herein by reference. ssDNA, and double-stranded DNA (dsDNA), which may be 0116 d. Other Assays used in accordance with the present invention. 0117. Other methods for genetic screening may be used 0108 PCT Appln. WO 89/06700 (incorporated herein by within the scope of the present invention, for example, to reference in its entirety) disclose a nucleic acid sequence detect mutations in genomic DNA, cDNA and/or RNA amplification scheme based on the hybridization of a pro samples. Methods used to detect point mutations include moter region/primer sequence to a target single-stranded denaturing gradient gel electrophoresis (“DGGE), restric DNA (“ssDNA) followed by transcription of many RNA tion fragment length polymorphism analysis (“RFLP”), copies of the sequence. This scheme is not cyclic, i.e., new chemical or enzymatic cleavage methods, direct sequencing templates are not produced from the resultant RNA tran of target regions amplified by PCRTM (see above), single US 2013/0203827 A1 Aug. 8, 2013

strand conformation polymorphism analysis (“SSCP) and (0.126 SNPs relating to ABCC2 can be characterized by the other methods well known in the art. use of any of these methods or suitable modification thereof. 0118. One method of screening for point mutations is Such methods include the director indirect sequencing of the based on RNase cleavage of base pair mismatches in RNA/ site, the use of restriction enzymes where the respective alle DNA or RNA/RNA heteroduplexes. As used herein, the term les of the site create or destroy a restriction site, the use of “mismatch’ is defined as a region of one or more unpaired or allele-specific hybridization probes, the use of antibodies that mispaired nucleotides in a double-stranded RNA/RNA, are specific for the proteins encoded by the differentalleles of RNA/DNA or DNA/DNA molecule. This definition thus the polymorphism, or any other biochemical interpretation. includes mismatches due to insertion/deletion mutations, as (O127 (1) DNA Sequencing well as single or multiple base point mutations. I0128. The most commonly used method of characterizing 0119 U.S. Pat. No. 4,946,773 describes an RNase A mis a polymorphism is direct DNA sequencing of the genetic match cleavage assay that involves annealing single-stranded locus that flanks and includes the polymorphism. Such analy DNA or RNA test samples to an RNA probe, and subsequent sis can be accomplished using either the "dideoxy-mediated treatment of the nucleic acid duplexes with RNase A. For the chain termination method also known as the "Sanger detection of mismatches, the single-stranded products of the Method’ (Sanger et al., 1975) or the “chemical degradation RNase A treatment, electrophoretically separated according method, also known as the "Maxam-Gilbert method to size, are compared to similarly treated control duplexes. (Maxam et al., 1977). Sequencing in combination with Samples containing Smaller fragments (cleavage products) genomic sequence-specific amplification technologies. Such not seen in the control duplex are scored as positive. as the polymerase chain reaction may be utilized to facilitate 0120. Other investigators have described the use of RNase the recovery of the desired genes (Mullis et al., 1986: Euro I in mismatch assays. The use of RNase I for mismatch pean Patent Applin. 50,424; European Patent Applin. 84,796, detection is described in literature from Promega Biotech. European Patent Applin. 258,017, European Patent Applin. Promega markets a kit containing RNase I that is reported to 237,362: European Patent Applin. 201,184; U.S. Pat. Nos. cleave three out of four known mismatches. Others have 4,683.202; 4,582,788; and 4,683,194), all of the above incor described using the MutS protein or other DNA-repair porated herein by reference. enzymes for detection of single-base mismatches. 0129 (2) Exonuclease Resistance 0121 Alternative methods for detection of deletion, inser 0.130. Other methods that can be employed to determine tion or substitution mutations that may be used in the practice the identity of a nucleotide present at a polymorphic site of the present invention are disclosed in U.S. Pat. Nos. 5,849, utilize a specialized exonuclease-resistant nucleotide deriva 483, 5,851,770, 5,866,337, 5,925,525 and 5,928,870, each of tive (U.S. Pat. No. 4,656,127). A primer complementary to an which is incorporated herein by reference in its entirety. allelic sequence immediately 3'- to the polymorphic site is 0122 e. Specific Examples of Polymorphism Nucleic hybridized to the DNA under investigation. If the polymor Acid Screening Methods phic site on the DNA contains a nucleotide that is comple 0123 Spontaneous mutations that arise during the course mentary to the particular exonucleotide-resistant nucleotide of evolution in the genomes of organisms are often not imme derivative present, then that derivative will be incorporated by diately transmitted throughout all of the members of the spe a polymerase onto the end of the hybridized primer. Such cies, thereby creating polymorphic alleles that co-exist in the incorporation makes the primer resistant to exonuclease species populations. Often polymorphisms are the cause of cleavage and thereby permits its detection. As the identity of genetic diseases. Several classes of polymorphisms have been the exonucleotide-resistant derivative is known one can deter identified. For example, variable nucleotide type polymor mine the specific nucleotide present in the polymorphic site phisms (VNTRS), arise from spontaneous tandem duplica of the DNA. tions of di- or trinucleotide repeated motifs of nucleotides. If 0131) (3) Microsequencing Methods Such variations alter the lengths of DNA fragments generated 0.132. Several other primer-guided nucleotide incorpora by restriction endonuclease cleavage, the variations are tion procedures for assaying polymorphic sites in DNA have referred to as restriction fragment length polymorphisms been described (Komheret al., 1989: Sokolov, 1990; Syvanen (RFLPs). RFLPs are been widely used in human and animal 1990; Kuppuswamy et al., 1991; Prezant et al., 1992; Ugoz genetic analyses. Zollet al., 1992; Nyrenet al., 1993). These methods rely on 0.124. Another class of polymorphisms are generated by the incorporation of labeled deoxynucleotides to discriminate the replacement of a single nucleotide. Such single nucleotide between bases at a polymorphic site. As the signal is propor polymorphisms (SNPs) rarely result in changes in a restric tional to the number of deoxynucleotides incorporated, poly tion endonuclease site. Thus, SNPs are rarely detectable morphisms that occur in runs of the same nucleotide result in restriction fragment length analysis. SNPs are the most com a signal that is proportional to the length of the run (Syvanen mon genetic variations and occur once every 100 to 300 bases et al., 1990). and several SNP mutations have been found that affect a (0.133 (4) Extension in Solution single nucleotide in a protein-encoding gene in a manner I0134) French Patent 2,650,840 and PCT Applin. WO91/ Sufficient to actually cause agenetic disease. SNP diseases are 02087 discuss a solution-based method for determining the exemplified by hemophilia, sickle-cell anemia, hereditary identity of the nucleotide of a polymorphic site. According to hemochromatosis, late-onset alzheimer disease etc. these methods, a primer complementary to allelic sequences 0.125 Several methods have been developed to screen immediately 3'- to a polymorphic site is used. The identity of polymorphisms and some examples are listed below. The the nucleotide of that site is determined using labeled reference of Kwok and Chen (2003) and Kwok (2001) pro dideoxynucleotide derivatives which are incorporated at the vide overviews of some of these methods; both of these ref end of the primer if complementary to the nucleotide of the erences are specifically incorporated by reference. polymorphic site. US 2013/0203827 A1 Aug. 8, 2013

0135 (5) Genetic Bit Analysis or Solid-Phase Extension 0144 (9) Other Methods to Detect SNPs 0.136 PCT Applin. WO92/15712 describes a method that 0145 Several other specific methods for polymorphism uses mixtures of labeled terminators and a primer that is detection and identification are presented below and may be complementary to the sequence 3' to a polymorphic site. The used as such or with Suitable modifications in conjunction labeled terminator that is incorporated is complementary to with identifying polymorphisms of the PDE3A gene in the the nucleotide present in the polymorphic site of the target present invention. Several other methods are also described molecule being evaluated and is thus identified. Here the on the SNP web site of the NCBI on the World WideWeb at primer or the target molecule is immobilized to a Solid phase. incbi.nlm.nih.gov/SNP, incorporated herein by reference. 0146 In a particular embodiment, extended haplotypes 0.137 (6) Oligonucleotide Ligation Assay (OLA) may be determined at any given locus in a population, which 0.138. This is another solid phase method that uses differ allows one to identify exactly which SNPs will be redundant ent methodology (Landegren et al., 1988). Two oligonucle and which will be essential in association studies. The latteris otides, capable of hybridizing to abutting sequences of a referred to as haplotype tag SNPs (htSNPs), markers that single strand of a target DNA are used. One of these oligo capture the haplotypes of a gene or a region of linkage dis nucleotides is biotinylated while the other is detectably equilibrium. See Johnson et al. (2001) and Ke and Cardon labeled. If the precise complementary sequence is found in a (2003), each of which is incorporated herein by reference, for target molecule, the oligonucleotides will hybridize such that exemplary methods. their termini abut, and create a ligation Substrate. Ligation 0147 The VDA-assay utilizes PCR amplification of permits the recovery of the labeled oligonucleotide by using genomic segments by long PCR methods using TakaRa LA avidin. Other nucleic acid detection assays, based on this Taq reagents and other standard reaction conditions. The long method, combined with PCR have also been described (Nick amplification can amplify DNA sizes of about 2,000-12,000 erson et al., 1990). Here PCR is used to achieve the exponen bp. Hybridization of products to variant detector array (VDA) tial amplification of target DNA, which is then detected using can be performed by a Affymetrix High Throughput Screen the OLA. ing Center and analyzed with computerized software. 0139 (7) Ligase/Polymerase-Mediated Genetic Bit 0.148. A method called Chip Assay uses PCR amplifica Analysis tion of genomic segments by standard or long PCR protocols. 0140 U.S. Pat. No. 5,952,174 describes a method that also Hybridization products are analyzed by VDA. Halushka et al. involves two primers capable of hybridizing to abutting (1999), incorporated herein by reference. SNPs are generally sequences of a target molecule. The hybridized product is classified as "Certain” or “Likely based on computer analy formed on a solid support to which the target is immobilized. sis of hybridization patterns. By comparison to alternative Here the hybridization occurs such that the primers are sepa detection methods such as nucleotide sequencing, "Certain' rated from one another by a space of a single nucleotide. SNPs have been confirmed 100% of the time; and “Likely” Incubating this hybridized product in the presence of a poly SNPs have been confirmed 73% of the time by this method. merase, a ligase, and a nucleoside triphosphate mixture con 0149 Other methods simply involve PCR amplification taining at least one deoxynucleoside triphosphate allows the following digestion with the relevant restriction enzyme. Yet ligation of any pair of abutting hybridized oligonucleotides. others involve sequencing of purified PCR products from Addition of a ligase results in two events required to generate known genomic regions. a signal, extension and ligation. This provides a higher speci 0150. In yet another method, individual exons or overlap ficity and lower “noise than methods using either extension ping fragments of large exons are PCR-amplified. Primers are or ligation alone and unlike the polymerase-based assays, this designed from published or database sequences and PCR method enhances the specificity of the polymerase step by amplification of genomic DNA is performed using the fol combining it with a second hybridization and a ligation step lowing conditions: 200 ng DNA template, 0.5 LM each for a signal to be attached to the Solid phase. primer, 80 uM each of dCTP, dATP, dTTP and dGTP, 5% formamide, 1.5 mMMgCl, 0.5 U of Taq polymerase and 0.1 0141 (8) Invasive Cleavage Reactions volume of the Taq buffer. Thermal cycling is performed and 0142 Invasive cleavage reactions can be used to evaluate resulting PCR-products are analyzed by PCR-single strand cellular DNA for a particular polymorphism. A technology conformation polymorphism (PCR-SSCP) analysis, under a called INVADER(R) employs such reactions (e.g., de Arruda et variety of conditions, e.g. 5 or 10% polyacrylamide gel with al., 2002; Stevens et al., 2003, which are incorporated by 15% urea, with or without 5% glycerol. Electrophoresis is reference). Generally, there are three nucleic acid molecules: performed overnight. PCR-products that show mobility shifts 1) an oligonucleotide upstream of the target site ("upstream are reamplified and sequenced to identify nucleotide varia oligo'), 2) a probe oligonucleotide covering the target site tion. (“probe'), and 3) a single-stranded DNA with the target site 0151. In a method called CGAP-GAI (DEMIGLACE), (“target”). The upstream oligo and probe do not overlap but sequence and alignment data (from a PHRAPace file), qual they contain contiguous sequences. The probe contains a ity scores for the sequence base calls (from PHRED quality donor fluorophore, such as fluoroscein, and an acceptor dye, files), distance information (from PHYLIP dnadistand neigh such as Dabcyl. The nucleotide at the 3' terminal end of the bour programs) and base-calling data (from PHRED -d upstream oligo overlaps (“invades”) the first base pair of a Switch) are loaded into memory. Sequences are aligned and probe-target duplex. Then the probe is cleaved by a structure examined for each vertical chunk (slice’) of the resulting specific 5' nuclease causing separation of the fluorophore/ assembly for disagreement. Any such slice is considered a quencher pair, which increases the amount of fluorescence candidate SNP (DEMIGLACE). A number of filters are used that can be detected. See Lu et al., 2004. by DEMIGLACE to eliminate slices that are not likely to 0143. In some cases, the assay is conducted on a solid represent true polymorphisms. These include filters that: (i) Surface or in an array format. exclude sequences in any given slice from SNP consideration US 2013/0203827 A1 Aug. 8, 2013

where neighboring sequence quality Scores drop 40% or 0158. In a method described as KWOK(1), SNPs are iden more; (ii) exclude calls in which peak amplitude is below the tified by comparing high quality genomic sequence data from fifteenth percentile of all base calls for that nucleotide type: four randomly chosen individuals by direct DNA sequencing (iii) disqualify regions of a sequence having a high number of of PCR products with dye-terminator chemistry (see Kwoket disagreements with the consensus from participating in SNP al., 1996). In a related method identified as KWOK(2) SNPs calculations; (iv) removed from consideration any base call are identified by comparing high quality genomic sequence with an alternative call in which the peak takes up 25% or data from overlapping large-insert clones such as bacterial more of the area of the called peak; (V) exclude variations that occur in only one read direction. PHRED quality scores were artificial chromosomes (BACs) or P1-based artificial chro converted into probability-of-error values for each nucleotide mosomes (PACs). An STS containing this SNP is then devel in the slice. Standard Baysian methods are used to calculate oped and the existence of the SNP in various populations is the posterior probability that there is evidence of nucleotide confirmed by pooled DNA sequencing (see Taillon-Miller et al., 1998). In another similar method called KWOK(3), SNPs heterogeneity at a given location. are identified by comparing high quality genomic sequence 0152. In a method called CU-RDF (RESEQ), PCR ampli fication is performed from DNA isolated from blood using data from overlapping large-insert clones BACs or PACs. The specific primers for each SNP, and after typical cleanup pro SNPs found by this approach represent DNA sequence varia tocols to remove unused primers and free nucleotides, direct tions between the two donor chromosomes but the allele sequencing using the same or nested primers. frequencies in the general population have not yet been deter 0153. In a method called DEBNICK (METHOD-B), a mined. In method KWOK(5), SNPs are identified by com comparative analysis of clustered EST sequences is per paring high quality genomic sequence data from a homozy formed and confirmed by fluorescent-based DNA sequenc gous DNA sample and one or more pooled DNA samples by ing. In a related method, called DEBNICK (METHOD-C), direct DNA sequencing of PCR products with dye-terminator comparative analysis of clustered EST sequences with phred chemistry. The STSs used are developed from sequence data quality >20 at the site of the mismatch, average phred quality found in publicly available databases. Specifically, these >=20 over 5 bases 5'-FLANK and 3' to the SNP, no mis STSs are amplified by PCR against a complete hydatidiform matches in 5 bases 5' and 3' to the SNP, at least two occur mole (CHM) that has been shown to be homozygous at all loci rences of each allele is performed and confirmed by examin and a pool of DNA samples from 80 CEPH parents (see Kwok ing traces. et al., 1994). 0154) In a method identified by ERO (RESEQ), new prim ers sets are designed for electronically published STSs and 0159. In another such method, KWOK (OverlapSnpDe used to amplify DNA from 10 different mouse strains. The tectionWithPolyBayes), SNPs are discovered by automated amplification product from each strain is then gel purified and computer analysis of overlapping regions of large-insert sequenced using a standard dideoxy, cycle sequencing tech human genomic clone sequences. For data acquisition, clone nique with P-labeled terminators. All the ddATP terminated sequences are obtained directly from large-scale sequencing reactions are then loaded in adjacent lanes of a sequencing gel centers. This is necessary because base quality sequences are followed by all of the ddCTP reactions and so on. SNPs are not present/available through GenBank. Raw data processing identified by visually scanning the radiographs. involves analyzed of clone sequences and accompanying (O155 In another method identified as ERO (RESEQ-HT), base quality information for consistency. Finished (base per new primers sets are designed for electronically published fect, error rate lower than 1 in 10,000 bp) sequences with no murine DNA sequences and used to amplify DNA from 10 associated base quality sequences are assigned a uniform different mouse strains. The amplification product from each base quality value of 40 (1 in 10,000 bp error rate). Draft strain is prepared for sequencing by treating with Exonu sequences without base quality values are rejected. Processed clease I and Shrimp Alkaline Phosphatase. Sequencing is sequences are entered into a local database. A version of each performed using ABI Prism Big Dye Terminator Ready Reac sequence with known human repeats masked is also stored. tion Kit (Perkin-Elmer) and sequence samples are run on the Repeat masking is performed with the program 3700 DNA Analyzer (96 Capillary Sequencer). “MASKERAID.” Overlap detection: Putative overlaps are 0156 FGU-CBT (SCA2-SNP) identifies a method where detected with the program “WUBLAST.” Several filtering the region containing the SNP were PCR amplified using the steps followed in order to eliminate false overlap detection primers SCA2-FP3 and SCA2-RP3. Approximately 100 ng results, i.e. similarities between a pair of clone sequences that of genomic DNA is amplified in a 50 ml reaction volume arise due to sequence duplication as opposed to true overlap. containing a final concentration of 5 mM Tris, 25 mM KCl, Total length of overlap, overall percent similarity, number of 0.75 mM MgCl, 0.05% gelatin, 20 pmol of each primer and sequence differences between nucleotides with high base 0.5 U of Taq DNA polymerase. Samples are denatured, quality value "high-quality mismatches. Results are also annealed and extended and the PCR product is purified from compared to results of restriction fragment mapping of a band cut out of the agarose gel using, for example, the genomic clones at Washington University Genome Sequenc QIAquick gel extraction kit (Qiagen) and is sequenced using ing Center, finisher's reports on overlaps, and results of the dye terminator chemistry on an ABI Prism 377 automated sequence contig building effort at the NCBI. SNP detection: DNA sequencer with the PCR primers. Overlapping pairs of clone sequence are analyzed for candi (O157. In a method identified as JBLACK (SEQ/RE date SNP Sites with the POLYBAYES SNP detection Soft STRICT), two independent PCR reactions are performed ware. Sequence differences between the pair of sequences are with genomic DNA. Products from the first reaction are ana scored for the probability of representing true sequence varia lyzed by sequencing, indicating a unique FspI restriction site. tion as opposed to sequencing error. This process requires the The mutation is confirmed in the product of the second PCR presence of base quality values for both sequences. High reaction by digesting with Fsp I. scoring candidates are extracted. The search is restricted to US 2013/0203827 A1 Aug. 8, 2013

Substitution-type single base pair variations. Confidence merase I. The reaction is stopped by adding EDTA, and unin score of candidate SNP is computed by the POLYBAYES corporated nucleotides are dephosphorylated by adding calf software. intestinal alkaline phosphatase. SSCP followed by electro 0160. In method identified by KWOK (TaqMan assay), phoresis is performed in a capillary using an ABI Prism 310 the TaqMan assay is used to determine genotypes for 90 Genetic Analyzer. Genescan softwares (P-E Biosystems). random individuals. In method identified by KYUGEN(Q1), DNA of individuals including those who showed different DNA samples of indicated populations are pooled and ana genotypes on DHPLC or SSCP are subjected for direct lyzed by PLACE-SSCP. Peak heights of each allele in the sequencing using big-dye terminator chemistry, on ABI pooled analysis are corrected by those in a heterozygote, and Prism 310 sequencer. Multiple sequence trace files obtained are Subsequently used for calculation of allele frequencies. from ABI Prism 310 are processed and aligned by Phred/ Allele frequencies higher than 10% are reliably quantified by Phrap and viewed using Consed viewer. SNPs are identified this method. Allele frequency=0 (zero) means that the allele by PolyPhred software and visual inspection. Trace chro was found among individuals, but the corresponding peak is matogram data of EST sequences in Unigene are processed not seen in the examination of pool. Allele frequency=0-0.1 with PHRED. To identify likely SNPs, single base mis indicates that minor alleles are detected in the pool but the matches are reported from multiple sequence alignments pro peaks are too low to reliably quantify. duced by the programs PHRAP, BRO and POA for each (0161. In yet another method identified as KYUGEN Unigene cluster. BRO corrected possible misreported EST (Method1), PCR products are post-labeled with fluorescent orientations, while POA identified and analyzed non-linear dyes and analyzed by an automated capillary electrophoresis alignment structures indicative of gene mixing/chimeras that system under SSCP conditions (PLACE-SSCP). Four or might produce spurious SNPs. Bayesian inference is used to more individual DNAs are analyzed with or without two weigh evidence for true polymorphism versus sequencing pooled DNA (Japanese pool and CEPH parents pool) in a error, misalignment or ambiguity, misclustering or chimeric series of experiments. Alleles are identified by visual inspec EST sequences, assessing data Such as raw chromatogram tion. Individual DNAs with different genotypes are height, sharpness, overlap and spacing; sequencing error sequenced and SNPs identified. Allele frequencies are esti rates; context-sensitivity; cDNA library origin, etc. mated from peak heights in the pooled samples after correc (0163. In method identified as MARSHFIELD(Method tion of signal bias using peak heights in heterozygotes. For B), overlapping human DNA sequences which contained the PCR primers are tagged to have 5'-ATT or 5'-GTT at their putative insertion/deletion polymorphisms are identified ends for post-labeling of both strands. Samples of DNA (10 through searches of public databases. PCR primers which ngful) are amplified in reaction mixtures containing the buffer flanked each polymorphic site are selected from the consen (10 mM Tris-HCl, pH 8.3 or 9.3, 50 mM KC1, 2.0 mM Sus sequences. Primers are used to amplify individual or MgCl), 0.25uM of each primer, 200 uM of each dNTP and pooled human genomic DNA. Resulting PCR products are 0.025 units/ul of Taq DNA polymerase premixed with anti resolved on a denaturing polyacrylamide gel and a Phospho Taq antibody. The two strands of PCR products are differen rImager is used to estimate allele frequencies from DNA tially labeled with nucleotides modified with R110 and R6G pools. by an exchange reaction of Klenow fragment of DNA poly 0164 f. Linkage Disequilibrium merase I. The reaction is stopped by adding EDTA, and unin 0.165 Polymorphisms in linkage disequilibrium with corporated nucleotides are dephosphorylated by adding calf another polymorphism in which identification of one poly intestinal alkaline phosphatase. For the SSCP: an aliquot of morphism is predictive of the identity of the linked polymor fluorescently labeled PCR products and TAMRA-labeled phism. “Linkage disequilibrium” (“LD” as used herein, internal markers are added to deionized formamide, and though also referred to as “LED in the art) refers to a situa denatured. Electrophoresis is performed in a capillary using tion where a particular combination of alleles (i.e., a variant an ABI Prism 310 Genetic Analyzer. Genescan softwares form of a given gene) or polymorphisms at two loci appears (P-E Biosystems) are used for data collection and data pro more frequently than would be expected by chance. “Signifi cessing. DNA of individuals (two to eleven) including those cant’ as used in respect to linkage disequilibrium, as deter who showed different genotypes on SSCP are subjected for mined by one of skill in the art, is contemplated to be a direct sequencing using big-dye terminator chemistry, on statistical p or C. value that may be 0.25 or 0.1 and may be 0.1, ABI Prism 310 sequencers. Multiple sequence trace files 0.05. 0.001, 0.00001 or less. Insertions/deletions in the obtained from ABI Prism 310 are processed and aligned by PDE3A promoter may be determined by evaluating the Phred/Phrap and viewed using Consed viewer. SNPs are nucleic acid sequence of a polymorphism in linkage disequi identified by PolyPhred software and visual inspection. librium with the insertion/deletion. The invention may be (0162. In yet another method identified as KYUGEN implemented in this manner with respect to one or more (Method2), individuals with different genotypes are searched polymorphisms so as to allow haplotype analysis. “Haplo by denaturing HPLC (DHPLC) or PLACE-SSCP (Inazuka et type' is used according to its plain and ordinary meaning to al., 1997) and their sequences are determined to identify one skilled in the art. It refers to a collective genotype of two SNPs. PCR is performed with primers tagged with 5'-ATT or or more alleles or polymorphisms along one of the homolo 5'-GTT at their ends for post-labeling of both strands. gous chromosomes. DHPLC analysis is carried out using the WAVE DNA frag 0166 The term “polymorphism’, as used herein, refers to ment analysis system (Transgenomic). PCR products are a difference in the nucleotide or amino acid sequence of a injected into DNASep column, and separated under the con given nucleotide oramino acid region as compared to a nucle ditions determined using WAVEMaker program (Transge otide or amino acid sequence in the corresponding region of nomic). The two strands of PCR products that are differen another individual of the same species. Preferably, the species tially labeled with nucleotides modified with R110 and R6G is human. A polymorphism is generally defined in relation to by an exchange reaction of Klenow fragment of DNA poly a “reference' sequence. In the subject application, “refer US 2013/0203827 A1 Aug. 8, 2013

ence' sequence and “wild type' sequence are used inter who will most benefit from the treatment and to avoid treat changeably. Nucleotide polymorphisms include single nucle ment of individuals who will experience symptomatic side otide differences, differences in sequence of more than one effects, in the case of a PDE3A inhibitor the adverse side nucleotide, and single or multiple nucleotide insertions, effect can be tolerance and toxicity to the PDE3A inhibitor. inversions, Substitutions, and deletions. Amino acid polymor Differences in metabolism of therapeutics can lead to severe phisms include single amino acid differences, differences in toxicity or therapeutic failure by altering the relation between sequence of more than one amino acid, and single or multiple dose and blood concentration of the pharmacologically active amino acid insertions, Substitutions, and deletions. drug. Thus, a physician or clinician may consider applying 0167 A “biological sample' encompasses a variety of knowledge obtained in relevant pharmacogenomics studies in sample types obtained from an individual and can be used in determining whether to administer a PDE3A inhibitor as well a diagnostic or monitoring assay. The definition encompasses as tailoring the dosage, regimen, and/or therapeutically effec blood and other liquid samples of biological origin, Solid tive amounts to be administered so as to attain the effect tissue samples Such as a biopsy specimen or tissue cultures or desired by treatment with the modulator. cells derived therefrom and the progeny thereof. The defini 0172 A determination of how a given PDE3A polymor tion also includes samples that have been manipulated in any phism is predictive of an individuals likelihood of respond way after their procurement, such as by treatment with ing to a given drug treatment for a condition relating to reagents, solubilization, or enrichment for certain compo abnormally high levels of circulating lipids can be accom nents, such as polynucleotides. The term biological sample plished by determining the genotype of the individual in the encompasses a clinical sample, and also includes cells in PDE3A gene, as described above. Information generated culture, cell Supernatants, cell lysates, serum, plasma, bio from one or more of these approaches can be used to deter logical fluid, and tissue samples. In one embodiment, the mine appropriate dosage and treatment regimens for prophy sample is collected by the individual. For example, an indi lactic or therapeutic treatment of an individual. This knowl vidual can collect a swap of tissue from the inside of the cheek edge, when applied to dosing or drug selection, can avoid for use as a nucleic acid sample. As known in the art, many adverse reactions or therapeutic failure and thus enhance types of samples can be used for the extraction of nucleic therapeutic or prophylactic efficiency when treating an indi acids. vidual with a niacin receptor modulator, such as niacin oran 0168 As used herein the term “treating in reference to a analog thereof. disorder means a reduction in severity of one or more symp 0173. In one embodiment, the invention provides a kit for toms associated with a particular disorder. Therefore, treating use in the methods of the invention, for example, a kit for a disorder does not necessarily mean a reduction in severity of determining a level of probability for an individual for a all symptoms associated with a disorder and does not neces condition responsiveness to PDE3A therapy, a kit for using a sarily mean a complete reduction in the severity of one or PDE3A Zygosity of an individual for determining a suitability more symptoms associated with a disorder. Treatment, as or an unsuitability of an individual for inclusion in a clinical used in this context, covers any treatment of a symptomatic trial, or a kit for determining a level of probability for a condition, such as an adverse reaction in a mammal, particu condition associated with heart failure. A kit can comprise larly in a human, and includes: (a) diagnosing and then pre reagents and instructions for performing the methods of the venting the adverse reaction from occurring in an individual invention. For example, a kit can include genotyping reagents which can be predisposed to the reaction but has not yet been Such as reagents for isolating nucleic acid molecules and diagnosed as having it; (b) inhibiting the adverse reaction, reagents for amplifying nucleic acid molecules such as prim i.e., arresting its development; and (c) relieving the adverse ers. A kit can also include, for example, a PDE3A assay such reaction, i.e., causing regression of the reaction. as an ELISA. In addition, a kit can contain control samples, 0169. The term “therapeutically effective amount’ means for example, to show that amplification reactions are not an amount that is effective in treating a particular disorder; contaminated. that is an amount that is effective for reducing the severity of 0.174. The contents of the kit are contained in packaging one or more symptoms associated with the particular disorder material, preferably to provide a sterile, contaminant-free for which treatment is sought. The term “ameliorate.” as used environment. In addition, the packaging material contains for instance in the amelioration of a particular condition instructions indicating how the materials within the kit can be means to make one or more symptoms of the condition at least employed. The instructions for use typically include a tan more tolerable, if not better. The term ameliorate does not gible expression describing the reagent concentration or at necessarily mean an increase in toleration of all symptoms least one assay method parameter, Such as the relative associated with a disorder and does not necessarily mean a amounts of reagent and sample to be admixed, maintenance complete reduction in the severity of one or more symptoms time periods for reagent/sample admixtures, temperature, associated with a disorder. buffer conditions, and the like. 0170 In another embodiment, a further step is added wherein a portion of the PDE3A gene spanning nucleotides III. METHODS OF TREATING CARDIAC 274 to 302 of SEQID NO:3 is amplified prior to the identi HYPERTROPHY fying step. In another embodiment, the identifying is per (0175. Once the PDE3A genotype of the individual is formed by a method selected from the group consisting of a determined a therapeutic course of treatment may be indi hybridization assay, a sequencing assay, a microSequencing vidualized. In a preferred embodiment of the method, the trait assay, a MALDI-TOF assay, and an allele-specific amplifica of interest is a clinical response exhibited by a patient to some tion assay. In a further embodiment, the identifying is per therapeutic treatment, for example, response to a drug such as formed by an antibody-based assay. but not limited to a PDE3A inhibitor. The term “clinical 0171 Pharmacogenomics allows a clinician or physician response' means a quantitative measure of the efficacy or to target prophylactic ortherapeutic treatments to individuals potency of the therapy and adverse events (i.e., side effects). US 2013/0203827 A1 Aug. 8, 2013

0176 Thus, individuals homozygous for an insertional conditions of storage and use, these preparations generally polymorphism in the PDE3A gene having or suspected of contain a preservative to prevent the growth of microorgan having or at risk of developing heart failure can be placed on isms. a therapy that includes PDE3A inhibitors such as but not limited to enoximone. The PDE3A inhibitor may be admin 0.184 The pharmaceutical forms suitable for injectable istered alone or in combination with at least one other agent, use include, for example, sterile aqueous solutions or disper Such as a stabilizing compound. sions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these 0177 A. Routes of Administration preparations are sterile and fluid to the extent that easy inject (0178 Administration of the PDE3A inhibitor may be by ability exists. Preparations should be stable under the condi any number of routes including, but not limited to oral, intra tions of manufacture and storage and should be preserved venous, intramuscular, intra-arterial, intramedullary, intrath against the contaminating action of microorganisms. Such as ecal, intraventricular, intradermal, intratracheal, intravesicle, bacteria and fungi. Appropriate solvents or dispersion media intraocular, transdermal, Subcutaneous, intraperitoneal, intra may contain, for example, water, ethanol, polyol (for nasal, enteral, topical, Sublingual, or rectal. Further details on example, glycerol, propylene glycol, and liquid polyethylene techniques for formulation and administration may be found glycol, and the like), Suitable mixtures thereof, and vegetable in the latest edition of Remington's Pharmaceutical Sciences oils. The proper fluidity can be maintained, for example, by (Maack Publishing Co., Easton, Pa.). In certain embodiments the use of a coating, such as lecithin, by the maintenance of enoximone or other PDE3A inhibitors are formulated for oral the required particle size in the case of dispersion and by the administration. use of surfactants. The prevention of the action of microor 0179 B. Formulations ganisms can be brought about by various antibacterial an 0180. Where clinical applications are contemplated, phar antifungal agents, for example, parabens, chlorobutanol, phe maceutical compositions will be prepared in a form appropri nol, Sorbic acid, thimerosal, and the like. In many cases, it will ate for the intended application. Generally, this will entail be preferable to include isotonic agents, for example, Sugars preparing compositions that are essentially free of pyrogens, or sodium chloride. Prolonged absorption of the injectable as well as other impurities that could be harmful to humans or compositions can be brought about by the use in the compo animals. sitions of agents delaying absorption, for example, aluminum 0181 Aqueous compositions of the present invention monostearate and gelatin. comprise an effective amount of the agent, dissolved or dis 0185. The compositions of the present invention generally persed in a pharmaceutically acceptable carrier or aqueous may be formulated in a neutral or salt form. Pharmaceuti medium. The phrase “pharmaceutically or pharmacologi cally-acceptable salts include, for example, acid addition cally acceptable' refer to molecular entities and compositions salts (formed with the free amino groups of the protein) that do not produce adverse, allergic, or other untoward reac derived from inorganic acids (e.g., hydrochloric or phospho tions when administered to an animal or a human. As used ric acids, or from organic acids (e.g., acetic, oxalic, tartaric, herein, “pharmaceutically acceptable carrier' includes sol mandelic, and the like. Salts formed with the free carboxyl vents, buffers, solutions, dispersion media, coatings, antibac groups of the protein can also be derived from inorganic bases terial and antifungal agents, isotonic and absorption delaying (e.g., sodium, potassium, ammonium, calcium, or ferric agents and the like acceptable for use informulating pharma hydroxides) or from organic bases (e.g., isopropylamine, tri ceuticals, such as pharmaceuticals suitable for administration methylamine, histidine, procaine and the like. to humans. The use of such media and agents for pharmaceu 0186. Upon formulation, solutions are preferably admin tically active substances is well known in the art. Except istered in a manner compatible with the dosage formulation insofar as any conventional media or agent is incompatible and in such amount as is therapeutically effective. The for with the active ingredients of the present invention, its use in mulations may easily be administered in a variety of dosage therapeutic compositions is contemplated. Supplementary forms such as injectable solutions, drug release capsules and active ingredients also can be incorporated into the composi the like. For parenteral administration in an aqueous solution, tions, provided they do not inactivate the vectors or cells of for example, the solution generally is suitably buffered and the compositions. the liquid diluent first rendered isotonic for example with 0182. The active compositions of the present invention Sufficient saline or glucose. Such aqueous solutions may be may include classic pharmaceutical preparations. Adminis used, for example, for intravenous, intramuscular, Subcutane tration of these compositions according to the present inven ous and intraperitoneal administration. Preferably, sterile tion may be via any common route so long as the target tissue aqueous media are employed as is known to those of skill in is available via that route. This includes oral, nasal, or buccal. the art, particularly in light of the present disclosure. By way Alternatively, administration may be by intradermal, Subcu of illustration, a single dose may be dissolved in 1 ml of taneous, intramuscular, intraperitoneal or intravenous injec isotonic NaCl solution and either added to 1000 ml of hypo tion, or by direct injection into cardiac tissue. Such compo dermoclysis fluid or injected at the proposed site of infusion, sitions would normally be administered as pharmaceutically (see for example, “Remington's Pharmaceutical Sciences acceptable compositions, as described Supra. 15th Edition, pages 1035-1038 and 1570-1580). Some varia 0183 The active compounds may also be administered tion in dosage will necessarily occur depending on the con parenterally or intraperitoneally. By way of illustration, solu dition of the subject being treated. The person responsible for tions of the active compounds as free base or pharmacologi administration will, in any event, determine the appropriate cally acceptable salts can be prepared in water Suitably mixed dose for the individual subject. Moreover, for human admin with a Surfactant, such as hydroxypropylcellulose. Disper istration, preparations should meet sterility, pyrogenicity, sions can also be prepared in glycerol, liquid polyethylene general safety and purity standards as required by FDA Office glycols, and mixtures thereof and in oils. Under ordinary of Biologics standards. US 2013/0203827 A1 Aug. 8, 2013

0187 1. Controlled/Extended/Sustained/Prolonged (0195 c. Transdermal Patch Device Release Administration 0196. Transdermal delivery involves delivery of a thera 0188 Another aspect of this invention provides methods peutic agent through the skin for distribution within the body of treating heart failure patients by delivering a PDE3A by circulation of the blood. Transdermal delivery can be inhibitor to a patient, having a homozygous insertional poly compared to continuous, controlled intravenous delivery of a morhism genotype, as a controlled release formulation. As drug using the skinas a port of entry instead of an intravenous used herein, the terms “controlled “extended,” “sustained, needle. The therapeutic agent passes through the outer layers or “prolonged release of the composition of the present of the skin, diffuses into the capillaries or tiny blood vessels in invention will collectively be referred to herein as “controlled the skin and then is transported into the main circulatory release.” and includes continuous or discontinuous, andlinear system. or non-linear release of the composition of the present inven 0.197 Transdermal patch devices which provide a con tion. trolled, continuous administration of a therapeutic agent (0189 a. Tablets through the skin are well known in the art. Such devices, for 0190. A controlled release tablet suitable for purposes of example, are disclosed in U.S. Pat. Nos. 4,627,429; 4,784, this invention is disclosed in U.S. Pat. No. 5,126,145, which 857; 5,662,925; 5,788,983; and 6,113,940, which are all is incorporated by reference herein. This tablet comprises, in incorporated herein by reference. Characteristically, these admixture, about 5-30% high viscosity hydroxypropyl devices contain a drug impermeable backing layer which methyl cellulose, about 2-15% of a water-soluble pharmaceu defines the outer surface of the device and a permeable skin tical binder, about 2-20% of a hydrophobic component such attaching membrane, Such as an adhesive layer, sealed to the as a waxy material, e.g., a fatty acid, and about 30-90% active barrier layer in such a way as to create a reservoir between ingredient. them in which the therapeutic agent is placed. In one embodi (0191 b. Films ment of the present invention, a formulation of the PDE3A 0.192 This invention further provides a prophylaxis for or inhibitor is introduced into the reservoir of a transdermal method of treating a patient having a homozygous insertional patch and used by a patient who is homozygous for an inser polymorphism in the PDE3A gene following an invasive tional polymorphism at the PDE3A gene. cardiac procedure comprising administering biodegradable, 0198 d. Medical Devices biocompatible polymeric film comprising a PDE3A inhibitor, 0199 Another embodiment contemplates the incorpora Such as enoXimone, to a patient. The polymeric films are thin tion of a PDE3A inhibitor into a medical device that is then compared to their length and breadth. The films typically have positioned to a desired target location within the body, where a uniform selected thickness between about 60 micrometers upon the PDE3A inhibitor elutes from the medical device. As and about 5 mm. Films of between about 600 micrometers used herein, "medical device' refers to a device that is intro and 1 mm and between about 1 mm and about 5 mm thick, as duced temporarily or permanently into a mammal for the well as films between about 60 micrometers and about 1000 prophylaxis or therapy of a medical condition. These devices micrometers, and between about 60 and about 300 microme include any that are introduced Subcutaneously, percutane ters are useful in the manufacture of therapeutic implants for ously or Surgically to rest within an organ, tissue or lumen. insertion into a patient’s body. The films can be administered Medical devices include, but are not limited to, stents, syn to the patient in a manner similar to methods used in adhesion thetic grafts, artificial heart valves, artificial hearts and fix surgeries. For example, a PDE3A inhibitor film formulation tures to connect the prosthetic organ to the vascular circula can be sprayed or dropped onto a cardiac tissue site or artery tion, Venous valves, abdominal aortic aneurysm (AAA) during Surgery, or a formed film can be placed over the grafts, inferior Venal caval filters, catheters including perma selected tissue site. In an alternative embodiment, the film can nent drug infusion catheters, embolic coils, embolic materials be used as controlled release coating on a medical device Such used in vascular embolization (e.g., PVA foams), mesh repair as a stent, as is discussed in further detail below. materials, a Dracon Vascular particle orthopedic metallic 0193 Either biodegradable or nonbiodegradable poly plates, rods and screws and vascular Sutures. mers may be used to fabricate implants in which the PDE3A 0200. In one embodiment, the medical device such as a inhibitor is uniformly distributed throughout the polymer stent or graft is coated with a matrix. The matrix used to coat matrix. A number of suitable biodegradable polymers for use the stent or graft according to this invention may be prepared in making the biodegradable films of this invention are known from a variety of materials. A primary requirement for the to the art, including polyanhydrides and aliphatic polyesters, matrix is that it be sufficiently elastic and flexible to remain preferably polylactic acid (PLA), polyglycolic acid (PGA) unruptured on the exposed Surfaces of the Stent or synthetic and mixtures and copolymers thereof, more preferably 50:50 graft. copolymers of PLA:PGA and most preferably 75:25 copoly 0201 B. Dosages mers of PLA:PGA. Single enantiomers of PLA may also be 0202 The amount of PDE3A inhibitor (e.g., enoximone) used, preferably L-PLA, either alone or in combination with that is administered or prescribed to the patient can be about, PGA. Polycarbonates, polyfumarates and caprolactones may at least about, or at most about 0.1, 0.5, 1,2,3,4,5,6,7,8,9, also be used to make the implants of this invention. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, (0194 The amount of the PDE3A inhibitor to be incorpo 27, 28, 29, 30, 31, 32,33, 34,35, 36, 37,38, 39, 40, 41,42, 43, rated into the polymeric films of this invention is an amount 44, 45,46, 47, 48,49, 50, 51, 52,53,54, 55,56, 57,58, 59, 60, effective to show a measurable effect in treating diseases 61, 62,63, 64, 65,66, 67,68, 69,70, 71, 72,73, 74, 75, 76, 77, having similar pathophysiological states. Such as but not lim 78, 79,80, 81, 82, 83, 84,85, 86, 87, 88, 89,90,91, 92,93, 94, ited to heart failure. The composition of the present invention 95, 96, 97,98,99, 100, 110, 120, 130, 140, 150, 160, 170, 180, can be incorporated into the film by various techniques such 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, as by solution methods, Suspension methods, or melt press 310,320, 330, 340, 350, 360, 370, 380,390, 400, 410, 420, 1ng. 430,440, 441, 450, 460, 470, 480,490,500 mg, or any range US 2013/0203827 A1 Aug. 8, 2013 17 derivable therein. This may be with respect to a single dose or one may also provide to the patient more 'standard pharma total dose in a day (24 hour period) or in a week. Alternatively, ceutical cardiac therapies. Examples of other therapies the amount administered or prescribed may be about, at least include, without limitation, beta blockers, anti-hypertensives, about, or at most about 0.001, 0.002, 0.003, 0.004, 0.005, cardiotonics, anti-thrombotics, vasodilators, hormone 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, antagonists, iontropes, diuretics, endothelin antagonists, cal 0.07, 0.08, 0.09, 0.1, 0.2,0.3, 0.4,0.5,0.6, 0.7, 0.8, 0.9, 1.0, cium channel blockers, phosphodiesterase inhibitors, ACE 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, inhibitors, angiotensin type 2 antagonists and cytokine block 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7. 3.8, ers/inhibitors, and HDAC inhibitors. 3.9, 4.0.4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 mg/kg, or any range derivable therein, with respect to the weight of the 0209 Combinations may be achieved by contacting car patient. diac cells with a single composition or pharmacological for 0203 When provided in a discrete amount, each intake of mulation that includes both agents, or by contacting the cell PDE3A inhibitor can be considered a “dose.” A medical prac with two distinct compositions or formulations, at the same titioner may prescribe or administer multiple doses of time, wherein one composition includes the expression con PDE3A inhibitor over a particular time course (treatment struct and the other includes the agent. Alternatively, the regimen) or indefinitely. therapy using a PDE3A inhibitor may precede or follow 0204 PDE3A inhibitor may be prescribed or administered administration of the other agent(s) by intervals ranging from 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, minutes to weeks. In embodiments where the other agent and 30, 40, 50, 60, 70, 80, or more times or any range derivable expression construct are applied separately to the cell, one therein. It is further contemplated that the drug may be taken would generally ensure that a significant period of time did for an indefinite period of time or for as long as the patient not expire between the time of each delivery, such that the exhibits symptoms of the medical condition for which agent and expression construct would still be able to exert an PDE3A inhibitor was prescribed or administered. Also, the advantageously combined effect on the cell. In Such drug may be administered every 2,3,4,5,6,7,8,9, 10, 15, 20, instances, it is contemplated that one would typically contact 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, the cell with both modalities within about 12-24 hours of each 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or other and, more preferably, within about 6-12 hours of each 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, other, with a delay time of only about 12 hours being most 7, 8, 9, 10, 11, 12 months or more, or any range derivable preferred. In some situations, it may be desirable to extend the therein. Alternatively, it may be administered systemically time period for treatment significantly, however, where sev over any such period of time and be extended beyond more eral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 than a year. Alternatively, a patient may be treated 1,2,3,4, 5, or 8) lapse between the respective administrations. or 6 times a day with the same or different doses. In some 0210. It also is conceivable that more than one adminis cases, the dose is up-titrated or down-titrated. tration of either a PDE3A inhibitor, or the other agent will be 0205 C. Other Therapeutic Options desired. In this regard, various combinations may be 0206. In certain embodiments of the invention, methods employed. By way of illustration, where the PDE3A inhibitor may involve administering a PDE3A inhibitor that is not is “A” and the other agent is “B”, the following permutations enoximnoe or that is a diuretic, ACE-I, AII antagonist, BNP. based on 3 and 4 total administrations are exemplary:

ABA BFAB B.B.A. AAB BAA AB,B B.B.B.A. B.BAB AABB ABAB ABBA BBiAA BABA BAAB B.B.B.A. AAAB BAAA ABAA AABA ABBB B.A.B.B. B.B.AB

Ca"-blocker, or an HDAC inhibitor. These agents may be Other combinations are likewise contemplated. prescribed or administered instead of or in addition to a 0211 1. Pharmacological Therapeutic Agents PDE3A inhibitor after the PDE3A polymorphisms are evalu 0212 Pharmacological therapeutic agents and methods of ated. administration, dosages, etc., are well known to those of skill 0207 As a second therapeutic regimen, the agent may be in the art (see for example, the “Physicians Desk Reference'. administered or taken at the same time as a PDE3A inhibitor, Klaassen’s “The Pharmacological Basis of Therapeutics'. or either before or after a PDE3A inhibitor. The treatment “Remington’s Pharmaceutical Sciences', and “The Merck may improve one or more symptoms of pathologic cardiac Index, Eleventh Edition', incorporated herein by reference in hypertrophy or heart failure Such as providing increased exer relevant parts), and may be combined with the invention in cise capacity, increased cardiac ejection Volume, decreased light of the disclosures herein. Some variation in dosage will left ventricular end diastolic pressure, decreased pulmonary necessarily occur depending on the condition of the Subject capillary wedge pressure, increased cardiac output or cardiac being treated. The person responsible for administration will, index, lowered pulmonary artery pressures, decreased left in any event, determine the appropriate dose for the individual Ventricular end systolic and diastolic dimensions, decreased Subject, and Such individual determinations are within the left and right ventricular wall stress, decreased wall tension skill of those of ordinary skill in the art. and wall thickness, increased quality of life, and decreased 0213 Non-limiting examples of a pharmacological thera disease-related morbidity and mortality. peutic agent that may be used in the present invention include 0208. In another embodiment, it is envisioned to use a an antihyperlipoproteinemic agent, an antiarteriosclerotic PDE3A inhibitor in combination with other therapeutic agent, an antithrombotic/fibrinolytic agent, a blood coagu modalities. Thus, in addition to the therapies described above, lant, an antiarrhythmic agent, an antihypertensive agent, a US 2013/0203827 A1 Aug. 8, 2013 vasopressor, a treatment agent for congestive heart failure, an 0233 (1) Anticoagulants antianginal agent, an antibacterial agent or a combination 0234. A non-limiting example of an anticoagulant include thereof. acenocoumarol, ancrod, anisindione, bromindione, clorindi 0214) a. Antihyperlipoproteinemics one, coumetarol, cyclocumarol, dextran Sulfate Sodium, dicu 0215. In certain embodiments, administration of an agent marol, diphenadione, ethyl biscoumacetate, ethylidene that lowers the concentration of one of more blood lipids dicoumarol, fluindione, heparin, hirudin, lyapolate Sodium, and/or lipoproteins, known herein as an “antihyperlipopro oxazidione, pentosan polysulfate, phenindione, phenprocou teinemic. may be combined with a cardiovascular therapy mon, phosvitin, picotamide, tioclomarol and warfarin. according to the present invention, particularly intreatment of 0235 (2) Antiplatelet Agents atherSclerosis and thickenings or blockages of vascular tis 0236 Non-limiting examples of antiplatelet agents Sues. In certain aspects, an antihyperlipoproteinemic agent include aspirin, a dextran, dipyridamole (persantin), heparin, may comprise an aryloxyalkanoic/fibric acid derivative, a Sulfinpyranone (anturane) and ticlopidine (ticlid). resin/bile acid sequesterant, a HMG CoA reductase inhibitor, 0237 (3) Thrombolytic Agents a nicotinic acid derivative, a thyroid hormone or thyroid hor 0238. Non-limiting examples of thrombolytic agents mone analog, a miscellaneous agent oracombination thereof. include tissue plaminogen activator (activase), plasmin, pro 0216 (1) Aryloxyalkanoic Acid/Fibric Acid Derivatives urokinase, urokinase (abbokinase) streptokinase (streptase), 0217 Non-limiting examples of aryloxyalkanoic/fibric anistreplase/APSAC (eminase). acid derivatives include beclobrate, enZafibrate, binifibrate, 0239 d. Blood Coagulants ciprofibrate, clinofibrate, clofibrate (atromide-S), clofibric 0240. In certain embodiments wherein a patient is suffer acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate, ing from a hemmorage oran increased likelihood of hemmor aging, an agent that may enhance blood coagulation may be pirifibrate, ronifibrate, simfibrate and theofibrate. used. Non-limiting examples of a blood coagulation promot 0218 (2) Resins/Bile Acid Sequesterants ing agent include thrombolytic agent antagonists and antico 0219 Non-limiting examples of resins/bile acid seques agulant antagonists. terants include cholestyramine (cholybar, questran), colesti 0241 (1) Anticoagulant Antagonists pol (colestid) and polidexide. 0242. Non-limiting examples of anticoagulantantagonists 0220 (3) HMG CoA Reductase Inhibitors include protamine and vitamine K1. 0221 Non-limiting examples of HMG CoA reductase 0243 (2) Thrombolytic Agent Antagonists and Anti inhibitors include lovastatin (mevacor), pravastatin (prav thrombotics ochol) or simvastatin (Zocor). 0244 Non-limiting examples of thrombolytic agent 0222 (4) Nicotinic Acid Derivatives antagonists include amiocaproic acid (amicar) and tranex 0223 Non-limiting examples of nicotinic acid derivatives amic acid (amstat). Non-limiting examples of antithrombot include nicotinate, acepimox, niceritrol, nicoclonate, nico ics include anagrelide, argatroban, cilstaZol, daltroban, defi mol and oxiniacic acid. brotide, enoxaparin, fraxiparine, indobufen, lamoparan, 0224 (5) Thyroid Hormones and Analogs oZagrel, picotamide, plafibride, tedelparin, ticlopidine and 0225. Non-limiting examples of thyroid hormones and triflusal. analogs thereof include etoroXate, thyropropic acid and thy 0245 e. Antiarrhythmic Agents roXine. 0246 Non-limiting examples of antiarrhythmic agents 0226 (6) Miscellaneous Antihyperlipoproteinemics include Class I antiarrythmic agents (sodium channel block 0227 Non-limiting examples of miscellaneous antihyper ers), Class II antiarrythmic agents (beta-adrenergic blockers), lipoproteinemics include acifran, azacosterol, benfluorex, Class II antiarrythmic agents (repolarization prolonging B-benzalbutyramide, carnitine, chondroitin Sulfate, clom drugs), Class IV antiarrhythmic agents (calcium channel estrone, detaXtran, dextran sulfate sodium, 5,8,11,14, 17 blockers) and miscellaneous antiarrythmic agents. eicosapentaenoic acid, eritadenine, furazabol, meglutol, 0247 (1) Sodium Channel Blockers melinamide, mytatrienediol, ornithine, Y-ory Zanol, pan 0248. Non-limiting examples of sodium channel blockers include Class IA, Class IB and Class IC antiarrhythmic tethine, pentaerythritol tetraacetate, C.-phenylbutyramide, agents. Non-limiting examples of Class IA antiarrhythmic pirozadil, probucol (lorelco), B-sitosterol, Sultosilic acid-pip agents include disppyramide (norpace), procainamide (pron erazine salt, tiadenol, triparanol and Xenbucin. estyl) and quinidine (quinidex). Non-limiting examples of 0228 b. Antiarteriosclerotics Class IB antiarrhythmic agents include lidocaine (Xylocaine), 0229 Non-limiting examples of an antiarteriosclerotic tocainide (tonocard) and mexiletine (mexitil). Non-limiting include pyridinol carbamate. examples of Class IC antiarrhythmic agents include encain 0230 c. Antithrombotic/Fibrinolytic Agents ide (enkaid) and flecamide (tambocor). 0231. In certain embodiments, administration of an agent 0249 (2) Beta Blockers that aids in the removal or prevention of blood clots may be 0250) Non-limiting examples of a beta blocker, otherwise combined with administration of a modulator, particularly in known as a B-adrenergic blocker, a B-adrenergic antagonist or treatment of athersclerosis and vasculature (e.g., arterial) a Class II antiarrhythmic agent, include acebutolol (sectral), blockages. Non-limiting examples of antithrombotic and/or alprenolol, amoSulalol, arotinolol, atenolol, befunolol, betax fibrinolytic agents include anticoagulants, anticoagulant olol, bevantolol, bisoprolol, bopindolol, bucumolol, bufe antagonists, antiplatelet agents, thrombolytic agents, throm tolol, bufuralol, bunitrolol, bupranolol, butidrine hydrochlo bolytic agent antagonists or combinations thereof. ride, butofilolol, carazolol, carteolol, carvedilol, celiprolol, 0232. In certain aspects, antithrombotic agents that can be cetamolol, cloranolol, dilevalol, epanolol, esmolol administered orally, Such as, for example, aspirin and wafarin (brevibloc), indenolol, labetalol, levobunolol, mepindolol, (coumadin), are preferred. metipranolol, metoprolol, moprolol, nadolol, nadoxolol. US 2013/0203827 A1 Aug. 8, 2013 nifenalol, nipradillol, oXprenolol, penbutolol, pindolol, prac tors) include alacepril, enalapril (vasotec), captopril, cilaza tolol, pronethalol, propanolol (inderal), Sotalol (betapace), pril, delapril, enalaprilat, fosinopril, lisinopril, moveltopril, sulfinalol, talinolol, tertatolol, timolol, toliprolol and xibi perindopril, quinapril and ramipril. Non-limiting examples of nolol. In certain aspects, the beta blocker comprises an ary an angiotensin II receptor blocker, also known as an angio loxypropanolamine derivative. Non-limiting examples of tension II receptor antagonist, an ANG receptor blocker oran aryloxypropanolamine derivatives include acebutolol, alpre ANG-II type-1 receptor blocker (ARBS), include angiocan nolol, arotinolol, atenolol, betaxolol, bevantolol, bisoprolol. desartan, eprosartan, irbesartan, losartan and Valsartan. bopindolol, bunitrolol, butofilolol, carazolol, carteolol. 0265 (4) Sympatholytics carvedilol, celiprolol, cetamolol, epanolol, indenolol, mepin 0266 Non-limiting examples of a sympatholytic include a dolol, metipranolol, metoprolol, moprolol, nadolol. centrally acting sympatholytic or a peripherially acting sym nipradillol, oXprenolol, penbutolol, pindolol, propanolol, tali patholytic. Non-limiting examples of a centrally acting sym nolol, tertatolol, timolol and toliprolol. patholytic, also known as an central nervous system (CNS) 0251 (3) Repolarization Prolonging Agents sympatholytic, include clonidine (catapres), guanabenz 0252. Non-limiting examples of an agent that prolong (wytensin) guanfacine (tenex) and methyldopa (aldomet). repolarization, also known as a Class III antiarrhythmic Non-limiting examples of a peripherally acting sym agent, include amiodarone (cordarone) and Sotalol (beta patholytic include a ganglion blocking agent, an adrenergic pace). neuron blocking agent, a B-adrenergic blocking agent or a 0253 (4) Calcium Channel Blockers/Antagonist alpha1-adrenergic blocking agent. Non-limiting examples of 0254 Non-limiting examples of a calcium channel a ganglion blocking agent include mecamylamine (inversine) blocker, otherwise known as a Class IV antiarrythmic agent, and trimethaphan (arfonad). Non-limiting of an adrenergic include an arylalkylamine (e.g., bepridile, diltiazem, neuron blocking agent include guanethidine (ismelin) and fendiline, gallopamil, prenylamine, terodiline, Verapamil), a reserpine (serpasil). Non-limiting examples of a B-adrenergic dihydropyridine derivative (felodipine, isradipine, nicar blocker include acenitolol (sectral), atenolol (tenormin), dipine, nifedipine, nimodipine, nisoldipine, nitrendipine) a betaxolol (kerlone), carteolol (cartrol), labetalol (normodyne, piperaZinde derivative (e.g., cinnarizine, flunarizine, lidofla trandate), metoprolol (lopressor), nadanol (corgard), penb Zine) or a miscellaneous calcium channel blocker Such as utolol (levatol), pindolol (visken), propranolol (inderal) and bencyclane, etafenone, magnesium, mibefradil or perhexy timolol (blocadren). Non-limiting examples of alpha1-adren line. In certain embodiments a calcium channel blocker com ergic blocker include praZosin (minipress), doxazocin (car prises a long-acting dihydropyridine (nifedipine-type) cal dura) and terazosin (hytrin). cium antagonist. 0255 (5) Miscellaneous Antiarrhythmic Agents 0267 (5) Vasodilators 0256 Non-limiting examples of miscellaneous antiarrhy 0268. In certain embodiments a cardiovasculator thera mic agents include adenosine (adenocard), digoxin (lanoxin), peutic agent may comprise a vasodilator (e.g., a cerebral acecamide, ajmaline, amoproxan, aprindine, bretylium tosy vasodilator, a coronary vasodilator or a peripheral vasodila late, bunaftine, butobendine, capobenic acid, cifenline, dis tor). In certain preferred embodiments, a vasodilator com opyramide, hydroquinidine, indecamide, ipatropium bro prises a coronary vasodilator. Non-limiting examples of a mide, lidocaine, lorajmine, lorcainide, meobentine, coronary vasodilator include amotriphene, bendaZol, benfu moricizine, pirmenol, prajmaline, propafenone, pyrinoline, rodil hemisuccinate, benziodarone, chloracizine, chromonar, quinidine polygalacturonate, quinidine Sulfate and Viquidil. clobenfurol, clonitrate, dilaZep, dipyridamole, droprenil 0257 f. Antihypertensive Agents amine, efloxate, erythrityl tetranitrane, etafenone, fendiline, 0258 Non-limiting examples of antihypertensive agents floredil, ganglefene, herestrol bis(B-diethylaminoethyl include sympatholytic, alpha/beta blockers, alpha blockers, ether), hexobendine, itramin tosylate, khellin, lidoflanine, anti-angiotensin II agents, beta blockers, calcium channel mannitol hexanitrane, medibazine, nicorglycerin, pen blockers, vasodilators and miscellaneous antihypertensives. taerythritol tetranitrate, pentrinitrol, perhexyline, pimefyl 0259 (1) Alpha Blockers line, trapidil, tricromyl, trimetazidine, trolnitrate phosphate 0260. Non-limiting examples of an alpha blocker, also and Visnadine. known as an O-adrenergic blocker oran C.-adrenergic antago 0269. In certain aspects, a vasodilator may comprise a nist, include amoSulalol, arotinolol, dapiprazole, doxazosin, chronic therapy vasodilator or a hypertensive emergency ergoloid mesylates, fenspiride, indoramin, labetalol, nicer vasodilator. Non-limiting examples of a chronic therapy goline, prazosin, teraZosin, tolazoline, trimaZosin and yohim vasodilator include hydralazine (apresoline) and minoxidil bine. In certain embodiments, an alpha blocker may comprise (loniten). Non-limiting examples of a hypertensive emer a quinazoline derivative. Non-limiting examples of quinazo gency vasodilator include nitroprusside (nipride), diaZoxide line derivatives include alfuZosin, bunaZosin, doxazosin, pra (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) Zosin, teraZosin and trimaZosin. and Verapamil. 0261 (2) Alpha/Beta Blockers 0270 (6) Miscellaneous Antihypertensives 0262. In certain embodiments, an antihypertensive agent 0271 Non-limiting examples of miscellaneous antihyper is both an alpha and beta adrenergic antagonist. Non-limiting tensives include ajmaline, Y-aminobutyric acid, bufeniode, examples of an alpha/beta blocker comprise labetalol (nor cicletainine, ciclosidomine, a cryptenamine tannate, modyne, trandate). fenoldopam, floSequinan, ketanserin, mebutamate, mecamy 0263 (3) Anti-Angiotension II Agents lamine, methyldopa, methyl 4-pyridylketone thiosemicarba 0264. Non-limiting examples of anti-angiotension II Zone, muZolimine, pargyline, pempidine, pinacidil, piper agents include angiotensin converting enzyme inhibitors and oxan, primaperone, a protoveratrine, raubasine, rescimetol, angiotension II receptor antagonists. Non-limiting examples rillmenidene, Saralasin, sodium nitrorusside, ticrynafen, tri of angiotension converting enzyme inhibitors (ACE inhibi methaphan camsylate, tyrosinase and urapidil. US 2013/0203827 A1 Aug. 8, 2013 20

0272. In certain aspects, an antihypertensive may com etillefrin, gepefrine, metaraminol, midodrine, norepineph prise an arylethanolamine derivative, a benzothiadiazine rine, pholedrine and synephrine. derivative, a N-carboxyalkyl (peptide/lactam) derivative, a 0295 g. Treatment Agents for Congestive Heart Failure dihydropyridine derivative, a guanidine derivative, a hydra 0296 Non-limiting examples of agents for the treatment Zines/phthalazine, an imidazole derivative, a quanternary of congestive heart failure include anti-angiotension II ammonium compound, a reserpine derivative or a Sulfona agents, afterload-preload reduction treatment, diuretics and mide derivative. inotropic agents. 0273 Arylethanolamine Derivatives. 0297 (1) Afterload-Preload Reduction 0274 Non-limiting examples of arylethanolamine deriva 0298. In certain embodiments, an animal patient that can tives include amosulalol, bufuralol, dilevalol, labetalol, not tolerate an angiotension antagonist may be treated with a pronethalol, Sotalol and Sulfinalol. combination therapy. Such therapy may combine administra 0275 Benzothiadiazine Derivatives. tion of hydralazine (apresoline) and isosorbide dinitrate (isor 0276 Non-limiting examples of benzothiadiazine deriva dil, sorbitrate). tives include althizide, bendroflumethiazide, benzthiazide, 0299 (2) Diuretics benzylhydrochlorothiazide, buthiazide, chlorothiazide, chlo 0300 Non-limiting examples of a diuretic include a thiaz rthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epi ide or benzothiadiazine derivative (e.g., althiazide, bendrof thiazide, ethiazide, fenguizone, hydrochlorothizide, hydrof lumethazide, benzthiazide, benzylhydrochlorothiazide, lumethizide, methyclothiazide, meticrane, metolaZone, buthiazide, chlorothiazide, chlorothiazide, chlorthalidone, paraflutizide, polythizide, tetrachlormethiazide and trichlo cyclopenthiazide, epithiazide, ethiazide, ethiazide, fen rmethiazide. quizone, hydrochlorothiazide, hydroflumethiazide, methy (0277 N-Carboxyalkyl (Peptide/Lactam) Derivatives. clothiazide, meticrane, metolaZone, paraflutizide, polythiz 0278. Non-limiting examples of N-carboxyalkyl (peptide/ ide, tetrachloromethiazide, trichlormethiazide), an lactam) derivatives include alacepril, captopril, cilaZapril, organomercurial (e.g., chlormerodrin, meralluride, mercam delapril, enalapril, enalaprilat, fosinopril, lisinopril, movel phamide, mercaptomerin Sodium, mercumallylic acid, mer tipril, perindopril, quinapril and ramipril. cumatilindodium, mercurous chloride, mersalyl), a pteridine (0279 Dihydropyridine Derivatives. (e.g., furtherene, triamterene), purines (e.g., acefylline, 0280. Non-limiting examples of dihydropyridine deriva 7-morpholinomethyltheophylline, pamobrom, protheobro tives include amlodipine, felodipine, isradipine, nicardipine, mine, theobromine), Steroids including aldosterone antago nifedipine, nilvadipine, nisoldipine and nitrendipine. nists (e.g., canrenone, oleandrin, Spironolactone), a Sulfona 0281 Guanidine Derivatives. mide derivative (e.g., acetazolamide, ambuside, aZosemide, 0282. Non-limiting examples of guanidine derivatives bumetanide, butazolamide, chloraminophenamide, clofena include bethanidine, debrisoquin, guanabenz, guanacline, mide, clopamide, clorexolone, diphenylmethane-4,4'-disul guanadrel, guanaZodine, guanethidine, guanfacine, gua fonamide, disulfamide, ethoXZolamide, furosemide, indapa nochlor, guanoXabenZ and guanoxan. mide, mefruside, methazolamide, piretanide, quinethaZone, (0283 Hydrazines/Phthalazines. torasemide, tripamide, Xipamide), a uracil (e.g., aminometra 0284. Non-limiting examples of hydrazines/phthalazines dine, amisometradine), a potassium sparing antagonist (e.g., include budralazine, cadralazine, dihydralazine, endralazine, amiloride, triamterene) or a miscellaneous diuretic Such as hydracarbazine, hydralazine, pheniprazine, pildralazine and aminozine, arbutin, chlorazanil, ethacrynic acid, etoZolin, todralazine. hydracarbazine, isosorbide, mannitol, metochalcone, 0285 Imidazole Derivatives. muZolimine, perhexyline, ticrnafen and urea. 0286 Non-limiting examples of imidazole derivatives 0301 (3) Inotropic Agents include clonidine, lofexidine, phentolamine, tiamenidine and 0302) Non-limiting examples of a positive inotropic agent, tolonidine. also known as a cardiotonic, includeacefylline, an acetyldigi 0287 Quanternary Ammonium Compounds. toxin, 2-amino-4-picoline, aminone, benfurodil hemisucci nate, bucladesine, cerberosine, camphotamide, convalla 0288. Non-limiting examples of quanternary ammonium toxin, cymarin, denopamine, deslanoside, digitalin, digitalis, compounds include azamethonium bromide, chlorison digitoxin, digoxin, dobutamine, dopamine, dopexamine, damine chloride, hexamethonium, pentacynium bis(methyl enoXimone, erythrophleine, fenalcomine, gitalin, gitoxin, Sulfate), pentamethonium bromide, pentolinium tartrate, glycocyamine, heptaminol, hydrastinine, ibopamine, a lana phenactropinium chloride and trimethidinium methosulfate. toside, metamivam, milrinone, nerifolin, oleandrin, ouabain, 0289 Reserpine Derivatives. oxyfedrine, prenalterol, proscillaridine, resilbufogenin, Scil 0290. Non-limiting examples of reserpine derivatives laren, Scillarenin, strphanthin, Sulmazole, theobromine and include bietaserpine, deserpidine, rescinnamine, reserpine Xamoterol. and Syrosingopine. 0303. In particular aspects, an intropic agent is a cardiac 0291 Suflonamide Derivatives. glycoside, a beta-adrenergic agonist or a phosphodiesterase 0292. Non-limiting examples of sulfonamide derivatives inhibitor. Non-limiting examples of a cardiac glycoside include ambuside, clopamide, furosemide, indapamide, includes digoxin (lanoxin) and digitoxin (crystodigin). Non quinethaZone, tripamide and Xipamide. limiting examples of a B-adrenergicagonist include albuterol, 0293 (7) Vasopressors bambuterol, bitolterol, carbuterol, clenbuterol, clorprenaline, 0294 Vasopressors generally are used to increase blood denopamine, dioxethedrine, dobutamine (dobutrex), dopam pressure during shock, which may occur during a Surgical ine (intropin), dopexamine, ephedrine, etafedrine, ethylnore procedure. Non-limiting examples of a vasopressor, also pinephrine, fenoterol, formoterol, hexoprenaline, ibopamine, known as an antihypotensive, include amezinium methyl Sul isoetharine, isoproterenol, mabuterol, metaproterenol, meth fate, angiotensin amide, dimetofrine, dopamine, etifelmin, oxyphenamine, oxyfedrine, pirbuterol, procaterol, protoky US 2013/0203827 A1 Aug. 8, 2013

lol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, tre Example 1 toquinol, tulobuterol and Xamoterol. Non-limiting examples of a phosphodiesterase inhibitor include enoXimone and ami A Polymorphism in the PDE3A Gene Promoter that none (inocor). Prevents cAMP-Induced Increases in Transcriptional 0304 (4) Antianginal Agents Activity, and Protects Against PDE3A Inhibitor Drug Tolerance 0305 Antianginal agents may comprise organonitrates, calcium channel blockers, beta blockers and combinations 0312 The phosphodiesterase (PDE3A) 3A protein gene thereof. product regulates contractile function, via its co-localization 0306 Non-limiting examples of organonitrates, also in the cardiac myocyte phospholamban-Serca2a micro domain. Drugs that inhibit PDE3A (PDEIs) are positive ino known as nitrovasodilators, include nitroglycerin (nitro-bid, tropic and lusitropic agents, but exhibit therapeutic response nitrostat), isosorbide dinitrate (isordil, sorbitrate) and amyl heterogeneity that may be explained by varying degrees of nitrate (aspirol, vaporole). PDEI tolerance. This led the inventors to search for function 0307 2. Surgical Therapeutic Agents ally important genetic variation in the PDE3A gene (FIG. 1). 0308. In certain aspects, the secondary therapeutic agent 0313. The inventors identified a 29 nt insertion/deletion may comprise a Surgery of Some type, which includes, for (Ins/Del, allele frequencies 0.41/0.59) polymorphism in the example, preventative, diagnostic or staging, curative and PDE3A promoter region, at -1120 from one of the start sites palliative Surgery. Surgery, and in particular a curative Sur (FIG. 2). The Ins contains a binding site for ectotropic viral gery, may be used in conjunction with other therapies, such as integration site-1 (Evi-1), a transcription factor that is mark the present invention and one or more other agents. edly induced (by 16-fold) by cAMP. Evi-1 typically functions 0309 Such surgical therapeutic agents for vascular and as a strong transcriptional repressor. In explanted left ven cardiovascular diseases and disorders are well known to those tricular samples (LVs) from heart failure (HF) patients treated of skill in the art, and may comprise, but are not limited to, with enoximone, mRNA expression of Evi-1 is increased performing Surgery on an organism, providing a cardiovas (FIG. 5). In LVs explanted from end-stage HF patients under cular mechanical prostheses, angioplasty, coronary artery going transplantation, patients treated with enoXimone who reperfusion, catheter ablation, providing an implantable car were either homozygous or heterozygous for PDE3A Del (Del carriers, n=9), PDE3A mRNA abundance was increased dioverter defibrillator to the subject, mechanical circulatory 2.1+0.3 fold compared to Del patients not receiving enoxi Support or a combination thereof. Non-limiting examples of a mone (n=19, p<0.001) (FIG. 7). In contrast, LV samples from mechanical circulatory Support that may be used in the patients treated with enoXimone who were homozygous for present invention comprise an intra-aortic balloon counter the Ins (Ins/Ins) did not exhibit an increase in PDE3A gene pulsation, left ventricular assist device or combination expression (1.1+0.3 fold, p=0.7) (FIG. 7). In neonatal rat thereof. cardiac myocytes (NRVMs) transfected with either Ins or Del PDE3A promoter-luciferase constructs, agents that increased IV. KITS cAMP markedly increased transcriptional activity in Del but not Ins (FIG.3). However, deletion of the CREB binding site 0310. In some embodiments, the present invention pro (CRE) at -1070 abolishes the promoter response to enoxi vides kits for the detection of PDE3A polymorphisms. In mone and dbcAMP in NRVMS transfected with the Del con Some embodiments, the kits contain reagents specific for the struct (FIG. 4). PDE3A mRNA levels are upregulated in detection or analysis of DNA (e.g., oligonucleotide probes or failing heart patients with the Ins polymorphism (FIG. 8). primers). In preferred embodiments, the kits contain all of the 0314. A 29 nt Ins/Del polymorphism in the PDE3A pro components necessary to perform a detection assay, includ moter regulates PDE3A gene expression, whereby the major ing all controls, directions for performing assays, and any allele, Del variant exhibits increased PDE3A expression in necessary Software for analysis and presentation of results. In response to agents that increase cAMP. This provided a Some embodiments, individual probes and reagents for detec molecular explanation of tolerance to PDE3 inhibitors. In tion of PDE3A polymorphisms are provided as analyte spe contrast, patients who are Ins/Ins exhibit no Such up-regula cific reagents. In other embodiments, the kits are provided as tion of PDE3A gene expression in response to cAMP aug in vitro diagnostics. menting agents, and as a result may not be predisposed to drug tolerance. V. EXAMPLES Example 2 0311. The following examples are given for the purpose of illustrating various embodiments of the invention and are not Clinical Use of Enoximone meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present inven 0315 Patients. tion is well adapted to carry out the objects and obtain the 0316 Inclusion criteria can include: age >18 years; heart ends and advantages mentioned, as well as those objects, ends failure (HF) caused by ischemic or nonischemic cardiomy and advantages inherent herein. The present examples, along opathy; LV systolic dysfunction shown by an ejection frac with the methods described herein are presently representa tion (EF) s30%, detected on radionuclide ventriculography, tive of preferred embodiments, are exemplary, and are not two-dimensional echocardiography, or nuclear magnetic intended as limitations on the scope of the invention. Changes resonance imaging; an echocardiographically determined LV therein and other uses which are encompassed within the end-diastolic diameter >3.2 cm/m or 26.0 cm; symptoms of spirit of the invention as defined by the scope of the claims dyspnoea or fatigue at rest or at minimal exertion New York will occur to those skilled in the art. Heart Association (NYHA) class III-IV for >2 months; at US 2013/0203827 A1 Aug. 8, 2013 22 least one hospitalization or two outpatient visits requiring 0348 U.S. Pat. No. 5,788,983 intravenous diuretic or vasodilator therapy within 12 months 0349 U.S. Pat. No. 5,840,873 before screening; and optimal medical therapy including 0350 U.S. Pat. No. 5,843,640 diuretics, beta-blockers, and angiotensin-converting enzyme 0351 U.S. Pat. No. 5,843,650 (ACE)-inhibitors or angiotensin receptor blockers (ARBs) 0352 U.S. Pat. No. 5,843,651 unless intolerant or contraindicated. 0353 U.S. Pat. No. 5,843,663 0317 Dosing. 0354 U.S. Pat. No. 5,846,708 0318 Drug dose can be 25-100 mg three times daily. 0355 U.S. Pat. No. 5,846,709 Patients are re-evaluated at 1 and 2 weeks. Drug dose can be 0356 U.S. Pat. No. 5,846,717 up-titrated to 50 mg or more three times daily in patients 0357 U.S. Pat. No. 5,846,726 weighing more than 50 kg without renal and hepatic dysfunc- 0358 U.S. Pat. No. 5,846,729 tion. Patients will undergo follow-up clinical visits at 1, 2, 4, 0359 U.S. Pat. No. 5,846,783 6, 8, 9, 12 months and, in the following years, every 4 months. 0360 U.S. Pat. No. 5,849,481 Each visit includes clinical examination and blood sampling 0361 U.S. Pat. No. 5,849,483 for analysis of serum bilirubin, creatinine, and potassium. 0362 U.S. Pat. No. 5,849,486 0319 All of the compositions and methods disclosed and 0363 U.S. Pat. No. 5,849,487 claimed herein can be made and executed without undue 0364 U.S. Pat. No. 5,849,497 experimentation in light of the present disclosure. While the 0365 U.S. Pat. No. 5,849,546 compositions and methods of this invention have been 0366 U.S. Pat. No. 5,849,547 described in terms of preferred embodiments, it will be appar- 0367 U.S. Pat. No. 5,851,770 ent to those of skill in the art that variations may be applied to 0368 U.S. Pat. No. 5,851,772 the compositions and methods and in the steps or in the 0369 U.S. Pat. No. 5,853,990 sequence of steps of the methods described herein without 0370 U.S. Pat. No. 5,853,992 departing from the concept, spirit and scope of the invention. 0371 U.S. Pat. No. 5,853,993 More specifically, it will be apparent that certain agents which 0372 U.S. Pat. No. 5,856,092 are both chemically and physiologically related may be Sub- 0373 U.S. Pat. No. 5,858,652 stituted for the agents described herein while the same or 0374 U.S. Pat. No. 5,861,244 similar results would beachieved. All such similar substitutes 0375 U.S. Pat. No. 5,863,732 and modifications apparent to those skilled in the art are 0376 U.S. Pat. No. 5,863,753 deemed to be within the spirit, scope and concept of the 0377 U.S. Pat. No. 5,866,331 invention as defined by the appended claims. 0378 U.S. Pat. No. 5,866,337 0379 U.S. Pat. No. 5,866,366 VI. REFERENCES 0380 U.S. Pat. No. 5,900,481 0320. The following references, to the extent that they 0381 U.S. Pat. No. 5,905,024 provide exemplary procedural or other details Supplementary 0382 U.S. Pat. No. 5,910.407 to those set forth herein, are specifically incorporated herein 0383 U.S. Pat. No. 5,912,124 by reference. 0384 U.S. Pat. No. 5,912,145 0321 U.S. Pat. No. 4,582,788 0385 U.S. Pat. No. 5,912,148 0322 U.S. Pat. No. 4,627,429 0386 U.S. Pat. No. 5,916,776 0323 U.S. Pat. No. 4,659,774 0387 U.S. Pat. No. 5,916,779 0324 U.S. Pat. No. 4,683,194 0388 U.S. Pat. No. 5,919,626 0325 U.S. Pat. No. 4,683,195 0389 U.S. Pat. No. 5,919,630 0326 U.S. Pat. No. 4,683.202 0390 U.S. Pat. No. 5,922,574 0327 U.S. Pat. No. 4,784,857 0391 U.S. Pat. No. 5,925,517 E: YS.S. EasPat. N.No. 4, s:s 0392 U.S. Pat. No. 5,925,525 0330 U.S. Pat. No. 4,883,750 E. S.E.N.E. 0331 U.S. Pat. No. 4,946,773 s- 1- Yos 0332 U.S. Pat. No. 4,959,463 0395 U.S. Pat. No. 5,928,870 0333 U.S. Pat. No. 4,965,188 0396 U.S. Pat. No. 5,928.905 0334 U.S. Pat. No. 5,126,145 0397 U.S. Pat. No. 5,928,906 0336 U.S. Pat No. 5,141,813 0399 U.S. Pat No. 5,932,413 0337 U.S. Pat. No. 5,169,766 04.00 U.S. Pat. No. 5,932,451 0338 U.S. Pat. No. 5,264,566 04.01 U.S. Pat. No. 5,935,791 0339 U.S. Pat. No. 5.279,721 04.02 U.S. Pat. No. 5,935,825 0340 U.S. Pat. No. 5,428,148 0403 U.S. Pat. No. 5,939,291 0341 U.S. Pat. No. 5,554,744 04.04 U.S. Pat. No. 5,942,391 0342 U.S. Pat. No. 5,574,146 04.05 U.S. Pat. No. 5,952,174 0343 U.S. Pat. No. 5,602,244 0406 U.S. Pat. No. 6,113,940 0344 U.S. Pat. No. 5,605,798 0407 U.S. Pat. No. 4,656,127 (0345 U.S. Pat. No. 5,645,897 0408 U.S. Pat. No. 4,682,195 0346 U.S. Pat. No. 5,662,925 04.09 Ausubel et al., Current Protocols in Molecular Biol 0347 U.S. Pat. No. 5,705,629 ogy, John Wiley & Sons, NY, 1989. US 2013/0203827 A1 Aug. 8, 2013

0410 Barany, et al., Proc. Natl. Acad. Sci. USA, 88:189 0446 Modrich, Ann. Rev. Genet., 25:229-253, 1991. 193, 1991. 0447 Movsesian, J. Am. Coll. Cardiol., 34:318-324, 0411 Bellus, J. Macromol. Sci. Pure Appl. Chem. A31 1999. (1): 1355-1376, 1994. 0448 Mullis et al., Cold Spring Harbor Symp. Ouant. 0412 de Arruda et al., Expert. Rev. Mol. Diagn., 2:487 Biol. 51:263-273, 1986. 496, 2002. 0449 Nickerson et al., Proc. Natl. Acad. Sci. USA, 0413 Ding et al., Circulation, 111:2469-2476, 2005. 87:8923-8927, 1990. 0414. Durand et al., Ann. Med., 27:311-317, 1995. 0450 Nyrenet al., Anal. Biochem. 208:171-175, 1993. 0415 European Applin. 201,184 0451. Ohara et al., Proc. Natl. Acad. Sci. USA, 86:5673 0416 European Applin. 237,362 5677, 1989. 0417 European Applin. 258,017 0452. Orita et al., Genomics, 5:874-879, 1989. 0418 European Applin. 266.032 0453 PCT Applin. PCT/US87/00880 04.19 European Applin. 320 308 0454 PCT Applin. PCT/US89/01025 0420 European Applin. 329,822 0455 PCT Appln. WO 88/10315 0421 European Applin. 50,424 0456 PCT Applin. WO 89/06700 0422 European Applin. 84,796 0457. PCT Appln. WO 93/22456 0423 French Patent 2,650,840 0458 PCT Appln. WO95/11995 0424. Froehler et al., Nucleic Acids Res., 14(13):5399 0459 PCT Appln. WO 89/06700 5407, 1986. 0460 PCT Applin. WO 90/01069 0425. Frohman, In: PCR Protocols. A Guide To Methods 0461 PCT Applin. WO 91/02087 And Applications, Academic Press, N.Y., 1990. 0462. PCT Applin. WO92/15712 0426 Great Britain Applin. 2 202328 0463 Physicians Desk Reference 0427 Halushka et al., Nat. Genet., 22(3):239-247, 1999. 0464 Prezant et al., Hum. Mutat., 1:159-164, 1992. 0428 Humphries, et al., In: Molecular Diagnosis of 0465 Reinhardt et al., J. Clin. Invest., 95:1528-1538, Genetic Diseases, Elles (Ed.), 321-340, 1996. 1995. 0429 Inazuka et al., Genome Res, 7(11):1094-1103, 1997. 0466 Remington's Pharmaceutical Sciences, 15th Edi 0430. Innis et al., Proc. Natl. Acad. Sci. USA, 85(24): tion, pages 1035-1038 and 1570-1580. 1990. 9436-9440, 1988. 0467 Ruano et al., Nucl. Acids Res., 19:6877-6882, 1991. 0431 Johnson et al., Nat. Genet., 29(2):233-237, 2001. 0468 Ruano et al., Nucl. Acids Res., 17:8392, 1989. 0432 Jones, Nature, 199:280-282, 1963. 0469 Sambrooketal. In: Molecular cloning, Cold Spring 0433 Ke and Cardon Bioinformatics, 19(2):287-288, Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. 2003. 0470 Sanger et al., J. Molec. Biol., 94:441, 1975. 0434 Klaassen's The Pharmacological Basis of Thera 0471 Shakur et al., Prog. Nucleic Acids Res. Mol. Biol., peutics 66:241-277, 2000. 0472. Sheffield et al., Proc. Natl. Acad. Sci. USA, 86:232 0435 Komher, et al., Nucl. Acids. Res. 17:7779-7784, 236, 1989. 1989. 0473. Small et al., Methods Enzymol, 343:459-75, 2002. 0436 Kuppuswamy et al., Proc. Natl. Acad. Sci. USA, 0474 Sokolov, Nucl. Acids Res. 18:3671, 1990. 88: 1143-1147, 1991. 0475 Stevens et al., Biotechniques, 34:198-203, 2003. 0437. Kwoh et al., Proc. Natl. Acad. Sci. USA, 86: 1173, 0476 Syvanen et al., Genomics 8:684-692, 1990. 1989. 0477 Taillon-Miller et al., Genome Res, 8(7):748-754, 0438 Kwok and Chen, Curr Issues Mol. Biol., 5(2):43-60. 1998. 2003. 0478. The Merck Index, Eleventh Edition 0439 Kwok et al., Genomics, 23(1):138-144, 1994. 0479. Turki et al., J. Clin. Invest., 95: 1635-1641, 1995. 0440 Kwok et al., Genomics, 31(1):123-6, 1996. 0480. Ugozzollet al., GATA 9:107-112, 1992. 0441 Kwok, Annu. Rev. Genomics Hum. Genet., 2:235 0481 Walker et al., Proc. Natl. Acad. Sci. USA, 89:392 258, 2001. 396, 1992. 0442 Landegren et al., Science 241:1077-1080, 1988. 0482 Wang and Dhalla, Mol. Cell Biochem., 214:131 0443) Lu et al., Biopolymers, 73:606–613, 2004. 155, 2000. 0444 Maxam et al., Proc. Natl. Acad. Sci. USA, 74:560, 0483 Wartellet al., Nucl. Acids Res., 18:2699-2706, 1990. 1977. 0484 Winter et al., Proc. Natl. Acad. Sci. USA, 82:7575, 0445 Meyers et al., Science, 230:1242, 1985. 1985.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 7

<21 Os SEQ ID NO 1 &211s LENGTH: 15 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer US 2013/0203827 A1 Aug. 8, 2013 24

- Continued <4 OOs, SEQUENCE: 1 tatic tactta tdtca 15

<210s, SEQ ID NO 2 &211s LENGTH: 29 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 2 ttct catat c tactitatgtc. ataat atta 29

<210s, SEQ ID NO 3 &211s LENGTH: 1874 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer <4 OOs, SEQUENCE: 3 gccaacagat atgaatgatt titccaatcaa gotttitt cac toccatgtgt cccagg tagg 6 O atctgg cata taatcat at gtctaaaaag gaaact catgaaagtatgtg togtgctag 12 O ggggct catt tdt ctittggc aaaga cattg ctgct cotgt titc cittaatt attaalacaga 18O tggtgcc.cac togc cattgac tagctg.ccca act catactic ctdtgtggta gcaca attct 24 O ggacatctga cagagagaag ctatacctitt at agtttctic at atc tactt atgtcataat 3OO attaatat cit gcc titc tact agaac aggct gct tcc catgaagctgttta aaacttaagt 360 catc.ttct co ccctgtcc cc ataaattaat tittat ct caa goat citcctt ttggctttitt 42O ttattgctitc atgtctacat acaaaataca gtggcatatt gaatcaatta t ctaaatgct 48O c taggctgta aatacactitt aacctggaat gtcaagataa ttalagacaaa attggattat 54 O talagattatt attagtgagg gcaaatgaca t ct aagatag ccatgttaala agtggagtat 6OO catattaaaa agacaactag atcc.caggga acatcaatag agtttaagtic cattgaacag 660 at actgaatt Ctttitt cata atctgccalaa aaaaggittag ctitgaaaatt ttcttittagt 72 O ttct caaata t cacactgct gcagtacacg aacct titact cattaataac taaggtoctd 78O attitttitt ca tatgctittgc ticgaagatgt agtattittgc agc catagac agt cittctaa 84 O gayc to tcct agtgttaa.cc caccitatic ct cacct ct coc ttgagattitt tottt attitt 9 OO ttgatgaact atctgggctt ttaaactttgtta accttitt ttgaggatac gigt cacttaa 96.O t ct caatgta attitt actitt coacagt caa aaact attgt gtaatactica tdcactggat O2O ttaaatgact gctgcct citc ctitcc tittct ttittatacta ttgtggit ct a ggtaaggctg O8O attct tccat catttgaacc aac aggc.cag gottgggttct cataaag.ca gacct tccag 14 O Caggagcgac Caaaggatga cactgtcacc taaattgga citgctgttgt acctgacttg 2OO ggaa catctt taatcagac agtagaagtg gctgt cattt to aggga cag tagaaagtat 26 O gttggct citc atctgccaag taggcaaaca caatctttitt tttitttittitt toctitccaac 32O gttctaggga gct cagcctic agggctagoc goagc.ccc.cc acaccc.cggg gctg.cggtgg 38O gctg.cgcggt ggat.ca.gc.ct cagcagcc cc tict c cagcc titagggtga accggcc.gct 44 O titcc.ca.gcaa aggagcaatc gagctgaggg tagcgc.cticc ticcgcaggag ggggcgggag SOO

Ctcggctgag aaa.gctitt Co. tagggagttg cct taaagaa agaaag.cgga attgtcgatc 560

US 2013/0203827 A1 Aug. 8, 2013 26

- Continued aaatagaagt gigggagggaa aaaaaaag.ca aatct cottc. tcc cttctica ccctic cctitt 168O cittcto acco tocct tcc to tct tactc.gc ticcittct coc tocct coctt citgcggctgc 1740 cgctagt citc. tcggtctggc tict ct citccg acggg actta gcaacttctt atttct cagc 18OO c ccttgtc.ca tttitttitttt to catcctitt gccatgaatt ggatt 1845

<210s, SEQ ID NO 5 &211s LENGTH: 15 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic peptide <4 OOs, SEQUENCE: 5 Asp Gly Ala Thr Ala Asp Gly Ala Asp Trp Ala Gly Ala Thr Ala 1. 5 1O 15

<210s, SEQ ID NO 6 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer

<4 OOs, SEQUENCE: 6 c cactgcc at tdactagotg

<210s, SEQ ID NO 7 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic primer

<4 OO > SEQUENCE: 7 gccaaaagga gatccttgag at 22

1. A method for treating a patient with heart failure com digestion, allele-specific nucleic acid amplification, single prising treating the patient with an effective amount of a Stranded conformational polymorphism analysis, or allele PDE3A inhibitor after the patient is determined to be specific hybridization analysis. homozygous for an ectotropic viral integration site-1 protein 11. The method of claim 7, further comprising preparing a (Evi-1) insertional polymorphism in the promoter of the report containing information regarding the genotype of one phosphodiesterase type 3A (PDE3A) gene. or more PDE3A genes of the patient. 2. The method of claim 1, wherein the PDE3A inhibitor is 12. The method of claim 7, wherein the patient has symp aminone, cilostazol, milrinone, quaZinone, SiguaZodan, tre toms of or has been diagnosed with heart failure. quinsin, or enoXimone. 13. The method of claim 7, wherein the patient is deter 3. The method of claim 1, wherein the PDE3A inhibitor is mined to be homozygous for the insertional polymorphism in enoXimone. the PDE3A gene and is subsequently treated with a PDE3A 4.-6. (canceled) inhibitor. 7. A method for evaluating a heart failure patient compris 14. The method of claim 13, wherein the PDE3A inhibitor ing analyzing a biological sample from the patient for the is aminone, cilostaZol, milrinone, quaZinone, siguaZodan, tre presence of an ectotropic viral integration site-1 protein (Evi quinsin, enoXimone. 1) deletion or insertional polymorphism in the promoter of 15. The method of claim 14, wherein the PDE3A inhibitor one or more phosphodiesterase type 3A (PDE3A) genes of is enoXimone. the patient, wherein a patient determined to be homozygous 16.-18. (canceled) for the insertional polymorphism is responsive to PDE3A 19. The method of claim 7, wherein the patient is deter inhibitor therapy. mined not to be homozygous for the insertional polymor 8. The method of claim 7, further comprising obtaining a phism in the PDE3A gene and is subsequently treated with a biological sample from the patient. non-PDE3A inhibitor treatment. 9. (canceled) 20. A method for evaluating a patient having or at risk of 10. The method of claim 7, wherein analyzing the sample developing heart failure for responsiveness to phosphodi comprises performing nucleic acid sequencing, restriction esterase inhibitor therapy comprising obtaining a determina US 2013/0203827 A1 Aug. 8, 2013 27 tion of the patient’s genotype for a deletion or insertional 34. The method of claim 33, wherein the allele specific polymorphism in a phosphodiesterase type 3A (PDE3A) amplification primer orallele specific nucleic acid probe spe gene, wherein homozygosity for the insertional polymor cifically hybridizes with SEQID NO:2 under high stringency phism is predictive of responsiveness to phosphodiesterase conditions. inhibitor therapy for treating heart failure in the patient. 35. The method of claim 30, wherein the nucleic acid 21.-29. (canceled) insertion is detected using nucleic acid amplification, nucleic 30. A method of determining efficacy of phosphodiesterase acid hybridization, restriction fragment length polymorphism type 3A (PDE3A) inhibition therapy comprising detecting a (RFLP) analysis, single stranded conformational polymor nucleic acid insertion having a nucleotide sequence of SEQ phism (SSCP) analysis, nucleic acid sequencing, denaturing ID NO:2 in a promoter of one or both phosphodiesterase type high performance liquid chromatography, comparative 3A (PDE3A) genes of a subject. genome hybridization, and/or Southern blotting. 31. The method of claim 30, wherein the nucleic acid insertion is within the 2000 nucleotides 5' of the transcription 36. The method of claim 35, wherein nucleic acid ampli start site. fication comprises polymerase chain reaction amplification 32. The method of claim 30, wherein detection of the or ligase chain reaction amplification. nucleic acid insertion uses at least one oligonucleotide that 37. The method of claim 35, wherein nucleic acid hybrid anneals to SEQID NO:3, SEQID NO:4, or SEQID NO:3 and ization detection method comprises an allele specific oligo SEQID NO:4. nucleotide probe or a microarray of nucleic acid probes. 33. The method of claim 30, wherein the detection method comprises at least one allele specific amplification primer or 38.-46. (canceled) allele specific nucleic acid probe.