Treatment of aortic stenosis with inhibitors of alkaline phosphatase (ALPL)

Abstract

The present disclosure relates generally to the treatment of subjects having aortic stenosis, or at risk of developing aortic stenosis, by administering an Alkaline Phosphatase (ALPL) inhibitor to the subject.

Description

[0001] Treatment Of Aortic Stenosis With Inhibitors Of Alkaline Phosphatase (ALPL)

[0002] Reference To Sequence Listing

[0003] This application includes a Sequence Listing filed electronically as an XML file named 381204407SEQ, created on June 10, 2025, with a size of 11,063,421 bytes. The Sequence Listing is incorporated herein by reference.

[0004] Field

[0005] The present disclosure relates generally to the treatment of subjects having aortic stenosis or at risk of developing aortic stenosis, by administering an Alkaline Phosphatase (ALPL) inhibitor to the subject.

[0006] Background

[0007] The aorta is the main artery that carries blood from the heart to the rest of the body. Blood flows out of the heart and into the aorta through the aortic valve. Aortic stenosis mainly occurs due to the buildup of calcium deposits that narrow the valve (i.e., calcific aortic stenosis). In aortic stenosis, the aortic valve does not open fully, which decreases blood flow from the heart. As the aortic valve narrows, the left ventricle has to work harder to pump blood out through the valve. To do this extra work, the muscles in the ventricle walls become thicker, which can lead to chest pain. As the pressure continues to rise, blood may back up into the lungs. Severe aortic stenosis can limit the amount of blood that reaches the brain and the rest of the body. Aortic stenosis may be present from birth (congenital), but most often it develops later in life. Children with aortic stenosis may have other conditions present from birth. Calcium buildup of the valve happens sooner in people who are born with abnormal aortic or bicuspid valves. In rare cases, calcium buildup can develop more quickly when a person has received chest radiation (such as for cancer treatment). Another cause of aortic stenosis is rheumatic fever. This condition can develop after strep throat or scarlet fever. Valve problems do not develop for 5 to 10 years or longer after rheumatic fever occurs. Aortic stenosis occurs in about 2% of people over 65 years of age, and it occurs more often in men than in women.

[0008] The Alkaline phosphatase (ALPL) gene is located on chromosome lp36.1 and consists of 12 exons distributed over 50 kb, encoding for a tissue non-specific alkaline phosphatase (TNSALP). TNSALP is a homodimeric enzyme where each monomer is composed of 524 amino acids (57.2 kDa). Alkaline phosphatase (ALP) is an enzyme that is found throughout the human body. ALP is often considered a liver enzyme because it is primarily found in the liver. High levels of ALP may indicate liver disease or certain bone disorders. High alkaline phosphatase levels in the liver may indicate the following conditions: cholestasis of pregnancy, cirrhosis of the liver, hepatitis, biliary atresia, biliary stricture, biliary obstruction due to cancer, and mononucleosis. High bone alkaline phosphatase levels may indicate the following conditions: bone metastasis, Paget's disease of the bone (osteitis derformans), osteogenic sarcoma, healing fractures, hyperparathyroidism, hyperthyroidism, osteomalacia, and untreated celiac disease.

[0009] Summary

[0010] The present disclosure provides methods of treating a subject having aortic stenosis, or at risk of developing aortic stenosis, the methods comprising administering an ALPL inhibitor to the subject.

[0011] The present disclosure also provides methods of treating a subject having aortic stenosis, or at risk of developing aortic stenosis by administering an aortic stenosis therapeutic agent or aortic stenosis therapy, the method comprising: determining or having determined whether the subject has an ALPL variant nucleic acid molecule by: obtaining or having obtained a biological sample from the subject; and performing or having performed a sequence analysis on the biological sample to determine if the subject has a genotype comprising an ALPL variant nucleic acid molecule; and administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in a standard dosage amount to a subject that is ALPL reference, and / or administering an ALPL inhibitor to the subject; administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount to a subject that is heterozygous for the ALPL variant nucleic acid molecule, and / or administering an ALPL inhibitor to the subject; or administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount to a subject that is homozygous for the ALPL variant nucleic acid molecule; wherein the presence of a genotype having the ALPL variant nucleic acid molecule indicates the subject has a decreased risk of developing aortic stenosis.

[0012] The present disclosure also provides methods of identifying a subject having an increased risk of developing aortic stenosis, the methods comprising: determining or having determined the presence or absence of an ALPL variant nucleic acid molecule in a biological sample obtained from the subject, wherein: when the subject is ALPL reference, then the subject has an increased risk of developing aortic stenosis; and when the subject is heterozygous or homozygous for the ALPL variant nucleic acid molecule, then the subject has a decreased risk of developing aortic stenosis.

[0013] The present disclosure also provides aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis for use in the treatment and / or prevention of aortic stenosis in a subject having an ALPL variant nucleic acid molecule.

[0014] The present disclosure provides ALPL inhibitors for use in the treatment and / or prevention of aortic stenosis in a subject that is ALPL reference or is heterozygous for an ALPL variant nucleic acid molecule.

[0015] Description

[0016] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0017] Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-expressed basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0018] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0019] As used herein, the term "about" means that the recited numerical value is approximate and small variations would not significantly affect the practice of the disclosed embodiments. Where a numerical value is used, unless indicated otherwise by the context, the term "about" means the numerical value can vary by ±10% and remain within the scope of the disclosed embodiments. As used herein, the term "comprising" may be replaced with "consisting" or "consisting essentially of" in particular embodiments as desired.

[0020] As used herein, the term "isolated", in regard to a nucleic acid molecule or a polypeptide, means that the nucleic acid molecule or polypeptide is in a condition other than its native environment, such as apart from blood and / or animal tissue. In some embodiments, an isolated nucleic acid molecule or polypeptide is substantially free of other nucleic acid molecules or other polypeptides, particularly other nucleic acid molecules or polypeptides of animal origin. In some embodiments, the nucleic acid molecule or polypeptide can be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. When used in this context, the term "isolated" does not exclude the presence of the same nucleic acid molecule or polypeptide in alternative physical forms, such as dimers or alternately phosphorylated or derivatized forms.

[0021] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "polynucleotide", or "oligonucleotide" can comprise a polymeric form of nucleotides of any length, can comprise DNA and / or RNA, and can be single-stranded, doublestranded, or multiple stranded. One strand of a nucleic acid also refers to its complement.

[0022] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (such as, for example, horses, cows, and pigs), companion animals (such as, for example, dogs and cats), laboratory animals (such as, for example, mice, rats, and rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under the care of a physician.

[0023] It has been observed in accordance with the present disclosure that ALPL variant nucleic acid molecules (whether these variants are homozygous or heterozygous in a particular subject) associate with a decreased risk of developing aortic stenosis. It is believed that ALPL variant nucleic acid molecules have not been associated with aortic stenosis in humans. Therefore, subjects that are ALPL reference or heterozygous for an ALPL variant nucleic acid molecule may be treated with an ALPL inhibitor such that aortic stenosis is inhibited or prevented, the symptoms thereof are reduced or prevented, and / or development of symptoms is repressed or prevented. It is also believed that such subjects having aortic stenosis may further be treated with aortic stenosis therapeutic agents or aortic stenosis therapy that treat or inhibit aortic stenosis. For purposes of the present disclosure, any particular subject, such as a human, can be categorized as having one of three ALPL genotypes: i) ALPL reference; ii) heterozygous for ALPL variant nucleic acid molecule; or iii) homozygous for an ALPL variant nucleic acid molecule. A subject is ALPL reference when the subject does not have a copy of an ALPL variant nucleic acid molecule. A subject is heterozygous for an ALPL variant nucleic acid molecule when the subject has a single copy of an ALPL variant nucleic acid molecule. A subject is homozygous for an ALPL variant nucleic acid molecule when the subject has two copies of an ALPL variant nucleic acid molecule.

[0024] An ALPL variant nucleic acid molecule can be any nucleic acid molecule (such as, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding a variant ALPL polypeptide having a partial loss-of-function, a complete loss-of-function, a predicted partial loss-of-function, or a predicted complete loss-of-function. In some embodiments, the ALPL variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the ALPL variant nucleic acid molecule comprises a variation in a coding region. In some embodiments, the ALPL variant nucleic acid molecule does not comprise a variation in a non-coding region, except for splice acceptor regions (two bases before the start of any exon except the first). A subject who has an ALPL polypeptide having a partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for ALPL. In some embodiments, the ALPL variant nucleic acid molecule is any nucleic acid molecule resulting in decreased or aberrant expression of ALPL mRNA or polypeptide. In some embodiments, the ALPL variant nucleic acid molecule is associated with a reduced in vitro response to ALPL ligands compared with reference ALPL. In some embodiments, the ALPL variant nucleic acid molecule is an ALPL variant nucleic acid molecule that results or is predicted to result in a premature truncation of an ALPL polypeptide compared to the human reference genome sequence. In some embodiments, the ALPL variant nucleic acid molecule is a variant that is predicted to be damaging to the protein function (and hence, in this case, protective to the human) by in vitro prediction algorithms such as Polyphen, SIFT, or similar algorithms. In some embodiments, the ALPL variant nucleic acid molecule is a variant that causes or is predicted to cause a nonsynonymous amino-acid substitution in an ALPL nucleic acid molecule and whose allele frequency is less than 1 / 100 alleles in the population from which the subject is selected. In some embodiments, the ALPL variant nucleic acid molecule is any rare missense variant (allele frequency < 0.1%; or 1 in 1,000 alleles), or any splice-site, stop-gain, start-loss, stop-loss, frameshift, or in-frame indel, or other frameshift ALPL variant.

[0025] In any of the embodiments described herein, the ALPL variant nucleic acid molecule can include variations at positions of chromosome 1 (positions 21,509,397-21,578,410 forward strand; GRCh38:CM000663.2) using the nucleotide sequence of the ALPL reference genomic nucleic acid molecule (see, ENSG00000162551 annotated in the in the Ensembl database (URL: world wide web at "uswest.ensembl.org / Homo_sapiens / Gene / )) as a reference sequence. The ALPL gene has 7 transcripts (e.g., ENST00000374840.8, ENST00000539907.5, ENST00000374832.5, EN5T00000540617.5, ENST00000374830.2, ENST00000374829.2, and ENST00000468526.1). The sequences provided in these transcripts for the ALPL genomic nucleic acid molecule are only an exemplary sequence. Other sequences for the ALPL genomic nucleic acid molecule are also possible. Exemplary ALPL variant nucleic acid molecules include, but are not limited to, those recited in Table 4.

[0026] For subjects that are genotyped or determined to be ALPL reference, such subjects have an increased risk of developing aortic stenosis. For subjects that are genotyped or determined to be either ALPL reference or heterozygous for an ALPL variant nucleic acid molecule, such subjects or subjects can be treated with an ALPL inhibitor.

[0027] In any of the embodiments described herein, the subject in whom aortic stenosis is prevented by administering the ALPL inhibitor can be anyone at risk for developing aortic stenosis including, but not limited to, subjects with a genetic predisposition for developing aortic stenosis. Additional risk factors for aortic stenosis include, but are not limited to, older age, certain heart conditions present at birth (congenital heart disease) such as a bicuspid aortic valve, a history of infections that can affect the heart, having cardiovascular risk factors (e.g., diabetes, high cholesterol, and high blood pressure), chronic kidney disease, history of radiation therapy to the chest. In some embodiments, administering an ALPL inhibitor to a subject having aortic stenosis may be carried out to prevent development of another of aortic stenosis in a subject who has already had aortic stenosis. In any of the embodiments described herein, the methods can be used to improve aortic stenosis.

[0028] In any of the embodiments described herein, the ALPL predicted loss-of-function polypeptide can be any ALPL polypeptide having a partial loss-of-function, a complete loss-of- function, a predicted partial loss-of-function, or a predicted complete loss-of-function. Any one or more (i.e., any combination) of the ALPL variant nucleic acid molecules described herein can be used within any of the methods described herein to determine whether a subject has an increased or decreased risk of developing aortic stenosis. The combinations of particular variants can form a mask used for statistical analysis of the particular correlation of ALPL and an increased or decreased risk of developing aortic stenosis. In some embodiments, the mask used for statistical analysis of the particular correlation of ALPL and an increased or decreased risk of developing aortic stenosis can exclude any one or more of these ALPL variant nucleic acid molecules described herein.

[0029] In any of the embodiments described herein, the subject can have aortic stenosis. In any of the embodiments described herein, the subject can be at risk of developing aortic stenosis.

[0030] In any of the embodiments described herein, the methods can be used to treat a complication or co-morbidity of aortic stenosis, or reduce the risk of developing the same. Complications and co-morbidities of aortic stenosis include, but are not limited to, heart failure, stroke, blood clots, bleeding, heart rhythm problems (arrhythmias), infections that affect the heart (e.g., endocarditis), and death.

[0031] The present disclosure provides methods of treating a subject having aortic stenosis or at risk of developing aortic stenosis, the methods comprising administering an ALPL inhibitor to the subject.

[0032] In some embodiments, the ALPL inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNAs (siRNAs), and short hairpin RNAs (shRNAs). Such inhibitory nucleic acid molecules can be designed to target any region of an ALPL nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within an ALPL genomic nucleic acid molecule or mRNA molecule and decreases expression of the ALPL polypeptide in a cell in the subject. In some embodiments, the ALPL inhibitor comprises an antisense molecule that hybridizes to an ALPL genomic nucleic acid molecule or mRNA molecule and decreases expression of the ALPL polypeptide in a cell in the subject. In some embodiments, the ALPL inhibitor comprises an siRNA that hybridizes to an ALPL genomic nucleic acid molecule or mRNA molecule and decreases expression of the ALPL polypeptide in a cell in the subject. In some embodiments, the ALPL inhibitor comprises an shRNA that hybridizes to an ALPL genomic nucleic acid molecule or mRNA molecule and decreases expression of the ALPL polypeptide in a cell in the subject.

[0033] In some embodiments, the antisense nucleic acid molecules comprise or consist of any of the nucleotide sequences represented by SEQ ID NOs: 1-2,666. In some embodiments, the siRNA molecules comprise or consist of any of the nucleotide sequences (sense and antisense strands presented one after the other) represented by SEQ ID NOs: 2,667-8,706 (e.g., the sense strand is, for example, SEQ ID NO: 2,667 and the corresponding antisense strand is SEQ ID NO: 2,668; the sense strand is, for example, SEQ ID NO: 8,705 and the corresponding antisense strand is SEQ ID NO: 8,706; etc.).

[0034] The inhibitory nucleic acid molecules can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence, such as in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecules can be within a vector or as an exogenous donor sequence comprising the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. The inhibitory nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (such as, for example, fluorophore) or indirectly detectable (such as, for example, hapten, enzyme, or fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal. The term "label" can also refer to a "tag" or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag along with an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and examined using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, an epitope tag, or the Fc portion of immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels.

[0035] The inhibitory nucleic acid molecules can comprise, for example, nucleotides or nonnatural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include a nucleotide that contains a modified base, sugar, or phosphate group, or that incorporates a non-natural moiety in its structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophor-labeled nucleotides.

[0036] The inhibitory nucleic acid molecules can also comprise one or more nucleotide analogs or substitutions. A nucleotide analog is a nucleotide which contains a modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include, but are not limited to, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (such as, for example, 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0037] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-a I ky l-O-al kyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted Cnoal ky I or Cz-ioalkenyl, and Cz-ioalkynyl. Exemplary 2' sugar modifications also include, but are not limited to, -O[(CH2)nO]mCH3, -O(CH2)nOCH3, -O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)n-ONH2, and -O(CH2)r>ON[(CH2)nCH3)]2, where n and m, independently, are from 1 to about 10. Other modifications at the 2' position include, but are not limited to, Ci-wa I kyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars can also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties in place of the pentofu ranosyl sugar.

[0038] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. These phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0039] In some embodiments, the antisense nucleic acid molecules are gapmers, whereby the first one to seven nucleotides at the 5' and 3' ends each have 2'-methoxyethyl (2'-MOE) modifications. In some embodiments, the first five nucleotides at the 5' and 3' ends each have 2'-MOE modifications. In some embodiments, the first one to seven nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, each of the backbone linkages between the nucleotides is a phosphorothioate linkage.

[0040] In some embodiments, the siRNA molecules have termini modifications. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, 5'-phosphate analogs that cannot be hydrolyzed, such as 5'-(E)-vinyl-phosphonate are used.

[0041] In some embodiments, the siRNA molecules have backbone modifications. In some embodiments, the modified phosphodiester groups that link consecutive ribose nucleosides have been shown to enhance the stability and in vivo bioavailability of siRNAs The non-ester groups (-OH, =0) of the phosphodiester linkage can be replaced with sulfur, boron, or acetate to give phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, substituting the phosphodiester group with a phosphotriester can facilitate cellular uptake of siRNAs and retention on serum components by eliminating their negative charge. In some embodiments, the siRNA molecules have sugar modifications. In some embodiments, the sugars are deprotonated (reaction catalyzed by exo- and endonucleases) whereby the 2'-hydroxyl can act as a nucleophile and attack the adjacent phosphorous in the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0042] In some embodiments, the siRNA molecules have base modifications. In some embodiments, the bases can be substituted with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0043] In some embodiments, the siRNA molecules are conjugated to lipids. Lipids can be conjugated to the 5' or 3' termini of siRNA to improve their in vivo bioavailability by allowing them to associate with serum lipoproteins. Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids, such as palmitate and tocopherol.

[0044] In some embodiments, a representative siRNA has the following formula:

[0045] Sense: mN*mN i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN /

[0046] Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N wherein: "N" is the base; "2F" is a 2'-F modification; "m" is a 2'-O-methyl modification, "I" is an internal base; and "*" is a phosphorothioate backbone linkage.

[0047] The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vectors comprise any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vectors can be viral or nonviral vectors capable of transporting a nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (such as, for example, a circular double-stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno- associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast artificial chromosomes (YACs), Epstein-Barr (EBV)-derived episomes, and other expression vectors known in the art. The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the compositions comprise a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic-acid) (PLGA) microspheres, liposomes, micelles, inverse micelles, lipid cochleates, and lipid microtubules. A carrier may comprise a buffered salt solution such as PBS, HBSS, etc.

[0048] In some embodiments, the ALPL inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks at a recognition sequence(s) or a DNA-binding protein that binds to a recognition sequence within an ALPL genomic nucleic acid molecule. The recognition sequence can be located within a coding region of the ALPL gene, or within regulatory regions that influence the expression of the gene. A recognition sequence of the DNA-binding protein or nuclease agent can be located in an intron, an exon, a promoter, an enhancer, a regulatory region, or any non-protein coding region. The recognition sequence can include or be proximate to the start codon of the ALPL gene. For example, the recognition sequence can be located about 10, about 20, about 30, about 40, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides from the start codon. As another example, two or more nuclease agents can be used, each targeting a nuclease recognition sequence including or proximate to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence including or proximate to the start codon, and one targeting a nuclease recognition sequence including or proximate to the stop codon, wherein cleavage by the nuclease agents can result in deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break into a desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.

[0049] Suitable nuclease agents and DNA-binding proteins for use herein include, but are not limited to, zinc finger protein or zinc finger nuclease (ZFN) pair, Transcription Activator-Like Effector (TALE) protein or Transcription Activator-Like Effector Nuclease (TALEN), or Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, and includes, for example, recognition sequences that are about 30-36 bp for a zinc finger protein or ZFN pair, about 15-18 bp for each ZFN, about 36 bp for a TALE protein or TALEN, and about 20 bp for a CRISPR / Cas guide RNA.

[0050] In some embodiments, CRISPR / Cas systems can be used to modify an ALPL genomic nucleic acid molecule within a cell. The methods and compositions disclosed herein can employ CRISPR-Cas systems by utilizing CRISPR complexes (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-directed cleavage of ALPL nucleic acid molecules.

[0051] Cas proteins generally comprise at least one RNA recognition or binding domain that can interact with gRNAs. Cas proteins can also comprise nuclease domains (such as, for example, DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, a wild type Cas9 protein and a wild type Cpfl protein (such as, for example, FnCpfl). A Cas protein can have full cleavage activity to create a double-strand break in an ALPL genomic nucleic acid molecule or it can be a nickase that creates a single-strand break in an ALPL genomic nucleic acid molecule. Additional examples of Cas proteins include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof. In some embodiments, a Cas system, such as Casl2a, can have multiple gRNAs encoded into a single crRNA. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain. Cas proteins can be provided in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternately, a Cas protein can be provided in the form of a nucleic acid molecule encoding the Cas protein, such as an RNA or DNA.

[0052] In some embodiments, targeted genetic modifications of ALPL genomic nucleic acid molecules can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize to one or more gRNA recognition sequences within a target genomic locus in the ALPL genomic nucleic acid molecule. The gRNA recognition sequence can include or be proximate to the start codon of an ALPL genomic nucleic acid molecule or the stop codon of an ALPL genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located from about 10, from about 20, from about 30, from about 40, from about 50, from about 100, from about 200, from about 300, from about 400, from about 500, or from about 1,000 nucleotides of the start codon or the stop codon.

[0053] The gRNA recognition sequences within a target genomic locus in an ALPL genomic nucleic acid molecule are located near a Protospacer Adjacent Motif (PAM) sequence, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. The canonical PAM is the sequence 5'-NGG-3' where "N" is any nucleobase followed by two guanine ("G") nucleobases. gRNAs can transport Cas9 to anywhere in the genome for gene editing, but no editing can occur at any site other than one at which Cas9 recognizes PAM. In addition, 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells. Generally, the PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the gRNA. The PAM can flank the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be flanked on the 3' end by the PAM. In some embodiments, the gRNA recognition sequence can be flanked on the 5' end by the PAM. For example, the cleavage site of Cas proteins can be about 1 to about 10, about 2 to about 5 base pairs, or three base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from S. pyogenes or a closely related Cas9 is used), the PAM sequence of the non- complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. As such, the PAM sequence of the complementary strand would be 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

[0054] A gRNA is an RNA molecule that binds to a Cas protein and targets the Cas protein to a specific location within an ALPL genomic nucleic acid molecule. An exemplary gRNA is a gRNA effective to direct a Cas enzyme to bind to or cleave an ALPL genomic nucleic acid molecule, wherein the gRNA comprises a DNA-targeting segment that hybridizes to a gRNA recognition sequence within the ALPL genomic nucleic acid molecule. Exemplary gRNAs comprise a DNA- targeting segment that hybridizes to a gRNA recognition sequence present within an ALPL genomic nucleic acid molecule that includes or is proximate to the start codon or the stop codon. For example, a gRNA can be selected such that it hybridizes to a gRNA recognition sequence that is located from about 5, from about 10, from about 15, from about 20, from about 25, from about 30, from about 35, from about 40, from about 45, from about 50, from about 100, from about 200, from about 300, from about 400, from about 500, or from about 1,000 nucleotides of the start codon or located from about 5, from about 10, from about 15, from about 20, from about 25, from about 30, from about 35, from about 40, from about 45, from about 50, from about 100, from about 200, from about 300, from about 400, from about 500, or from about 1,000 nucleotides of the stop codon. Suitable gRNAs can comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNAs can comprise 20 nucleotides.

[0055] The Cas protein and the gRNA form a complex, and the Cas protein cleaves the target ALPL genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site within or outside of the nucleic acid sequence present in the target ALPL genomic nucleic acid molecule to which the DNA-targeting segment of a gRNA will bind. For example, formation of a CRISPR complex (comprising a gRNA hybridized to a gRNA recognition sequence and complexed with a Cas protein) can result in cleavage of one or both strands in or near (such as, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the nucleic acid sequence present in the ALPL genomic nucleic acid molecule to which a DNA-targeting segment of a gRNA will bind.

[0056] Such methods can result, for example, in an ALPL genomic nucleic acid molecule in which a region of the ALPL genomic nucleic acid molecule is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within the target genomic locus in the ALPL genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (such as, for example, a second gRNA that hybridizes to a second gRNA recognition sequence), cleavage by the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0057] In some embodiments, the methods of treatment and / or prevention further comprise detecting the presence or absence of an ALPL variant nucleic acid molecule in a biological sample from the subject. In some embodiments, the ALPL variant nucleic acid molecule can be any of the ALPL variant nucleic acid molecules disclosed herein.

[0058] The present disclosure also provides methods of treating a subject with an aortic stenosis therapeutic agent or aortic stenosis therapy that treats or inhibits aortic stenosis, wherein the subject has aortic stenosis or is at risk of developing aortic stenosis. In some embodiments, the methods comprise determining whether the subject has an ALPL variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject and performing or having performed a sequence analysis on the biological sample to determine if the subject has a genotype comprising the ALPL variant nucleic acid molecule. In some embodiments, the methods further comprise administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in a standard dosage amount to a subject that is ALPL reference, and / or administering an ALPL inhibitor to the subject. In some embodiments, the methods further comprise administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount to a subject that is heterozygous for the ALPL variant nucleic acid molecule, and / or administering an ALPL inhibitor to the subject. In some embodiments, the methods further comprise administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount to a subject that is homozygous for the ALPL variant nucleic acid molecule. The presence of a genotype having the ALPL variant nucleic acid molecule indicates the subject has a decreased risk of developing aortic stenosis. In some embodiments, the subject is ALPL reference. In some embodiments, the subject is heterozygous for an ALPL variant nucleic acid molecule.

[0059] For subjects that are genotyped or determined to be either ALPL reference or heterozygous for an ALPL variant nucleic acid molecule, such subjects can be administered an ALPL inhibitor, as described herein.

[0060] Detecting the presence or absence of an ALPL variant nucleic acid molecule in a biological sample from a subject and / or determining whether a subject has an ALPL variant nucleic acid molecule can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject.

[0061] In some embodiments, when the subject is ALPL reference, the subject is administered an aortic stenosis therapeutic agent or aortic stenosis therapy that treats, prevents, or inhibits aortic stenosis in a standard dosage amount. In some embodiments, when the subject is heterozygous for an ALPL variant nucleic acid molecule, the subject is administered an aortic stenosis therapeutic agent or aortic stenosis therapy that treats, prevents, or inhibits aortic stenosis in a dosage amount that is the same as or less than a standard dosage amount.

[0062] In some embodiments, the treatment and / or prevention methods comprise detecting the presence or absence of an ALPL predicted loss-of-function polypeptide in a biological sample from the subject. In some embodiments, when the subject does not have an ALPL predicted loss-of-function polypeptide, the subject is administered an aortic stenosis therapeutic agent or aortic stenosis therapy that treats, prevents, or inhibits aortic stenosis in a standard dosage amount. In some embodiments, when the subject has an ALPL predicted loss- of-function polypeptide, the subject is administered an aortic stenosis therapeutic agent or aortic stenosis therapy that treats, prevents, or inhibits aortic stenosis in a dosage amount that is the same as or less than a standard dosage amount.

[0063] The present disclosure also provides methods of treating a subject with an aortic stenosis therapeutic agent or aortic stenosis therapy that treats or inhibits aortic stenosis, wherein the subject has aortic stenosis or is at risk of developing aortic stenosis. In some embodiments, the method comprises determining whether the subject has an ALPL predicted loss-of-function polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine if the subject has an ALPL predicted loss-of-function polypeptide. When the subject does not have an ALPL predicted loss-of-function polypeptide, the aortic stenosis therapeutic agent or aortic stenosis therapy is administered or continued to be administered to the subject in a standard dosage amount, and / or an ALPL inhibitor is administered to the subject. When the subject has an ALPL predicted loss-of-function polypeptide, the aortic stenosis therapeutic agent or aortic stenosis therapy is administered or continued to be administered to the subject in an amount that is the same as or less than a standard dosage amount, and / or an ALPL inhibitor is administered to the subject. The presence of an ALPL predicted loss-of-function polypeptide indicates the subject has a decreased risk of developing aortic stenosis. In some embodiments, the subject has an ALPL predicted loss-of-function polypeptide. In some embodiments, the subject does not have an ALPL predicted loss-of-function polypeptide.

[0064] The present disclosure also provides methods of preventing a subject from developing aortic stenosis by administering an aortic stenosis therapeutic agent or aortic stenosis therapy that prevents aortic stenosis. In some embodiments, the method comprises determining whether the subject has an ALPL predicted loss-of-function polypeptide by obtaining or having obtained a biological sample from the subject and performing or having performed an assay on the biological sample to determine if the subject has an ALPL predicted loss-of-function polypeptide. When the subject does not have an ALPL predicted loss-of-function polypeptide, the aortic stenosis therapeutic agent or aortic stenosis therapy is administered or continued to be administered to the subject in a standard dosage amount, and / or an ALPL inhibitor is administered to the subject. When the subject has an ALPL predicted loss-of-function polypeptide, the aortic stenosis therapeutic agent or aortic stenosis therapy is administered or continued to be administered to the subject in an amount that is the same as or less than a standard dosage amount, and / or an ALPL inhibitor is administered to the subject. The presence of an ALPL predicted loss-of-function polypeptide indicates the subject has a decreased risk of developing aortic stenosis. In some embodiments, the subject has an ALPL predicted loss-of- function polypeptide. In some embodiments, the subject does not have an ALPL predicted loss- of-function polypeptide.

[0065] Detecting the presence or absence of an ALPL predicted loss-of-function polypeptide in a biological sample from a subject and / or determining whether a subject has an ALPL predicted loss-of-function polypeptide can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the polypeptide can be present within a cell obtained from the subject.

[0066] In some embodiments, the ALPL inhibitor is a small molecule. In some embodiments, the small molecule is low molecular weight (< 900 daltons) organic compound.

[0067] In some embodiments, the ALPL inhibitor is an antibody, or antigen-binding fragment thereof. In some embodiments, the antibody, or antigen-binding fragment thereof, binds specifically to human ALPL. In some embodiments, the antibody is a fully human monoclonal antibody (mAb), or antigen-binding fragment thereof, that specifically binds and neutralizes, inhibits, blocks, abrogates, reduces, or interferes with, at least one activity of ALPL, in particular, human ALPL. In some embodiments, an antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with, an activity of ALPL by binding to an epitope of ALPL that is directly involved in the targeted activity of ALPL. In some embodiments, an antibody or fragment thereof can neutralize, inhibit, block, abrogate, reduce, or interfere with, an activity of ALPL by binding to an epitope of ALPL that is not directly involved in the targeted activity of ALPL, but the antibody or fragment binding thereto sterically or conformationally inhibits, blocks, abrogates, reduces, or interferes with, the targeted activity of ALPL. In some embodiments, an antibody or fragment thereof binds to an epitope of ALPL that is not directly involved in the targeted activity of ALPL (i.e., a non-blocking antibody), but the antibody or fragment binding thereto results in the enhancement of the clearance of ALPL from the circulation, compared to the clearance of ALPL in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, abrogating, reducing, or interfering with, an activity of ALPL. Clearance of ALPL from the circulation can be particularly enhanced by combining two or more different non-blocking antibodies that do not compete with one another for specific binding to ALPL.

[0068] The antibodies can be full-length (for example, an IgGl or lgG4 antibody) or may comprise only an antigen-binding portion (for example, a Fab, F(ab')2 or scFv fragment), and may be modified to affect functionality, e.g., to eliminate residual effector functions (Reddy et al., J. Immunol., 2000, 164, 1925-1933).

[0069] The present disclosure also provides nucleic acid molecules that encode anti-ALPL antibodies or fragments thereof, in particular, any one of those described above. Recombinant expression vectors carrying these nucleic acids, and host cells, e.g., bacterial cells, such as E. coli, or mammalian cells, such as CHO cells, into which such vectors have been introduced, are also encompassed herein, as are methods of producing the antibodies by culturing the host cells under conditions permitting production of the antibodies, and recovering the antibodies produced.

[0070] In some embodiments, the anti-ALPL antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) encoded by nucleotide sequence segments derived from VH, DH and JH germline sequences, and a light chain variable region (LCVR) encoded by nucleotide sequence segments derived from VK and JK germline sequences, wherein the HCVR and the LCVR are encoded by nucleotide sequence segments derived from a germline gene combination selected from the group consisting of: (i) VH3-43, DH3-3, 43, VK1-5 and JK2; (ii) VH3-11, DH1-1, JH4, VK1-39 and JK4; (iii) VH3-30, DHl-7, JH6, VKl-5 and JK1; (iv) VH3-30, DHl-26, JH6, VK1-12 and JK3; (v) VH3-30, DH3-10, JH6, VK1-12 and JK3; and (vi) VH3-23, DH3-10, JH4, VKl-5 and JKI. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to ALPL with an equilibrium dissociation constant (KD) of about 7 nM or less, about 6 nM or less, about 5 nM or less, about 4 nM or less, about 3 nM or less, about 2 nM or less, or about 1 nM or less, as measured by surface plasmon resonance assay (for example, BIACORE™). In some embodiments, the antibody exhibits a KD of about 800 pM or less, about 700 pM or less; about 600 pM or less; about 500 pM or less; about 400 pM or less; about 300 pM or less; about 200 pM or less; about 100 pM or less; or about 50 pM or less.

[0071] In some embodiments, the anti-ALPL antibodies have a modified glycosylation pattern. In some applications, modification to remove undesirable glycosylation sites may be useful, or e.g., removal of a fucose moiety to increase antibody dependent cellular cytotoxicity (ADCC) function (see, Shield et al., J. Biol. Chem., 2002, 277, 26733). In other applications, removal of N-glycosylation site may reduce undesirable immune reactions against the therapeutic antibodies or increase affinities of the antibodies. In yet other applications, modification of galactosylation can be made in order to modify complement dependent cytotoxicity (CDC).

[0072] The present disclosure also provides compositions comprising a combination of an antibody or antigen-binding fragment thereof and an aortic stenosis therapeutic agent.

[0073] In some embodiments, the aortic stenosis therapeutic agents include, but are not limited to, an angiotensin-converting enzyme (ACE) inhibitor, a beta-blocker (e.g., INDERAL® (propranolol)), a diuretic, a nitrate, an anti-hypertensive agent, and an anti-arrhythmia, or any combination thereof. Additional aortic stenosis therapies include any therapy used to reduce or manage general cardiovascular disease risk or risk factors for cardiovascular disease. In some embodiments, the aortic stenosis therapeutic agent or aortic stenosis therapy can be combined with an ALPL inhibitor. In some embodiments, the treatment for aortic stenosis is surgery, including but not limited to, aortic valve repair, balloon valvuloplasty, aortic valve replacement, and transcatheter aortic valve replacement (TAVR). These surgeries may be delayed or avoided altogether by treatment with an ALPL inhibitor as described herein.

[0074] In some embodiments, the dose of the aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis can be decreased by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, or by about 90% for subjects that are heterozygous for an ALPL variant nucleic acid molecule (i.e., a less than the standard dosage amount) compared to subjects that are ALPL reference (who may receive a standard dosage amount). In some embodiments, the dose of the aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis can be decreased by about 10%, by about 20%, by about 30%, by about 40%, or by about 50%. In addition, the subjects that are heterozygous for an ALPL variant nucleic acid molecule can be administered less frequently compared to subjects that are ALPL reference.

[0075] In some embodiments, the dose of the aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis can be decreased by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, for subjects that are homozygous for an ALPL variant nucleic acid molecule compared to subjects that are heterozygous for an ALPL variant nucleic acid molecule. In some embodiments, the dose of the aortic stenosis therapeutic agents can be decreased by about 10%, by about 20%, by about 30%, by about 40%, or by about 50%. In addition, the dose of aortic stenosis therapeutic agents in subjects that are homozygous for an ALPL variant nucleic acid molecule can be administered less frequently compared to subjects that are heterozygous for an ALPL variant nucleic acid molecule.

[0076] Administration of the aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis and / or ALPL inhibitors can be repeated, for example, after one day, two days, three days, five days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, eight weeks, two months, or three months. The repeated administration can be at the same dose or at a different dose. The administration can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, according to certain dosage regimens a subject can receive therapy for a prolonged period of time such as, for example, 6 months, 1 year, or more.

[0077] Administration of the aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis and / or ALPL inhibitors can occur by any suitable route including, but not limited to, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile and substantially isotonic and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or auxiliaries. The formulation depends on the route of administration chosen. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof. The terms "treat", "treating", and "treatment" and "prevent", "preventing", and "prevention" as used herein, refer to eliciting the desired biological response, such as a therapeutic and prophylactic effect, respectively. In some embodiments, a therapeutic effect comprises one or more of a decrease / reduction in aortic stenosis, a decrease / reduction in the severity of aortic stenosis (such as, for example, a reduction or inhibition of development of aortic stenosis), a decrease / reduction in symptoms and disease-related effects, delaying the onset of symptoms and disease-related effects, reducing the severity of symptoms of disease- related effects, reducing the number of symptoms and disease-related effects, reducing the latency of symptoms and disease-related effects, an amelioration of symptoms and disease- related effects, reducing secondary symptoms, reducing secondary infections, preventing relapse to aortic stenosis, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics, and / or an increased survival time of the affected host animal, following administration of the agent or composition comprising the agent. A prophylactic effect may comprise a complete or partial avoidance / inhibition or a delay of aortic stenosis development / progression (such as, for example, a complete or partial avoidance / inhibition or a delay), and an increased survival time of the affected host animal, following administration of a therapeutic protocol. Treatment of aortic stenosis encompasses the treatment of a subject already diagnosed as having any form of aortic stenosis at any clinical stage or manifestation, the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of aortic stenosis, and / or preventing and / or reducing the severity of aortic stenosis.

[0078] In some embodiments, the ALPL inhibitor and the aortic stenosis therapeutic agent are disposed within a pharmaceutical composition. In some embodiments, the ALPL inhibitor is disposed within a first pharmaceutical composition and the aortic stenosis therapeutic agent is disposed within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered afterthe second pharmaceutical composition.

[0079] In some embodiments, the ALPL predicted loss-of-function variant nucleic acid molecule comprises a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, a missense variant, an in-frame indel variant, and / or a variant that encodes a truncated ALPL predicted loss-of-function polypeptide.

[0080] The present disclosure also provides methods of identifying a subject having an increased risk of developing aortic stenosis. In some embodiments, the method comprises determining or having determined in a biological sample obtained from the subject the presence or absence of an ALPL variant nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, and / or cDNA molecule). When the subject lacks an ALPL variant nucleic acid molecule (i.e., the subject is genotypically categorized as ALPL reference), then the subject has an increased risk of developing aortic stenosis. When the subject has an ALPL variant nucleic acid molecule (i.e., the subject is heterozygous or homozygous for an ALPL variant nucleic acid molecule), then the subject has a decreased risk of developing aortic stenosis.

[0081] Having a single copy of an ALPL variant nucleic acid molecule is more protective of a subject from developing aortic stenosis than having no copies of an ALPL variant nucleic acid molecule. Without intending to be limited to any particular theory or mechanism of action, it is believed that a single copy of an ALPL variant nucleic acid molecule (i.e., heterozygous for an ALPL variant nucleic acid molecule) is protective of a subject from developing aortic stenosis and it is also believed that having two copies of an ALPL variant nucleic acid molecule (i.e., homozygous for an ALPL variant nucleic acid molecule) may be more protective of a subject from developing aortic stenosis, relative to a subject with a single copy. Thus, in some embodiments, a single copy of an ALPL variant nucleic acid molecule may not be completely protective, but instead, may be partially or incompletely protective of a subject from developing aortic stenosis. While not desiring to be bound by any particular theory, there may be additional factors or molecules involved in the development of aortic stenosis that are still present in a subject having a single copy of an ALPL variant nucleic acid molecule, thus resulting in less than complete protection from the development of aortic stenosis.

[0082] Determining whether a subject has an ALPL variant nucleic acid molecule in a biological sample from a subject and / or determining whether a subject has an ALPL variant nucleic acid molecule can be carried out by any of the methods described herein. In some embodiments, these methods can be carried out in vitro. In some embodiments, these methods can be carried out in situ. In some embodiments, these methods can be carried out in vivo. In any of these embodiments, the nucleic acid molecule can be present within a cell obtained from the subject. In some embodiments, when a subject is identified as having an increased risk of developing aortic stenosis, the subject is administered an aortic stenosis therapeutic agent or aortic stenosis therapy and / or an ALPL inhibitor, as described herein. For example, when the subject is ALPL reference, and therefore has an increased risk of developing aortic stenosis, the subject is administered an ALPL inhibitor. In some embodiments, such a subject is also administered an aortic stenosis therapeutic agent or aortic stenosis therapy. In some embodiments, when the subject is heterozygous for an ALPL variant nucleic acid molecule, the subject is administered the aortic stenosis therapeutic agent or aortic stenosis therapy in a dosage amount that is the same as or less than a standard dosage amount, and is also administered an ALPL inhibitor. In some embodiments, such a subject is also administered an aortic stenosis therapeutic agent or aortic stenosis therapy. In some embodiments, when the subject is homozygous for an ALPL variant nucleic acid molecule, the subject is administered the aortic stenosis therapeutic agent or aortic stenosis therapy that in a dosage amount that is the same as or less than a standard dosage amount. In some embodiments, the subject is ALPL reference. In some embodiments, the subject is heterozygous for an ALPL variant nucleic acid molecule. In some embodiments, the subject is homozygous for an ALPL variant nucleic acid molecule.

[0083] In some embodiments, any of the methods described herein can further comprise determining the subject's aggregate burden of having an ALPL variant nucleic acid molecule, and / or an ALPL predicted loss-of-function variant polypeptide associated with a decreased risk of developing aortic stenosis. The aggregate burden is the sum of all variants in the ALPL gene, which can be carried out in an association analysis with aortic stenosis. In some embodiments, the subject is homozygous for one or more ALPL variant nucleic acid molecules associated with a decreased risk of developing aortic stenosis. In some embodiments, the subject is heterozygous for one or more ALPL variant nucleic acid molecules associated with a decreased risk of developing aortic stenosis. The result of the association analysis suggests that ALPL variant nucleic acid molecules are associated with decreased risk of developing aortic stenosis. When the subject has a lower aggregate burden, the subject is at a higher risk of developing aortic stenosis and the subject is administered or continued to be administered the aortic stenosis therapeutic agent or aortic stenosis therapy in a standard dosage amount, and / or an ALPL inhibitor. When the subject has a greater aggregate burden, the subject is at a lower risk of developing aortic stenosis and the subject is administered or continued to be administered the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than the standard dosage amount. The greater the aggregate burden, the lower the risk of developing aortic stenosis. The gene burden analysis can comprise any of the ALPL variant nucleic acid molecules disclosed herein, such as those listed in Table 4.

[0084] In some embodiments, the subject's aggregate burden of having any one or more ALPL variant nucleic acid molecules represents a weighted sum of a plurality of any of the ALPL variant nucleic acid molecules. In some embodiments, the aggregate burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about or more than 1,000,000 genetic variants present in or around (up to 10 Mb) the ALPL gene where the genetic burden is the number of alleles multiplied by the association estimate with aortic stenosis or related outcome for each allele (e.g., a weighted polygenic burden score). In some embodiments, when the subject has an aggregate burden above a desired threshold score, the subject has a decreased risk of developing aortic stenosis. In some embodiments, when the subject has an aggregate burden below a desired threshold score, the subject has an increased risk of developing aortic stenosis.

[0085] In some embodiments, the aggregate burden may be divided into quintiles, e.g., top quintile, intermediate quintile, and bottom quintile, wherein the top quintile of aggregate burden corresponds to the lowest risk group and the bottom quintile of aggregate burden corresponds to the highest risk group. In some embodiments, a subject having a greater aggregate burden comprises the highest weighted aggregate burdens, including, but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% of aggregate burdens from a subject population. In some embodiments, the genetic variants comprise the genetic variants having association with aortic stenosis in the top 10%, top 20%, top 30%, top 40%, or top 50% of p- value range for the association. In some embodiments, each of the identified genetic variants comprise the genetic variants having association with aortic stenosis with p-value of no more than about 10'2, about 10'3, about 10'4, about 10'5, about 10'6, about 10'7, about 10'8, about IO-9, about IO10, about 10n, about 1012, about 1013, about 1014, about or 1015. In some embodiments, the identified genetic variants comprise the genetic variants having association with aortic stenosis with p-value of less than 5 x 10‘8. In some embodiments, the identified genetic variants comprise genetic variants having association with aortic stenosis in high-risk subjects as compared to the rest of the reference population with odds ratio (OR) about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, or about 2.25 or greater for the top 20% of the distribution; or about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, about 2.25 or greater, about 2.5 or greater, or about 2.75 or greater. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, from about 1.5 to about 2.0, from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, from about 4.5 to about 5.0, from about 5.0 to about 5.5, from about 5.5 to about 6.0, from about 6.0 to about 6.5, from about 6.5 to about 7.0, or greater than 7.0. In some embodiments, high-risk subjects comprise subjects having aggregate burdens in the bottom decile, quintile, or tertile in a reference population. The threshold of the aggregate burden is determined on the basis of the nature of the intended practical application and the risk difference that would be considered meaningful for that practical application.

[0086] In some embodiments, when a subject is identified as having an increased risk of developing aortic stenosis, the subject is further administered an aortic stenosis therapeutic agent or aortic stenosis therapy and / or an ALPL inhibitor, as described herein. For example, when the subject is ALPL reference, and therefore has an increased risk of developing aortic stenosis, the subject is administered an ALPL inhibitor. In some embodiments, such a subject is also administered an aortic stenosis therapeutic agent or aortic stenosis therapy. In some embodiments, when the subject is heterozygous for an ALPL variant nucleic acid molecule, the subject is administered the aortic stenosis therapeutic agent or aortic stenosis therapy in a dosage amount that is the same as or less than a standard dosage amount, and is also administered an ALPL inhibitor. In some embodiments, the subject is ALPL reference. In some embodiments, the subject is heterozygous for an ALPL variant nucleic acid molecule. Furthermore, when the subject has a lower aggregate burden for having an ALPL variant nucleic acid molecule, and therefore has an increased risk of developing aortic stenosis, the subject is administered an aortic stenosis therapeutic agent or aortic stenosis therapy. In some embodiments, when the subject has a lower aggregate burden for having an ALPL variant nucleic acid molecule, the subject is administered the aortic stenosis therapeutic agent or aortic stenosis therapy in a dosage amount that is the same as or greater than the standard dosage amount administered to a subject who has a greater aggregate burden for having an ALPL variant nucleic acid molecule.

[0087] The present disclosure also provides methods of detecting the presence or absence of an ALPL variant nucleic acid molecule (i.e., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule produced from an mRNA molecule) in a biological sample from a subject. It is understood that gene sequences within a population and mRNA molecules encoded by such genes can vary due to polymorphisms such as single-nucleotide polymorphisms.

[0088] The biological sample can be derived from any cell, tissue, or biological fluid from the subject. The biological sample may comprise any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of bodily fluid, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascitic fluid, cystic fluid, or urine. In some cases, the sample comprises a buccal swab. The biological sample used in the methods disclosed herein can vary based on the assay format, nature of the detection method, and the tissues, cells, or extracts that are used as the sample. A biological sample can be processed differently depending on the assay being employed. For example, when detecting any ALPL variant nucleic acid molecule, preliminary processing designed to isolate or enrich the biological sample for the genomic DNA can be employed. A variety of techniques may be used for this purpose. When detecting the level of any ALPL variant nucleic acid molecule, different techniques can be used enrich the biological sample with mRNA molecules. Various methods to detect the presence or level of an mRNA molecule or the presence of a particular variant genomic DNA locus can be used.

[0089] In some embodiments, detecting an ALPL variant nucleic acid molecule in a subject comprises performing a sequence analysis on a biological sample obtained from the subject to determine whether ALPL genomic nucleic acid molecule in the biological sample, and / or an ALPL mRNA molecule in the biological sample, and / or an ALPL cDNA molecule produced from an mRNA molecule in the biological sample, comprises one or more variations that cause a loss- of-function (partial or complete) or are predicted to cause a loss-of-fu notion (partial or complete).

[0090] In some embodiments, the methods of detecting the presence or absence of an ALPL variant nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule produced from an mRNA molecule) in a subject, comprise performing an assay on a biological sample obtained from the subject. The assay determines whether a nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0091] In some embodiments, the biological sample comprises a cell or cell lysate. Such methods can further comprise, for example, obtaining a biological sample from the subject comprising an ALPL genomic nucleic acid molecule or mRNA molecule, and if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can comprise, for example determining the identity of these positions of the particular ALPL nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0092] In some embodiments, the determining step, detecting step, or sequence analysis comprises sequencing at least a portion of the nucleotide sequence of the ALPL genomic nucleic acid molecule, the ALPL mRNA molecule, or the ALPL cDNA molecule in the biological sample, wherein the sequenced portion comprises one or more variations that cause a loss-of- function (partial or complete) or are predicted to cause a loss-of-function (partial or complete).

[0093] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only an ALPL genomic nucleic acid molecule is analyzed. In some embodiments, only an ALPL mRNA is analyzed. In some embodiments, only an ALPL cDNA obtained from ALPL mRNA is analyzed.

[0094] Alteration-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in a nucleic acid sequence. Alteration-specific primers can be used because the DNA polymerase will not extend when a mismatch with the template is present.

[0095] In some embodiments, the nucleic acid molecule in the sample is mRNA and the mRNA is reverse-transcribed into a cDNA prior to the amplifying step. In some embodiments, the nucleic acid molecule is present within a cell obtained from the subject.

[0096] In some embodiments, the assay comprises contacting the biological sample with a primer or probe, such as an alteration-specific primer or alteration-specific probe, that specifically hybridizes to an ALPL variant genomic sequence, variant mRNA sequence, or variant cDNA sequence and not the corresponding ALPL reference sequence under stringent conditions and determining whether hybridization has occurred.

[0097] In some embodiments, the determining step, detecting step, or sequence analysis comprises: a) amplifying at least a portion of the ALPL nucleic acid molecule that encodes the ALPL polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support comprising an alteration-specific probe; and d) detecting the detectable label.

[0098] In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assays also comprise reverse transcribing mRNA into cDNA, such as by the reverse transcriptase polymerase chain reaction (RT-PCR).

[0099] In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify a polynucleotide comprising an ALPL variant genomic nucleic acid molecule, variant mRNA molecule, or variant cDNA molecule. The hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length may be any length that is sufficient for use in a detection method of choice, including any assay described or exemplified herein. Such probes and primers can hybridize specifically to a target nucleotide sequence under high stringency hybridization conditions. Probes and primers may have complete nucleotide sequence identity of contiguous nucleotides within the target nucleotide sequence, although probes differing from the target nucleotide sequence and that retain the ability to specifically detect and / or identify a target nucleotide sequence may be designed by conventional methods. Probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity with the nucleotide sequence of the target nucleic acid molecule.

[0100] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, including using labeled primers or probes directed against purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, a target nucleic acid molecule may be amplified prior to or simultaneous with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0101] In hybridization techniques, stringent conditions can be employed such that a probe or primer will specifically hybridize to its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a detectably greater degree than to other non-target sequences, such as, at least 2-fold, at least 3-fold, at least 4- fold, or more over background, including over 10-fold over background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by at least 4-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a detectably greater degree than to other nucleotide sequences by over 10-fold over background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0102] Appropriate stringency conditions which promote DNA hybridization, for example, 6X sodium chloride / sodium citrate (SSC) at about 45°C., followed by a wash of 2X SSC at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection will be those in which the salt concentration is less than about 1.5 M Na+ion, typically about 0.01 to 1.0 M Na+ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (such as, for example, 10 to 50 nucleotides) and at least about 60°C for longer probes (such as, for example, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

[0103] In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, or at least about 5000 nucleotides. In some embodiments, such isolated nucleic acid molecules comprise or consist of at least about 5, at least about 8, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, or at least about 25 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 12 to about 30, from about 12 to about 28, from about 12 to about 24, from about 15 to about 30, from about 15 to about 25, from about 18 to about 30, from about 18 to about 25, from about 18 to about 24, or from about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 18 to about 30 nucleotides. In some embodiments, the isolated nucleic acid molecules comprise or consist of at least about 15 nucleotides to at least about 35 nucleotides.

[0104] In some embodiments, such isolated nucleic acid molecules hybridize to ALPL variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules) under stringent conditions. Such nucleic acid molecules can be used, for example, as probes, primers, alteration-specific probes, or alteration-specific primers as described or exemplified herein, and include, without limitation primers, probes, antisense RNAs, shRNAs, and siRNAs, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0105] In some embodiments, the isolated nucleic acid molecules hybridize to at least about 15 contiguous nucleotides of a nucleic acid molecule that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to ALPL variant nucleic acid molecules. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 15 to about 100 nucleotides, or from about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecules consist of or comprise from about 15 to about 35 nucleotides.

[0106] In some embodiments, the alteration-specific probes and alteration-specific primers comprise DNA. In some embodiments, the alteration-specific probes and alteration-specific primers comprise RNA.

[0107] In some embodiments, the probes and primers described herein (including alterationspecific probes and alteration-specific primers) have a nucleotide sequence that specifically hybridizes to any of the nucleic acid molecules disclosed herein, or the complement thereof. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0108] In some embodiments, the primers, including alteration-specific primers, can be used in second generation sequencing or high throughput sequencing. In some instances, the primers, including alteration-specific primers, can be modified. In particular, the primers can comprise various modifications that are used at different steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 Pyrosequencing. Modified primers can be used at several steps of the process, including biotinylated primers in the cloning step and fluorescently labeled primers used at the bead loading step and detection step. Polony sequencing is generally performed using a paired-end tags library wherein each molecule of DNA template is about 135 bp in length. Biotinylated primers are used at the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonamer oligonucleotides are used at the detection step. An adaptor can contain a 5'-biotin tag for immobilization of the DNA library onto streptavidin-coated beads.

[0109] The probes and primers described herein can be used to detect a nucleotide variation within any of the ALPL variant nucleic acid molecules disclosed herein. The primers described herein can be used to amplify any ALPL variant nucleic acid molecule, or a fragment thereof.

[0110] In the context of the disclosure "specifically hybridizes" means that the probe or primer (such as, for example, the alteration-specific probe or alteration-specific primer) does not hybridize to a nucleic acid sequence encoding an ALPL reference genomic nucleic acid molecule, an ALPL reference mRNA molecule, and / or an ALPL reference cDNA molecule.

[0111] In some embodiments, the probes (such as, for example, an alteration-specific probe) comprise a label. In some embodiments, the label is a fluorescent label, a radiolabel, or biotin. The present disclosure also provides supports comprising a substrate to which any one or more of the probes disclosed herein is attached. Solid supports are solid-state substrates or supports with which molecules, such as any of the probes disclosed herein, can be associated. A form of solid support is an array. Another form of solid support is an array detector. An array detector is a solid support to which multiple different probes have been coupled in an array, grid, or other organized pattern. A form for a solid-state substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a multiwell glass slide can be employed that normally contains one array per well.

[0112] The genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be from any organism. For example, the genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or an ortholog from another organism, such as a non-human mammal, a rodent, a mouse, or a rat. It is understood that gene sequences within a population can vary due to polymorphisms such as single-nucleotide polymorphisms.

[0113] Also provided herein are functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences. The functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional polynucleotides can possess a de novo activity independent of any other molecules.

[0114] The isolated nucleic acid molecules disclosed herein can comprise RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to a heterologous nucleic acid sequence, such as in a vector, or a heterologous label. For example, the isolated nucleic acid molecules disclosed herein can be within a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to a heterologous label. The label can be directly detectable (such as, for example, fluorophore) or indirectly detectable (such as, for example, hapten, enzyme, or fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radiolabels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The label can also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal. The term "label" can also refer to a "tag" or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, biotin can be used as a tag along with an avidin or streptavidin conjugate of horseradish peroxidate (HRP) to bind to the tag, and examined using a calorimetric substrate (such as, for example, tetramethylbenzidine (TMB)) or a fluorogenic substrate to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6Xhis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, an epitope tag, or the Fc portion of immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorogenic and chemiluminescent substrates and other labels.

[0115] Percent identity (or percent complementarity) between particular stretches of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Herein, if reference is made to percent sequence identity, the higher percentages of sequence identity are preferred over the lower ones.

[0116] The present disclosure also provides aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis for use in the treatment and / or prevention of aortic stenosis in a subject having an ALPL variant nucleic acid molecule. Any of the aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis described herein can be used in these methods. Any of the ALPL variant nucleic acid molecules disclosed herein can be used in these methods.

[0117] The present disclosure also provides uses of aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis for use in the preparation of a medicament for treating and / or preventing aortic stenosis in a subject having an ALPL variant nucleic acid molecule. Any of the aortic stenosis therapeutic agents that treat, prevent, or inhibit aortic stenosis described herein can be used in these methods. Any of the ALPL variant nucleic acid molecules disclosed herein can be used in these methods. The present disclosure also provides ALPL inhibitors for use in the treatment and / or prevention of aortic stenosis in a subject that is ALPL reference or is heterozygous for an ALPL variant nucleic acid molecule. Any of the ALPL inhibitors described herein can be used in these methods. Any of the ALPL variant nucleic acid molecules disclosed herein can be used in these methods.

[0118] The present disclosure also provides ALPL inhibitors in the preparation of a medicament for treating and / or preventing aortic stenosis in a subject that is ALPL reference or is heterozygous for an ALPL variant nucleic acid molecule. Any of the ALPL inhibitors described herein can be used in these methods. Any of the ALPL variant nucleic acid molecules disclosed herein can be used in these methods.

[0119] In some embodiments, the ALPL inhibitor and the aortic stenosis therapeutic agent are disposed within a pharmaceutical composition. In some embodiments, the ALPL inhibitor is disposed within a first pharmaceutical composition and the aortic stenosis therapeutic agent is disposed within a second pharmaceutical composition. In some embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered afterthe second pharmaceutical composition.

[0120] In some embodiments, the ALPL predicted loss-of-function variant nucleic acid molecule comprises a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, an in-frame indel variant, and / or a variant that encodes a truncated ALPL predicted loss-of-function polypeptide.

[0121] All patent documents, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the present disclosure can be used in combination with any other feature, step, element, embodiment, or aspect unless specifically indicated otherwise. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

[0122] The following examples are provided to describe the embodiments in greater detail. They are intended to illustrate, not to limit, the claimed embodiments. The following examples provide those of ordinary skill in the art with a disclosure and description of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (such as, for example, amounts, temperature, etc.), but some errors and deviations may be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.

[0123] Examples

[0124] Example 1: General Methods

[0125] Participating cohorts

[0126] The UKB is a population-based cohort study of people aged between 40 and 69 years recruited through 22 testing centers in the UK between 2006-2010. A total of 477,181 European ancestry participants from UKB with available whole-exome sequencing and phenotype data were included. The MyCode Community Health Initiative cohort from the Geisinger Health System (GHS) (Carey et al., Genet. Med., 2016, 18, 906-913) is a health system-based cohort of patients from Central and Eastern Pennsylvania (USA) recruited in 2007-2019. A total of 136,239 European ancestry participants from GHS with available whole-exome sequencing and phenotype data were included. The Mount Sinai's BioMe Personalized Medicine Cohort (SINAI) (Gottesman et al., Genet. Med., 2013, 15, 761-771) is an electronic health record-linked clinical care cohort of 30,619 individuals. The University of Pennsylvania Medicine BioBank (UPENN- PMBB) is a health system-based cohort based in Pennsylvania. The Malmo Diet and Cancer Study (MDCS) is a cohort study based in Malmo, Sweden (Berglund et al., J. Intern. Med., 1993, 233, 45-51). The Indiana University School of Medicine Biobank (INDIANA) data is a biobank of 50,000 linked to their electronic medical records. The number of cases and controls included in each of the analyzed outcomes are shown in Table 1.

[0127] Phenotype definitions

[0128] Clinical laboratory measurements for alkaline phosphatase activity and circulating phosphate were measured from blood at UKB recruitment centers. Alkaline Phosphatase was measured by IFCC (International Federation of Clinical Chemistry) analysis and phosphate levels were measured using a Phophomolybdate complex on a Beckman Coulter AU5800. Prior to genetic association analysis, continues phenotype values were transformed by the inverse standard normal function, applied within each ancestry group and separately in men and women.

[0129] In GHS, UKB, MDCS, UPENN-PMBB, INDIANA, and SINAI, prevalent cases for aortic stenosis were defined according to the International Classification of Diseases, Ninth and Tenth Revision (ICD-9 and ICD-10), outpatient and operation procedure codes stored in EHRs, and self-reports were used when available; all of which and combined into single variables to classify individuals into cases or controls. Rheumatic valve disease patients were excluded from the analysis, individuals with heart murmurs were excluded from the controls. Genotype data

[0130] High coverage whole exome sequencing was performed as previously described (Dewey et al., Science, 2016, 354, 6319, aaf6814, "Distribution and clinical impact of functional variants in 50,726 whole-exome sequences from the DiscovEHR study"; and Van Hout et al., Nature, 2020, 586, 749-756) and as summarized below. NimbleGen probes (VCRome; for part of the GHS cohort) or a modified version of the xGen design available from Integrated DNA Technologies (IDT; for the rest of GHS and other cohorts) were used for target sequence capture of the exome. A unique 6 base pair (bp) barcode (VCRome) or 10 bp barcode (IDT) was added to each DNA fragment during library preparation to facilitate multiplexed exome capture and sequencing. Equal amounts of sample were pooled prior to exome capture. Sequencing was performed using 75 bp paired-end reads on Illumina v4 HiSeq 2500 (for part of the GHS cohort) or NovaSeq (for the rest of GHS and other cohorts) instruments. Sequencing had a coverage depth (i.e., number of sequence-reads covering each nucleotide in the target areas of the genome) sufficient to provide greater than 20x coverage over 85% of targeted bases in 96% of VCRome samples and 20x coverage over 90% of targeted bases in 99% of IDT samples. Data processing steps included sample de-multiplexing using Illumina software, alignment to the GRCh38 Human Genome reference sequence including generation of binary alignment and mapping files (BAM), processing of BAM files (e.g., marking of duplicate reads and other read mapping evaluations). Variant calling was performed using the GLNexus system (DOI: 10.1101 / 343970). Variant mapping and annotation were based on the GRCh38 Human Genome reference sequence and Ensembl v85 gene definitions using the snpEff software. The snpEff predictions that involve protein-coding transcripts with an annotated start and stop were then combined into a single functional impact prediction by selecting the most deleterious functional effect class for each gene. The hierarchy (from most to least deleterious) for these annotations was frameshift, stop-gain, stop-loss, splice acceptor, splice donor, stop-lost, in-frame indel, missense, other annotations. Predicted LOF genetic variants included: a) insertions or deletions resulting in a frameshift, b) insertions, deletions or single nucleotide variants resulting in the introduction of a premature stop codon or in the loss of the transcription start site or stop site, and c) variants in donor or acceptor splice sites. Missense variants were classified for likely functional impact according to the number of in silico prediction algorithms that predicted deleteriousness using SIFT (Adzhubei et al., Nat. Methods, 2010, 7, 248-9) and Polyphen2_HVAR (Adzhubei et al., Nat. Methods, 2010, 7, 248-9), LRT (Chun et al., Genome Res., 2009, 19, 1553-61) and MutationTaster (Schwarz et al., Nat. Methods, 2010, 7, 575-6). For each gene, the alternative allele frequency (AAF) and functional annotation of each variant determined inclusion into these 2 gene burden exposures: 1) pLOF variants with AAF < 1%; 2) pLOF or any missense variants with AAF < 0.1%.

[0131] Association analysis of gene burden of rare loss of function variation

[0132] Association between the burden of rare predicted loss-of-function or missense variants in a given gene and phenotype was examined by fitting a linear (for quantitative traits) or firth bias-corrected logistic (for binary traits) regression model adjusted for a polygenic score that approximates a genomic kinship matrix using REGENIE vl.O (world wide web at "doi.org / 10.1101 / 2020.06.19.162354"). Analyses were stratified by ancestry and adjusted for age, age2, sex, age-by-sex and age2-by-sex interaction terms, experimental batch-related covariates, 10 common variant-derived principal components, and 20 rare variant-derived principal components. Results across cohorts for each variant-phenotype association were combined using fixed effects inverse variance weighted meta-analysis. In gene burden tests, all individuals were labeled as heterozygotes if they carry one or more qualifying rare variant (as described above based on frequency and functional annotation) and as homozygotes if they carry any qualifying variant in the homozygous state. This "composite genotype" was then used to test for association.

[0133] Gl / VAS of common variants

[0134] Associated common variants were identified by performing a genome-wide association study including over 12 million common-to-low-frequency genetic variants imputed using the Haplotype Reference Consortium panel. In the GHS study, imputation was performed separately in samples genotyped with the Illumina Human Omni Express Exome array (OMNI set) and the Global Screening array (GSA set). Dosage data from imputed variants were then merged across the two GHS sets, to obtain a combined dataset for association analysis. Genome-wide association analyses were performed in each cohort separately by fitting whole genome regression models using REGENIE (Mbatchou et al., , bioRxiv, 2020.06.19.162354). As described above for burden tests, within each cohort analyses were stratified by ancestry and adjusted for age, age2, sex, age-by-sex and age2-by-sex interaction terms, experimental batch- related covariates, and 10 common variant-derived principal components. Results from the individual cohort analyses were then combined by inverse variance-weighted meta-analysis to obtain a genome-wide meta-analysis.

[0135] Identification of multiple single rare variants driving the association with aortic stenosis

[0136] A iterative leave-one-variant-out analysis scheme was used to generate the list of individual variant sites that contributed to the observed association with decreased odds of aortic stenosis for rare coding variants in ALPL. In successive iterations, the variant site whose removal maximally attenuated the gene-burden association signal (i.e., resulting in the largest p-value for association) after meta-analyses across all participating cohorts and ancestries was generated. In the following iteration, the identified variant was removed. This was repeated until the gene burden based on the remaining list of variants had an association at P > 0.05.

[0137] Example 2: Loss of Function Variants in the Gene Encoding Alkaline Phosphatase are Associated with Protection Against Aortic Stenosis

[0138] To identify genetic factors contributing to aortic stenosis, imputed genotype data, exome sequence data were analyzed on participants of the UK Biobank cohort (UKB), the Geisinger Health System MyCode Community Health Initiative cohort study (GHS), Mount Sinai's BioMe Personalized Medicine Cohort (SINAI), the Malmo Diet and Cancer Study (MDCS), Indiana University School of Medicine Biobank (INDIANA), and the University of Pennsylvania Penn Medicine BioBank (UPENN-PMBB). Individuals with aortic were defined using electronic health records and questionnaires. A genome wide analysis study (GWAS) of aortic stenosis was then carried out using an imputed dataset of 11,336,546 genetic variants and 665,739 individuals (11,626 cases of aortic stenosis and 654,113 controls). The GWAS identified rsl2141569 (C->T, alternative allele frequency of 53%) near the ALPL gene to be significantly associated with lower odds of aortic stenosis (OR (95% confidence interval)=0.92 (0.90, 0.95), P=1.5xl0'8). Associations for rare coding variants in the ALPL gene, which can provide greater confidence for the implication of the gene in aortic stenosis, were investigated next. Using exome sequence data, statistical ly-sign ifica nt associations were observed between the burden of rare (AAF<0.1%) predicted loss-of-fu notion (pLOF) plus missense variants in the ALPL gene and lower alkaline phosphatase, higher phosphate, consistent with ALPL loss-of-function. These variants were also associated with protection against aortic stenosis (OR=0.68; p=8.2xl0-5) (Table 1).

[0139] Table 1: Associations between pLOF burden plus missense variants in the ALPL gene and alkaline phosphatase (ALP) activity, phosphate levels and aortic stenosis

[0140] RR, number of individuals carrying no rare pLoF or missense variants in ALPL (homozygous noncarriers); RA, number of individuals carrying a rare pLoF or missense variant in a single ALPL allele (heterozygous carriers); AA, number of carrying rare pLoF or missense variants in both ALPL alleles (homozygous carriers); AAF, the alternative allele frequency reported as a fraction of 1; SD, standard deviation; OR, odds ratio; Cl, confidence interval; pLOF, predicted loss of function; ALP, Alkaline phosphatase. Missense mutations that inactivate ALPL are known to cause autosomal dominant or recessive hypophosphatasia, a disease characterized by defective mineralization of the teeth and bones with variable penetrance and severity (Whyte, Nat. Rev. Endocrinol., 2016, 12, 233- 246). It was observed that missense variants previously identified for hypophosphatasia drive the observed association with aortic stenosis (Table 2). These results indicate that the protective association with aortic stenosis are driven by a loss of function (i.e., inactivation) of the ALPL gene.

[0141] Table 2: Hypophosphatasia variants drive most of the observed association with aortic stenosis RR, number of individuals carrying no rare pLoF or missense variants in ALPL (homozygous noncarriers); RA, number of individuals carrying a rare pLoF or missense variant in a single ALPL allele (heterozygous carriers); AA, number of carrying rare pLoF or missense variants in both ALPL alleles (homozygous carriers); AAF, alternative allele frequency reported as a fraction of 1; Cl, confidence interval; pLOF, predicted loss of function; HGMD, Human Gene Mutation Database.

[0142] Multiple rare coding variants that contributes to the association with aortic stenosis were identified, several of which are reported as causative variants for hypophosphatasia (Table 3).

[0143] Table 3: Multiple rare coding variants contribute to the association with aortic stenosis, several of which are reported as causative variants for hypophosphatasia

[0144] RR, number of individuals carrying no rare pLoF or missense variants in ALPL (homozygous non- carriers); RA, number of individuals carrying a rare pLoF or missense variant in a single ALPL allele (heterozygous carriers); AA, number of carrying rare pLoF or missense variants in both ALPL alleles (homozygous carriers); AAF, alternative allele frequency reported as a fraction of 1.

[0145] Table 4 shows all pLoF and missense variants included in the ALPL gene burden analyses of aortic stenosis. The Variant, Effect on gene, and RSID column indicates the chromosome, physical genomic position in base pairs, reference allele, and alternative allele for each variant according to build 38 of the Human Genome sequence by the Human Genome Reference Consortium, its functional impact on ALPL transcripts, and its rsid. Coding DNA and protein changes are provided according to the Human Genome Variation Society nomenclature, and refer to four protein coding (ENST00000374840, ENST00000374832, ENST00000539907, ENST00000540617) ALPL transcripts, in the order shown in the Affected transcripts column; as annotated in the Ensembl database (Howe, Achuthan et al., Nuc. Acids Res., 2021, 8, 49(D1), D884-D891). AAF, alternative allele frequency of variants included in this analysis.

[0146] Table 4: ALPL pLoF or missense variants identified by WES and included in the gene burden association analysis

[0147] Various modifications of the described subject matter, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such mod ifications are also intended to fall within the scope of the appended claims. Each reference (including, but not limited to, journal articles, U.S. and non-U. S. patents, patent application publications, international patent application publications, gene bank accession numbers, and the like) cited in the present application is incorporated herein by reference in its entirety and for all purposes.

Claims

What is Claimed is:

1. A method of treating a subject having aortic stenosis, or at risk of developing aortic stenosis, the method comprising administering an Alkaline Phosphatase (ALPL) inhibitor to the subject.

2. The method of claim 1, wherein the ALPL inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an ALPL nucleic acid molecule.

3. The method of claim 2, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

4. The method of claim 3, wherein the inhibitory nucleic acid molecule comprises an siRNA.

5. The method of claim 3, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

6. The method of claim 1, wherein the ALPL inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an ALPL genomic nucleic acid molecule.

7. The method of claim 6, wherein the Cas protein comprises a Cas9 and / or a Cpfl.

8. The method of claim 6 or claim 7, wherein the gRNA comprises from about 17 to about 23 nucleotides.

9. The method of any one of claims 1 to 8, wherein the subject is also administered an aortic stenosis therapeutic agent or aortic stenosis therapy.

10. The method of any one of claims 1 to 9, wherein the ALPL inhibitor and the aortic stenosis therapeutic agent are disposed within the same pharmaceutical composition.

11. The method of any one of claims 1 to 9, wherein the ALPL inhibitor is disposed within a first pharmaceutical composition and the aortic stenosis therapeutic agent is disposed within a second pharmaceutical composition.

12. The method of claim 11, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously.

13. The method of claim 11, wherein the first pharmaceutical composition is administered before the second pharmaceutical composition.

14. The method of claim 11, wherein the first pharmaceutical composition is administered after the second pharmaceutical composition.

15. The method of any one of claims 1 to 14, further comprising detecting the presence or absence of an ALPL variant nucleic acid molecule in a biological sample from the subject.

16. The method of claim 15, further comprising administering an aortic stenosis therapeutic agent or aortic stenosis therapy in a standard dosage amount to the subject when the ALPL variant nucleic acid molecule is absent from the biological sample.

17. The method of claim 15, further comprising administering an aortic stenosis therapeutic agent or aortic stenosis therapy in a dosage amount that is the same as or less than a standard dosage amount to the subject when the subject is heterozygous for the ALPL variant nucleic acid molecule.

18. The method of any one of claims 15 to 17, wherein the ALPL variant nucleic acid molecule comprises a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, a missense variant, an in-frame indel variant, and / or a variant that encodes a truncated ALPL predicted loss-of-function polypeptide.

19. A method of treating a subject having aortic stenosis, or at risk of developing aortic stenosis by administering an aortic stenosis therapeutic agent or aortic stenosis therapy, the method comprising: determining or having determined whether the subject has an Alkaline Phosphatase (ALPL) variant nucleic acid molecule, by: obtaining or having obtained a biological sample from the subject; and performing or having performed a sequence analysis on the biological sample to determine if the subject has a genotype comprising an ALPL variant nucleic acid molecule; and administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in a standard dosage amount to a subject that is ALPL reference, and / or administering an ALPL inhibitor to the subject; administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount to a subject that is heterozygous for the ALPL variant nucleic acid molecule, and / or administering an ALPL inhibitor to the subject; or administering or continuing to administer the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount to a subject that is homozygous for the ALPL variant nucleic acid molecule;wherein the presence of a genotype having the ALPL variant nucleic acid molecule indicates the subject has a decreased risk of developing aortic stenosis.

20. The method of claim 19, wherein the ALPL inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an ALPL nucleic acid molecule.

21. The method of claim 20, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

22. The method of claim 21, wherein the inhibitory nucleic acid molecule comprises an siRNA.

23. The method of claim 21, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

24. The method of claim 19, wherein the ALPL inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an ALPL genomic nucleic acid molecule.

25. The method of claim 24, wherein the Cas protein comprises a Cas9 and / or a Cpfl.

26. The method of claim 24 or claim 25, wherein the gRNA comprises from about 17 to about 23 nucleotides.

27. The method of any one of claims 19 to 26, wherein the ALPL inhibitor and the aortic stenosis therapeutic agent are disposed within the same pharmaceutical composition.

28. The method of any one of claims 19 to 26, wherein the ALPL inhibitor is disposed within a first pharmaceutical composition and the aortic stenosis therapeutic agent is disposed within a second pharmaceutical composition.

29. The method of claim 28, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously.

30. The method of claim 28, wherein the first pharmaceutical composition is administered before the second pharmaceutical composition.

31. The method of claim 28, wherein the first pharmaceutical composition is administered after the second pharmaceutical composition.

32. The method of any one of claims 19 to 31, wherein the subject is heterozygous for the ALPL variant nucleic acid molecule, and the subject is administered or continued to be administered the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount.

33. The method of any one of claims 19 to 32, wherein the ALPL variant nucleic acid molecule comprises a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, a missense variant, an in-frame indel variant, and / or a variant that encodes a truncated ALPL predicted loss-of-function polypeptide.

34. A method of identifying a subject having an increased risk of developing aortic stenosis, the method comprising: determining or having determined the presence or absence of an ALPL variant nucleic acid molecule in a biological sample obtained from the subject; wherein: when the subject is ALPL reference, then the subject has an increased risk of developing aortic stenosis; and when the subject is heterozygous or homozygous for the ALPL variant nucleic acid molecule, then the subject has a decreased risk of developing aortic stenosis.

35. The method of claim 34, wherein the ALPL variant nucleic acid molecule is a splice-site variant, a stop-gain variant, a start-loss variant, a stop-loss variant, a frameshift variant, a missense variant, an in-frame indel variant, or a variant that encodes a truncated ALPL predicted loss-of-function polypeptide.

36. The method of claim 34 or claim 35, further comprising administering an aortic stenosis therapeutic agent or aortic stenosis therapy in a standard dosage amount and / or administering an ALPL inhibitor to a subject that is ALPL reference.

37. The method of claim 34 or claim 35, further comprising administering an aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount and / or administering an ALPL inhibitor to a subject that is heterozygous for an ALPL variant nucleic acid molecule.

38. The method of claim 36 or claim 37, wherein the ALPL inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an ALPL nucleic acid molecule.

39. The method of claim 38, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

40. The method of claim 39, wherein the inhibitory nucleic acid molecule comprises an siRNA.

41. The method of claim 39, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

42. The method of claim 36 or claim 37, wherein the ALPL inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an ALPL genomic nucleic acid molecule.

43. The method of claim 42, wherein the Cas protein comprises a Cas9 and / or a Cpfl.

44. The method of claim 42 or claim 43, wherein the gRNA comprises from about 17 to about 23 nucleotides.

45. The method of any one of claims 36 to 44, wherein the ALPL inhibitor and the aortic stenosis therapeutic agent are disposed within the same pharmaceutical composition.

46. The method of any one of claims 36 to 44, wherein the ALPL inhibitor is disposed within a first pharmaceutical composition and the aortic stenosis therapeutic agent is disposed within a second pharmaceutical composition.

47. The method of claim 46, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously.

48. The method of claim 46, wherein the first pharmaceutical composition is administered before the second pharmaceutical composition.

49. The method of claim 46, wherein the first pharmaceutical composition is administered after the second pharmaceutical composition.

50. The method of any one of claims 36 to 49, wherein the subject is ALPL reference, and the subject is administered or continued to be administered the aortic stenosis therapeutic agent or aortic stenosis therapy in a standard dosage amount, and the subject is administered the ALPL inhibitor.

51. The method of any one of claims 36 to 49, wherein the subject is heterozygous for the ALPL variant nucleic acid molecule, and the subject is administered or continued to be administered the aortic stenosis therapeutic agent or aortic stenosis therapy in an amount that is the same as or less than a standard dosage amount, and the subject is administered the ALPL inhibitor.

52. An aortic stenosis therapeutic agent for use in the treatment and / or prevention of aortic stenosis in a subject having an ALPL variant nucleic acid molecule.

53. The aortic stenosis therapeutic agent according to claim 52, wherein the ALPL variant nucleic acid molecule is a splice-site variant, a stop-gain variant, a start-loss variant, a stop-lossvariant, a frameshift variant, a missense variant, an in-frame indel variant, or a variant that encodes a truncated ALPL predicted loss-of-function polypeptide.

54. An ALPL inhibitor for use in the treatment and / or prevention of aortic stenosis in a subject that is ALPL reference or is heterozygous for an ALPL variant nucleic acid molecule.

55. The ALPL inhibitor of claim 54, wherein the ALPL inhibitor comprises an inhibitory nucleic acid molecule that hybridizes to an ALPL nucleic acid molecule.

56. The ALPL inhibitor of claim 55, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

57. The ALPL inhibitor of claim 56, wherein the inhibitory nucleic acid molecule comprises an siRNA.

58. The ALPL inhibitor of claim 56, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

59. The ALPL inhibitor of claim 54, wherein the ALPL inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an ALPL genomic nucleic acid molecule.

60. The ALPL inhibitor of claim 59, wherein the Cas protein comprises a Cas9 and / or a Cpfl.

61. The ALPL inhibitor of claim 59 or claim 60, wherein the gRNA comprises from about 17 to about 23 nucleotides.