Methods for treating metabolic disorders with mitogen-activated protein kinase kinase kinase 15 (MAP3K15) inhibitors

Abstract

The present disclosure provides methods for treating or preventing a subject having or at risk of developing a metabolic disorder from developing a metabolic disorder, and methods for identifying subjects at high risk of developing a metabolic disorder.

Description

[Technical field]

[0001] The present disclosure relates generally to the treatment of subjects having or at risk of developing a metabolic disorder with mitogen-activated protein kinase kinase kinase 15 (MAP3K15) inhibitors, and to methods of identifying subjects at high risk of developing a metabolic disorder. [Background technology]

[0002] The global epidemic of type 2 diabetes is a major public health problem as the disease is the fifth leading cause of death worldwide and a leading cause of morbidity, premature coronary heart disease, stroke, peripheral vascular disease, renal failure, and amputation. The number of people with diabetes worldwide is projected to increase from 366 million in 2011 to 552 million in 2030. Type 2 diabetes is a non-insulin-dependent form of diabetes mellitus, characterized by hyperglycemia due to impaired insulin secretion and insulin resistance in target tissues. Type 2 diabetes is usually diagnosed after the age of 40 and is caused by the combined effects of genetic susceptibility and environmental factors. Type 2 diabetes is associated with obesity and is also a multifactorial genetic disease.

[0003] Mitogen-activated protein kinase kinase kinase 15 (MAP3K15) encodes a ubiquitously expressed mitogen-activated protein kinase involved in apoptotic cell death (Non-Patent Document 1), but its involvement in type 2 diabetes has not been suggested until now. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Kaji et al.,Biochem.Biophys.Res.Commun.,2010,395,213-218 Summary of the Invention

[0005] The present disclosure provides a method of treating a subject having or at risk of developing a metabolic disorder, the method comprising administering to the subject a MAP3K15 inhibitor. The disclosure also provides a method of treating a subject having or at risk of developing type 2 diabetes, the method comprising administering to the subject a MAP3K15 inhibitor.

[0006] The disclosure also provides a method of treating a subject having or at risk of developing elevated hemoglobin A1c, the method comprising administering to the subject a MAP3K15 inhibitor.

[0007] The disclosure also provides a method of treating a subject having or at risk of developing elevated serum glucose, the method comprising administering to the subject a MAP3K15 inhibitor.

[0008] The disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing a metabolic disorder, wherein the subject has or is at risk of developing a metabolic disorder, the method comprising the steps of determining whether the subject has a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in MAP3K15 by obtaining or obtaining a biological sample from the subject; performing or performing sequence analysis on the biological sample to determine whether the subject has a genotype that includes a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in MAP3K15; i) administering or continuing to administer a standard dose of a therapeutic agent for treating or preventing a metabolic disorder to the subject who is MAP3K15 normative; and and / or administering a MAP3K15 inhibitor to the subject; ii) administering or continuing to administer to the subject who is heterozygous for the MAP3K15 missense mutant nucleic acid molecule a therapeutic agent for treating or preventing a metabolic disorder at the same or a lower standard dosage, and / or administering a MAP3K15 inhibitor to the subject; or iii) administering or continuing to administer to the subject who is homozygous for the MAP3K15 missense mutant nucleic acid molecule a therapeutic agent for treating or preventing a metabolic disorder at the same or a lower standard dosage, wherein the presence of a genotype having a MAP3K15 missense mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 indicates that the subject is at low risk of developing a metabolic disorder.

[0009] The present disclosure also provides a method for identifying a subject at high risk of developing a metabolic disorder, the method comprising determining or having determined the presence or absence of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 in a biological sample obtained from the subject; if the subject meets the MAP3K15 criteria, the subject has a high risk of developing a metabolic disorder; and if the subject is heterozygous or homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, the subject has a low risk of developing a metabolic disorder.

[0010] The present disclosure also provides a therapeutic agent for treating or preventing a metabolic disorder for use in treating or preventing a metabolic disorder in a subject having a MAP3K15 missense mutant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, a MAP3K15 missense mutant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15, or a MAP3K15 missense mutant cDNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15.

[0011] The present disclosure also provides a MAP3K15 inhibitor for use in treating or preventing a metabolic disorder in a subject that is a) reference to a MAP3K15 genomic nucleic acid molecule, a MAP3K15 mRNA molecule, or a MAP3K15 cDNA molecule, or b) heterozygous for i) a MAP3K15 missense variant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, ii) a MAP3K15 missense variant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15, or iii) a MAP3K15 missense variant cDNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Various terms relating to the aspects of the present disclosure are used throughout the specification and claims. Unless otherwise indicated, such terms are to be given their ordinary meaning in the art. Other terms that are specifically defined are to be interpreted in a manner consistent with the definitions set forth herein.

[0013] Unless expressly stated otherwise, any method or embodiment set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically specifies in the claim or description that the steps are to be limited to a particular order, it is in no way intended to dictate order in any respect. This also applies to any possible implicit criteria of interpretation, including logical matters regarding the arrangement of steps or work flow, general meanings derived from grammatical construction or punctuation, or the number or type of embodiments described herein.

[0014] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. As used herein, the term "about" means that a cited numerical value is approximate, and small variations do not significantly affect the practice of the disclosed embodiments. When a numerical value is used, unless otherwise indicated by context, the term "about" means that the numerical value can vary by ±10% and remain within the range of the disclosed embodiments.

[0015] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting" or "consisting essentially of," as desired.

[0016] As used herein, with respect to a nucleic acid molecule or polypeptide, the term "isolated" means that the nucleic acid molecule or polypeptide is in a state other than its native environment, e.g., away 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., more than 95% pure or more 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 alternatively phosphorylated or derivatized forms.

[0017] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complement.

[0018] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs), pet animals (e.g., dogs, cats), laboratory animals (e.g., mice, rats, 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.

[0019] According to the present disclosure, it has been observed that MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15 (regardless of whether these variants are homozygous or heterozygous in a particular subject) are associated with a reduced risk of developing a metabolic disorder. MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15 are not believed to be associated with metabolic disorders, such as type 2 diabetes. Furthermore, the identification of the association of additional variants with the gene burden mask according to the present disclosure indicates that MAP3K15 itself (rather than linkage disequilibrium with variants of another gene) is responsible for the protective effect in metabolic disorders, such as type 2 diabetes. Thus, subjects who are heterozygous for the MAP3K15 standard or MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15 can be treated with a MAP3K15 inhibitor so that the metabolic disorder is suppressed or prevented, the symptoms thereof are alleviated or prevented, and / or the onset of the symptoms is suppressed or prevented. It is also believed that such subjects with a metabolic disorder may be further treated with a therapeutic agent that treats or prevents the metabolic disorder.

[0020] For the purpose of this disclosure, any particular subject, for example, a human, can be classified as having one of three MAP3K15 genotypes: i) MAP3K15-based; ii) heterozygous for MAP3K15 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by MAP3K15; or iii) homozygous for MAP3K15 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by MAP3K15.If the subject does not have a copy of MAP3K15 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by MAP3K15, the subject is MAP3K15-based.If the subject has a single copy of MAP3K15 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by MAP3K15, the subject is heterozygous for MAP3K15 missense variant nucleic acid molecule encoding loss-of-function polypeptide predicted by MAP3K15. MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15 is any nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding a variant MAP3K15 polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. A subject having a MAP3K15 polypeptide with partial loss-of-function (or predicted partial loss-of-function) is hypomorphic for MAP3K15. If a subject has two copies (same or different) of a MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15, the subject is homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15.

[0021] For subjects who are genotyped or determined to be MAP3K15-based, such subjects are at high risk of developing metabolic disorders such as type 2 diabetes, elevated hemoglobin A1c, or elevated serum glucose.For subjects who are genotyped or determined to be MAP3K15-based or heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a MAP3K15-predicted loss-of-function polypeptide, such subjects can be treated with a MAP3K15 inhibitor.

[0022] In any of the embodiments described herein, the MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15 can be any nucleic acid molecule (e.g., genomic nucleic acid molecule, mRNA molecule, or cDNA molecule) encoding a MAP3K15 variant polypeptide with partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function. In some embodiments, the MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15 is associated with a reduced in vitro response to a MAP3K15 ligand compared to a reference MAP3K15. In some embodiments, the MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15 is a MAP3K15 variant that causes or is predicted to cause early truncation of the MAP3K15 polypeptide compared to a human reference genome sequence. In some embodiments, the MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15 is a variant predicted to be damaging by an in vitro prediction algorithm such as Polyphen, SIFT, or similar algorithm. In some embodiments, the MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15 is a variant that causes or is predicted to cause a non-synonymous amino acid substitution in MAP3K15, and its allele frequency is less than 1 / 100 alleles in the population that the subject is selected from. In some embodiments, the MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in MAP3K15 is any rare missense variant (allele frequency less than 0.1%; or 1 allele in 1,000), or any splice site, stop gain, start loss, stop loss, frame shift, or in-frame indel, or other frame shift MAP3K15 variant.

[0023] In any of the embodiments described herein, the MAP3K15 predicted loss-of-function polypeptide can be any MAP3K15 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.

[0024] In any of the embodiments described herein, a MAP3K15 missense mutant nucleic acid molecule encoding a MAP3K15 predicted loss-of-function polypeptide can contain a mutation at any position on the X chromosome using the nucleotide sequence of the MAP3K15 reference genomic nucleic acid molecule (SEQ ID NO:1; ENSG00000180815.14 of the GRCh38 / hg38 human genome assembly) as the reference sequence.

[0025] Any one or more (i.e., any combination) of MAP3K15 missense variant nucleic acid molecules encoding MAP3K15 predicted loss-of-function polypeptides can be used within any of the methods described herein to determine whether a subject has an increased risk of developing a metabolic disorder, such as type 2 diabetes. A particular variant combination can form a mask that is used to statistically analyze a particular correlation between MAP3K15 and a reduced risk of developing a metabolic disorder, such as type 2 diabetes.

[0026] In any of the embodiments described herein, the metabolic disorder is type 2 diabetes, elevated hemoglobin A1c, or elevated serum glucose.In some embodiments, the metabolic disorder is type 2 diabetes.In some embodiments, the metabolic disorder is elevated hemoglobin A1c.In some embodiments, the metabolic disorder is elevated serum glucose.

[0027] Symptoms of type 2 diabetes include, but are not limited to, any one or more of hyperglycemia, insulin resistance, and reduced insulin levels, or a combination thereof. In some embodiments, symptoms of type 2 diabetes further include polyuria, polydipsia, hypereating, weight loss, blurred vision, pruritus, peripheral neuropathy, recurrent vaginal infections, and fatigue, or any combination thereof.

[0028] The present disclosure provides a method of treating a subject having or at risk of developing a metabolic disorder, the method comprising administering to the subject a MAP3K15 inhibitor. The disclosure also provides a method of treating a subject having or at risk of developing type 2 diabetes, the method comprising administering to the subject a MAP3K15 inhibitor.

[0029] The disclosure also provides a method of treating a subject having or at risk of developing elevated hemoglobin A1c, the method comprising administering to the subject a MAP3K15 inhibitor.

[0030] The disclosure also provides a method of treating a subject having or at risk of developing elevated serum glucose, the method comprising administering to the subject a MAP3K15 inhibitor.

[0031] The disclosure also provides a method for preventing a subject from developing a metabolic disorder, the method comprising administering to the subject a MAP3K15 inhibitor. The disclosure also provides a method of preventing a subject from developing type 2 diabetes, the method comprising administering to the subject a MAP3K15 inhibitor.

[0032] The disclosure also provides a method for preventing a subject from developing elevated hemoglobin A1c, the method comprising administering to the subject a MAP3K15 inhibitor. The disclosure also provides a method of preventing a subject from developing elevated serum glucose, the method comprising administering to the subject a MAP3K15 inhibitor.

[0033] In some embodiments, the MAP3K15 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 RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be designed to target any region of the MAP3K15 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes with a sequence within the MAP3K15 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the MAP3K15 polypeptide in the cells of the subject. In some embodiments, the MAP3K15 inhibitor comprises an antisense molecule that hybridizes with the MAP3K15 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the MAP3K15 polypeptide in the cells of the subject. In some embodiments, the MAP3K15 inhibitor comprises an siRNA that hybridizes with the MAP3K15 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the MAP3K15 polypeptide in the cells of the subject. In some embodiments, the MAP3K15 inhibitor comprises an shRNA that hybridizes to a MAP3K15 genomic nucleic acid molecule or mRNA molecule and reduces expression of a MAP3K15 polypeptide in cells of a subject.

[0034] The inhibitory nucleic acid molecule can comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous nucleic acid sequence, for example in a vector, or a heterologous label. For example, the inhibitory nucleic acid molecule can be present as an exogenous donor sequence in or containing a vector that contains the inhibitory nucleic acid molecule and the heterologous nucleic acid sequence. The inhibitory nucleic acid molecule can also be linked or fused to a heterologous label. The label can be directly detectable (e.g., a fluorophore) or indirectly detectable (e.g., a hapten, an enzyme, or a fluorophore quencher). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, 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, whereby an enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or 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, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0035] Inhibitory nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0036] The inhibitory nucleic acid molecule can also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains a modification to either the base, sugar, or phosphate moiety. 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 various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthin-9-yl (I), and 2-aminoadenin-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, 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-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[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 ribose and deoxyribose, 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-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10 Exemplary 2' sugar modifications include -O[(CH 2 ) n O] m CH 3 , -O(CH 2 ) n OCH 3 , -O(CH 2 ) n NH 2 , -O(CH 2 ) n CH 3 , -O(CH 2 ) n -ONH 2 , and -O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1~10 Alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2Modified sugars include, but are not limited to, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may 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 include, but are not limited to, CH 2 and S. Nucleotide sugar analogs can also include those containing modifications at the bridging ring oxygen, such as S. Nucleotide sugar analogs can also have sugar mimetics such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0038] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse 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 molecule is a gapmer, whereby the first 1-7 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end, respectively, have a 2'-MOE modification. In some embodiments, the first 1-7 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, the first 5 nucleotides of the 5'-end and the 3'-end are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0040] In some embodiments, the siRNA molecule has terminal modification.In some embodiments, the 5'-end of the antisense strand is phosphorylated.In some embodiments, a 5'-phosphate analog that cannot be hydrolyzed, such as 5'-(E)-vinyl-phosphonate, is used.

[0041] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to increase the stability and bioavailability of siRNA in vivo. Non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate to obtain phosphorothioate, boranophosphate, and phosphonoacetate linkages. In addition, the phosphodiester group can be replaced with a phosphotriester to facilitate cellular uptake of the siRNA and retention in serum components by removing its negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), allowing the 2'-hydroxyl to act as a nucleophile and attack the adjacent phosphorus of the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0042] In some embodiments, the siRNA molecule has base modifications, in some embodiments, the bases may be replaced with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0043] In some embodiments, siRNA molecules are bound to lipid.Lipid can be bound to 5'-end or 3'-end of siRNA, and can improve their bioavailability in vivo by associating with serum lipoprotein.Representative lipids include, but are not limited to, cholesterol and vitamin E, and fatty acids such as palmitic acid and tocopherol.

[0044] In some embodiments, an exemplary siRNA has the following formula: Sense: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N where "N" is a 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.

[0045] The present disclosure also provides a vector comprising any one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double-stranded DNA to which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector to which 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.

[0046] 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 composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. The carrier may comprise a buffered salt solution such as PBS, HBSS, and the like.

[0047] In some embodiments, the MAP3K15 inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks in the recognition sequence(s) in the MAP3K15 genomic nucleic acid molecule or a DNA binding protein that binds to the recognition sequence. The recognition sequence can be located in the coding region of the MAP3K15 gene or in a regulatory region that affects the expression of the gene. The recognition sequence of the DNA binding protein or nuclease agent can be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence can include or be adjacent to the start codon of the MAP3K15 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 of which targets a nuclease recognition sequence that includes or is adjacent to the start codon. As another example, two nuclease agents can be used, one targeting a nuclease recognition sequence containing or adjacent to a start codon, and the other targeting a nuclease recognition sequence containing or adjacent to a stop codon, and cleavage by these nuclease agents can result in the deletion of the coding region between the two nuclease recognition sequences.Any nuclease agent that induces a nick or double-strand break at the desired recognition sequence can be used in the methods and compositions disclosed herein.Any DNA binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0048] Suitable nuclease agents and DNA binding proteins for use herein include, but are not limited to, zinc finger proteins or zinc finger nuclease (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The length of the recognition sequence can vary, but includes, for example, recognition sequences that are about 30-36 bp for zinc finger proteins or ZFN pairs, about 15-18 bp for each ZFN, about 36 bp for TALE proteins or TALEN, and about 20 bp for CRISPR / Cas guide RNA.

[0049] In some embodiments, the CRISPR / Cas system can be used to modify the MAP3K15 genomic nucleic acid molecule in a cell. The methods and compositions disclosed herein can employ the CRISPR-Cas system by utilizing a CRISPR complex (comprising a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of the MAP3K15 nucleic acid molecule.

[0050] Cas proteins generally contain at least one RNA recognition domain or RNA binding domain that can interact with gRNA. Cas proteins can also contain nuclease domains (e.g., DNase or RNase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (e.g., FnCpf1). Cas proteins can have full cleavage activity to create double-stranded breaks in MAP3K15 genomic nucleic acid molecules, or can be nickases that create single-stranded breaks in MAP3K15 genomic nucleic acid molecules. Additional examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cas proteins include, but are not limited to, Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as homologs or variants thereof. Cas proteins can also be operably linked to heterologous polypeptides as fusion proteins. For example, Cas proteins 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, the Cas protein can be provided in the form of a protein, e.g., a Cas protein complexed with a gRNA.Alternatively, the Cas protein can be provided in the form of a nucleic acid molecule, e.g., RNA or DNA, encoding the Cas protein.

[0051] In some embodiments, targeted genetic modification of a MAP3K15 genomic nucleic acid molecule can be generated by contacting a cell with a Cas protein and one or more gRNAs that hybridize with one or more gRNA recognition sequences in a target genomic locus in a MAP3K15 genomic nucleic acid molecule. For example, the gRNA recognition sequence can be located within the region of SEQ ID NO: 1. The gRNA recognition sequence can include or be adjacent to the start codon of the MAP3K15 genomic nucleic acid molecule or the stop codon of the MAP3K15 genomic nucleic acid molecule. For example, the gRNA 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 away from the start codon or the stop codon.

[0052] The gRNA recognition sequence in the target genomic locus in the MAP3K15 genomic nucleic acid molecule is located near a protospacer adjacent motif (PAM) sequence, which is a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease. A canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. The gRNA can transport Cas9 anywhere in the genome for gene editing, but cannot edit at sites other than the site where Cas9 recognizes the 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 be adjacent to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 3' end. In some embodiments, the gRNA recognition sequence can be adjacent to the PAM at the 5' end. For example, the cleavage site of the Cas protein can be about 1 to about 10, about 2 to about 5, or 3 base pairs upstream or downstream of the PAM sequence. In some embodiments (e.g., when using Cas9 from S. pyogenes or a closely related Cas9), the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is immediately 3' to the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand is 5'-CCN-3', where N is any DNA nucleotide and is immediately 5' to the gRNA recognition sequence of the complementary strand of the target DNA.

[0053] gRNA is an RNA molecule that binds to Cas protein and targets Cas protein to a specific location in MAP3K15 genomic nucleic acid molecule.Exemplary gRNA is an effective gRNA for inducing Cas enzyme to bind to or cleave MAP3K15 genomic nucleic acid molecule, wherein gRNA comprises a DNA targeting segment that hybridizes with gRNA recognition sequence in MAP3K15 genomic nucleic acid molecule.Exemplary gRNA comprises a DNA targeting segment that hybridizes with gRNA recognition sequence present in MAP3K15 genomic nucleic acid molecule that includes or is adjacent to start codon or stop codon. For example, a gRNA can be selected to hybridize with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a start codon, or with a gRNA recognition sequence located about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 200, about 300, about 400, about 500, or about 1,000 nucleotides away from a stop codon. Suitable gRNAs can include about 17 to about 25 nucleotides, about 17 to about 23 nucleotides, about 18 to about 22 nucleotides, or about 19 to about 21 nucleotides. In some embodiments, the gRNA can include 20 nucleotides.

[0054] Examples of suitable gRNA recognition sequences located within the human MAP3K15 reference gene are set forth in Table 1 as SEQ ID NOs: 19-38.

[0055] [Table 1]

[0056] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target MAP3K15 genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at a site inside or outside the nucleic acid sequence present in the target MAP3K15 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds. For example, the formation of a CRISPR complex (including the gRNA hybridized with the gRNA recognition sequence and complexed with the Cas protein) can result in the cleavage of one or both strands within or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 50 or more base pairs) the nucleic acid sequence present in the MAP3K15 genomic nucleic acid molecule to which the DNA targeting segment of the gRNA binds.

[0057] Such a method can result in a MAP3K15 genomic nucleic acid molecule in which, for example, a region of SEQ ID NO:1 is destroyed, the start codon is destroyed, the stop codon is destroyed, or the coding sequence is destroyed or deleted. Optionally, the cell can be further contacted with one or more additional gRNAs that hybridize with additional gRNA recognition sequences in the target genomic locus of the MAP3K15 genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes with a second gRNA recognition sequence), the cleavage by the Cas protein can create two or more double-strand breaks or two or more single-strand breaks.

[0058] In some embodiments, the treatment or prevention method further comprises detecting the presence or absence of a MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide in a biological sample of a subject. As used throughout this disclosure, a "MAP3K15 missense variant nucleic acid molecule encoding a predicted loss-of-function polypeptide" is any MAP3K15 nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes a MAP3K15 polypeptide having partial loss-of-function, complete loss-of-function, predicted partial loss-of-function, or predicted complete loss-of-function.

[0059] The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing a metabolic disorder, wherein the subject has or is at risk of developing a metabolic disorder. In some embodiments, the subject has a metabolic disorder. In some embodiments, the subject is at risk of developing a metabolic disorder. The present disclosure also provides a method of preventing a subject from developing a metabolic disorder by administering a therapeutic agent for preventing a metabolic disorder. In some embodiments, the method includes determining whether the subject has a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in MAP3K15 by obtaining or obtaining a biological sample from the subject and performing or performing a sequence analysis on the biological sample to determine whether the subject has a genotype that includes a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted in MAP3K15. In some embodiments, the method further includes administering or continuing to administer a standard dose of a therapeutic agent for treating or preventing a metabolic disorder to the subject who is a MAP3K15 standard, and / or administering a MAP3K15 inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent for treating or preventing a metabolic disorder to the subject heterozygous for the MAP3K15 missense variant nucleic acid molecule at a standard dose or less, and / or administering a MAP3K15 inhibitor to the subject. In some embodiments, the method further comprises administering or continuing to administer a therapeutic agent for treating or preventing a metabolic disorder to the subject homozygous for the MAP3K15 missense variant nucleic acid molecule at a standard dose or less. The presence of a genotype with a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 indicates that the subject is at low risk of developing a metabolic disorder, such as type 2 diabetes. In some embodiments, the subject is MAP3K15 normative. In some embodiments, the subject is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15.

[0060] For subjects who have been genotyped or determined to be heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a MAP3K15-based or MAP3K15-predicted loss-of-function polypeptide, such subjects can be administered a MAP3K15 inhibitor as described herein.

[0061] Detecting the presence or absence of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 in a biological sample from a subject and / or determining whether a subject has a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0062] In some embodiments, if the subject is MAP3K15 normative, the subject is administered a standard dose of a therapeutic agent for treating or preventing a metabolic disorder. In some embodiments, if the subject is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a MAP3K15 predicted loss-of-function polypeptide, the subject is administered a standard dose or a lower dose of a therapeutic agent for treating or preventing a metabolic disorder.

[0063] In some embodiments, the treatment or prevention method further comprises detecting the presence or absence of a MAP3K15 predicted loss-of-function polypeptide in the subject's biological sample. In some embodiments, if the subject does not have a MAP3K15 predicted loss-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing a metabolic disorder. In some embodiments, if the subject has a MAP3K15 predicted loss-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing a metabolic disorder. In some embodiments, if the subject has a MAP3K15 predicted loss-of-function polypeptide, the subject is administered a standard dose of a therapeutic agent for treating or preventing a metabolic disorder.

[0064] The present disclosure also provides a method of treating a subject with a therapeutic agent for treating or preventing a metabolic disorder, wherein the subject has or is at risk of developing a metabolic disorder. In some embodiments, the method includes determining whether the subject has a loss-of-function polypeptide predicted by MAP3K15 by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a loss-of-function polypeptide predicted by MAP3K15. If the subject does not have a loss-of-function polypeptide predicted by MAP3K15, the therapeutic agent for treating or preventing the metabolic disorder is administered or continues to be administered to the subject at a standard dosage, and / or a MAP3K15 inhibitor is administered to the subject. If the subject has a loss-of-function polypeptide predicted by MAP3K15, the therapeutic agent for treating or preventing the metabolic disorder is administered or continues to be administered to the subject at a standard dosage or less, and / or a MAP3K15 inhibitor is administered to the subject. The presence of a MAP3K15 predicted loss-of-function polypeptide indicates that the subject is at low risk of developing a metabolic disorder. In some embodiments, the subject has a MAP3K15 predicted loss-of-function polypeptide. In some embodiments, the subject does not have a MAP3K15 predicted loss-of-function polypeptide.

[0065] The present disclosure also provides a method of preventing a subject from developing a metabolic disorder by administering a therapeutic agent that prevents the metabolic disorder. In some embodiments, the method includes determining whether the subject has a loss-of-function polypeptide predicted by MAP3K15 by obtaining or obtaining a biological sample from the subject and performing or performing an assay on the biological sample to determine whether the subject has a loss-of-function polypeptide predicted by MAP3K15. If the subject does not have a loss-of-function polypeptide predicted by MAP3K15, the therapeutic agent that prevents the metabolic disorder is administered or continues to be administered to the subject at a standard dose, and / or a MAP3K15 inhibitor is administered to the subject. If the subject has a loss-of-function polypeptide predicted by MAP3K15, the therapeutic agent that prevents the metabolic disorder is administered or continues to be administered to the subject at a standard dose or less, and / or a MAP3K15 inhibitor is administered to the subject. The presence of a loss-of-function polypeptide predicted by MAP3K15 indicates that the subject is at low risk of developing a metabolic disorder. In some embodiments, the subject has a MAP3K15 predicted loss-of-function polypeptide, hi some embodiments, the subject does not have a MAP3K15 predicted loss-of-function polypeptide.

[0066] Detecting the presence or absence of a MAP3K15 predicted loss-of-function polypeptide in a biological sample from a subject and / or determining whether a subject has a MAP3K15 predicted loss-of-function polypeptide can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide can be present in a cell obtained from the subject.

[0067] In some embodiments, the MAP3K15 inhibitor is a small molecule. In some embodiments, the MAP3K15 inhibitor is staurosporine, lestaurtinib, NVP-TAE684, ruxolitinib, SU-14813, sunitinib, JNJ-28312141, crizotinib, linifanib, quizartinib, axitinib, motesanib, AST-487, AT-7519, barasertib-h QPA, cediranib, selumetinib, BI-2536, afatinib, dramapimod, BMS-345541, BMS-387032, brivanib, CHIR-265, canertinib, CI-1040, tofacitinib, dasatinib, foretinib, alvocidib, GDC-0879, pictilisib, GSK-1838705A, GSK-461364A, GW-2580, neratinib, imatinib, Ki-20227, KW-2449, lapatinib, enzastaurin, MLN-120B, tandutinib, MLN-8054, nilotinib, pazopanib, PD-173955, PHA-665752, PI-103, midostaurin, PLX-4720, Talanib, tamatinib, R547, SGX-523, bosutinib, sorafenib, TG-100-115, fedratinib, vandetanib, tozasertib, neflamapimod, dovitinib, erlotinib, gefitinib, GSK690693, ruboxistaurin, SB203580, A-674563, or masitinib. In some embodiments, the MAP3K15 inhibitor is staurosporine, lestaurtinib, NVP-TAE684, ruxolitinib, SU-14813, sunitinib, JNJ-28312141, crizotinib, SB203580, or ruboxistaurin. In some embodiments, the MAP3K15 inhibitor is staurosporine. In some embodiments, the MAP3K15 inhibitor is lestaurtinib. In some embodiments, the MAP3K15 inhibitor is NVP-TAE684. In some embodiments, the MAP3K15 inhibitor is ruxolitinib. In some embodiments, the MAP3K15 inhibitor is SU-14813. In some embodiments, the MAP3K15 inhibitor is sunitinib. In some embodiments, the MAP3K15 inhibitor is JNJ-28312141.In some embodiments, the MAP3K15 inhibitor is crizotinib. In some embodiments, the MAP3K15 inhibitor is linifanib. In some embodiments, the MAP3K15 inhibitor is quizartinib. In some embodiments, the MAP3K15 inhibitor is axitinib. In some embodiments, the MAP3K15 inhibitor is motesanib. In some embodiments, the MAP3K15 inhibitor is AST-487. In some embodiments, the MAP3K15 inhibitor is AT-7519. In some embodiments, the MAP3K15 inhibitor is barasertib-hQPA. In some embodiments, the MAP3K15 inhibitor is cediranib. In some embodiments, the MAP3K15 inhibitor is selumetinib. In some embodiments, the MAP3K15 inhibitor is BI-2536. In some embodiments, the MAP3K15 inhibitor is afatinib. In some embodiments, the MAP3K15 inhibitor is doramapimod. In some embodiments, the MAP3K15 inhibitor is BMS-345541. In some embodiments, the MAP3K15 inhibitor is BMS-387032. In some embodiments, the MAP3K15 inhibitor is brivanib. In some embodiments, the MAP3K15 inhibitor is CHIR-265. In some embodiments, the MAP3K15 inhibitor is canertinib. In some embodiments, the MAP3K15 inhibitor is CI-1040. In some embodiments, the MAP3K15 inhibitor is tofacitinib. In some embodiments, the MAP3K15 inhibitor is dasatinib. In some embodiments, the MAP3K15 inhibitor is foretinib. In some embodiments, the MAP3K15 inhibitor is alvocidib. In some embodiments, the MAP3K15 inhibitor is GDC-0879. In some embodiments, the MAP3K15 inhibitor is pictilisib. In some embodiments, the MAP3K15 inhibitor is GSK-1838705A. In some embodiments, the MAP3K15 inhibitor is GSK-461364A. In some embodiments, the MAP3K15 inhibitor is GW-2580. In some embodiments, the MAP3K15 inhibitor is neratinib.In some embodiments, the MAP3K15 inhibitor is imatinib. In some embodiments, the MAP3K15 inhibitor is Ki-20227. In some embodiments, the MAP3K15 inhibitor is KW-2449. In some embodiments, the MAP3K15 inhibitor is lapatinib. In some embodiments, the MAP3K15 inhibitor is enzastaurin. In some embodiments, the MAP3K15 inhibitor is MLN-120B. In some embodiments, the MAP3K15 inhibitor is tandutinib. In some embodiments, the MAP3K15 inhibitor is MLN-8054. In some embodiments, the MAP3K15 inhibitor is nilotinib. In some embodiments, the MAP3K15 inhibitor is pazopanib. In some embodiments, the MAP3K15 inhibitor is PD-173955. In some embodiments, the MAP3K15 inhibitor is PHA-665752. In some embodiments, the MAP3K15 inhibitor is PI-103. In some embodiments, the MAP3K15 inhibitor is midostaurin. In some embodiments, the MAP3K15 inhibitor is PLX-4720. In some embodiments, the MAP3K15 inhibitor is vatalanib. In some embodiments, the MAP3K15 inhibitor is tamatinib. In some embodiments, the MAP3K15 inhibitor is R547. In some embodiments, the MAP3K15 inhibitor is SGX-523. In some embodiments, the MAP3K15 inhibitor is bosutinib. In some embodiments, the MAP3K15 inhibitor is sorafenib. In some embodiments, the MAP3K15 inhibitor is TG-100-115. In some embodiments, the MAP3K15 inhibitor is fedratinib. In some embodiments, the MAP3K15 inhibitor is vandetanib. In some embodiments, the MAP3K15 inhibitor is tozasertib. In some embodiments, the MAP3K15 inhibitor is neflamapimod. In some embodiments, the MAP3K15 inhibitor is dovitinib. In some embodiments, the MAP3K15 inhibitor is erlotinib.In some embodiments, the MAP3K15 inhibitor is gefitinib.In some embodiments, the MAP3K15 inhibitor is GSK690693. In some embodiments, the MAP3K15 inhibitor is ruboxistaurin. In some embodiments, the MAP3K15 inhibitor is SB203580. In some embodiments, the MAP3K15 inhibitor is A-674563. In some embodiments, the MAP3K15 inhibitor is masitinib.

[0068] Examples of therapeutic agents that treat or prevent type 2 diabetes, treat or prevent elevated hemoglobin A1c, and / or treat or prevent elevated serum glucose include, but are not limited to, metformin, insulin, sulfonylureas (e.g., glyburide, glipizide, and glimepiride), meglitinides (e.g., repaglinide and nateglinide), thiazolidinediones (e.g., rosiglitazone and pioglitazone), DPP-4 inhibitors (e.g., sitagliptin, saxagliptin, and linagliptin), GLP-1 receptor agonists (e.g., exenatide, liraglutide, and semaglutide), and SGLT2 inhibitors (e.g., canagliflozin, dapagliflozin, and empagliflozin). In some embodiments, the therapeutic agent is metformin, insulin, glyburide, glipizide, glimepiride, repaglinide, nateglinide, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, semaglutide, canagliflozin, dapagliflozin, or empagliflozin. In some embodiments, the therapeutic agent is metformin. In some embodiments, the therapeutic agent is insulin. In some embodiments, the therapeutic agent is glyburide. In some embodiments, the therapeutic agent is glipizide. In some embodiments, the therapeutic agent is glimepiride. In some embodiments, the therapeutic agent is repaglinide. In some embodiments, the therapeutic agent is nateglinide. In some embodiments, the therapeutic agent is rosiglitazone. In some embodiments, the therapeutic agent is pioglitazone. In some embodiments, the therapeutic agent is sitagliptin. In some embodiments, the therapeutic agent is saxagliptin. In some embodiments, the therapeutic agent is linagliptin. In some embodiments, the therapeutic agent is exenatide. In some embodiments, the therapeutic agent is liraglutide. In some embodiments, the therapeutic agent is semaglutide. In some embodiments, the therapeutic agent is canagliflozin. In some embodiments, the therapeutic agent is dapagliflozin. In some embodiments, the therapeutic agent is empagliflozin.

[0069] In some embodiments, the therapeutic agent is GLUCOPHAGE® or GLUMETZA® (metformin), sulfonylureas (DIABETA® or GLYNASE® (glyburide), GLCOTROL® (glipizide), and AMARYL® (glimepiride)), meglitinides (PRANDIN® (repaglinide) and STARLIX® (nateglinide)), thiazolidinediones (AVANDIA® (rosiglitazone) and ACTOS® (pioglitazone)), dipeptidyl peptidase-4 (DPP-4) inhibitors (JANUVIA® (sitagliptin), ONGLYZA® (saxagliptin), and TRADJENTA® (linagliptin). )), glucagon-like peptide-1 (GLP-1) receptor agonists (BYETTA® (exenatide) and VICTOZA® (liraglutide)), SGLT2 inhibitors (INVOKANA® (canagliflozin) and FARXIGA® (dapagliflozin)), or APIDRA® (insulin glulisine), HUMALOG® (insulin lispro), NOVOLOG® (insulin aspart), LANTUS® (insulin glargine), LEVEMIR® (insulin detemir), or HUMULIN® N or NOVOLIN® N (insulin isophane), PRALUENT® (alirocumab), or any combination thereof. In some embodiments, the therapeutic agent is PRALUENT® (alirocumab).

[0070] In some embodiments, the therapeutic agent is metformin, a sulfonylurea (glyburide, glipizide, or glimepiride), a meglitinide (repaglinide or nateglinide), a thiazolidinedione (rosiglitazone or pioglitazone), a dipeptidyl peptidase-4 (DPP-4) inhibitor (sitagliptin, saxagliptin, or linagliptin), a glucagon-like peptide-1 (GLP-1) receptor agonist (exenatide or liraglutide), an SGLT2 inhibitor (canagliflozin or dapagliflozin), or an insulin (gululisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, or insulin isophane), or alirocumab, or any combination thereof. In some embodiments, the therapeutic agent is alirocumab.

[0071] In some embodiments, the dose of a therapeutic agent for treating or preventing a metabolic disorder can be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% for a subject who is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 (i.e., receiving a lower standard dose) compared to a subject who is MAP3K15-based (which may receive a standard dose). In some embodiments, the dose of a therapeutic agent for treating or preventing a metabolic disorder can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, a subject who is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 can be administered less frequently compared to a subject who is MAP3K15-based.

[0072] In some embodiments, the dosage of a therapeutic agent for treating or preventing a metabolic disorder can be reduced by about 10%, about 20%, about 30%, about 40%, about 50% for a subject who is homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 compared to a subject who is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15. In some embodiments, the dosage of a therapeutic agent for treating or preventing a metabolic disorder can be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, the dosage of a therapeutic agent for treating or preventing a metabolic disorder in a subject who is homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 can be administered less frequently compared to a subject who is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15.

[0073] Administration of the therapeutic agent for treating or preventing metabolic disorders and / or MAP3K15 inhibitor can be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. Repeated administration can be the same dose or different doses. Administration can be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a particular dosing regimen, a subject can be treated for an extended period of time, for example, 6 months, 1 year, or more.

[0074] Administration of the therapeutic agent and / or MAP3K15 inhibitor for treating or preventing metabolic disorders can occur by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a single administration dose). Pharmaceutical compositions can be formulated using one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0075] As used herein, the terms "treat", "treating", and "treatment" and "prevent", "prevention", and "prevention" refer to eliciting a desired biological response, such as a therapeutic effect and a prophylactic effect, respectively. In some embodiments, the therapeutic effect includes one or more of the following after administration of the agent or a composition comprising the agent: reduction / reduction of metabolic disorder, reduction / reduction in the severity of metabolic disorder (e.g., reduction or inhibition of onset of metabolic disorder), reduction / reduction in symptoms and metabolic disorder-related effects, delay in onset of symptoms and metabolic disorder-related effects, reduction in the severity of symptoms of metabolic disorder-related effects, reduction in the number of symptoms and metabolic disorder-related effects, reduction in the latency period of symptoms and metabolic disorder-related effects, amelioration of symptoms and metabolic disorder-related effects, reduction in secondary symptoms, reduction in secondary infections, prevention of recurrence of metabolic disorder, reduction in the number or frequency of recurrent episodes, increase in the latency period between symptomatic episodes, increase in the time to sustained progression, accelerated recovery, or increased efficacy or reduced resistance to alternative therapeutic agents, and / or increased survival time of the affected host animal. The prophylactic effect may include complete or partial avoidance / suppression or delay (e.g., complete or partial avoidance / suppression or delay) of the onset / progression of a metabolic disorder following administration of a treatment protocol, and increased survival time of the affected host animal. Treatment of a metabolic disorder, such as type 2 diabetes, includes treatment of a subject already diagnosed with some form of metabolic disorder, either at a clinical stage or clinical symptoms, delaying the onset or progression or worsening or aggravation of symptoms or signs of a metabolic disorder, and / or preventing and / or reducing the severity of the metabolic disorder. In some embodiments, the metabolic disorder is type 2 diabetes, elevated hemoglobin A1c, or elevated serum glucose.

[0076] The present disclosure also provides a method for identifying a subject at high risk of developing a metabolic disorder. In some embodiments, the method includes determining or having determined the presence or absence of a MAP3K15 missense variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule and / or a cDNA molecule) encoding a loss-of-function polypeptide predicted by MAP3K15 in a biological sample obtained from the subject. If the subject lacks a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 (i.e., the subject is classified as MAP3K15-standard by genotyping), the subject has a high risk of developing a metabolic disorder. If the subject has a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 (i.e., the subject is heterozygous or homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15), the subject has a low risk of developing a metabolic disorder.

[0077] Having a single copy of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 further protects a subject from developing a metabolic disorder than not having a copy of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15. Without intending to be limited to a particular theory or mechanism of action, it is believed that a single copy of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 (i.e., heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15) protects a subject from developing a metabolic disorder, and it is also believed that having two copies of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 (i.e., homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15) may further protect a subject from developing a metabolic disorder compared to a subject having a single copy. Thus, in some embodiments, a single copy of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 may not completely protect a subject from developing a metabolic disorder, but may instead provide partial or incomplete protection. Without wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of a metabolic disorder that are still present in a subject having a single copy of a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, resulting in less than complete protection from developing a metabolic disorder.

[0078] Determining whether a subject has a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 in a biological sample from the subject and / or determining whether a subject has a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15 can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a cell obtained from the subject.

[0079] In some embodiments, once a subject is identified as having a high risk of developing a metabolic disorder, the subject is administered a therapeutic agent for treating or preventing the metabolic disorder and / or a MAP3K15 inhibitor as described herein. For example, if a subject is MAP3K15-based and therefore at high risk of developing a metabolic disorder, the subject is administered a MAP3K15 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing the metabolic disorder. In some embodiments, if a subject is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, the subject is administered a therapeutic agent for treating or preventing the metabolic disorder at a dose equal to or less than the standard dose, and also administered a MAP3K15 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing the metabolic disorder. In some embodiments, if the subject is homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, the subject is administered a therapeutic agent for treating or preventing a metabolic disorder at a dose equal to or less than the standard dose. In some embodiments, the subject is MAP3K15 norm. In some embodiments, the subject is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15. In some embodiments, the subject is homozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15.

[0080] In some embodiments, any of the methods described herein may further comprise determining the total burden of the subject having a MAP3K15 missense variant nucleic acid molecule encoding a MAP3K15 predicted loss-of-function polypeptide and / or a MAP3K15 predicted loss-of-function variant polypeptide associated with a reduced risk of developing a metabolic disorder. The total burden is the sum of all variants in the MAP3K15 gene, which may be performed in an association analysis with a metabolic disorder. In some embodiments, the subject is homozygous for one or more of the MAP3K15 missense variant nucleic acid molecules encoding a MAP3K15 predicted loss-of-function polypeptide associated with a reduced risk of developing a metabolic disorder. In some embodiments, the subject is heterozygous for one or more of the MAP3K15 missense variant nucleic acid molecules encoding a MAP3K15 predicted loss-of-function polypeptide associated with a reduced risk of developing a metabolic disorder. The results of the association analysis suggest that the MAP3K15 missense variant nucleic acid molecule encoding a MAP3K15 predicted loss-of-function polypeptide is associated with a reduced risk of developing a metabolic disorder. When a subject has a lower total load, the subject is at higher risk of developing metabolic disorder, and the subject is administered or continues to be administered a standard dose of a therapeutic agent for treating or preventing metabolic disorder and / or a MAP3K15 inhibitor.When a subject has a higher total load, the subject is at lower risk of developing metabolic disorder, and the subject is administered or continues to be administered a standard dose or a lower dose of a therapeutic agent for treating or preventing metabolic disorder.The higher the total load, the lower the risk of developing metabolic disorder.

[0081] MAP3K15 variants that can be used in the total loading analysis include any one or more or any combination of the following:

[0082] [Table 2-1]

[0083] [Table 2-2]

[0084]

Table 2-3

[0085]

Table 2-4

[0086]

Table 2-5

[0087]

Table 2-6

[0088]

Table 2-7

[0089]

Table 2-8

[0090]

Table 2-9

[0091]

Table 2-10

[0092]

Table 2-11

[0093]

Table 2-12

[0094]

Table 2-13

[0095]

Table 2-14

[0096]

Table 2-15

[0097]

Table 2-16

[0098]

Table 2-17

[0099]

Table 2-18

[0100]

Table 2-19

[0101]

Table 2-20

[0102]

Table 2-21

[0103]

Table 2-22

[0104]

Table 2-23

[0105]

Table 2-24

[0106]

Table 2-25

[0107]

Table 2-26

[0108]

Table 2-27

[0109]

Table 2-28

[0110]

Table 2-29

[0111]

Table 2-30

[0112]

Table 2-31

[0113]

Table 2-32

[0114]

Table 2-33

[0115]

Table 2-34

[0116]

Table 2-35

[0117]

Table 2-36

[0118]

Table 2-37

[0119]

Table 2-38

[0120]

Table 2-39

[0121]

Table 2-40

[0122]

Table 2-41

[0123]

Table 2-42

[0124]

Table 2-43

[0125]

Table 2-44

[0126]

Table 2-45

[0127]

Table 2-46

[0128]

Table 2-47

[0129]

Table 2-48

[0130]

Table 2-49

[0131]

Table 2-50

[0132]

Table 2-51

[0133]

Table 2-52

[0134]

Table 2-53

[0135]

Table 2-54

[0136]

Table 2-55

[0137]

Table 2-56

[0138]

Table 2-57

[0139]

Table 2-58

[0140]

Table 2-59

[0141]

Table 2-60

[0142]

Table 2-61

[0143]

Table 2-62

[0144]

Table 2-63

[0145]

Table 2-64

[0146]

Table 2-65

[0147]

Table 2-66

[0148]

Table 2-67

[0149]

Table 2-68

[0150] In some embodiments, the total burden of subjects having any one or more MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15 represents a weighted sum of any multiple MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15. In some embodiments, the total 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 1,000,000 or more genetic variants present in or surrounding the MAP3K15 gene (up to 10 Mb), where the genetic burden is the number of alleles multiplied by the estimated association for each allele with a metabolic disorder or related outcome (e.g., a weighted polygenic burden score). This can include any genetic variants that are close to the MAP3K15 gene (up to 10Mb around the gene) and show non-zero association with metabolic disorder-related traits in gene association analysis, regardless of genome annotation.In some embodiments, if a subject has a total load higher than the desired threshold score, the subject has a low risk of developing metabolic disorder.In some embodiments, if a subject has a total load lower than the desired threshold score, the subject has a high risk of developing metabolic disorder.

[0151] In some embodiments, the total burden can be divided into quintiles, for example, top quintile, middle quintile, and bottom quintile, with the top quintile of total burden corresponding to the lowest risk group and the bottom quintile of total burden corresponding to the highest risk group. In some embodiments, subjects with a larger total burden include the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% total burden from the subject population. In some embodiments, the genetic variants include genetic variants that have an association with metabolic disorders in the top 10%, top 20%, top 30%, top 40%, or top 50% of the p-value range for the association. In some embodiments, each of the identified genetic variants is about 10 -2 , about 10 -3 , about 10 -4 , about 10 -5 , about 10 -6 , about 10 -7 , about 10 -8 , about 10 -9 , about 10 -10 , about 10 -11 , about 10 -12 , about 10 -13 , about 10 -14 , or about 10 -15 In some embodiments, the identified genetic variants include those with a p-value of 5×10 -8In some embodiments, the identified genetic variants include genetic variants that are associated with metabolic disorders in subjects at high risk with an odds ratio (OR) of about 1.5 or more, about 1.75 or more, about 2.0 or more, or about 2.25 or more for the top 20% of the distribution; or about 1.5 or more, about 1.75 or more, about 2.0 or more, about 2.25 or more, about 2.5 or more, or about 2.75 or more for the remainder of the reference population. In some embodiments, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or may be greater than 7.0. In some embodiments, high-risk subjects include subjects with a total burden in the bottom decile, quintile, or tertile in the reference population. The total burden threshold is determined based on the nature of the intended practical application and the risk difference that is considered meaningful for that practical application.

[0152] In some embodiments, once a subject is identified as having a high risk of developing a metabolic disorder, the subject is further administered a therapeutic agent for treating or preventing the metabolic disorder and / or a MAP3K15 inhibitor as described herein. For example, if a subject is MAP3K15 based and therefore at high risk of developing a metabolic disorder, the subject is administered a MAP3K15 inhibitor. In some embodiments, such a subject is also administered a therapeutic agent for treating or preventing the metabolic disorder. In some embodiments, if the subject is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, the subject is administered a therapeutic agent for treating or preventing the metabolic disorder at a dose equal to or less than the standard dose, and also administered a MAP3K15 inhibitor. In some embodiments, the subject is MAP3K15 based. In some embodiments, the subject is heterozygous for a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15. Furthermore, if a subject has a lower overall burden of having MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15 and therefore has a higher risk of developing a metabolic disorder, the subject is administered a therapeutic agent for treating or preventing the metabolic disorder. In some embodiments, if a subject has a lower overall burden of having MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15, the subject is administered a therapeutic agent for treating or preventing the metabolic disorder at a dose equal to or higher than the standard dose administered to a subject with a higher overall burden of having MAP3K15 missense variant nucleic acid molecules encoding loss-of-function polypeptides predicted by MAP3K15.

[0153] The present disclosure also provides a method for detecting the presence or absence of MAP3K15 missense variant genomic nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules generated from mRNA molecules) that code for a loss-of-function polypeptide predicted by MAP3K15 in a biological sample of a subject. It is understood that gene sequences within a population, and the mRNA molecules encoded by such genes, may differ due to polymorphisms, such as single nucleotide polymorphisms. The sequences provided herein for MAP3K15 variant genomic nucleic acid molecules, MAP3K15 variant mRNA molecules, and MAP3K15 variant cDNA molecules are merely exemplary sequences. Other sequences for MAP3K15 variant genomic nucleic acid molecules, variant mRNA molecules, and variant cDNA molecules are also possible.

[0154] The biological sample can be derived from any cell, tissue, or biological fluid of a subject. The biological sample may include any clinically relevant tissue, such as, for example, a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a sample of a bodily fluid, such as, for example, blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine. In some cases, the sample includes a buccal swab. The biological sample used in the methods disclosed herein may vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. The biological sample may be treated differently depending on the assay employed. For example, when detecting MAP3K15 missense mutant nucleic acid molecules that encode predicted loss-of-function polypeptides in MAP3K15, a pretreatment designed to isolate or enrich the biological sample for genomic DNA may be employed. For this purpose, various techniques may be used. When detecting the level of MAP3K15 mutant mRNA molecules, various techniques may be used to enrich the biological sample containing mRNA molecules. Various methods may be used to detect the presence or level of mRNA molecules, or the presence of a particular mutant genomic DNA locus.

[0155] In some embodiments, detecting a MAP3K15 missense variant nucleic acid molecule encoding a loss-of-function polypeptide predicted for MAP3K15 in a subject comprises performing sequence analysis on a biological sample obtained from the subject to determine whether a MAP3K15 genomic nucleic acid molecule in the biological sample, and / or a MAP3K15 mRNA molecule in the biological sample, and / or a MAP3K15 cDNA molecule generated from the mRNA molecule in the biological sample causes loss of function (partial or complete) or contains one or more mutations predicted to cause loss of function (partial or complete).

[0156] In some embodiments, a method for detecting the presence or absence of a MAP3K15 missense variant nucleic acid molecule (e.g., a genomic nucleic acid molecule, an mRNA molecule, and / or a cDNA molecule generated from an mRNA molecule) encoding a predicted loss-of-function polypeptide of MAP3K15 in a subject comprises performing an assay on a biological sample obtained from the subject, the assay determining whether the nucleic acid molecule in the biological sample comprises a particular nucleotide sequence.

[0157] In some embodiments, the biological sample includes cells or cell lysates. Such methods can further include, for example, obtaining a biological sample from a subject that includes a MAP3K15 genomic nucleic acid molecule or an mRNA molecule, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays can include, for example, determining the identity of these positions of a particular MAP3K15 nucleic acid molecule. In some embodiments, the methods are in vitro methods.

[0158] In some embodiments, the determining, detecting, or sequence analysis includes sequencing at least a portion of the nucleotide sequence of a MAP3K15 genomic nucleic acid molecule, a MAP3K15 mRNA molecule, or a MAP3K15 cDNA molecule in a biological sample, wherein the sequenced portion contains one or more mutations that cause or are predicted to cause a loss of function (partial or complete).

[0159] In some embodiments, the assay comprises sequencing the entire nucleic acid molecule. In some embodiments, only the MAP3K15 genomic nucleic acid molecule is analyzed. In some embodiments, only the MAP3K15 mRNA is analyzed. In some embodiments, only the MAP3K15 cDNA obtained from the MAP3K15 mRNA is analyzed.

[0160] Mutation-specific polymerase chain reaction techniques can be used to detect mutations such as SNPs in nucleic acid sequences. Mutation-specific primers can be used because DNA polymerase will not extend if there is a mismatch with the template.

[0161] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present in a cell obtained from the subject.

[0162] In some embodiments, the assay involves contacting a biological sample with a primer or probe, such as a mutation-specific primer or a mutation-specific probe, that specifically hybridizes to a MAP3K15 mutant genomic sequence, mutant mRNA sequence, or mutant cDNA sequence, but not to a corresponding MAP3K15 reference sequence, under stringent conditions, and determining whether hybridization occurs.

[0163] In some embodiments, the determining, detecting, or sequence analyzing step includes: a) amplifying at least a portion of a nucleic acid molecule encoding a MAP3K15 polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a support containing a mutation-specific probe; and d) detecting the detectable label.

[0164] In some embodiments, the assay involves RNA sequencing (RNA-Seq). In some embodiments, the assay also involves reverse transcribing mRNA into cDNA, for example, by reverse transcription polymerase chain reaction (RT-PCR).

[0165] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to the target nucleotide sequence and specifically detect and / or identify polynucleotides comprising mutant genomic nucleic acid molecules, mutant mRNA molecules, or mutant cDNA molecules of MAP3K15. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length that is sufficient for use in the detection method of choice, including any assay described or exemplified herein. Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. The probes and primers can have complete nucleotide sequence identity to consecutive nucleotides within the target nucleotide sequence, although probes that are different from the target nucleotide sequence and that retain the ability to specifically detect and / or identify the target nucleotide sequence can be designed by conventional methods. The 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 to the nucleotide sequence of the target nucleic acid molecule.

[0166] Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing (fluorescence in situ hybridization (FISH)), including the use of labeled primers or labeled probes on purified DNA, amplified DNA, and fixed cell preparations. In some methods, the target nucleic acid molecule may be amplified prior to or simultaneously 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).

[0167] Hybridization techniques can employ stringent conditions so that the probe or primer specifically hybridizes with its target. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence detectably higher than other non-target sequences, for example, at least 2-fold, at least 3-fold, at least 4-fold or more above background, including more than 10-fold above background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 2-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences at least 3-fold. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence detectably higher than other nucleotide sequences 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 extent than other nucleotide sequences, more than 10-fold above background. Stringent conditions are sequence-dependent and will be different in different circumstances.

[0168] Suitable stringent conditions that promote DNA hybridization, such as 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by a 2x SSC wash at 50°C, are known or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Typically, stringent conditions for hybridization and detection include conditions in which the salt concentration is less than about 1.5 M NaCl at pH 7.0-8.3. +ion, usually about 0.01 to 1.0 M Na + The conditions will be ionic concentration (or other salts) and temperature of at least about 30° C. for short probes (e.g., 10-50 nucleotides) and at least about 60° C. for longer probes (e.g., more than 50 nucleotides). Stringent conditions may be achieved by the addition of destabilizing agents such as formamide. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash will be at least long enough to reach equilibrium.

[0169] In some embodiments, such isolated nucleic acid molecules comprise 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 molecule comprises or consists of at least about 18 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 10 to about 35, about 10 to about 30, about 10 to about 25, about 12 to about 30, about 12 to about 28, about 12 to about 24, about 15 to about 30, about 15 to about 25, about 18 to about 30, about 18 to about 25, about 18 to about 24, or about 18 to about 22 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 18 to about 30 nucleotides.In some embodiments, the isolated nucleic acid molecule comprises or consists of at least about 15 nucleotides to at least about 35 nucleotides.

[0170] In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to MAP3K15 missense mutant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules). Such nucleic acid molecules can be used, for example, as probes, primers, mutation-specific probes, or mutation-specific primers as described or exemplified herein, including, but not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each of which is described in more detail elsewhere herein and can be used in any of the methods described herein.

[0171] In some embodiments, the isolated nucleic acid molecule hybridizes 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 a MAP3K15 missense variant genomic nucleic acid molecule, a MAP3K15 missense variant mRNA molecule, and / or a MAP3K15 missense variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides, or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule consists of or comprises about 15 to about 35 nucleotides.

[0172] In some embodiments, the mutation-specific probe and the mutation-specific primer comprise DNA. In some embodiments, the mutation-specific probe and the mutation-specific primer comprise RNA.

[0173] In some embodiments, the probes and primers described herein (including mutation-specific probes and mutation-specific primers) have nucleotide sequences that specifically hybridize to any of the nucleic acid molecules disclosed herein or their complements. In some embodiments, the probes and primers specifically hybridize to any of the nucleic acid molecules disclosed herein under stringent conditions.

[0174] In some embodiments, the primers can be used in second generation or high throughput sequencing, including mutation specific primers. Sometimes the primers can be modified, including mutation specific primers. In particular, the primers can include various modifications used in various steps of, for example, Massive Parallel Signature Sequencing (MPSS), Polony sequencing, and 454 pyrosequencing. Modified primers can be used in several steps of the process, including biotinylated primers in the cloning step, and fluorescently labeled primers used in the bead loading and detection steps. Polony sequencing is typically performed using paired-end tag libraries, where each molecule of DNA template is about 135 bp in length. Biotinylated primers are used in the bead loading and emulsion PCR steps. Fluorescently labeled degenerate nonamer oligonucleotides are used in the detection step. The adapters can contain 5'-biotin tags for immobilizing the DNA library on streptavidin-coated beads.

[0175] The probes and primers described herein can be used to detect nucleotide variations within any of the MAP3K15 variant missense genomic nucleic acid molecules, MAP3K15 variant mRNA molecules, and / or MAP3K15 variant cDNA molecules disclosed herein. The primers described herein can be used to amplify MAP3K15 variant missense genomic nucleic acid molecules, MAP3K15 variant mRNA molecules, or MAP3K15 variant cDNA molecules, or fragments thereof.

[0176] In the context of the present disclosure, "specifically hybridizes" means that a probe or primer (e.g., a mutation-specific probe or mutation-specific primer) does not hybridize to a nucleic acid sequence encoding a MAP3K15-based genomic nucleic acid molecule, a MAP3K15-based mRNA molecule, and / or a MAP3K15-based cDNA molecule.

[0177] In some embodiments, the probe (e.g., the mutation-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin. The present disclosure also provides a support comprising a substrate to which any one or more of the probes disclosed herein are attached. A solid support is a solid-state substrate or support to which a molecule such as any of the probes disclosed herein can associate. 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 a plurality of different probes are attached in an array, grid, or other organized pattern. A form of solid-state substrate is a microtiter dish such as a standard 96-well format. In some embodiments, a multi-well glass slide can be used, usually containing one array per well.

[0178] The nucleotide sequence of the MAP3K15 reference genomic nucleic acid molecule is set forth in SEQ ID NO:1. The nucleotide sequence of a MAP3K15-based mRNA molecule is set forth in SEQ ID NO: 2. The nucleotide sequence of another MAP3K15-based mRNA molecule is set forth in SEQ ID NO: 3. The nucleotide sequence of another MAP3K15-based mRNA molecule is set forth in SEQ ID NO: 4. The nucleotide sequence of another MAP3K15-based mRNA molecule is set forth in SEQ ID NO: 5. The nucleotide sequence of another MAP3K15-based mRNA molecule is set forth in SEQ ID NO: 6. The nucleotide sequence of another MAP3K15-based mRNA molecule is set forth in SEQ ID NO: 7.

[0179] The nucleotide sequence of a MAP3K15-based cDNA molecule is set forth in SEQ ID NO:8. The nucleotide sequence of another MAP3K15-based cDNA molecule is set forth in SEQ ID NO:9. The nucleotide sequence of another MAP3K15-based cDNA molecule is set forth in SEQ ID NO:10. The nucleotide sequence of another MAP3K15-based cDNA molecule is set forth in SEQ ID NO:11. The nucleotide sequence of another MAP3K15-based cDNA molecule is set forth in SEQ ID NO:12. The nucleotide sequence of another MAP3K15-based cDNA molecule is set forth in SEQ ID NO:13.

[0180] The amino acid sequence of a MAP3K15 reference polypeptide is set forth in SEQ ID NO: 14 and is 1,313 amino acids in length. Another MAP3K15 reference polypeptide has an amino acid sequence set forth in SEQ ID NO: 15 and is 788 amino acids in length. Another MAP3K15 reference polypeptide has an amino acid sequence set forth in SEQ ID NO: 16 and is 748 amino acids in length. Another MAP3K15 reference polypeptide has an amino acid sequence set forth in SEQ ID NO: 17 and is 247 amino acids in length. Another MAP3K15 reference polypeptide has an amino acid sequence set forth in SEQ ID NO: 18 and is 1,145 amino acids in length.

[0181] The genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be derived from any organism. For example, the genomic nucleic acid molecule, mRNA molecule, and cDNA molecule can be an ortholog from human or another organism (e.g., non-human mammal, rodent, mouse, or rat). It is understood that gene sequences within a population can differ due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are only exemplary sequences. Other sequences are also possible.

[0182] 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. Functional polynucleotides can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional polynucleotides can have de novo activity independent of any other molecules.

[0183] The isolated nucleic acid molecules disclosed herein can include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules can also be linked or fused to heterologous nucleic acid sequences, for example in a vector, or heterologous labels. For example, the isolated nucleic acid molecules disclosed herein can be present as exogenous donor sequences in or containing a vector that includes the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules can also be linked or fused to heterologous labels. The labels can be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such labels can be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioactive labels, pigments, dyes, chromogens, spin labels, and fluorescent labels. The labels can also be, for example, chemiluminescent materials; metal-containing materials; or enzymes, where enzyme-dependent secondary generation of a signal occurs. The term "label" can also refer to a "tag" or hapten that can be selectively attached to a binding molecule such that the binding molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag together with an avidin or streptavidin conjugate of horseradish peroxidase (HRP) to bind to the tag and probed using a colorimetric (e.g., tetramethylbenzidine (TMB)) or 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, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes and their colorimetric, fluorescent and chemiluminescent substrates, as well as other labels.

[0184] The isolated nucleic acid molecule or its complement can be present in a host cell. In some embodiments, the host cell can comprise a vector comprising any of the nucleic acid molecules described herein or their complements. In some embodiments, the nucleic acid molecule is operably linked to a promoter active in the host cell. In some embodiments, the promoter is an exogenous promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the host cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In some embodiments, the host cell is a bacterial cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is a mammalian cell.

[0185] The disclosed nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as, for example, nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0186] The nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or nucleotide substitutes. A nucleotide analog is a nucleotide that contains modifications to either the base, sugar, or phosphate moiety. 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 various purine or pyrimidine bases, such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, 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-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. Examples of uracils and cytosines include, but are not limited to, 4-isopropyl uracil, 4-isopropyl uracil, 8 ...

[0187] Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose, 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-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C-alkyl groups. 1~10 Alkyl or C 2~10 Alkenyl, and C 2~10Exemplary 2' sugar modifications include -O[(CH 2 ) n O] m CH 3 , -O(CH 2 ) n OCH 3 , -O(CH 2 ) n NH 2 , -O(CH 2 ) n CH 3 , -O(CH 2 ) n -ONH 2 , and -O(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 wherein n and m are independently 1 to about 10. Other modifications at the 2' position include, but are not limited to, C 1~10 Alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 Modified sugars include, but are not limited to, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications may 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 include, but are not limited to, CH 2and S. Nucleotide sugar analogs can also include those containing modifications at the bridging ring oxygen, such as S. Nucleotide sugar analogs can also have sugar mimetics such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0188] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those in which the linkage between two nucleotides can be modified to contain phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Such phosphate or modified phosphate linkages between two nucleotides can be via 3'-5' or 2'-5' linkages, and the linkages can contain reverse 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).

[0189] The present disclosure also provides a vector comprising any one or more of the nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double stranded DNA into which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector in which 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.

[0190] Desirable regulatory sequences for mammalian host cell expression may include, for example, viral elements directing high levels of polypeptide expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), Simian Virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), polyoma derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters and actin promoters. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known. The promoter may be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or tissue-specific promoter).

[0191] Percent identity (%) (or percent complementarity) between specific stretches of nucleotide sequences in nucleic acid molecules or amino acid sequences in polypeptides can be routinely determined using the BLAST program (basic sequence comparison search tool) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), or the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using default settings that use the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489). When referring to percent sequence identity in this specification, a higher percent sequence identity is preferred over a lower one.

[0192] As used herein, the phrase "corresponding to" or grammatical variations thereof, when used in the context of numbering a particular nucleotide or sequence of nucleotides or position, refers to the numbering of a specified reference sequence when the particular nucleotide or sequence of nucleotides is compared to a reference sequence (e.g., SEQ ID NO:1). In other words, the residue (e.g., nucleotide or amino acid) number or residue (e.g., nucleotide or amino acid) position of a particular polymer is specified with reference to a reference sequence, not by the actual position number of the residue within the particular nucleotide or nucleotide sequence. For example, a particular nucleotide sequence can be aligned to a reference sequence by introducing gaps to optimize residue matching between the two sequences. In these cases, although gaps exist, the numbering of the residues in the particular nucleotide or nucleotide sequence is done with reference to the reference sequence to which it is aligned.

[0193] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5'-end of the sequence and proceeding toward the 3'-end (i.e., from left to right in each sequence). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by reference to the shown strand. The amino acid sequences follow the standard convention of beginning at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each sequence).

[0194] The present disclosure also provides a therapeutic agent for treating or preventing a metabolic disorder for use in treating and / or preventing a metabolic disorder in a subject having a MAP3K15 missense variant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, a MAP3K15 missense variant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15, or a MAP3K15 missense variant cDNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15. Any of the therapeutic agents for treating or preventing a metabolic disorder described herein can be used in these methods. The metabolic disorder can be type 2 diabetes, elevated hemoglobin A1c, or elevated serum glucose.

[0195] The present disclosure also provides the use of a therapeutic agent for treating or preventing a metabolic disorder for use in the preparation of a medicament for treating and / or preventing a metabolic disorder in a subject having a MAP3K15 missense variant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, a MAP3K15 missense variant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15, or a MAP3K15 missense variant cDNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15. Any of the therapeutic agents for treating or preventing a metabolic disorder described herein can be used in these methods. The metabolic disorder can be type 2 diabetes, elevated hemoglobin A1c, or elevated serum glucose.

[0196] The present disclosure also provides a MAP3K15 inhibitor for use in treating and / or preventing a metabolic disorder in a subject that is a) a reference for a MAP3K15 genomic nucleic acid molecule, a MAP3K15 mRNA molecule, or a MAP3K15 cDNA molecule, or b) heterozygous for i) a MAP3K15 missense variant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, ii) a MAP3K15 missense variant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15, or iii) a MAP3K15 missense variant cDNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15. Any of the MAP3K15 inhibitors described herein can be used in these methods. The metabolic disorder can be type 2 diabetes, elevated hemoglobin A1c, or elevated serum glucose.

[0197] The present disclosure also provides the use of a MAP3K15 inhibitor in the preparation of a medicament for treating and / or preventing a metabolic disorder in a subject that is a) a reference for a MAP3K15 genomic nucleic acid molecule, a MAP3K15 mRNA molecule, or a MAP3K15 cDNA molecule, or b) is heterozygous for i) a MAP3K15 missense variant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, ii) a MAP3K15 missense variant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15, or iii) a MAP3K15 missense variant cDNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15. Any of the MAP3K15 inhibitors described herein can be used in these methods. The metabolic disorder can be type 2 diabetes, elevated hemoglobin A1c, or elevated serum glucose.

[0198] All patent documents, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual document was specifically and individually indicated to be so incorporated by reference. Where various versions of a sequence are associated with accession numbers at different times, the version associated with the accession number at the effective filing date of this application is meant. Effective filing date means the earlier of the actual filing date or the filing date of the priority application to which the accession number refers, if applicable. Similarly, where different versions of publications, websites, etc. have been published at different times, the version last 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 may be used in combination with any other feature, step, element, embodiment, or aspect, unless otherwise indicated. 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.

[0199] The following examples are provided to further illustrate the embodiments. They are intended to illustrate, not limit, the claimed embodiments. The following examples provide those skilled in the art with a disclosure and explanation of how the compounds, compositions, articles, devices and / or methods described herein are made and evaluated, and are intended to be merely illustrative and are not intended to limit the scope of any claims. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some error and deviation can be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is °C or is at ambient temperature, and pressure is at or near atmospheric pressure.

[0200] [Example] Example 1: A novel association between MAP3K15 and protection from type 2 diabetes The exomes of 454,787 UKB study participants were sequenced, covering 95.8% of the target bases at a depth of more than 20x, as previously described (Szustakowski, Advancing Human Genetics Research and Drug Discovery through Exome Sequencing of the UK Biobank. bioRxiv, 2021; and Van Hout et al., Nature, 2020). 12 million variants were identified in 39 million base pairs across the coding regions of 18,659 genes (data not shown). Among the variants identified, there were 3,375,252 (median 10,260 per individual) synonymous variants, 7,689,495 (9,284 per individual) missense variants, and 889,957 (212 per individual) putative loss-of-function (pLOF) variants (data not shown), of which approximately half were observed only once in this dataset (singleton variants; data not shown).

[0201] The burden of predicted loss-of-function (pLOF) and deleterious missense variants in MAP3K15 was associated with lower hemoglobin A1c levels (7,551 carriers; effect = -0.09 SD, 95% CI -0.10 to -0.073, P = 2 × 10 -31 ) and low serum glucose (6,885 carriers; effect = -0.090, 95% CI -0.110 to -0.073, P = 1.7 × 10 -25 In addition, pLOF and deleterious missense variant burden in MAP3K15 were also associated with protection from type 2 diabetes (7,863 carriers; OR = 0.80, 95% CI 0.74 to 0.87, P = 1 × 10 -7Furthermore, in the GHS study (a cohort based in a health system in central and eastern Pennsylvania (USA) that has been continuously recruiting participants since 2006), there were no significant differences in hemoglobin A1c (1,304 carriers; effect = -0.040 SD units, 95% CI -0.079 to -0.002, P = 0.038), glucose (1,754 carriers; effect = -0.097 SD units, 95% CI -0.130 to -0.064, P = 1.3 × 10 -8 ), and type 2 diabetes (2,455 carriers; OR = 0.91, 95% CI 0.84 to 0.98, P = 0.018) for all three phenotypes.

[0202] In addition to those described herein, various modifications of the described subject matter will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (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, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. An in vitro method for identifying the susceptibility to the onset of a metabolic disorder in a subject, the method comprising: determining, or having determined, the presence or absence of a MAP3K15 missense mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by mitogen-activated protein kinase kinase kinase 15 (MAP3K15) in a biological sample obtained from the subject; wherein the subject being a MAP3K15 reference indicates that the subject has a high risk of developing the metabolic disorder; the subject being heterozygous or homozygous for the MAP3K15 missense mutant nucleic acid molecule encoding the loss-of-function polypeptide predicted by MAP3K15 indicates that the subject has a low risk of developing the metabolic disorder, the method.

2. The method according to claim 1, wherein the MAP3K15 missense mutant nucleic acid molecule is a splice site mutant, a stop gain mutant, a start loss mutant, a stop loss mutant, a frameshift mutant, or an in-frame indel mutant, or a mutant encoding a loss-of-function polypeptide predicted by truncated MAP3K15.

3. The method according to claim 2, wherein the MAP3K15 missense mutant nucleic acid molecule encodes a loss-of-function polypeptide predicted by truncated MAP3K15.

4. Use of a therapeutic agent for treating or preventing a metabolic disorder in the preparation of a drug for treating or preventing the metabolic disorder in a subject, wherein the subject has a MAP3K15 missense mutant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by mitogen-activated protein kinase kinase kinase 15 (MAP3K15); or is heterozygous for a MAP3K15 missense mutant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15.

5. Use of a MAP3K15 inhibitor in the preparation of a drug for treating or preventing a metabolic disorder in a subject, wherein the subject a) is a reference for a mitogen-activated protein kinase kinase kinase 15 (MAP3K15) genomic nucleic acid molecule or a MAP3K15 mRNA molecule, or b) i) a MAP3K15 missense mutant genomic nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15; or ii) Use of a MAP3K15 inhibitor that is heterozygous with respect to a MAP3K15 missense mutant mRNA molecule encoding a loss-of-function polypeptide predicted by MAP3K15. **Claim 6** The use according to claim 5, wherein the MAP3K15 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with a MAP3K15 nucleic acid molecule. **Claim 7** The use according to claim 6, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA). **Claim 8** The use according to claim 5, wherein the MAP3K15 inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes with a guide RNA (gRNA) recognition sequence in a MAP3K15 genomic nucleic acid molecule. **Claim 9** The use according to claim 8, wherein the Cas protein is Cas9 or Cpf1. **Claim 10** The use according to claim 8 or 9, wherein the gRNA recognition sequence is located within SEQ ID NO:

1. **Claim 11** The use according to claim 8 or 9, wherein the protospacer adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence. **Claim 12** The use according to claim 8 or 9, wherein the gRNA comprises about 17 to about 23 nucleotides. **Claim 13** The use according to claim 8 or 9, wherein the gRNA recognition sequence comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 19 to 38. **Claim 14** The method according to any one of claims 1 to 3, wherein the metabolic disorder comprises type 2 diabetes, an increase in hemoglobin A1c, or an increase in serum glucose. **Claim 15** The use according to claim 4, wherein the metabolic disorder comprises type 2 diabetes, an increase in hemoglobin A1c, or an increase in serum glucose. **Claim 16** The use according to claim 4 or 15, wherein the therapeutic agent comprises metformin, insulin, sulfonylurea, meglitinide, thiazolidinedione, DPP-4 inhibitor, GLP-1 receptor agonist, SGLT2 inhibitor, or alirocumab. **Claim 17** Insulin comprises insulin glulisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, or insulin isophane; The sulfonylurea includes glibenclamide, glipizide, or glimepiride; The meglitinide includes repaglinide or nateglinide; The thiazolidinedione includes rosiglitazone or pioglitazone; The DPP-4 inhibitor includes sitagliptin, saxagliptin, or linagliptin; The GLP-1 receptor agonist includes exenatide, liraglutide, or semaglutide; The SGLT2 inhibitor includes canagliflozin, dapagliflozin, or empagliflozin, The use according to claim 16.

18. The use according to any one of claims 5 to 9, wherein the metabolic disorder includes type 2 diabetes, an increase in hemoglobin A1c, or an increase in serum glucose.

19. A pharmaceutical composition for treating a metabolic disorder in a patient who is heterozygous for a MAP3K15 missense mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, the pharmaceutical composition comprising, as an active ingredient, a therapeutic agent for treating or preventing a metabolic disorder.

20. A pharmaceutical composition for treating a metabolic disorder in a patient who is a) a reference for a MAP3K15 genomic nucleic acid molecule or a MAP3K15 mRNA molecule, or b) heterozygous for a MAP3K15 missense mutant nucleic acid molecule encoding a loss-of-function polypeptide predicted by MAP3K15, the pharmaceutical composition comprising, as an active ingredient, a MAP3K15 inhibitor.

21. The pharmaceutical composition according to claim 19 or 20, wherein the metabolic disorder includes type 2 diabetes, an increase in hemoglobin A1c, or an increase in serum glucose.