CN116583291A

CN116583291A - Methods of treating metabolic disorders and cardiovascular diseases with inhibitors of inhibin subunit beta E (INHBE)

Info

Publication number: CN116583291A
Application number: CN202180084054.9A
Authority: CN
Inventors: L·A·洛塔; P·阿克巴里; O·索西纳; M·A·R·费雷拉; A·巴拉斯
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2020-12-14
Filing date: 2021-12-13
Publication date: 2023-08-11

Abstract

The present disclosure provides methods of treating a subject suffering from a metabolic disorder and/or cardiovascular disease, methods of identifying a subject at increased risk of developing a metabolic disorder and/or cardiovascular disease, and methods of detecting human inhibin subunit βe variant nucleic acid molecules and variant polypeptides.

Description

Methods of treating metabolic disorders and cardiovascular diseases with inhibitors of inhibin subunit beta E (INHBE)

Reference to sequence Listing

The application includes a sequence listing submitted electronically as a text file, named 18923805702SEQ, created at 2021, 12, 11, 28 kilobytes in size. The sequence listing is incorporated herein by reference.

Technical Field

The present disclosure relates generally to methods of treating subjects suffering from metabolic disorders and/or cardiovascular diseases with inhibin subunit βe inhibitors, identifying subjects at increased risk of developing metabolic disorders and/or cardiovascular diseases, and detecting INHBE variant nucleic acid molecules and variant polypeptides.

Background

Body fat distribution is an important risk factor for cardiovascular and metabolic diseases, irrespective of overall obesity. Body fat distribution characterized by more fat accumulation around the waist (such as more abdominal fat or greater waist circumference) and/or less fat accumulation around the buttocks (such as less buttocks thigh fat or less hip circumference) resulting in a greater waist-to-buttocks ratio (WHR) is associated with higher cardiovascular metabolic risk and is independent of Body Mass Index (BMI). Metabolic conditions associated with body fat distribution include, but are not limited to: type 2 diabetes, hyperlipidemia or dyslipidemia (low density lipoprotein cholesterol (LDL-C), triglycerides, very low density lipoprotein cholesterol (VLDL-C), circulating levels of apolipoprotein B or other lipid fraction are high or altered), obesity (in particular abdominal obesity), lipodystrophy (such as inability to deposit fat locally in the fat reservoir (partial lipodystrophy) or to deposit fat systemically (lipoatrophy)), insulin resistance or higher or altered insulin levels during fasting or metabolic challenges, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, liver enzyme levels or liver injury, inflammation or other markers of fat deposition in the liver are higher or elevated or altered, blood pressure is higher and/or hypertension, blood glucose or blood glucose is higher or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerosis, and complications of each of the above conditions. Identifying genetic variants that are associated with more favorable fat profiles (such as lower WHR, particularly when adjusted for BMI) may be a way to identify mechanisms that can be used therapeutically to benefit these cardiovascular metabolic diseases.

Inhibin subunit βe (INHBE) is a member of the TGF- β (transforming growth factor β) protein superfamily. Inhibin is involved in the regulation of a variety of cellular processes including cell proliferation, apoptosis, immune responses and hormone secretion. Inhibin and activin inhibit and activate, respectively, the secretion of follitropin by the pituitary gland. Inhibin/activin is involved in regulating a variety of different functions depending on its subunit composition, such as hypothalamic and pituitary hormone secretion, gonadal hormone secretion, germ cell development and maturation, erythrocyte differentiation, insulin secretion, neural cell survival, embryonic axial development or bone growth. Inhibin appears to be functionally opposite to activin. In addition, INHBE can be up-regulated under conditions of endoplasmic reticulum stress, and the protein can inhibit cell proliferation and growth in the pancreas and liver.

Disclosure of Invention

The present disclosure provides methods of treating a subject having or at risk of developing a metabolic disorder comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides a method of treating a subject having or at risk of developing type 2 diabetes, the method comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject suffering from or at risk of developing obesity comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing elevated triglyceride levels (hypertriglyceridemia), comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing lipodystrophy, comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides a method of treating a subject having or at risk of developing liver inflammation, the method comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides a method of treating a subject having or at risk of developing fatty liver disease, the method comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing hypercholesterolemia comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing an elevated liver enzyme, such as, for example, alanine Aminotransferase (ALT) and/or aspartate Aminotransferase (AST), comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing non-alcoholic steatohepatitis (NASH), the method comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing a cardiovascular disease comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing cardiomyopathy comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject suffering from or at risk of developing heart failure, comprising administering to the subject an INHBE inhibitor.

The present disclosure also provides methods of treating a subject suffering from or at risk of developing hypertension, comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits a metabolic disorder, wherein the subject has a metabolic disorder, the method comprising the steps of: determining whether a subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide by: obtaining or having obtained a biological sample from a subject; and genotyping the biological sample to determine whether the subject has a genotype comprising an INHBE variant nucleic acid molecule; and when the subject is an INHBE reference, then administering or continuing to administer a therapeutic agent that treats or inhibits the metabolic disorder to the subject in a standard dose amount, and administering an INHBE inhibitor to the subject; and when the subject is heterozygous for the INHBE variant nucleic acid molecule, then administering or continuing to administer to the subject a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or less than the standard dose amount, and administering to the subject an INHBE inhibitor; when the subject is homozygous for the INHBE variant nucleic acid molecule, then administering or continuing to administer a therapeutic agent that treats or inhibits the metabolic disorder to the subject in an amount equal to or less than the standard dose amount; wherein the presence of a genotype having an INHBE variant nucleic acid molecule that encodes an INHBE predictive loss of function polypeptide indicates that the subject is at reduced risk of developing a metabolic disorder.

The present disclosure also provides a method of treating a subject with a therapeutic agent that treats or inhibits a cardiovascular disease, wherein the subject has a cardiovascular disease, the method comprising the steps of: determining whether a subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide by: obtaining or having obtained a biological sample from a subject; and genotyping the biological sample to determine whether the subject has a genotype comprising an INHBE variant nucleic acid molecule; and when the subject is an INHBE reference, then administering or continuing to administer a therapeutic agent that treats or inhibits the cardiovascular disease to the subject in a standard dose amount, and administering an INHBE inhibitor to the subject; and when the subject is heterozygous for the INHBE variant nucleic acid molecule, then administering or continuing to administer to the subject a therapeutic agent that treats or inhibits the cardiovascular disease in an amount equal to or less than the standard dose amount, and administering to the subject an INHBE inhibitor; when the subject is homozygous for the INHBE variant nucleic acid molecule, then administering or continuing to administer a therapeutic agent that treats or inhibits the cardiovascular disease to the subject in an amount equal to or less than the standard dose amount; wherein the presence of a genotype having an INHBE variant nucleic acid molecule that encodes an INHBE predictive loss of function polypeptide indicates that the subject is at reduced risk of developing cardiovascular disease.

The present disclosure also provides a method of identifying a subject at increased risk of developing a metabolic disorder, wherein the method comprises: determining or having determined the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from a subject; wherein: when the subject is an INHBE reference, then the subject is at increased risk of developing a metabolic disorder; and when the subject is heterozygous for the INHBE variant nucleic acid molecule or homozygous for the INHBE variant nucleic acid molecule, then the subject is at reduced risk of developing a metabolic disorder.

The present disclosure also provides a method of identifying a subject at increased risk of developing a cardiovascular disease, wherein the method comprises: determining or having determined the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from a subject; wherein: when the subject is an INHBE reference, then the subject is at increased risk of developing a cardiovascular disease; and when the subject is heterozygous for the INHBE variant nucleic acid molecule or homozygous for the INHBE variant nucleic acid molecule, then the subject's risk of developing a cardiovascular disease is reduced.

The present disclosure also provides a therapeutic agent for treating or inhibiting a metabolic disorder, for treating a metabolic disorder in a subject having: an INHBE variant genomic nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

The present disclosure also provides a therapeutic agent for treating or inhibiting a cardiovascular disease for treating a cardiovascular disease in a subject having: an INHBE variant genomic nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

The present disclosure also provides an INHBE inhibitor for treating or inhibiting a metabolic disorder, for use in treating a metabolic disorder in a subject having: an INHBE variant genomic nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

The present disclosure also provides an INHBE inhibitor for treating or inhibiting a cardiovascular disease for use in treating a cardiovascular disease in a subject having: an INHBE variant genomic nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or an INHBE variant cDNA molecule encoding an INHBE predicted loss-of-function polypeptide.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description, serve to explain the principles of the disclosure.

Figure 1 shows the association of INHBE predictive loss of function (pLOF) variants with favorable fat distribution (i.e., lower BMI-adjusted WHR) in an exome sequencing analysis of over 525,000 people from multiple studies; using REGENIE software, estimating a correlation analysis by fitting a mixed effect linear regression model in consideration of correlation and population stratification; abbreviations: confidence interval, CI; standard deviation, SD; body mass index, BMI; waist-to-hip ratio adjusted for BMI, WHRadjBMI; reference-reference allele, RR; reference-alternative allele, RA; substitution-substitution allele, AA; UK biological sample library (UK Biobank) cohort, UKB; european ancestry, EUR; a prospective study cohort, MCPS, from mexico; loss of predicted function, plofs.

FIG. 2 depicts a gene model of INHBE showing the position of the pLOF variants (upper panel) and the phenotype profile of BMI-adjusted WHR for the carrier of each variant; blue bars show the median BMI-adjusted WHR of non-carriers, while red bars show the median BMI-adjusted WHR of carriers; two variants highlighted in dark red were each individually associated with lower BMI-adjusted WHR; data were from the UKB biological sample library (UKB) and the mexico market prospective study (MCPS) cohort; abbreviations: body mass index, BMI; waist-hip ratio, WHR.

FIG. 3 shows the results of computer prediction of INHBE c.299-1G:C (12:57456093:G:C) splice variants with function; top sequence = original exon 2 (SEQ ID NO: 28); bottom sequence = predicted exon 2 (SEQ ID NO: 29).

FIG. 4 shows the wild-type INHBE protein sequence (top; SEQ ID NO: 8) and the computer predicted protein sequence of the c.299-1G: C acceptor splice variant (bottom; SEQ ID NO:8 shows the changes in the non-highlighted region).

FIG. 5 shows Chinese Hamster Ovary (CHO) cell experiments against c.299-1G > C variants. Variants occur in the splice acceptor site that is also the first and only one splice junction in the INHBE gene (panel a). In CHO cells, the c.299-1g > c variant resulted in the expression of lower molecular weight variants that were present in the cell lysate but not in the culture medium, consistent with loss of function (panel B).

Figure 6 shows the correlation of INHBE ploflf variants with body fat and indices of lean body mass, percentage and body surface adjustment as measured by bioelectrical impedance in 423,418 participants from the UKB study.

Fig. 7 shows INHBE expression patterns across tissue (left) and hepatocyte type (right). The first plot shows normalized mRNA expression values in Counts Per Million (CPM) for INHBEs of each organization obtained by web access "gtexport. Org/") using data from the genotype organization expression (GTEx) alliance (GTEx portal2021.2021, 6-month 1). The second panel shows normalized cell type-specific expression levels in the liver obtained from human protein profiles (human protein atlas, HPA), expressed in transcripts per million of protein-encoding genes (pTPM) (Uhlen et al, nat. Biotechnol.2010,28,1248-50). The box plot depicts the median (black thick bars), quartile range, and minimum and maximum CPM values for each tissue of the individual.

Fig. 8 shows that liver mRNA expression of INHBE is upregulated in steatosis and nonalcoholic steatohepatitis (NASH) patients compared to normal liver individuals in bariatric surgery patients from GHS. In the top panel, the graph shows liver mRNA expression levels of INHBE in normal liver (control), liver steatosis (simple steatosis) and nonalcoholic steatohepatitis (NASH) patients, expressed in terms of transcripts per million (TPM; normalization of RNA molecules per 100 ten thousand molecules detected in a certain experiment). The base graph is statistical data of the inter-group comparisons. The simplex steatosis group showed higher liver INHBE expression than the control group. NASH group showed higher expression compared to both control group and simple steatosis group. All differences in expression between groups were statistically significant.

Detailed Description

Various terms relating to 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 specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

Unless explicitly stated otherwise, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Therefore, in the claims or the specification, when a method claim does not explicitly state that the steps are limited to a particular order, it is in no way intended that the order be inferred. This applies to any possible non-expressed interpretation base including logical matters with respect to arrangement of steps or operational flow, ordinary meanings derived from grammatical organization or punctuation, or numbering or types of aspects described in the specification.

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

As used herein, the term "about" means that the recited values are approximations, and that the minor variations do not significantly affect the practice of the disclosed embodiments. Where numerical values are used, the term "about" means that the numerical values can vary by + -10% and still be within the scope of the disclosed embodiments unless the context indicates otherwise.

As used herein, the term "comprising" may be replaced with "consisting of … …" or "consisting essentially of … …" in particular embodiments, as desired.

As used herein, the term "isolated" with respect to a nucleic acid molecule or polypeptide refers to a nucleic acid molecule or polypeptide that is under conditions different from its natural environment, such as away from blood and/or animal tissue. In some embodiments, the 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 may be in a highly purified form, i.e., greater than 95% pure or greater than 99% pure. The term "isolated" as used in this context 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.

As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" may include polymeric forms of nucleotides of any length, may include DNA and/or RNA, and may be single-stranded, double-stranded, or multi-stranded. One strand of a nucleic acid also refers to its complementary sequence.

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

According to the present disclosure, loss of function variants in INHBE (whether these variants are homozygous or heterozygous in a particular subject) have been observed to be associated with reduced risk of developing metabolic disorders such as type 2 diabetes, obesity, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (such as ALT and/or AST, for example), NASH, and/or elevated triglyceride levels, and/or cardiovascular diseases such as cardiomyopathy, heart failure, and hypertension. It is believed that no loss of function variants in the INHBE gene or protein have been correlated with metabolic disorders and/or cardiovascular disease in whole genome or whole exome correlation studies. Thus, subjects homozygous or heterozygous for the reference INHBE variant nucleic acid molecule may be treated with an INHBE inhibitor such that metabolic disorders and/or cardiovascular disease are inhibited, symptoms thereof are alleviated, and/or development of symptoms is suppressed. It is also believed that such subjects suffering from metabolic disorders and/or cardiovascular diseases may be further treated with therapeutic agents that treat or inhibit metabolic disorders such as type 2 diabetes, obesity, hypertension, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (such as, for example, ALT and/or AST), NASH, and/or elevated triglyceride levels, and/or cardiovascular diseases such as cardiomyopathy, heart failure, and hypertension.

For purposes of this disclosure, any particular subject, such as a human, may be categorized as having one of the following three INHBE genotypes: i) INHBE reference; ii) heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide; or iii) homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss of function polypeptide. The subject is an INHBE reference when the subject does not have a copy of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide. When the subject has a single copy of an INHBE variant nucleic acid molecule that encodes an INHBE predicted loss-of-function polypeptide, the subject is heterozygous for the INHBE variant nucleic acid molecule. An INHBE variant nucleic acid molecule is any nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an INHBE polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. Subjects with INHBE polypeptides that contain partial loss of function (or predicted partial loss of function) are sub-potent alleles (hypomorphs) of INHBE. When a subject has two copies (the same or different) of an INHBE variant nucleic acid molecule that encodes an INHBE predicted loss-of-function polypeptide, the subject is homozygous for the INHBE variant nucleic acid molecule that encodes an INHBE predicted loss-of-function polypeptide.

For subjects genotyped or identified as INHBE-referenced, such subjects have an increased risk of developing metabolic disorders such as type 2 diabetes, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (such as, for example, ALT and/or AST), obesity, hypertension, and/or elevated triglyceride levels (hypertriglyceridemia), and/or cardiovascular diseases such as cardiomyopathy, heart failure, and hypertension. For subjects that are genotyped or determined to be INHBE-referenced or heterozygous for INHBE variant nucleic acid molecules, such subjects or subjects may be treated with INHBE inhibitors.

In any of the embodiments described herein, an INHBE variant nucleic acid molecule may be any nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) encoding an INHBE polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. In some embodiments, the INHBE variant nucleic acid molecule is associated with a reduced in vitro response to an INHBE ligand as compared to a reference INHBE. In some embodiments, the INHBE variant nucleic acid molecule is an INHBE variant that results in or is predicted to result in premature truncation of the INHBE polypeptide compared to the reference genomic sequence. In some embodiments, the INHBE variant nucleic acid molecule is a variant predicted to be destructive by an in vitro prediction algorithm such as Polyphen, SIFT or similar algorithm. In some embodiments, the INHBE variant nucleic acid molecule is a variant that results in or is predicted to result in a non-synonymous amino acid substitution in INHBE and has an allele frequency of less than 1/100 of the alleles in the population from which the subject is selected. In some embodiments, the INHBE variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%; or 1 out of 1,000 alleles), or any splice site, stop-gain, start-loss, stop-loss, frameshift, or in-frame insertion deletion (indel) or other frameshift INHBE variant.

In any of the embodiments described herein, an INHBE predicted loss of function polypeptide may be any INHBE polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.

In any of the embodiments described herein, an INHBE variant nucleic acid molecule encoding a variation in protein sequence may include a variation at chromosome 12 position using the nucleotide sequence of the INHBE reference genomic nucleic acid molecule (SEQ ID NO:1; ENST00000266646.3chr12:57455307-57458025 in the GRCH38/hg38 human genome assembly) as a reference sequence.

There are many genetic variants in INHBE that result in subsequent changes in the polypeptide sequence of INHBE, including but not limited to: gln7fs, arg18STOP, gln37STOP, arg40STOP, leu55fs, cys139fs, arg144STOP, cys192fs, arg224STOP, arg233fs, arg250STOP, asp251fs, tyr253STOP, tyr275 STOP, ser293fs, trp308fs, pro309fs, arg320STOP, leu323fs and Ter351 Tyrext? . Additional variant genomic nucleic acid molecules in the presence of INHBE, including but not limited to (using human genome reference version (human genome reference build) GRch 38): C298+1G:T (12:57455835:G:T), c.299-2A:G, c.299-1G:C (12:57456093:G:C) and 12:57259799:A:C. Additional variant INHBE polypeptides exist, including but not limited to INHBE polypeptides with the methionine at position 1 removed.

Variants of INHBE plofs of any one or more (i.e., any combination) can be used in any of the methods described herein to determine whether a subject has an increased risk of developing a metabolic disorder and/or cardiovascular disease. Combinations of specific variants may form masks (masks) for statistical analysis of specific correlations of INHBE with increased type 2 diabetes/BMI risk and/or cardiovascular disease.

In any of the embodiments described herein, the metabolic disorder is type 2 diabetes, obesity, NASH, and/or elevated triglyceride levels. In any of the embodiments described herein, the metabolic disorder is type 2 diabetes. In any of the embodiments described herein, the metabolic disorder is obesity. In any of the embodiments described herein, the metabolic disorder is NASH. In any of the embodiments described herein, the metabolic disorder is elevated triglyceride levels. In any of the embodiments described herein, the metabolic disorder is lipodystrophy. In any of the embodiments described herein, the metabolic disorder is liver inflammation. In any of the embodiments described herein, the metabolic disorder is fatty liver disease. In any of the embodiments described herein, the metabolic disorder is hypercholesterolemia. In any of the embodiments described herein, the metabolic disorder is an increase in liver enzymes (such as, for example, ALT and/or AST).

Metabolic disorders/conditions associated with body fat distribution also include, but are not limited to: type 2 diabetes, hyperlipidemia or dyslipidemia (low density lipoprotein cholesterol (LDL-C), triglycerides, very low density lipoprotein cholesterol (VLDL-C), elevated or altered circulating levels of apolipoprotein B or other lipid fraction), obesity (in particular abdominal obesity), lipodystrophy (such as inability to deposit fat locally in the fat reservoir (partial lipodystrophy) or to deposit fat systemically (lipoatrophy)), insulin resistance or elevated or altered insulin levels during fasting or glucose or insulin challenge, liver fat deposition or fatty liver disease and its complications (such as, for example, cirrhosis, fibrosis or liver inflammation), elevated or altered liver enzyme levels or liver injury, elevated or altered blood pressure, elevated or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions, and complications of each of the foregoing conditions.

In any of the embodiments described herein, the cardiovascular disease is cardiomyopathy, heart failure, or hypertension. In any of the embodiments described herein, the cardiovascular disease is cardiomyopathy. In any of the embodiments described herein, the cardiovascular disease is heart failure. In any of the embodiments described herein, the cardiovascular disease is hypertension.

The present disclosure provides methods of treating a subject having or at risk of developing a metabolic disorder, the method comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing elevated triglyceride levels, comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having NASH or at risk of developing, the method comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing fatty liver disease comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing elevated liver enzymes (such as, for example, ALT and/or AST) comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject having or at risk of developing a cardiovascular disease, comprising administering an INHBE inhibitor to the subject.

The present disclosure also provides methods of treating a subject suffering from heart failure or at risk of developing, the method comprising administering an INHBE inhibitor to the subject.

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

In some embodiments, the antisense nucleic acid molecule comprises or consists of a nucleotide sequence set forth in table 1.

TABLE 1

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In some embodiments, the antisense nucleic acid molecule comprises or consists of a nucleotide sequence set forth in table 2.

TABLE 2

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In some embodiments, the siRNA molecule comprises or consists of the nucleotide sequences shown in table 3 (sense strand and antisense strand).

TABLE 3 Table 3

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In some embodiments, the siRNA molecule comprises or consists of the nucleotide sequences shown in table 4 (sense strand and antisense strand).

TABLE 4 Table 4

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

The disclosed inhibitory nucleic acid molecules can include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified base, sugar or phosphate groups, or nucleotides incorporating non-natural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

The inhibitory nucleic acid molecules disclosed herein may also comprise one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides that contain modifications to the base, sugar or phosphate moiety. Modifications to the base moiety include, but are not limited to A, C, G and T/U as well as natural and synthetic modifications of different purine or pyrimidine bases such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I) and 2-aminoadenine-9-yl. Modified bases include, but are not limited to, 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-Thymine and 2-Thytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine,

6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,

8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (such as, for example, 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine,

7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine,

7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

Nucleotide analogs may also include modifications of the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, modifications at the 2' positions: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C _1-10 Alkyl or C _2-10 Alkenyl and C _2-10 Alkynyl groups. Exemplary 2' sugar modifications also include, but are not limited to, -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, alkylaryl, arylalkyl,

o-alkylaryl or O-aralkyl, SH, SCH ₃ 、OCN、Cl、Br、CN、CF ₃ 、OCF ₃ 、SOCH ₃ 、SO ₂ CH ₃ 、ONO ₂ 、NO ₂ 、N ₃ 、NH ₂ A heterocycloalkyl group, a heterocycloalkyl aryl group, an aminoalkylamino group, a polyalkylamino group, a substituted silyl group, an RNA cleavage group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, as well as other substituents having similar properties. Similar can also be made elsewhere on the sugarModifications, in particular the 3 'position of the sugar on the 3' terminal nucleotide or in the 2'-5' linked oligonucleotide and the 5 'position of the 5' terminal nucleotide. Modified sugars may also include those containing modifications at the bridging epoxy, such as CH ₂ And S. Nucleotide sugar analogs may also have sugar mimics, such as cyclobutyl moieties in place of pentose (pentofuranosyl sugar).

Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, those that can be modified such that the linkage between two nucleotides contains: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3 '-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3' -phosphoramidates and aminoalkyl phosphoramidates), phosphorothioates, phosphorothioate alkyl phosphonates, phosphorothioate alkyl phosphotriesters and borane phosphates. These phosphate or modified phosphate linkages between two nucleotides may be through a 3'-5' linkage or a 2'-5' linkage, and the linkages may comprise inverted polarities such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Nucleotide substitutions also include Peptide Nucleic Acids (PNAs).

In some embodiments, the antisense nucleic acid molecule is a gapmer (gapmer), whereby the first 1 to 7 nucleotides at the 5 'and 3' ends each have a 2 '-methoxyethyl (2' -MOE) modification. In some embodiments, the first 5 nucleotides of the 5' and 3' ends each have a 2' -MOE modification. In some embodiments, the first 1 to 7 nucleotides at the 5 'and 3' ends are RNA nucleotides. In some embodiments, the first 5 nucleotides at the 5 'and 3' ends are RNA nucleotides. In some embodiments, each backbone linkage between nucleotides is a phosphorothioate linkage.

In some embodiments, the siRNA molecule has a terminal modification. In some embodiments, the 5' end of the antisense strand is phosphorylated. In some embodiments, non-hydrolyzable 5 '-phosphate analogs are used, such as 5' - (E) -vinyl phosphonate.

In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups attached to successive ribonucleosides have been shown to enhance stability and in vivo bioavailability of siRNA. The non-ester groups (-OH, =o) of the phosphodiester linkage may be replaced with sulfur, boron or acetate to give phosphorothioate, boranephosphate and phosphonoacetate linkages. In addition, substitution of phosphodiester groups with phosphotriesters can promote cellular uptake of siRNA and retain it on serum components by eliminating its negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (reactions catalyzed by exo-and endonucleases), whereby the 2' -hydroxyl group can act as a nucleophile and attack adjacent phosphorus in the phosphodiester bond. Such alternatives include 2' -O-methyl, 2' -O-methoxyethyl and 2' -fluoro modifications.

In some embodiments, the siRNA molecule has a base modification. In some embodiments, the base may be substituted with modified bases such as pseudouridine, 5' -methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

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

In some embodiments, the representative siRNA has the formula:

sense: mN 2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2 FN/mN/32 FN%

Antisense: 52 FN/i 2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN/i2FN/mN

Wherein: "N" is a base; "2F" is a 2' -F modification; "m" is a 2' -O-methyl modification and "I" is an internal base; and "×" is phosphorothioate backbone linkage.

The present disclosure also provides vectors comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector may be a viral or non-viral vector capable of transporting the nucleic acid molecule. In some embodiments, the vector is a plasmid or cosmid (such as, for example, circular double stranded DNA into which additional DNA segments may be ligated). In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus and tobacco mosaic virus, yeast Artificial Chromosomes (YACs), epstein-Barr (EBV) -derived episomes, and other expression vectors known in the art.

The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. 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 helices, and lipid microtubules. The carrier may include a buffered saline solution such as PBS, HBSS, and the like.

In some embodiments, the INHBE inhibitor comprises a nuclease agent that induces one or more nicks or double-strand breaks at one or more recognition sequences or a DNA binding protein that binds to a recognition sequence within an INHBE genomic nucleic acid molecule. The recognition sequence may be located within the coding region of the INHBE gene, or within regulatory regions that affect gene expression. The recognition sequence for 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 may include or be adjacent to the start codon of the INHBE gene. For example, the recognition sequence may 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 may be used, each of which targets a nuclease recognition sequence comprising or adjacent to the initiation codon. As another example, two nuclease agents may be used, one targeting a nuclease recognition sequence comprising or adjacent to a start codon and one targeting a nuclease recognition sequence comprising or adjacent to a stop codon, wherein cleavage of the nuclease agent may result in a deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break in the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA binding protein that binds to a desired recognition sequence can be used in the methods and compositions disclosed herein.

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 (TALENs), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems. The length of the recognition sequences can vary and include, for example, about 30-36bp for zinc finger proteins or ZFN pairs, about 15-18bp for each ZFN, about 36bp for TALE proteins or TALENs, and about 20bp for CRISPR/Cas guide RNAs.

In some embodiments, the CRISPR/Cas system can be used to modify an INHBE genomic nucleic acid molecule within a cell. The methods and compositions disclosed herein can use a CRISPR-Cas system for site-directed cleavage of INHBE nucleic acid molecules by utilizing CRISPR complexes comprising guide RNAs (grnas) complexed with Cas proteins.

Cas proteins typically comprise at least one RNA recognition or binding domain that can interact with gRNA. Cas proteins may also comprise nuclease domains (such as, for example, dnase or rnase domains), DNA binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 proteins and wild-type Cpf1 proteins (such as, for example, fnCpf 1). The Cas protein may have full cleavage activity to create a double-strand break in the INHBE genomic nucleic acid molecule, or it may be a nickase that creates a single-strand break in the INHBE genomic nucleic acid molecule. Additional examples of Cas proteins include, but are not limited to, cas1B, cas2, cas3, cas4, cas5e (CasD), cas6e, cas6f, cas7, cas8a1, cas8a2, cas8b, cas8c, cas9 (Csn 1 or Csx 12), cas10d, casF, casG, casH, csy1, csy2, csy3, cse1 (CasA), cse2 (CasB), cse3 (CasE), cse4 (CasC), csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, ax, x3, csx1, csx15, csf1, csf2, csf4, and homologs of the like modifications. In some embodiments, a Cas system, such as Cas12a, may have multiple grnas encoded into a single crRNA. Cas proteins may also be operably linked to heterologous polypeptides as fusion proteins. For example, the Cas protein may be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repression domain. The Cas protein may be provided in any form. For example, the Cas protein may be provided in the form of a protein, such as a Cas protein complexed with a gRNA. Alternatively, the Cas protein may be provided in the form of a nucleic acid molecule encoding a Cas protein, such as RNA or DNA.

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

The gRNA recognition sequence within the target genomic locus in the INHBE genomic nucleic acid molecule is located near the prosomain sequence adjacent motif (PAM) sequence, which is a DNA sequence of 2-6 base pairs immediately following the Cas9 nuclease-targeted DNA sequence. Classical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleobases. gRNA can transport Cas9 to any location in the genome for gene editing, but editing cannot occur at any site other than the site where Cas9 recognizes PAM. Furthermore, 5'-NGA-3' may be a highly potent non-classical PAM of human cells. Typically, PAM is about 2-6 nucleotides downstream of the gRNA-targeted DNA sequence. PAM may flank the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence may be flanked on the 3' end by PAM. In some embodiments, the gRNA recognition sequence may be flanked on the 5' end by PAM. For example, the cleavage site of the Cas protein may be about 1 to about 10, about 2 to about 5, or three base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when Cas9 from streptococcus pyogenes(s) or closely related Cas9 is used), the PAM sequence of the non-complementary strand may be 5' -NGG-3', where N is any DNA nucleotide and immediately 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. Thus, the PAM sequence of the complementary strand will be 5' -CCN-3', where N is any DNA nucleotide, and immediately 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

gRNA is an RNA molecule that binds to and targets a Cas protein to a specific location within the INHBE genomic nucleic acid molecule. Exemplary grnas are grnas effective to guide Cas enzymes to bind or cleave an INHBE genomic nucleic acid molecule, wherein the grnas comprise a DNA targeting segment that hybridizes to a gRNA recognition sequence within the INHBE genomic nucleic acid molecule. Exemplary grnas comprise DNA targeting segments that hybridize to a gRNA recognition sequence present within an INHBE genomic nucleic acid molecule that includes or is adjacent to a start codon or a stop codon. For example, the gRNA can be selected to hybridize to 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 from the start codon or at 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 from the stop codon. Suitable grnas may comprise from about 17 to about 25 nucleotides, from about 17 to about 23 nucleotides, from about 18 to about 22 nucleotides, or from about 19 to about 21 nucleotides. In some embodiments, the gRNA may comprise 20 nucleotides.

Examples of suitable gRNA recognition sequences located within the human INHBE reference gene are shown in Table 5 as SEQ ID NOS.9-27.

Table 5: guide RNA recognition sequences near one or more INHBE variants

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

Such methods can result in, for example, INHBE genomic nucleic acid molecules in which the region of SEQ ID NO. 1 is disrupted, the start codon is disrupted, the stop codon is disrupted, or the coding sequence is disrupted or deleted. Optionally, the cell may be further contacted with one or more additional grnas that hybridize to additional gRNA recognition sequences within the target genomic locus in the INHBE genomic nucleic acid molecule. By contacting the cell with one or more additional grnas (such as, for example, a second gRNA that hybridizes to a second gRNA recognition sequence), two or more double strand breaks or two or more single strand breaks can be generated by Cas protein cleavage.

The methods and compositions disclosed herein can utilize exogenous donor sequences (e.g., targeting vectors or repair templates) to modify the INHBE gene without cleaving the INHBE gene or after cleavage of the INHBE gene with a nuclease agent. Exogenous donor sequence refers to any nucleic acid or vector that includes the elements necessary to achieve site-specific recombination with the target sequence. The use of exogenous donor sequences in combination with nuclease agents can result in more precise modifications within the INHBE gene by facilitating homology-directed repair.

In such methods, the nuclease agent cleaves the INHBE gene to produce a single-strand break (nick) or double-strand break, and the exogenous donor sequence recombines the INHBE gene by non-homologous end joining (NHEJ) -mediated ligation or by a homology-directed repair event. Optionally, repair with an exogenous donor sequence removes or disrupts the nuclease cleavage site such that the allele that has been targeted is not retargetable by the nuclease agent.

The exogenous donor sequence may comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), which may be single-stranded or double-stranded, and which may be in linear or circular form. For example, the exogenous donor sequence may be a single stranded oligodeoxynucleotide (ssODN). See, e.g., yoshimi et al, nat. Exemplary exogenous donor sequences are from about 50 nucleotides to about 5kb in length, from about 50 nucleotides to about 3kb in length, or from about 50 to about 1,000 nucleotides in length. Other exemplary exogenous donor sequences are from about 40 to about 200 nucleotides in length. For example, the exogenous donor sequence can be about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150, about 150 to about 160, about 160 to about 170, about 170 to about 180, about 180 to about 190, or about 190 to about 200 nucleotides in length. Alternatively, the exogenous donor sequence may be about 50 to about 100, about 100 to about 200, about 200 to about 300, about 300 to about 400, about 400 to about 500, about 500 to about 600, about 600 to about 700, about 700 to about 800, about 800 to about 900, or about 900 to about 1,000 nucleotides in length. Alternatively, the exogenous donor sequence may be about 1kb to about 1.5kb, about 1.5kb to about 2kb, about 2kb to about 2.5kb, about 2.5kb to about 3kb, about 3kb to about 3.5kb, about 3.5kb to about 4kb, about 4kb to about 4.5kb, or about 4.5kb to about 5kb in length. Alternatively, the length of the exogenous donor sequence may be, for example, no more than 5kb, 4.5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 900 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, or 50 nucleotides.

In some examples, the exogenous donor sequence is a ssODN that is about 80 nucleotides and about 200 nucleotides in length (e.g., about 120 nucleotides in length). In another example, the exogenous donor sequence is a ssODN of about 80 nucleotides and about 3kb in length. Such ssODN may have, for example, homology arms of about 40 nucleotides and about 60 nucleotides each in length. Such ssODN may also have homology arms of, for example, about 30 nucleotides and 100 nucleotides each in length. The homology arms may be symmetrical (e.g., 40 nucleotides in length each or 60 nucleotides in length each), or they may be asymmetrical (e.g., one homology arm of 36 nucleotides in length and one homology arm of 91 nucleotides in length).

The exogenous donor sequence may comprise modifications or sequences that provide additional desired characteristics (e.g., altered or modulated stability; tracked or detected with fluorescent markers; binding sites for proteins or protein complexes; etc.). The exogenous donor sequence may comprise one or more fluorescent labels, purification tags, epitope tags, or a combination thereof. For example, the exogenous donor sequence may comprise one or more fluorescent labels (e.g., fluorescent proteins or other fluorophores or dyes), such as at least 1, at least 2, at least 3, at least 4, or at least 5 fluorescent labels. Exemplary fluorescent labels include fluorophores such as fluorescein (e.g., 6-carboxyfluorescein (6-FAM)), texas Red (Texas Red), HEX, cy3, cy5, cy5.5, pacific Blue (Pacific Blue), 5- (and-6) -carboxytetramethyl rhodamine (TAMRA), and Cy7. A wide variety of fluorescent dyes for labeling oligonucleotides are commercially available (e.g., from Integrated DNA Technologies). Such fluorescent markers (e.g., internal fluorescent markers) can be used, for example, to detect exogenous donor sequences that have been directly integrated into a cleaved INHBE gene having an overhang compatible with the end of the exogenous donor sequence . The tag or label may be located at the 5 'end, the 3' end, or within the exogenous donor sequence. For example, the exogenous donor sequence may be 5 'terminal to an IR700 fluorophore from Integrated DNA Technologies (5'700 A) conjugation.

The exogenous donor sequence may also comprise a nucleic acid insertion sequence comprising a segment of DNA to be integrated into the INHBE gene. Integration of a nucleic acid insertion sequence in an INHBE gene may result in addition of a nucleic acid sequence of interest in the INHBE gene, deletion of a nucleic acid sequence of interest in the INHBE gene, or substitution (i.e., deletion and insertion) of a nucleic acid sequence of interest in the INHBE gene. Some exogenous donor sequences are designed to insert nucleic acid insertion sequences into the INHBE gene without causing any corresponding deletions in the INHBE gene. Other exogenous donor sequences are designed to delete the nucleic acid sequence of interest in the INHBE gene without any corresponding insertion of the nucleic acid insertion sequence. Still other exogenous donor sequences are designed to delete a nucleic acid sequence of interest in the INHBE gene and replace the nucleic acid sequence of interest with a nucleic acid insertion sequence.

The nucleic acid insertion sequences or the corresponding nucleic acids deleted and/or replaced in the INHBE gene may have different lengths. Exemplary nucleic acid insertion sequences or corresponding nucleic acids deleted and/or replaced in the INHBE gene are from about 1 nucleotide to about 5kb in length or from about 1 nucleotide to about 1,000 nucleotides in length. For example, the length of the nucleic acid insertion sequence or the corresponding nucleic acid deleted and/or replaced in the INHBE gene may be from about 1 to about 10, from about 10 to about 20, from about 20 to about 30, from about 30 to about 40, from about 40 to about 50, from about 50 to about 60, from about 60 to about 70, from about 70 to about 80, from about 80 to about 90, from about 90 to about 100, from about 100 to about 110, from about 110 to about 120, from about 120 to about 130, from about 130 to about 140, from about 140 to about 150, from about 150 to about 160, from about 160 to about 170, from about 170 to about 180, from about 180 to about 190, or from about 190 to about 200 nucleotides. Likewise, the length of the nucleic acid insert or the corresponding nucleic acid deleted and/or replaced in the INHBE gene may be from about 1 to about 100, from about 100 to about 200, from about 200 to about 300, from about 300 to about 400, from about 400 to about 500, from about 500 to about 600, from about 600 to about 700, from about 700 to about 800, from about 800 to about 900, or from about 900 to about 1,000 nucleotides. Likewise, the length of the nucleic acid insert or the corresponding nucleic acid deleted and/or replaced in the INHBE gene may be from about 1kb to about 1.5kb, from about 1.5kb to about 2kb, from about 2kb to about 2.5kb, from about 2.5kb to about 3kb, from about 3kb to about 3.5kb, from about 3.5kb to about 4kb, from about 4kb to about 4.5kb, or from about 4.5kb to about 5kb.

The nucleic acid insertion sequence may comprise genomic DNA or any other type of DNA. For example, the nucleic acid insertion sequence may comprise a cDNA.

The nucleic acid insertion sequence may comprise a sequence homologous to all or a portion of the INHBE gene (e.g., a portion of a gene encoding a particular motif or region of the INHBE protein). For example, the nucleic acid insertion sequence may comprise a sequence comprising one or more point mutations (e.g., 1, 2, 3, 4, 5 or more) or one or more nucleotide insertions or deletions compared to the sequence in the INHBE gene that is targeted for replacement. The nucleic acid insertion sequence or the corresponding nucleic acid deleted and/or replaced in the INHBE gene may be a coding region, such as an exon; non-coding regions, such as introns, untranslated regions or regulatory regions (e.g., promoters, enhancers or transcription repressor binding elements); or any combination thereof.

The nucleic acid insertion sequence may also comprise a conditional allele. The conditional allele may be a multifunctional allele as described in US 2011/0104799. For example, a conditional allele may comprise: a) An actuation sequence (actuating sequence) in a sense orientation relative to transcription of the target gene; b) A Drug Selection Cassette (DSC) in sense or antisense orientation; c) A Nucleotide Sequence of Interest (NSI) in an antisense orientation; and d) a conditional inversion module in reverse orientation (conditional by inversion module, spin, which utilizes exonically split introns and invertible gene trap-like modules). See, for example, US 2011/0104799. The conditional allele may further comprise a recombinable unit that recombines upon exposure to the first recombinase to form a conditional allele that i) lacks the actuation sequence and DSC; and ii) comprises an NSI in the sense orientation and a COIN in the antisense orientation. See, for example, US 2011/0104799.

The nucleic acid insertion sequence may also comprise a polynucleotide encoding a selectable marker. Alternatively, the nucleic acid insertion sequence may lack a polynucleotide encoding a selectable marker. The selection marker may be contained in a selection cassette. Optionally, the selection cassette may be a self-deleting cassette. See, for example, US 8,697,851 and US 2013/0312129. For example, the self-deletion cassette can comprise a Cre gene (comprising two exons encoding Cre recombinase separated by an intron) operably linked to a mouse Prm1 promoter and a neomycin resistance gene operably linked to a human ubiquitin promoter. Exemplary selectable markers include neomycin phosphotransferase (neo ^r ) Hygromycin B phosphotransferase (hyg) ^r ) puromycin-N-acetyltransferase (puro) ^r ) Blasticidin S deaminase (bsr) ^r ) Xanthine/guanine phosphoribosyl transferase (gpt) or herpes simplex virus thymidine kinase (HSV-k), or a combination thereof. The polynucleotide encoding the selectable marker may be operably linked to a promoter active in the cell being targeted. Examples of promoters are described elsewhere herein.

The nucleic acid insertion sequence may also comprise a reporter gene. Exemplary reporter genes include those encoding: luciferase, beta-galactosidase, green Fluorescent Protein (GFP), enhanced green fluorescent protein (eGFP), cyan Fluorescent Protein (CFP), yellow Fluorescent Protein (YFP), enhanced yellow fluorescent protein (eYFP), blue Fluorescent Protein (BFP), enhanced blue fluorescent protein (eBFP), dsRed, zsGreen, mmGFP, mPlum, mCherry, tdTomato, mStrawberry, J-Red, mOrange, mKO, mCitrine, venus, YPet, emerald, cyPet, cerulean, T-saphire, and alkaline phosphatase. Such reporter genes may be operably linked to a promoter active in the cell being targeted. Examples of promoters are described elsewhere herein.

The nucleic acid insertion sequence may also comprise one or more expression cassettes or deletion cassettes. A given cassette may comprise one or more of a nucleotide sequence of interest, a polynucleotide encoding a selectable marker and a reporter gene, and various regulatory components that affect expression. Examples of selectable markers and reporter genes that may be included are discussed in detail elsewhere herein.

The nucleic acid insertion sequence may comprise a nucleic acid flanked by site-specific recombination target sequences. Alternatively, the nucleic acid insertion sequence may comprise one or more site-specific recombination target sequences. Although the entire nucleic acid insertion sequence may be flanked by such site-specific recombination target sequences, any region within the nucleic acid insertion sequence or a separate polynucleotide of interest may also be flanked by such sites. The site-specific recombination target sequences that can flank the nucleic acid insert or any polynucleotide of interest in the nucleic acid insert can include, for example, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT11, FRT71, attp, att, FRT, rox, or a combination thereof. In some examples, the site-specific recombination site is flanked by polynucleotides encoding selectable markers and/or reporter genes contained within the nucleic acid insertion sequence. After integration of the nucleic acid insertion sequence in the INHBE gene, the sequence between the site-specific recombination sites can be removed. Optionally, two exogenous donor sequences, each having a nucleic acid insertion sequence comprising a site-specific recombination site, can be used. Exogenous donor sequences can be targeted to the 5 'and 3' regions flanking the nucleic acid of interest. After integration of the two nucleic acid insertion sequences into the target genomic locus, the nucleic acid of interest between the two inserted site-specific recombination sites can be removed.

The nucleic acid insertion sequence may also comprise one or more restriction sites for restriction endonucleases (i.e., restriction enzymes), including type I, type II, type III and type IV endonucleases. Type I and type III restriction endonucleases recognize specific recognition sequences, but typically cleave at variable positions from the nuclease binding site, which can be hundreds of base pairs from the cleavage site (recognition sequence). In type II systems, the restriction activity is independent of any methylase activity, and cleavage typically occurs at specific sites within or near the binding site. Most type II enzymes cleave palindromic sequences, but type IIa enzymes recognize non-palindromic recognition sequences and cleave outside the recognition sequence, type IIb enzyme cleaves sequences twice, both sites being outside the recognition sequence, and type IIs enzymes recognize asymmetric recognition sequences and cleave on one side and at a defined distance of about 1-20 nucleotides from the recognition sequence. Type IV restriction enzymes target methylated DNA. Restriction enzymes are further described and classified, for example, in REBASE database (web pages on REBASE. Neb. Com; roberts et al, nucleic Acids Res.,2003,31,418-420; roberts et al, nucleic Acids Res.,2003,31,1805-1812; and Belfort et al, edited by Craigie et al, (ASM Press, washington, DC)), pp.761-783, in Mobile DNA II, 2002.

Some exogenous donor sequences have short single stranded regions at the 5 'end and/or the 3' end that are complementary to one or more overhangs at the target genomic locus that result from nuclease-mediated or Cas protein-mediated cleavage (e.g., in the INHBE gene). These overhangs may also be referred to as 5 'and 3' homology arms. For example, some exogenous donor sequences have short single stranded regions at the 5 'end and/or 3' end that are complementary to one or more overhangs generated by Cas protein-mediated cleavage at the 5 'and/or 3' target sequences at the target genomic locus. Some such exogenous donor sequences have complementary regions only at the 5 'end or only at the 3' end. For example, some such exogenous donor sequences have complementary regions at the 5 'end only that are complementary to overhangs generated at the 5' target sequence at the target genomic locus, or complementary regions at the 3 'end only that are complementary to overhangs generated at the 3' target sequence at the target genomic locus. Other such exogenous donor sequences have complementary regions at both the 5 'and 3' ends. For example, other such exogenous donor sequences have complementary regions at both the 5 'and 3' ends, e.g., complementary to the first and second overhangs, respectively, produced by Cas-mediated cleavage at the target genomic locus. For example, if the exogenous donor sequence is double-stranded, the single-stranded complementary region may extend from the 5' end of the top strand of the donor sequence and the 5' end of the bottom strand of the donor sequence, thereby creating a 5' overhang at each end. Alternatively, the single stranded complementary region may extend from the 3' end of the top strand of the donor sequence and the 3' end of the bottom strand of the template, resulting in a 3' overhang.

The complementary region may have any length sufficient to facilitate ligation between the exogenous donor sequence and the INHBE gene. Exemplary complementary regions are about 1 to about 5 nucleotides in length, about 1 to about 25 nucleotides in length, or about 5 to about 150 nucleotides in length. For example, the complementary region can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. Alternatively, the length of the complementary region may be about 5 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, about 120 to about 130, about 130 to about 140, about 140 to about 150 nucleotides or more.

Such complementary regions may be complementary to overhangs generated by two pairs of nicking enzymes. Two double strand breaks with staggered ends can be created by using first and second nicking enzymes that cleave opposite strands of DNA to create a first double strand break, and third and fourth nicking enzymes that cleave opposite strands of DNA to create a second double strand break. For example, cas proteins may be used to nick first, second, third, and fourth guide RNA recognition sequences corresponding to the first, second, third, and fourth guide RNAs. The first and second guide RNA recognition sequences may be positioned to create a first cleavage site such that the nicks created by the first and second nicking enzymes on the first and second strands of DNA create a double-strand break (i.e., the first cleavage site comprises the nicks within the first and second guide RNA recognition sequences). Likewise, the third and fourth guide RNA recognition sequences may be positioned to create a second cleavage site such that the nicks created by the third and fourth nicking enzymes on the first and second strands of DNA create a double-strand break (i.e., the second cleavage site comprises nicks within the third and fourth guide RNA recognition sequences). Preferably, the nicks within the first and second guide RNA recognition sequences and/or the third and fourth guide RNA recognition sequences may be offset nicks (off-set nicks) that create overhangs. The offset window may be, for example, at least about 5bp, 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp or more. See Ran et al, cell,2013,154,1380-1389; mali et al, nat. Biotech.,2013,31,833-838; and Shen et al, nat. Methods,2014,11,399-404. In such cases, the double-stranded exogenous donor sequence may be designed to have a single-stranded complementary region that is complementary to the overhangs created by the nicks in the first and second guide RNA recognition sequences and the nicks in the third and fourth guide RNA recognition sequences. Such exogenous donor sequences can then be inserted through non-homologous end joining mediated ligation.

Some exogenous donor sequences (i.e., targeting vectors) comprise homology arms. If the exogenous donor sequence further comprises a nucleic acid insertion sequence, the homology arms may flank the nucleic acid insertion sequence. For ease of reference, homology arms are referred to herein as 5 'and 3' (i.e., upstream and downstream) homology arms. The term relates to the relative position of the homology arm and the nucleic acid insertion sequence within the exogenous donor sequence. The 5 'and 3' homology arms correspond to regions within the INHBE gene, which are referred to herein as the "5 'target sequence" and the "3' target sequence", respectively.

A homology arm and target sequence "correspond" or "correspond" to each other when the two regions share a sufficient level of sequence identity with each other to serve as substrates for homologous recombination reactions. The term "homology" includes DNA sequences that are identical to or share sequence identity with the corresponding sequence. The sequence identity between a given target sequence and the corresponding homology arms present in the exogenous donor sequence may be any degree of sequence identity that allows homologous recombination to occur. For example, the homology arm (or fragment thereof) of the exogenous donor sequence may share an amount of sequence identity with the target sequence (or fragment thereof) of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity such that homologous recombination of the sequences occurs. Furthermore, the respective region of homology between the homology arms and the respective target sequences may be of any length sufficient to promote homologous recombination. Exemplary homology arms are from about 25 nucleotides to about 2.5kb in length, from about 25 nucleotides to about 1.5kb in length, or from about 25 to about 500 nucleotides in length. For example, a given homology arm (or each homology arm) and/or corresponding target sequence may comprise a corresponding homology region of about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 300 to about 350, about 350 to about 400, about 400 to about 450, or about 450 to about 500 nucleotides in length such that the homology arm has sufficient homology to the corresponding target sequence within the INHBE gene for homologous recombination to occur. Alternatively, a given homology arm (or each homology arm) and/or corresponding target sequence may comprise a corresponding homology region of about 0.5kb to about 1kb, about 1kb to about 1.5kb, about 1.5kb to about 2kb, or about 2kb to about 2.5kb in length. For example, homology arms may each be about 750 nucleotides in length. The homology arms may be symmetrical (the respective lengths are approximately the same) or asymmetrical (one longer than the other).

The homology arms may correspond to loci native to the cell (e.g., targeting loci). Alternatively, for example, they may correspond to regions of heterologous or exogenous DNA segments integrated into the genome of a cell, including, for example, transgenes, expression cassettes, or heterologous or exogenous DNA regions. Alternatively, the homology arm of the targeting vector may correspond to a region of a Yeast Artificial Chromosome (YAC), a Bacterial Artificial Chromosome (BAC), a human artificial chromosome, or any other engineered region comprised in a suitable host cell. Furthermore, the homology arm of the targeting vector may correspond to or be derived from a region of a BAC library, cosmid library, or P1 phage library, or may be derived from synthetic DNA.

When a nuclease agent is used in combination with an exogenous donor sequence, the 5 'and 3' target sequences are preferably positioned close enough to the nuclease cleavage site to facilitate a homologous recombination event between the target sequences and the homology arms when a single strand break (nick) or double strand break occurs at the nuclease cleavage site. The term "nuclease cleavage site" includes a DNA sequence at which a nick or double-strand break is created by a nuclease agent (e.g., cas9 protein complexed with a guide RNA). Target sequences within the INHBE gene corresponding to the 5 'and 3' homology arms of the exogenous donor sequence are "positioned sufficiently close to the nuclease cleavage site" if the distance is capable of promoting a homologous recombination event between the 5 'and 3' target sequences and the homology arms when a single-strand break or double-strand break occurs at the nuclease cleavage site. Thus, the target sequence corresponding to the 5 'and/or 3' homology arm of the exogenous donor sequence may be, for example, within at least 1 nucleotide of the given nuclease cleavage site, or within at least 10 nucleotides to about 1,000 nucleotides of the given nuclease cleavage site. For example, the nuclease cleavage site can be in close proximity to at least one or both of the target sequences.

The spatial relationship of the target sequence and nuclease cleavage site corresponding to the homology arm of the exogenous donor sequence can vary. For example, the target sequence may be located 5 'to the nuclease cleavage site, the target sequence may be located 3' to the nuclease cleavage site, or the target sequence may flank the nuclease cleavage site.

Also provided are methods of treatment and methods of using the methods disclosed herein for modifying or altering expression of the endogenous INHBE gene to treat or prevent a subject suffering from or at risk of a metabolic disorder. Also provided are methods of treatment and methods of treating or preventing a subject suffering from or at risk of a metabolic disorder using methods for reducing expression of an INHBE mRNA transcript or using methods for providing a recombinant nucleic acid encoding an INHBE protein, providing an mRNA encoding an INHBE protein, or providing an INHBE protein to a subject. The method may comprise introducing one or more nucleic acids or proteins into the subject, into the liver of the subject, or into a cell (e.g., a hepatocyte) of the subject (e.g., in vivo or ex vivo).

Also provided are methods of treatment and methods of treating or preventing cardiovascular disease in a subject suffering from or at risk of cardiovascular disease using the methods disclosed herein for modifying or altering expression of the endogenous INHBE gene. Also provided are methods of treatment and methods of treating or preventing cardiovascular disease in a subject suffering from or at risk of cardiovascular disease using methods for reducing expression of an INHBE mRNA transcript or using methods for providing a recombinant nucleic acid encoding an INHBE protein, providing an mRNA encoding an INHBE protein, or providing an INHBE protein to a subject. The method may comprise introducing one or more nucleic acids or proteins into the subject, into the liver of the subject, or into a cell (e.g., a hepatocyte) of the subject (e.g., in vivo or ex vivo).

Such methods may include genome editing or gene therapy. For example, the endogenous INHBE gene that does not encode a loss-of-function variant may be modified to include any of the loss-of-function variants described herein. For another example, the endogenous INHBE gene that does not encode a loss-of-function variant may be knocked out or inactivated. Likewise, endogenous INHBE genes that do not encode a loss-of-function variant may be knocked out or inactivated, and INHBE genes comprising any one or any combination of the INHBE loss-of-function variants described herein may be introduced and expressed. Similarly, endogenous INHBE genes that do not encode a loss-of-function variant may be knocked out or inactivated, and recombinant DNA encoding any one of the INHBE loss-of-function variants described herein, or any combination thereof, may be introduced and expressed, mRNA encoding any one of the INHBE loss-of-function variants (or fragments thereof) described herein, or any combination thereof (e.g., intracellular protein replacement therapy), or cDNA encoding any one of the INHBE loss-of-function variants (or fragments thereof) described herein, or any combination thereof, may be introduced (e.g., protein replacement therapy).

Other such methods may comprise introducing and expressing a recombinant INHBE gene comprising any one of the INHBE loss-of-function variants described herein, or any combination thereof (e.g., an intact INHBE variant or comprising a modified minigene), introducing and expressing a recombinant nucleic acid (e.g., DNA) encoding any one of the INHBE loss-of-function variants described herein, or any combination thereof, introducing and expressing one or more mRNA (e.g., intracellular protein replacement therapy) encoding any one of the INHBE loss-of-function variants described herein, or any combination thereof (e.g., protein replacement therapy) without knocking out or inactivating the endogenous INHBE gene that does not encode the loss-of-function variants.

The INHBE gene or minigene or DNA encoding any one of the INHBE loss-of-function variants described herein or fragments thereof, or any combination thereof, may be introduced and expressed in the form of an expression vector that does not alter the genome, it may be introduced in the form of a targeting vector such that it is genomically integrated into the INHBE locus, or it may be introduced such that it is genomically integrated into loci other than the INHBE locus, such as the safe harbor locus (safe harbor locus). The genomically integrated INHBE gene may be operably linked to an INHBE promoter or another promoter, such as an endogenous promoter at the site of integration. The safe harbor locus is a chromosomal locus where transgenes can be stably and reliably expressed in all tissues of interest without adversely affecting gene structure or expression. The safe harbor locus may have, for example, one or more or all of the following features: a distance greater than 50kb from the 5' end of any gene; a distance of greater than 300kb from any cancer-associated gene; a distance greater than 300kb from any microRNA; outside the gene transcription unit, and outside the super-conserved region. Examples of suitable safe harbor loci include the adeno-associated viral locus 1 (AAVS 1), the locus of the chemokine (CC motif) receptor 5 (CCR 5) gene, and human orthologs of the mouse ROSA26 locus.

Combinations of INHBE protein isoforms or nucleic acids encoding INHBE protein isoforms that may be introduced and expressed include any one or any combination of the proteins or mRNA isoforms described herein. For example, INHBE, which is a nucleic acid encoding isoform 1 (SEQ ID NO: 2), is introduced or expressed, which encodes any one or any combination of the loss-of-function variants described herein, alone or in combination with other isoforms. Exemplary sequences for each of these isoforms and transcripts are provided elsewhere herein. However, it is understood that the sequence of genes within a population, the sequence of mRNAs transcribed from such genes, and the proteins translated from such mRNAs may vary due to polymorphisms, such as single nucleotide polymorphisms. The sequences provided herein for each transcript and isoform are merely exemplary sequences. Other sequences are also possible.

In some embodiments, the method comprises treating a subject that is not a carrier of any of the INHBE variant nucleic acid molecules described herein (or is simply a hybrid carrier of any one of the variant nucleic acid molecules described herein or any combination thereof) and that has or is susceptible to developing a metabolic disorder and/or cardiovascular disease, comprising introducing into the subject or into hepatocytes of the subject: a) A nuclease agent (or a nucleic acid encoding the same) that binds to a nuclease recognition sequence within an INHBE gene, wherein the nuclease recognition sequence comprises or is adjacent to a position of one of the INHBE variant nucleic acid molecules described herein; and b) an exogenous donor sequence comprising a 5 'homology arm that hybridizes to a target sequence 5' of a position of one of the INHBE variant nucleic acid molecules described herein, a 3 'homology arm that hybridizes to a target sequence 3' of the same INHBE variant nucleic acid molecule, and a nucleic acid insertion sequence comprising one or more variant nucleotides flanked by a 5 'homology arm and a 3' homology arm. The nuclease agent can cleave an INHBE gene in a hepatocyte of the subject, and the exogenous donor sequence can recombine with the INHBE gene in the hepatocyte, wherein upon recombination of the exogenous donor sequence with the INHBE gene, a nucleic acid insert sequence encoding a loss-of-function variant is introduced, thereby replacing the wild-type nucleotide. Examples of nuclease agents (e.g., cas9 proteins and guide RNAs) that can be used in such methods are disclosed elsewhere herein. Examples of suitable guide RNAs and guide RNA recognition sequences are disclosed elsewhere herein. Examples of exogenous donor sequences that can be used in such methods are disclosed elsewhere herein.

As another example, the method may comprise treating a subject who is not a carrier of any INHBE variant nucleic acid molecule described herein (or is simply a hybrid carrier of any one of the variant nucleic acid molecules described herein or any combination thereof) and who has or is susceptible to developing a metabolic disorder and/or cardiovascular disease, comprising introducing into a subject or into a liver cell of a subject an exogenous donor sequence comprising a 5 'homology arm that hybridizes to a target sequence 5' of one of the INHBE variant nucleic acid molecules described herein, a 3 'homology arm that hybridizes to a target sequence 3' of the same INHBE variant nucleic acid molecule, and a nucleic acid insertion sequence comprising one or more variant nucleotides flanking the 5 'homology arm and the 3' homology arm. The exogenous donor sequence may be recombined with an INHBE gene in a hepatocyte, wherein upon recombination of the exogenous donor sequence with the INHBE gene, a nucleic acid insert encoding a loss-of-function variant is introduced, thereby replacing the wild type nucleotide. Examples of exogenous donor sequences that can be used in such methods are disclosed elsewhere herein.

In some embodiments, the method comprises treating a subject that is not a carrier of any of the INHBE variant nucleic acid molecules described herein (or is simply a hybrid carrier of any one of the variant nucleic acid molecules described herein or any combination thereof) and that has or is susceptible to developing a metabolic disorder and/or cardiovascular disease, comprising introducing into the subject or into hepatocytes of the subject: a) A nuclease agent (or a nucleic acid encoding the same) that binds to a nuclease recognition sequence within an INHBE gene, wherein the nuclease recognition sequence comprises a start codon of the INHBE gene or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon. Nuclease agents can cleave the INHBE gene in hepatocytes of a subject and disrupt its expression. In some embodiments, the method comprises treating a subject that is not a carrier of any of the INHBE variant nucleic acid molecules described herein (or is simply a hybrid carrier of any one of the INHBE variant nucleic acid molecules described herein or any combination thereof) and that has or is susceptible to developing a metabolic disorder and/or cardiovascular disease, comprising introducing into the subject or into hepatocytes of the subject: a) A nuclease agent (or a nucleic acid encoding the same) that binds to a nuclease recognition sequence within the INHBE gene, wherein the nuclease recognition sequence comprises the start codon of the INHBE gene or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 1,000 nucleotides of the start codon or is selected from the group consisting of SEQ ID NOs 1-7; and b) an expression vector comprising a recombinant INHBE gene comprising any one of the loss-of-function variants described herein, or any combination thereof. The expression vector may be a vector that does not undergo genomic integration. Alternatively, a targeting vector (i.e., an exogenous donor sequence) comprising a recombinant INHBE gene comprising any one or any combination of the loss-of-function variants described herein may be introduced. The nuclease agent can cleave and disrupt expression of the INHBE gene in the hepatocytes of the subject, and the expression vector can express the recombinant INHBE gene in the hepatocytes of the subject. Alternatively, the genomically integrated recombinant INHBE gene may be expressed in hepatocytes of the subject. Examples of nuclease agents (e.g., cas9 proteins and guide RNAs having nuclease activity) that can be used in such methods are disclosed elsewhere herein. Examples of suitable guide RNAs and guide RNA recognition sequences are disclosed elsewhere herein. Step b) may alternatively comprise introducing an expression vector or targeting vector comprising a nucleic acid (e.g., DNA) encoding an INHBE protein that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE isoform or fragment thereof described herein and comprises any one or any combination of the INHBE variant nucleic acid molecules described herein. Likewise, step b) may alternatively comprise introducing an mRNA encoding an INHBE protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE mRNA isoform or fragment thereof described herein and comprising any one or any combination of the INHBE variant nucleic acid molecules described herein. Likewise, step b) may alternatively comprise introducing a protein comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE protein isoform or fragment thereof described herein and including any one or any combination of the loss-of-function variant polypeptides described herein.

In some embodiments, a second nuclease agent is also introduced into the subject or a hepatocyte of the subject, wherein the second nuclease agent binds to a second nuclease recognition sequence within the INHBE gene, wherein the second nuclease recognition sequence comprises a stop codon of the INHBE gene or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the stop codon, wherein the nuclease agent cleaves the INHBE gene in the hepatocyte within both the first nuclease recognition sequence and the second nuclease recognition sequence, wherein the hepatocyte is modified to comprise a deletion between the first nuclease recognition sequence and the second nuclease recognition sequence. For example, the second nuclease agent can be a Cas9 protein and a guide RNA. Suitable guide RNAs and guide RNA recognition sequences adjacent to a stop codon are disclosed elsewhere herein.

Such methods may also include methods of treating a subject who is not a carrier of any of the INHBE variant nucleic acid molecules described herein (or is simply a hybrid carrier of any of the INHBE variant nucleic acid molecules described herein or any combination thereof) and who has or is susceptible to developing a metabolic disorder and/or cardiovascular disease, comprising introducing into the subject or into hepatocytes of the subject: a) A DNA binding protein (or a nucleic acid encoding the same) that binds to a DNA binding protein recognition sequence within the INHBE gene, wherein the DNA binding protein recognition sequence comprises the start codon of the INHBE gene or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon. The DNA binding protein can alter (e.g., decrease) INHBE gene expression in hepatocytes of a subject. Such methods may also include methods of treating a subject who is not a carrier of any of the INHBE variant nucleic acid molecules described herein (or is simply a hybrid carrier of any of the INHBE variant nucleic acid molecules described herein or any combination thereof) and who has or is susceptible to developing a metabolic disorder and/or cardiovascular disease, comprising introducing into the subject or into hepatocytes of the subject: a) A DNA binding protein (or a nucleic acid encoding the same) that binds to a DNA binding protein recognition sequence within the INHBE gene, wherein the DNA binding protein recognition sequence comprises the start codon of the INHBE gene or is within about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides of the start codon; and b) an expression vector comprising a recombinant INHBE gene comprising any one of the loss-of-function variants described herein, or any combination thereof. The expression vector may be a vector that does not undergo genomic integration. Alternatively, a targeting vector (i.e., an exogenous donor sequence) comprising a recombinant INHBE gene comprising any one or any combination of the INHBE variant nucleic acid molecules described herein may be introduced. The DNA binding protein can alter (e.g., reduce) the expression of the INHBE gene in the hepatocytes of the subject, and the expression vector can express the recombinant INHBE gene in the hepatocytes of the subject. Alternatively, the genomically integrated recombinant INHBE gene may be expressed in hepatocytes of the subject. Examples of DNA binding proteins suitable for use in such methods are disclosed elsewhere herein. Such DNA-binding proteins (e.g., cas9 proteins and guide RNAs) may be fused or operably linked to a transcriptional repression domain. For example, the DNA-binding protein may be a catalytically inactive Cas9 protein fused to a transcription repression domain. Examples of suitable guide RNAs and guide RNA recognition sequences are disclosed elsewhere herein. Step b) may alternatively comprise introducing an expression vector or targeting vector comprising a nucleic acid (e.g., DNA) encoding an INHBE protein that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE isoform or fragment thereof described herein and comprises any one or any combination of the INHBE variant nucleic acid molecules described herein. Likewise, step b) may alternatively comprise introducing an mRNA encoding an INHBE protein having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE mRNA isoform or fragment thereof described herein and comprising any one or any combination of the INHBE variant nucleic acid molecules described herein. Likewise, step b) may alternatively comprise introducing a protein comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE protein isoform or fragment thereof described herein and including any one or any combination of the loss-of-function variant polypeptides described herein.

Other such methods may include a method of treating a subject who is not a carrier of any of the INHBE variant nucleic acid molecules described herein (or is simply a hybrid carrier of any of the INHBE variant nucleic acid molecules described herein or any combination thereof) and who has or is susceptible to developing a metabolic disorder and/or cardiovascular disease, comprising introducing into the subject or into hepatocytes of the subject an expression vector, wherein the expression vector comprises a recombinant INHBE gene comprising any of the loss-of-function variants described herein or any combination thereof, wherein the expression vector expresses the recombinant INHBE gene in hepatocytes of the subject. The expression vector may be a vector that does not undergo genomic integration. Alternatively, a targeting vector (i.e., an exogenous donor sequence) comprising a recombinant INHBE gene comprising any one or any combination of the INHBE variant nucleic acid molecules described herein may be introduced. In methods of using the expression vectors, the expression vectors can express the recombinant INHBE gene in hepatocytes of a subject. Alternatively, in a method of genomic integration of a recombinant INHBE gene, the recombinant INHBE gene may be expressed in hepatocytes of a subject. Such methods may alternatively comprise introducing an expression vector or targeting vector comprising a nucleic acid (e.g., DNA) encoding an INHBE protein that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE isoform or fragment thereof described herein and comprises any one or any combination of the loss of function variants described herein. Likewise, such methods may alternatively comprise introducing an mRNA encoding an INHBE protein that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE mRNA isoform or fragment thereof described herein and comprises any one or any combination of the INHBE variant nucleic acid molecules described herein. Likewise, such methods may alternatively comprise introducing a protein comprising a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any INHBE protein isoform or fragment thereof described herein and including any one or any combination of the loss-of-function variant polypeptides described herein.

Suitable expression vectors and recombinant INHBE genes for use in any of the above methods are disclosed elsewhere herein. For example, the recombinant INHBE gene may be a full length variant gene or may be an INHBE minigene in which one or more non-essential segments of the gene have been deleted relative to the corresponding wild-type INHBE gene. For example, the deleted segment may include one or more intron sequences. Examples of complete INHBE genes are genes that have at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO. 1 when optimally aligned with SEQ ID NO. 1.

In some embodiments, the method comprises modifying cells (e.g., hepatocytes) of a subject suffering from or susceptible to developing a chronic liver disease. In some embodiments, the methods comprise modifying cells (e.g., cardiomyocytes) of a subject suffering from or susceptible to developing a cardiovascular disease. In such methods, the nuclease agent and/or exogenous donor sequence and/or recombinant expression vector can be introduced into the cells by administration in an effective regimen, which refers to a dose, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further exacerbation, and/or improves at least one sign or symptom of the disease being treated. The term "symptom" refers to subjective evidence of a disease perceived by a subject, and "sign" refers to objective evidence of a disease observed by a physician. If the subject already has a disease, then the regimen may be referred to as a therapeutically effective regimen. If the subject has an elevated risk of suffering from the disease relative to the general population but has not yet developed symptoms, then the regimen is referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy can be observed in individual subjects relative to historical controls or past experiences of the same patient. In other cases, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial of a population of treated subjects relative to a control population of untreated subjects.

Delivery may be by any suitable method, as disclosed elsewhere herein. For example, the nuclease agent or exogenous donor sequence or recombinant expression vector may be delivered by vector delivery, viral delivery, particle-mediated delivery, nanoparticle-mediated delivery, liposome-mediated delivery, exosome-mediated delivery, lipid-nanoparticle-mediated delivery, cell penetrating peptide-mediated delivery, or implantable device-mediated delivery. Some specific examples include hydrodynamic delivery, virus-mediated delivery, and lipid nanoparticle-mediated delivery. Administration may be by any suitable route including, for example, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. A specific example often used for e.g. protein replacement therapy is intravenous infusion. The frequency and number of administrations may depend on factors such as the half-life of the nuclease agent or exogenous donor sequence or recombinant expression vector, the condition of the subject, and the route of administration. The pharmaceutical compositions for administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage form (i.e., a single administration dose). The pharmaceutical compositions may be formulated with one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration selected. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

Other such methods include ex vivo methods performed in cells from subjects suffering from or susceptible to developing chronic liver disease and/or cardiovascular disease. Cells with targeted genetic modifications can then be transplanted back into the subject.

In some embodiments, the INHBE inhibitor comprises a small molecule. In some embodiments, the INHBE inhibitor is any inhibitory nucleic acid molecule described herein. In some embodiments, the INHBE inhibitor comprises an antibody.

In some embodiments, the method of treatment further comprises detecting the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predictive loss of function polypeptide, or the presence of a corresponding INHBE polypeptide, or quantification of an INHBE polypeptide or nucleic acid (such as RNA) in a biological sample from the subject. As used throughout this disclosure, an "INHBE variant nucleic acid molecule" is any INHBE nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, or a cDNA molecule) that encodes an INHBE polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.

The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits a metabolic disorder, wherein the subject has the metabolic disorder. In some embodiments, the method comprises determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide by: a biological sample from the subject is obtained or has been obtained, and a genotyping assay is or has been performed on the biological sample to determine whether the subject has a genotype comprising an INHBE variant nucleic acid molecule. When the subject is an INHBE reference, the therapeutic agent that treats or inhibits the metabolic disorder is administered or continues to be administered to the subject in standard dosage amounts, and the INHBE inhibitor is administered to the subject. When the subject is heterozygous for the INHBE variant nucleic acid molecule, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or less than the standard dose amount, and the INHBE inhibitor is administered to the subject. When the subject is homozygous for the INHBE variant nucleic acid molecule, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or less than the standard dose amount. The presence of a genotype with an INHBE variant nucleic acid molecule encoding an INHBE predictive loss of function polypeptide is indicative of a reduced risk of a subject developing a metabolic disorder. In some embodiments, the subject is an INHBE reference. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide.

For subjects that are genotyped or determined to be INHBE-referenced or heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide, such subjects may be treated with an INHBE inhibitor as described herein.

The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits a cardiovascular disease, wherein the subject has the cardiovascular disease. In some embodiments, the method comprises determining whether the subject has an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide by: a biological sample from the subject is obtained or has been obtained, and a genotyping assay is or has been performed on the biological sample to determine whether the subject has a genotype comprising an INHBE variant nucleic acid molecule. When the subject is an INHBE reference, the therapeutic agent that treats or inhibits the cardiovascular disease is administered or continues to be administered to the subject in standard dosage amounts, and the INHBE inhibitor is administered to the subject. When the subject is heterozygous for the INHBE variant nucleic acid molecule, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits cardiovascular disease in an amount equal to or less than the standard dose amount, and the INHBE inhibitor is administered to the subject. When the subject is homozygous for the INHBE variant nucleic acid molecule, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits the cardiovascular disease in an amount equal to or less than the standard dose amount. The presence of a genotype with an INHBE variant nucleic acid molecule encoding an INHBE predictive loss of function polypeptide is indicative of a reduced risk of a subject developing cardiovascular disease. In some embodiments, the subject is an INHBE reference. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide.

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

In some embodiments, when the subject is an INHBE reference, the therapeutic agent that treats or inhibits the metabolic disorder is also administered to the subject in a standard dose amount. In some embodiments, when the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predictive loss-of-function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits a metabolic disorder in a dosage amount equal to or less than the standard dosage amount.

In some embodiments, when the subject is an INHBE reference, the therapeutic agent that treats or inhibits the cardiovascular disease is also administered to the subject in a standard dose amount. In some embodiments, when the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predictive loss-of-function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits cardiovascular disease in a dose amount equal to or less than the standard dose amount.

In some embodiments, the method of treatment further comprises detecting the presence or absence of an INHBE predictive loss of function polypeptide in a biological sample from the subject. In some embodiments, when the subject does not have an INHBE predicted loss of function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits a metabolic disorder in a standard dose amount. In some embodiments, when the subject has an INHBE-predicted loss of function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits a metabolic disorder in a dosage amount equal to or less than the standard dosage amount.

In some embodiments, the method of treatment further comprises detecting the presence or absence of an INHBE predictive loss of function polypeptide in a biological sample from the subject. In some embodiments, when the subject does not have an INHBE predicted loss of function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits a cardiovascular disease in a standard dose amount. In some embodiments, when the subject has an INHBE-predictive loss of function polypeptide, the subject is also administered a therapeutic agent that treats or inhibits a cardiovascular disease in a dosage amount that is equal to or less than the standard dosage amount.

The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits a metabolic disorder, wherein the subject has the metabolic disorder. In some embodiments, the method comprises determining whether the subject has an INHBE-predicted loss-of-function polypeptide by: a biological sample from the subject is obtained or has been obtained, and an assay is performed or has been performed on the biological sample to determine whether the subject has an INHBE predicted loss of function polypeptide. When the subject does not have an INHBE-predictive loss-of-function polypeptide, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits the metabolic disorder in a standard dose amount, and an INHBE inhibitor is administered to the subject. When the subject has an INHBE-predictive loss-of-function polypeptide, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or less than the standard dose amount, and an INHBE inhibitor is administered to the subject. The presence of an INHBE-predicted loss of function polypeptide is indicative of a reduced risk of the subject developing a metabolic disorder. In some embodiments, the subject has an INHBE predicted loss of function polypeptide. In some embodiments, the subject does not have an INHBE predicted loss of function polypeptide.

The present disclosure also provides methods of treating a subject with a therapeutic agent that treats or inhibits a cardiovascular disease, wherein the subject has the cardiovascular disease. In some embodiments, the method comprises determining whether the subject has an INHBE-predicted loss-of-function polypeptide by: a biological sample from the subject is obtained or has been obtained, and an assay is performed or has been performed on the biological sample to determine whether the subject has an INHBE predicted loss of function polypeptide. When the subject does not have an INHBE-predictive loss-of-function polypeptide, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits the cardiovascular disease in a standard dose amount, and an INHBE inhibitor is administered to the subject. When the subject has an INHBE-predictive loss-of-function polypeptide, the subject is administered or continues to be administered a therapeutic agent that treats or inhibits cardiovascular disease in an amount equal to or less than the standard dose amount, and an INHBE inhibitor is administered to the subject. The presence of an INHBE-predicted loss of function polypeptide is indicative of a reduced risk of a subject developing a cardiovascular disease. In some embodiments, the subject has an INHBE predicted loss of function polypeptide. In some embodiments, the subject does not have an INHBE predicted loss of function polypeptide.

Detecting the presence or absence of an INHBE-predicted loss-of-function polypeptide in a biological sample from a subject and/or determining whether a subject has an INHBE-predicted loss-of-function polypeptide may be performed by any of the methods described herein. In some embodiments, these methods may be performed in vitro. In some embodiments, these methods may be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the polypeptide may be present within a cell or blood sample obtained from the subject, or may be extrapolated from other information about the subject that has previously been generated from the collection of a cell or blood sample from the subject or a biological relative of the subject. In any of these embodiments, determining by quantifying the amount of the INHBE polypeptide to determine loss of function due to an effective deficiency or reduction in the amount of the INHBE polypeptide may be included. In any of these embodiments, detection, sequencing and/or quantification of INHBE DNA and RNA can be used as a method for determining loss of INHBE function or complete lack of INHBE.

Examples of therapeutic agents that treat or inhibit type 2 diabetes include, but are not limited to: metformin, insulin, sulfonylureas such as glibenclamide, glipizide and glimepiride, meglitinides such as repaglinide and nateglinide, thiazolidinediones such as rosiglitazone and pioglitazone, DPP-4 inhibitors such as sitagliptin, saxagliptin and linagliptin, GLP-1 receptor agonists such as exenatide, linagliptin and cable Ma Lutai, and SGLT2 inhibitors such as canaglizin and dapagliflozin. In some embodiments, the therapeutic agent is metformin, insulin, glibenclamide, glipizide, glimepiride, repaglinide, nateglinide, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, so Ma Lutai, canagliflozin, dapagliflozin, or enggliflozin. In some embodiments, the therapeutic agent is metformin. In some embodiments, the therapeutic agent is insulin. In some embodiments, the therapeutic agent is glibenclamide. 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 cord Ma Lutai. In some embodiments, the therapeutic agent is canagliflozin. In some embodiments, the therapeutic agent is dapagliflozin. In some embodiments, the therapeutic agent is enggliflozin.

Examples of therapeutic agents that treat or inhibit obesity include, but are not limited to: orlistat (orlistat), phentermine, topiramate, bupropion (bupropion), naltrexone (naltrexone) and liraglutide (liraglutide). In some embodiments, the therapeutic agent is orlistat. In some embodiments, the therapeutic agent is phentermine. In some embodiments, the therapeutic agent is topiramate. In some embodiments, the therapeutic agent is bupropion. In some embodiments, the therapeutic agent is naltrexone. In some embodiments, the therapeutic agent is liraglutide.

Examples of therapeutic agents that treat or inhibit elevated triglycerides include, but are not limited to: statins such as rosuvastatin (rosuvastatin), simvastatin (simvastatin) and atorvastatin (atorvastatin), fibrates such as fenofibrate (fenofibrate), gemfibrozil and fenofibrate acid (fenofibric acid), nicotinic acids such as nicotinic acid, and fatty acids such as omega-3 fatty acids. In some embodiments, the therapeutic agent is a statin.

Examples of therapeutic agents that treat or inhibit lipodystrophy include, but are not limited to:(temorelin (tesamorelin)),)>(II) Metformin),%>(Poly-L-lactic acid),(calcium hydroxyapatite), polymethyl methacrylate (e.g. PMMA), -poly (methyl methacrylate) (PMMA)>(bovine collagen),>(human collagen), silicone, glitazone (glitazone) and hyaluronic acid. In some embodiments, therapeutic agents that treat or inhibit lipodystrophy include, but are not limited to: temorelin, metformin, poly-l-lactic acid, calcium hydroxyapatite, polymethyl methacrylate, bovine collagen, human collagen, silicone and hyaluronic acid.

Examples of therapeutic agents that treat or inhibit liver inflammation include, but are not limited to, hepatitis therapeutic agents and hepatitis vaccines.

Examples of therapeutic agents or procedures for treating or inhibiting fatty liver disease include, but are not limited to, bariatric surgery and/or dietary intervention.

Examples of therapeutic agents that treat or inhibit hypercholesterolemia include, but are not limited to: the statin(s) (e.g.,(atorvastatin) and->(fluvastatin), lovastatin (lovastatin), and +.>(pitavastatin) and ++>(pravastatin) and->(rosuvastatin calcium) and +.>(simvastatin); bile acid sequestrants (e.g.)>(cholestyramine) >(colesevelam) and +.>(colestipol); PCSK9 inhibitors (e.g.)>(alikumab) and +.>(evokumab (evolocumab)); nicotinic acid (e.g., nissan and nisac); fibrates (e.g., fenofibrate and +.>(gemfibrozil)); ATP Citrate Lyase (ACL) inhibitors (e.g.,(Bei Peiduo (bempedoic))). In some embodiments, therapeutic agents that treat or inhibit hypercholesterolemia include, but are not limited to: statins (e.g., atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, and simvastatin); bile acid sequestrants (e.g., cholestyramine, colesevelam and colestipol); the PCSK9 inhibitor (e.g.,aliskirab and ibritumomab; nicotinic acid (e.g., nisetum and nisetum); fibrates (e.g., fenofibrate and gemfibrozil); and ACL inhibitors (e.g., bei Peiduo). In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is aliskiren or avokuzumab. In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is aliskiren. In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is avokuzumab.

Examples of therapeutic agents that treat or inhibit the elevation of liver enzymes (such as, for example, ALT and/or AST) include, but are not limited to, coffee, folic acid, potassium, vitamin B6, statin, and fiber, or any combination thereof.

Examples of therapeutic agents that treat or inhibit NASH include, but are not limited to(obeticholic acid), pioglitazone or other glitazones, span Long Se (Selonsertib), elabuno (elafimbranor), cinmivir (Cenicriviroc), gr_md_02, mgl_3196, imm124E, eicosanoyl-amino-cholanic acid (amamchol) ^TM ) GS0976, enlicasan (Emricasan), wo Liba Tex (Volixibat), NGM282, GS9674, trapezium (Tropifanor), MN_001, LMB763, BI_1467335, MSDC_0602, PF_05221304, DF102, sha Luoge column bundle (Saroglitazar), BMS986036, rankine (Lanibrchor), soxhaust Ma Lutai, nitazoxanide (Nitazoxanide), GRI_0621, EYP001, VK2809, nalmefene (Nalmefene), LIK066, MT_3995, elofexibat (Elobixibat), namodenoson), fu Lei Lushan anti (Foralumab), SAR425899, sotagirizin, soglison EDP_305, exobute (Isosabate), jicabin (Gemcabene), TERN_101, KBP_042, PF_06865571, DUR928, PF_06835919, NGM313, BMS_986171, na Ma Xizuo mab (Namacizumab), CER_209, ND_L02_s0201, RTU_1096, DRX_065, IONIS_DGAT2Rx, INT_767, NC_001, seladepar, PXL770, TE RN_201, NV556, AZD2693, SP_1373, VK0214, hepastein (Hepast em), TGFTX4, RL1127, GKT_137831, RYI _018, CB4209-CB4211 and JH_09 20。

In some embodiments, the therapeutic agent for treating a metabolic disorder is a melanocortin 4 receptor (MC 4R) agonist. In some embodiments, the MC4R agonist comprises a protein, peptide, nucleic acid molecule, or small molecule. In some embodiments, the protein is a peptide analog of MC 4R. In some embodiments, the peptide is semelandide (setmelanotide). In some embodiments, the therapeutic agent that treats or inhibits type 2 diabetes and/or reduces BMI is a combination of semaphorin and one or more of the following: sibutramine (sibutramine), orlistat, phentermine, lorcaserin (lorcaserin), naltrexone, liraglutide, diethylpropion (diethyl propion), bupropion, metformin, pramlintide (pramlintide), topiramate and zonisamide (zonisamide). In some embodiments, the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp. In some embodiments, the small molecule is 1,2,3r, 4-tetrahydroisoquinoline-3-carboxylic acid. In some embodiments, the MC4R agonist is ALB-127158 (a).

Examples of therapeutic agents that treat or inhibit cardiomyopathy include, but are not limited to: 1) Antihypertensives such as ACE inhibitors, angiotensin II receptor blockers, beta blockers and calcium channel blockers; 2) Heart rate slowing agents such as beta blockers, calcium channel blockers, and digoxin; 3) Agents that maintain the heart beating at normal rhythms, such as antiarrhythmic agents; 4) Electrolyte balancing agents such as aldosterone blockers; 5) Agents that remove excess fluid and sodium from the body, such as diuretics; 6) Agents that prevent clot formation, such as anticoagulants or blood diluents; and 7) agents that reduce inflammation, such as corticosteroids.

Examples of therapeutic agents that treat or inhibit heart failure include, but are not limited to: ACE inhibitors, angiotensin 2 receptor blockers, beta blockers, mineralocorticoid receptor antagonists, diuretics, ivabradine (ivabradine), sarcandesartan (sacubitril valsartan), nitrate-containing hydralazine, and digoxin.

Examples of therapeutic agents that treat or inhibit hypertension include, but are not limited to: diuretics such as chlorthalidone (chlorthalidone), chlorthiazine, hydrochlorothiazide, indapamide (indapamide) and metolazone (metazone); beta blockers (such as acebutolol, atenolol, betaxolol, bisoprolol fumarate (bisoprolol fumar ate), carteolol hydrochloride (carteolol hydrochloride), metoprolol tartrate (metop rolol tartrate), metoprolol succinate, nadolol (nadolol), etc.; ACE inhibitors such as benazepril (benazepril hydrochloride) hydrochloride, captopril (captopril), enalapril maleate (enalapril maleate), fosinopril sodium (fosinopril sodium), lisinopril (lisinopril), moexipril (moexipril), perindopril (perindopril), quinapril (quinapril hydrochloride) hydrochloride, ramipril (ramipril), and trandolapril (trandolapril); angiotensin II receptor blockers such as candesartan (candesartan), eprosartan mesylate (eprosartan mesylate), irbesartan, losartan potassium (Losartan potassium), telmisartan (telmesartan) and valsartan (valsartan); calcium channel blockers such as amlodipine besylate, benpridil (bepridil), diltiazem hydrochloride (diltiazem hydrochloride), felodipine (felodipine), veradipine (isradipine), nicardipine (nicaripine), nifedipine (nifedipine), nisoldipine (nisoldine), and verapamil hydrochloride (verapamil hydrochloride); alpha blockers such as doxazosin mesylate (doxazosin mesylate), prazosin hydrochloride (prazosin hydrochloride) and terazosin hydrochloride (terazosin hydrochloride), alpha-2 receptor agonists such as methyldopa, combined alpha and beta blockers such as carvedilol and labetalol hydrochloride, central agonists such as alpha methyldopa, clonidine hydrochloride, guanabene acetate and guanfacine hydrochloride, peripheral adrenergic inhibitors such as guanabene (guazadril), guanethidine monosulfate and reserpine (reserpine), and vasodilators such as hydrazobenzodiol hydrochloride and minoxidil.

In some embodiments, the dose of the therapeutic agent to treat or inhibit the metabolic disorder and/or cardiovascular disease may be reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% (i.e., less than the standard dose amount) in a subject heterozygous for the INHBE predictive loss of function variant as compared to the subject (which can receive the standard dose amount) to which the INHBE is referenced. In some embodiments, the dosage of the therapeutic agent to treat or inhibit a metabolic disorder and/or cardiovascular disease may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. In addition, subjects who are heterozygous for the INHBE predictive loss of function variant may be administered less frequently than subjects who are referenced to INHBE.

In some embodiments, the dose of therapeutic agent for treating a metabolic disorder and/or cardiovascular disease may be reduced by about 10%, about 20%, about 30%, about 40%, about 50% in a subject homozygous for a predicted loss of function variant INHBE nucleic acid molecule as compared to a subject heterozygous for the predicted loss of function variant INHBE nucleic acid molecule. In some embodiments, the dosage of the therapeutic agent to treat or inhibit a metabolic disorder and/or cardiovascular disease may be reduced by about 10%, about 20%, about 30%, about 40%, or about 50%. Furthermore, the dose of therapeutic agent to treat or inhibit a metabolic disorder and/or cardiovascular disease may be administered less frequently in subjects homozygous for the predicted loss of function variant INHBE nucleic acid molecule than subjects heterozygous for the predicted loss of function variant INHBE nucleic acid molecule.

Administration of therapeutic agents and/or INHBE inhibitors to treat or inhibit metabolic disorders and/or cardiovascular diseases may be repeated, for example, after one, two, three, five, one, two, three, one, five, six, seven, eight, two or three months. Repeated administration may be the same dose or different doses. The administration may be repeated one, two, three, four, five, six, seven, eight, nine, ten or more times. For example, according to certain dosage regimens, a subject may receive treatment for an extended period of time, such as, for example, 6 months, 1 year, or more.

Administration of the therapeutic agent and/or INHBE inhibitor to treat or inhibit a metabolic disorder and/or cardiovascular disease may be by any suitable route including, but not limited to, parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. The pharmaceutical compositions for administration are desirably sterile and substantially isotonic and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage form (i.e., a single administration dose). The pharmaceutical compositions may be formulated with one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration selected. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient or adjuvant is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof.

As used herein, the terms "treatment", "treatment" 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 comprises one or more of the following: upon administration of the agent or a composition comprising the agent, the metabolic disorder and/or cardiovascular disease is reduced/reduced, the severity of the metabolic disorder and/or cardiovascular disease is reduced/reduced (such as, for example, reducing or inhibiting the development of the metabolic disorder and/or cardiovascular disease), the symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects is reduced/reduced, the symptoms and metabolic disorder-related effects and/or the onset of the cardiovascular disease-related effects is delayed, the severity of the symptoms of the metabolic disorder-related effects and/or cardiovascular disease-related effects is reduced, the number of symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects is reduced, the latency of symptoms and metabolic disorder-related effects and/or cardiovascular disease-related effects is reduced, the secondary symptoms are reduced, the secondary infections are reduced, the recurrence of metabolic disorder and/or cardiovascular disease, the number or frequency of recurrence of the episodes is reduced, the latency between symptomatic episodes is increased, the time to sustained progression is accelerated, or the efficacy of the replacement therapy is increased or the resistance to the replacement therapy is reduced, and/or the survival time of the host animal is affected. The prophylactic effect can include a complete or partial avoidance/inhibition or delay of progression/progression of the metabolic disorder and/or cardiovascular disease (such as, for example, a complete or partial avoidance/inhibition or delay) following administration of the treatment regimen, as well as an increase in survival time of the affected host animal. Treatment of a metabolic disorder encompasses treatment of a subject, delay in onset or evolution or exacerbation or worsening of symptoms or signs of a metabolic disorder and/or cardiovascular disease, and/or prevention and/or reduction of the severity of a metabolic disorder and/or cardiovascular disease, of any form of metabolic disorder and/or cardiovascular disease that has been diagnosed as being in any clinical stage or manifestation.

The present disclosure also provides methods of identifying a subject at increased risk of developing a metabolic disorder. In some embodiments, the methods comprise determining or having determined the presence or absence of an INHBE variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and/or a cDNA molecule) encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from a subject. When the subject lacks an INHBE variant nucleic acid molecule encoding an INHBE predictive loss of function polypeptide (i.e., the subject is genotyped as an INHBE reference), then the subject is at increased risk of developing a metabolic disorder. When the subject has an INHBE variant nucleic acid molecule that encodes an INHBE predictive loss-of-function polypeptide (i.e., the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule that encodes an INHBE predictive loss-of-function polypeptide), then the subject's risk of developing a metabolic disorder is reduced. In some embodiments, liver expression quantitative trait loci (eQTL) may be analyzed.

The present disclosure also provides methods of identifying a subject at increased risk of developing a cardiovascular disease. In some embodiments, the methods comprise determining or having determined the presence or absence of an INHBE variant nucleic acid molecule (such as a genomic nucleic acid molecule, an mRNA molecule, and/or a cDNA molecule) encoding an INHBE predicted loss-of-function polypeptide in a biological sample obtained from a subject. When the subject lacks an INHBE variant nucleic acid molecule encoding an INHBE predictive loss of function polypeptide (i.e., the subject is genotyped as an INHBE reference), then the subject is at increased risk of developing a cardiovascular disease. When the subject has an INHBE variant nucleic acid molecule that encodes an INHBE predictive loss-of-function polypeptide (i.e., the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule that encodes an INHBE predictive loss-of-function polypeptide), then the subject's risk of developing a cardiovascular disease is reduced. In some embodiments, liver expression quantitative trait loci (eQTL) may be analyzed.

A single copy of an INHBE variant nucleic acid molecule that encodes an INHBE predictive loss-of-function polypeptide is more capable of protecting a subject from developing a metabolic disorder and/or cardiovascular disease than a copy of an INHBE variant nucleic acid molecule that does not encode an INHBE predictive loss-of-function polypeptide. Without wishing to be bound by any particular theory or mechanism of action, it is believed that a single copy of the INHBE variant nucleic acid molecule (i.e., heterozygous for the INHBE variant nucleic acid molecule) protects the subject from developing a metabolic disorder and/or cardiovascular disease, and it is also believed that having two copies of the INHBE variant nucleic acid molecule (i.e., homozygous for the INHBE variant nucleic acid molecule) may be more likely to protect the subject from developing a metabolic disorder and/or cardiovascular disease relative to a subject having a single copy. Thus, in some embodiments, a single copy of an INHBE variant nucleic acid molecule may not be fully protective, but may partially or incompletely protect a subject from developing a metabolic disorder and/or cardiovascular disease. While not wishing to be bound by any particular theory, there may be additional factors or molecules involved in the development of metabolic disorders and/or cardiovascular disease that are still present in subjects with a single copy of the INHBE variant nucleic acid molecule, thus resulting in less than complete protection from developing metabolic disorders and/or cardiovascular disease.

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

In some embodiments, when the subject is identified as having an increased risk of developing a metabolic disorder, the subject is further treated with a therapeutic agent and/or INHBE inhibitor as described herein that treats or inhibits the metabolic disorder. For example, when the subject is an INHBE reference, and thus the risk of developing a metabolic disorder is increased, an INHBE inhibitor is administered to the subject. In some embodiments, a therapeutic agent that treats or inhibits a metabolic disorder is also administered to such a subject. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predictive loss-of-function polypeptide, the subject is administered a therapeutic agent that treats or inhibits a metabolic disorder at a dose amount equal to or less than the standard dose amount, and an INHBE inhibitor is also administered. In some embodiments, a therapeutic agent that treats or inhibits a metabolic disorder is also administered to such a subject. In some embodiments, when the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss of function polypeptide, the subject is administered a therapeutic agent that treats or inhibits a metabolic disorder in a dosage amount equal to or less than the standard dosage amount. In some embodiments, the subject is an INHBE reference. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide. In some embodiments, the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide.

In some embodiments, when the subject is identified as having an increased risk of developing a cardiovascular disease, the subject is further treated with a therapeutic agent and/or INHBE inhibitor as described herein that treats or inhibits the cardiovascular disease. For example, when the subject is an INHBE reference, and thus the risk of developing cardiovascular disease is increased, an INHBE inhibitor is administered to the subject. In some embodiments, a therapeutic agent that treats or inhibits a cardiovascular disease is also administered to such a subject. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predictive loss-of-function polypeptide, the subject is administered a therapeutic agent that treats or inhibits cardiovascular disease in a dosage amount equal to or less than the standard dosage amount, and an INHBE inhibitor is also administered. In some embodiments, a therapeutic agent that treats or inhibits a cardiovascular disease is also administered to such a subject. In some embodiments, when the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss of function polypeptide, the subject is administered a therapeutic agent that treats or inhibits cardiovascular disease in a dosage amount equal to or less than the standard dosage amount. In some embodiments, the subject is an INHBE reference. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide. In some embodiments, the subject is homozygous for an INHBE variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide.

In some embodiments, any of the methods described herein may further comprise determining that the subject has an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide and/or a genetic burden of an INHBE-predicted loss-of-function variant polypeptide associated with reduced risk of developing a metabolic disorder and/or cardiovascular disease. The genetic burden is the collection (aggregation) of all variants in the INHBE gene, which can be performed in association analysis with metabolic disorders and/or cardiovascular diseases. In some embodiments, the subject is homozygous for one or more INHBE variant nucleic acid molecules encoding an INHBE predictive loss of function polypeptide associated with reduced risk of developing a metabolic disorder and/or cardiovascular disease. In some embodiments, the subject is heterozygous for one or more INHBE variant nucleic acid molecules encoding an INHBE-predictive loss-of-function polypeptide associated with reduced risk of developing a metabolic disorder and/or cardiovascular disease. The results of the association analysis indicate that the INHBE variant nucleic acid molecules encoding INHBE predictive loss of function polypeptides are associated with reduced risk of developing metabolic disorders and/or cardiovascular disease. When the subject has a lower genetic burden, the subject is at a higher risk of developing a metabolic disorder and/or cardiovascular disease and the subject is administered or continues to be administered a therapeutic agent that treats, prevents, or inhibits the metabolic disorder and/or cardiovascular disease, and/or an INHBE inhibitor, in standard dosage amounts. When the subject has a greater genetic burden, the subject is at a lower risk of developing a metabolic disorder and/or cardiovascular disease and the subject is administered or continues to be administered a therapeutic agent that treats, prevents, or inhibits the metabolic disorder and/or cardiovascular disease in an amount equal to or less than the standard dose amount. The greater the genetic burden, the lower the risk of developing metabolic disorders and/or cardiovascular disease.

In some embodiments, the genetic burden of a subject with any one or more INHBE variant nucleic acid molecules encoding an INHBE predicted loss-of-function polypeptide represents a weighted sum of a plurality of any INHBE variant nucleic acid molecules encoding INHBE predicted loss-of-function polypeptides. In some embodiments, a genetic burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about or more than 1,000,000 genetic variants present in or around (up to 10 Mb) the INHBE gene, wherein the genetic burden is the number of alleles multiplied by an estimate (e.g., weighted burden score) of each allele's association with a metabolic disorder or related outcome. This may include any genetic variant of a neighboring INHBE gene (up to 10Mb around the gene) that exhibits a non-zero association with a metabolic disorder-related trait and/or a cardiovascular disease-related trait in a genetic association analysis, regardless of its genome annotation. In some embodiments, when the subject has a genetic burden above a desired threshold score, the subject's risk of developing a metabolic disorder and/or cardiovascular disease is reduced. In some embodiments, the subject's risk of developing a metabolic disorder and/or cardiovascular disease is increased when the subject has a genetic burden below a desired threshold score.

In some embodiments, the genetic burden can be divided into five-digits, e.g., a highest five-digit, a middle five-digit, and a lowest five-digit, wherein the highest five-digit of the genetic burden corresponds to the lowest risk group and the lowest five-digit of the genetic burden corresponds to the highest risk group. In some embodiments, the subject with a greater genetic burden comprises the highest weighted genetic burden, including but not limited toFrom the top 10%, top 20%, top 30%, top 40% or top 50% of the subject population. In some embodiments, the genetic variants include genetic variants associated with a metabolic disorder and/or cardiovascular disease in the first 10%, first 20%, first 30%, first 40%, or first 50% of the associated P-value range. In some embodiments, each of the identified genetic variants includes a p-value associated with a metabolic disorder and/or cardiovascular disease of no more than about 10 ^-2 About 10 ^-3 About 10 ^-4 About 10 ^-5 About 10 ^-6 About 10 ^-7 About 10 ^-8 About 10 ^-9 About 10 ^-10 About 10 ^-11 About 10 ^-12 About 10 ^-13 About 10 ^-14 Or about 10 ^-15 Is a genetic variant of (a). In some embodiments, the identified genetic variants include those having a p-value less than 5 x 10 associated with a metabolic disorder and/or cardiovascular disease ^-8 Is a genetic variant of (a). In some embodiments, the identified genetic variants comprise an Odds Ratio (OR) for the first 20% of distribution of about 1.5 OR greater, about 1.75 OR greater, about 2.0 OR greater, OR about 2.25 OR greater, relative to the association of the remaining subjects in the reference population with the metabolic disorder and/OR cardiovascular disease in the high risk subject; or about 1.5 or greater, about 1.75 or greater, about 2.0 or greater, about 2.25 or greater, about 2.5 or greater, or about 2.75 or greater. In some embodiments, the Odds Ratio (OR) may be in the range of 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 greater than 7.0. In some embodiments, the high risk subject comprises a subject having a genetic burden at the lowest ten, five, or three digits in the reference population. The threshold value of the genetic burden is determined based on the nature of the intended actual application and the risk differences that will be considered significant to the actual application.

In some embodiments, when the subject is identified as having an increased risk of developing a metabolic disorder, the subject is further administered a therapeutic agent and/or an INHBE inhibitor as described herein to treat, prevent, or inhibit the metabolic disorder. For example, when the subject is an INHBE reference, and thus the risk of developing a metabolic disorder is increased, an INHBE inhibitor is administered to the subject. In some embodiments, therapeutic agents that treat, prevent, or inhibit a metabolic disorder are also administered to such subjects. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predictive loss-of-function polypeptide, the subject is administered a therapeutic agent that treats, prevents, or inhibits a metabolic disorder at a dose amount equal to or less than the standard dose amount, and an INHBE inhibitor is also administered. In some embodiments, the subject is an INHBE reference. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide. In addition, when the subject has a lower genetic burden of the INHBE variant nucleic acid molecule encoding an INHBE predictive function loss polypeptide and thus an increased risk of developing a metabolic disorder, a therapeutic agent that treats, prevents or inhibits the metabolic disorder is administered to the subject. In some embodiments, when the subject has a lower genetic burden of an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats, prevents, or inhibits a metabolic disorder in a dose amount that is equal to or greater than the standard dose amount administered to a subject having a greater genetic burden of an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide.

In some embodiments, when the subject is identified as having an increased risk of developing a cardiovascular disease, the subject is further administered a therapeutic agent and/or INHBE inhibitor as described herein to treat, prevent, or inhibit the cardiovascular disease. For example, when the subject is an INHBE reference, and thus the risk of developing cardiovascular disease is increased, an INHBE inhibitor is administered to the subject. In some embodiments, therapeutic agents that treat, prevent, or inhibit cardiovascular disease are also administered to such subjects. In some embodiments, when the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE predictive loss-of-function polypeptide, the subject is administered a therapeutic agent that treats, prevents, or inhibits cardiovascular disease in a dosage amount equal to or less than the standard dosage amount, and an INHBE inhibitor is also administered. In some embodiments, the subject is an INHBE reference. In some embodiments, the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide. In addition, when the subject has a lower genetic burden of the INHBE variant nucleic acid molecule encoding an INHBE predictive function loss polypeptide and thus an increased risk of developing cardiovascular disease, a therapeutic agent that treats, prevents or inhibits cardiovascular disease is administered to the subject. In some embodiments, when the subject has a lower genetic burden of an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide, the subject is administered a therapeutic agent that treats, prevents, or inhibits a cardiovascular disease in a dose amount that is equal to or higher than the standard dose amount administered to a subject having a greater genetic burden of an INHBE variant nucleic acid molecule encoding an INHBE-predicted loss-of-function polypeptide.

The present disclosure also provides methods of diagnosing a metabolic disorder in a subject. The method comprises determining or has determined whether a subject has any one or more of the INHBE variant nucleic acid molecules described herein or a polypeptide produced therefrom. When the subject is an INHBE reference and has one or more symptoms of a metabolic disorder, the subject is diagnosed as having a metabolic disorder. In some embodiments, the subject is homozygous for the reference INHBE nucleic acid molecule. In some embodiments, the subject is homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predictive loss-of-function INHBE polypeptide. In some embodiments, when the subject is identified as having a metabolic disorder (such as having one or more symptoms of the metabolic disorder and being homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predictive loss-of-function INHBE polypeptide), the subject is further treated with a therapeutic agent that treats or inhibits the metabolic disorder (such as any of those described herein).

The present disclosure also provides methods of diagnosing cardiovascular disease in a subject. The method comprises determining or has determined whether a subject has any one or more of the INHBE variant nucleic acid molecules described herein or a polypeptide produced therefrom. When the subject is an INHBE reference and has one or more symptoms of a cardiovascular disease, the subject is diagnosed as having a cardiovascular disease. In some embodiments, the subject is homozygous for the reference INHBE nucleic acid molecule. In some embodiments, the subject is homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predictive loss-of-function INHBE polypeptide. In some embodiments, when the subject is identified as having a cardiovascular disease (such as having one or more symptoms of a cardiovascular disease and being homozygous or heterozygous for an INHBE variant nucleic acid molecule encoding a predictive loss of function INHBE polypeptide), the subject is further treated with a therapeutic agent that treats or inhibits the cardiovascular disease (such as any of those described herein).

The present disclosure also provides a method of identifying a subject at increased risk of developing a metabolic disorder, wherein the method comprises determining or has determined the presence or absence of an INHBE predictive loss of function polypeptide in a biological sample obtained from the subject. In some embodiments, the method is a blood-based quantitative assay, such as a somalogic assay for quantifying inhibin E.

The present disclosure also provides a method of identifying a subject at increased risk of developing a cardiovascular disease, wherein the method comprises determining or has determined the presence or absence of an INHBE predictive loss of function polypeptide in a biological sample obtained from the subject. In some embodiments, the method is a blood-based quantitative assay, such as a somalogic assay for quantifying inhibin E.

The presence of an INHBE polypeptide in a suitable fluid sample, such as blood, plasma and/or serum, can be determined by detecting an INHBE polypeptide using a variety of methods for measuring INHBE or INHBE activity. For example, an INHBE polypeptide may be detected by an immunoassay using antibodies specific for INHBE. The antibody is capable of selectively binding INHBE polypeptides and/or CEA. The antibodies can be used, for example, in western blots of one-or two-dimensional gels, high throughput methods such as enzyme-linked immunosorbent assays, and/or dot blot (antibody sandwich) assays of total cellular proteins or partially purified proteins. In some embodiments, the concentration of INHBE in a suitable fluid is measured by an enzyme-linked immunosorbent assay (ELISA). In one example of this assay, serum samples are diluted 400-fold and plated onto plates to which INHBE polypeptide antibodies (primary antibodies) from an animal source are attached. If sufficient INHBE is present in the serum, INHBE can bind to these INHBE antibodies. The plates were then washed to remove all other components of serum. A specially prepared "secondary antibody", such as from an animal source other than the primary antibody, is then coated onto the plate, one antibody that binds to the primary antibody, and then another wash is performed. This secondary antibody is chemically linked to, for example, an enzyme in advance. Thus, the plate will contain an enzyme proportional to the amount of secondary antibody bound to the plate. The substrate of the enzyme is coated and the catalytic action of the enzyme results in a change in color or fluorescence. Samples that generated stronger signals than known healthy samples were "positive". Those that generated weaker signals than known healthy samples were "negative".

Alternatively, the concentration of the INHBE polypeptide in a suitable fluid may be determined by using a spectroscopic method such as LC-MS/MS mass spectrometer, GCMS mass spectrometer, SDS PAGE method followed by detection of the INHBE polypeptide by densitometry or mass spectrometry or any similar method to quantify the protein. Additional methods of quantifying polypeptide levels include, but are not limited to, HPLC (high performance liquid chromatography), SEC (size exclusion chromatography), modified Lowry assay, spectrophotometry, SEC-MALLS (size exclusion chromatography/multi-angle laser light scattering), and NMR (nuclear magnetic resonance).

Aptamers specific for the INHBE polypeptides may also be used. Suitable aptamers are capable of selectively binding to the INHBE polypeptide for use in measuring blood, plasma or serum concentrations of the INHBE polypeptide, or for use in detecting the presence of a variant INHBE. Recombinant or chemically synthetically produced INHBE polypeptides, as well as fragments or other derivatives or analogs thereof, including fusion proteins, may be used as immunogens to produce aptamers that recognize the INHBE polypeptides. The term "aptamer" refers to a non-naturally occurring oligonucleotide chain or peptide molecule that has a specific effect on a target compound, such as a specific epitope, therapeutic drug marker, or surrogate marker. Specific effects include, but are not limited to, binding to a target compound, catalytically altering a target compound, and/or reacting with a target compound in a manner that modifies/alters the functional activity of the target compound or target compound. By reversing Multiple rounds of in vitro selection or SELEX ^TM Aptamers are engineered (by ligand system evolution through exponential enrichment) to bind various molecular targets, such as small molecules. The production/synthesis method is described, for example, in: ellington et al, nature,1990,346,818-822; and Tuerk et al Science,1990,249,505-510."SELEX ^TM The "method" involves a combination of selected nucleic acid ligands that interact with a particular epitope, e.g., bind to a protein, with a desired effect, and amplify those selected nucleic acids. The optional iterative loop of the selection/amplification step allows for selection of one or a small number of nucleic acids from a pool comprising a very large number of nucleic acids, which interact most strongly with a specific epitope. The cycling of the selection/amplification procedure is continued until the selected goal is achieved. The SELEX process is described in the following U.S. Pat. nos. 5,475,096 and 5,270,163.

The present disclosure also provides methods of identifying subjects having a disease, such as a metabolic disorder, that may respond differently to treatment with INHBE inhibitors or other therapeutic agents that affect fat distribution. In some embodiments, the method comprises determining or has determined the presence or absence of INHBE plofs or pGOF in a biological sample (liver, plasma, serum and/or whole blood) obtained from the subject or which correlates with measurement of INHBE in liver expression or circulation of INHBE or expression in liver. When a subject lacks such an INHBE variant (i.e., the subject is genotyped as an INHBE reference), then the subject is at increased risk of developing a metabolic disorder, and may be amenable to treatment with an INHBE inhibitor or other therapeutic agent that affects fat distribution. When a subject has such an INHBE variant nucleic acid molecule (i.e., the subject is heterozygous for INHBE plofpgof or homozygous for INHBE plofpgof), then the subject's risk of developing a metabolic disorder is reduced.

The present disclosure also provides methods of detecting the presence or absence of an INHBE variant nucleic acid molecule (genomic, mRNA, or cDNA) encoding a predictive loss-of-function INHBE polypeptide in a biological sample from a subject. It will be appreciated that the sequence of genes within a population and the mRNA molecules encoded by such genes may vary due to polymorphisms, such as single nucleotide polymorphisms.

The biological sample may be derived from any cell, tissue or biological fluid from the subject. The sample may comprise any clinically relevant tissue, such as a bone marrow sample, a tumor biopsy, a fine needle aspirate, or a body fluid sample, such as blood, gingival crevicular fluid, plasma, serum, lymph fluid, ascites fluid, cyst fluid, or urine. In some cases, the sample comprises an oral swab. The samples used in the methods disclosed herein will vary based on the assay format, the nature of the detection method, and the tissue, cells, or extract used as the sample. Biological samples may be processed differently depending on the assay employed. For example, when detecting any predicted loss-of-function variant INHBE nucleic acid molecules, a preliminary treatment designed to isolate or enrich a sample for genomic DNA may be employed. A variety of techniques may be used for this purpose. When detecting the level of any predictive loss of function variant INHBE mRNA, different techniques can be used to enrich the biological sample for mRNA. Various methods of detecting the presence or level of mRNA or the presence of a particular variant genomic DNA locus may be used.

In some embodiments, detecting an INHBE variant nucleic acid molecule encoding a predicted loss-of-function INHBE polypeptide in a subject comprises assaying or genotyping a biological sample obtained from the subject to determine INHBE genomic nucleic acid molecules in the biological sample, and/or whether an INHBE mRNA molecule in the biological sample, and/or whether an INHBE cDNA molecule produced from an mRNA molecule in the biological sample comprises one or more variations that result in loss of function (partial or complete) or are predicted to result in loss of function (partial or complete), such as any of the INHBE variant nucleic acid molecules encoding predicted loss-of-function INHBE polypeptides described herein.

In some embodiments, a method of detecting the presence or absence of an INHBE variant nucleic acid molecule (such as, for example, a genomic nucleic acid molecule, an mRNA molecule, and/or a cDNA molecule produced from an mRNA molecule) in a subject comprises assaying a biological sample obtained from the subject. The assay determines whether a nucleic acid molecule in a biological sample comprises a particular nucleotide sequence.

In some embodiments, the biological sample comprises cells or cell lysates. Such methods may also include, for example, obtaining a biological sample from the subject comprising an INHBE genomic nucleic acid molecule or an mRNA molecule, and if mRNA, optionally reverse transcribing the mRNA into cDNA. Such assays may include, for example, determining the identity of these locations of a particular INHBE nucleic acid molecule. In some embodiments, the method is an in vitro method.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an INHBE genomic nucleic acid molecule, an INHBE mRNA molecule, or an INHBE cDNA molecule in the biological sample, wherein the sequenced portion comprises one or more variations that result in loss of function (partial or complete) or are predicted to result in loss of function (partial or complete), such as any of the predicted loss of function variant INHBE nucleic acid molecules described herein.

In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an INHBE genomic nucleic acid molecule in the biological sample, the nucleotide sequence of an INHBE mRNA molecule in the biological sample, or the nucleotide sequence of an INHBE cDNA molecule produced from an INHBE mRNA in the biological sample. In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the INHBE genomic nucleic acid molecule in the biological sample. In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of the INHBE mRNA molecule in the biological sample. In some embodiments, the determining step, detecting step, or genotyping assay comprises sequencing at least a portion of the nucleotide sequence of an INHBE cDNA molecule produced from an INHBE mRNA molecule in the biological sample.

In some embodiments, the determining comprises sequencing the entire nucleic acid molecule. In some embodiments, only INHBE genomic nucleic acid molecules are analyzed. In some embodiments, only INHBE mRNA is analyzed. In some embodiments, only INHBE cDNA obtained from INHBE mRNA is analyzed.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: a) Amplifying at least a portion of a nucleic acid molecule encoding an INHBE polypeptide; b) Labeling the amplified nucleic acid molecules with a detectable label; c) Contacting the labeled nucleic acid molecules with a support comprising a probe that alters the specificity; and d) detecting the detectable label.

In some embodiments, the nucleic acid molecule is an mRNA, and the determining step further comprises reverse transcribing the mRNA into cDNA prior to the amplifying step.

In some embodiments, the determining step, the detecting step, or the genotyping assay comprises: contacting a nucleic acid molecule in a biological sample with a change-specific probe comprising a detectable label, wherein the change-specific probe comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the amplified nucleic acid molecule; and detecting the detectable label. Altering specific polymerase chain reaction techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Because the DNA polymerase will not extend when there is a mismatch with the template, modified specific primers can be used.

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

In some embodiments, the assay comprises contacting the biological sample with a primer or probe, such as a change-specific primer or change-specific probe, that hybridizes specifically under stringent conditions to an INHBE variant nucleic acid molecule (genome, mRNA, or cDNA) but not to a corresponding INHBE reference sequence, and determining whether hybridization has occurred. In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay further comprises reverse transcription of the mRNA into cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

In some embodiments, the methods utilize probes and primers of sufficient nucleotide length to bind to a target nucleotide sequence and specifically detect and/or identify polynucleotides comprising an INHBE variant nucleic acid molecule (genomic, mRNA, or cDNA) encoding a predicted loss-of-function INHBE polypeptide. Hybridization conditions or reaction conditions can be determined by the operator to achieve this result. The nucleotide length may be any length sufficient for the detection method selected, including any of the assays described or exemplified herein. Such probes and primers can specifically hybridize to a target nucleotide sequence under high stringency hybridization conditions. Probes and primers can have complete nucleotide sequence identity to consecutive nucleotides within a target nucleotide sequence, but probes that differ from the target nucleotide sequence and retain the ability to specifically detect and/or identify the target nucleotide sequence can be designed by conventional methods. Probes and primers can have about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% sequence identity or complementarity to the nucleotide sequence of the target nucleic acid molecule.

Illustrative examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, which involve the use of labeled primers or probes (fluorescence in situ hybridization (FISH)) for purified DNA, amplified DNA, and immobilized cell preparations. In some methods, the target nucleic acid molecule can be amplified prior to or concurrent 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).

In hybridization techniques, stringent conditions may be employed such that probes or primers will specifically hybridize to their targets. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target sequence to a degree that is detectably greater than hybridization to other non-target sequences, such as at least 2-fold, at least 3-fold, at least 4-fold or more relative to background, including more than 10-fold greater relative to background. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is at least 2-fold greater than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is at least 3-fold greater than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is at least 4-fold greater than hybridization to other nucleotide sequences. In some embodiments, a polynucleotide primer or probe under stringent conditions will hybridize to its target nucleotide sequence to a degree that is detectably greater than hybridization to other nucleotide sequences by a factor of more than 10 relative to background. Stringent conditions are sequence-dependent and will be different in different circumstances.

Suitable stringency conditions for promoting DNA hybridization, such as 6 Xsodium chloride/sodium citrate (SSC), are known and can be found in Current Protocols in Molecular Biology, john Wiley, by washing 2 XSSC at about 45℃followed by 50 ℃&Sons, n.y. (1989), 6.3.1-6.3.6. In general, stringent conditions for hybridization and detection will be those in which: salt concentration at pH 7.0 to 8.3 is less than about 1.5M Na ⁺ Ions, typically about 0.01 to 1.0M Na ⁺ Ion concentration (or other salt), and temperature is at least about 30 ℃ for short probes (such as, for example, 10 to 50 nucleotides), and at least about 60 ℃ for longer probes (such as, for example, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Optionally, the wash buffer may comprise about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, typically about 4 to about 12 hours. The duration of the washing time will be at least a length of time sufficient to reach equilibrium.

The present disclosure also provides methods of detecting the presence of a human INHBE predicted loss of function polypeptide comprising assaying a sample obtained from a subject to determine whether the INHBE polypeptide in the subject comprises one or more variations that result in the polypeptide having lost function (partial or complete) or predicted lost function (partial or complete).

In some embodiments, the detecting step comprises sequencing at least a portion of the polypeptide. In some embodiments, the detecting step comprises an immunoassay for detecting the presence of a polypeptide.

In some embodiments, when the subject does not have an INHBE predictive function loss polypeptide, then the subject has an increased risk of developing any of a metabolic disorder or type 2 diabetes, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (such as, for example, ALT and/or AST), obesity, hypertension, NASH, and/or elevated triglyceride levels. In some embodiments, when the subject has an INHBE predictive function-depriving polypeptide, then the subject is at reduced risk of developing a metabolic disorder or any of type 2 diabetes, obesity, lipodystrophy, liver inflammation, fatty liver disease, hypercholesterolemia, elevated liver enzymes (such as, for example, ALT and/or AST), elevated hypertension, NASH, and/or elevated triglyceride levels.

In some embodiments, when the subject does not have an INHBE predictive loss of function polypeptide, then the subject is at increased risk of developing any of cardiovascular disease or cardiomyopathy, heart failure, and hypertension. In some embodiments, when the subject has an INHBE-predictive loss of function polypeptide, then the subject is at reduced risk of developing any one of cardiovascular disease or cardiomyopathy, heart failure, and hypertension.

The present disclosure also provides for the use of an isolated nucleic acid molecule that hybridizes to an INHBE variant genomic nucleic acid molecule, an INHBE variant mRNA molecule, and/or an INHBE variant cDNA molecule (such as any of the genomic variant nucleic acid molecules, mRNA variant molecules, and cDNA variant molecules disclosed herein) in any of the methods described herein.

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

In some embodiments, such isolated nucleic acid molecules hybridize under stringent conditions to INHBE variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and/or cDNA molecules). Such nucleic acid molecules may be used as, for example, probes, primers, altered specific probes, or altered specific primers as described or exemplified herein, and include, but are not limited to, primers, probes, antisense RNAs, shrnas, and sirnas, each of which is described in more detail elsewhere herein, and may be used in any of the methods described herein.

In some embodiments, the isolated nucleic acid molecule hybridizes to at least about 15 consecutive nucleotides of a nucleic acid molecule having 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% identity to an INHBE variant genomic nucleic acid molecule, an INHBE variant mRNA molecule, and/or an INHBE variant cDNA molecule. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides or about 15 to about 35 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 100 nucleotides. In some embodiments, the isolated nucleic acid molecule comprises or consists of about 15 to about 35 nucleotides.

In some embodiments, the altering specific probe and altering specific primer comprise DNA. In some embodiments, the altering specific probe and altering specific primer comprise RNA.

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

In some embodiments, the primers, including the altered specific primers, may be used in second generation sequencing or high throughput sequencing. In some cases, primers, including altering specific primers, may be modified. In particular, the primers may comprise various modifications used in different steps such as large-scale parallel signature sequencing (Massive Parallel Signature Sequencing, MPSS), polymerase clone sequencing (Polony sequencing) and 454 pyrosequencing. Modified primers may be used in several steps of the process, including biotinylated primers in the cloning step and fluorescently labeled primers in the bead loading step and detection step. Polymerase clone sequencing is typically performed using a library of double-ended sequencing tags, wherein each DNA template molecule is about 135bp in length. Biotinylated primers were used in the bead loading step and emulsion PCR (emulsion PCR). Fluorescent-labeled degenerate nona-oligonucleotides were used in the detection step. The adaptors may contain 5' -biotin tags for immobilization of the DNA library onto streptavidin coated beads.

The probes and primers described herein can be used to detect nucleotide variations within any of the INHBE variant genomic nucleic acid molecules, INHBE variant mRNA molecules, and/or INHBE variant cDNA molecules disclosed herein. The primers described herein may be used to amplify an INHBE variant genomic nucleic acid molecule, an INHBE variant mRNA molecule or an INHBE variant cDNA molecule or fragment thereof.

In the context of the present disclosure, "specifically hybridizing" means that a probe or primer (such as, for example, a change in a specific probe or a change in a specific primer) does not hybridize to a nucleic acid sequence encoding an INHBE reference genomic nucleic acid molecule, an INHBE reference mRNA molecule, and/or an INHBE reference cDNA molecule.

In some embodiments, the probe (such as, for example, a change-specific probe) comprises a label. In some embodiments, the label is a fluorescent label, a radiolabel, or biotin.

The present disclosure also provides a support comprising any one or more of the attached matrices of the probes disclosed herein. A solid support is a solid matrix or support with which molecules, such as any of the probes disclosed herein, can associate. One form of solid support is an array. Another form of solid support is an array detector. Array detectors are solid supports to which a variety of different probes are coupled in an array, grid, or other organized pattern. One form of solid state matrix is a microtiter dish, such as a standard 96 well type. In some embodiments, porous glass slides may be employed that typically contain an array per well.

The nucleotide sequence of the INHBE reference genomic nucleic acid molecule is shown in SEQ ID NO. 1 (ENST 00000266646.3, which contains chr12:57455307-57458025 in the GRCh38/hg38 human genome assembly).

The nucleotide sequence of the INHBE reference mRNA molecule is shown in SEQ ID NO. 2. The nucleotide sequence of the other INHBE reference mRNA molecule is shown in SEQ ID NO. 3. The nucleotide sequence of the other INHBE reference mRNA molecule is shown in SEQ ID NO. 4.

The nucleotide sequence of the INHBE reference cDNA molecule is shown in SEQ ID NO. 5. The nucleotide sequence of the other INHBE reference cDNA molecule is shown in SEQ ID NO. 6. The nucleotide sequence of the other INHBE reference cDNA molecule is shown in SEQ ID NO. 7.

The amino acid sequence of the INHBE reference polypeptide is shown in SEQ ID NO. 8. With reference to SEQ ID NO. 8, the INHBE reference polypeptide is 350 amino acids in length.

Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules may be from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules may be human or orthologs from another organism (such as a non-human mammal, rodent, mouse, or rat). It will be appreciated that the sequence of genes within a population may vary due to polymorphisms, such as single nucleotide polymorphisms. The examples provided herein are merely exemplary sequences. Other sequences are also possible.

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

The disclosed nucleic acid molecules can include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified base, sugar or phosphate groups, or nucleotides incorporating non-natural moieties in their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, aminated, deaminated, alkylated, benzylated, and fluorophore-labeled nucleotides.

The nucleic acid molecules disclosed herein may also comprise one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides that contain modifications to the base, sugar or phosphate moiety. Modifications to the base moiety include, but are not limited to A, C, G and T/U as well as natural and synthetic modifications of different purine or pyrimidine bases such as, for example, pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I) and 2-aminoadenine-9-yl. Modified bases include, but are not limited to, 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 (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,

8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (such as, for example, 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine,

7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine,

7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

-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 from 1 to about 10. Other modifications at the 2' position include, but are not limited to, C _1-10 Alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH ₃ 、OCN、Cl、Br、CN、CF ₃ 、OCF ₃ 、SOCH ₃ 、SO ₂ CH ₃ 、ONO ₂ 、NO ₂ 、N ₃ 、NH ₂ A heterocycloalkyl group, a heterocycloalkyl aryl group, an aminoalkylamino group, a polyalkylamino group, a substituted silyl group, an RNA cleavage group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, specifically at the 3 'position of the sugar and at the 5' position of the 5 'terminal nucleotide on the 3' terminal nucleotide or in the 2'-5' linked oligonucleotide. Modified sugars may also include those containing modifications at the bridging epoxy, such as CH ₂ And S. Nucleotide sugar analogues may also have sugar mouldsA mimetic, such as a cyclobutyl moiety, replaces pentose.

The present disclosure also provides a therapeutic agent for treating or inhibiting a metabolic disorder, for treating a metabolic disorder in a subject having: an INHBE variant genomic nucleic acid molecule encoding a predictive loss-of-function INHBE polypeptide; an INHBE variant mRNA molecule encoding a predictive loss-of-function INHBE polypeptide; or an INHBE variant cDNA molecule encoding a predictive loss-of-function INHBE polypeptide.

In some embodiments, the metabolic disorder is type 2 diabetes and the therapeutic agent is selected from the group consisting of metformin, insulin, glibenclamide, glipizide, glimepiride, repaglinide, nateglinide, thiazolidinedione, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, so Ma Lutai, canagliflozin, dapagliflozin, and engagliflozin.

In some embodiments, the metabolic disorder is obesity and the therapeutic agent is selected from orlistat, phentermine, topiramate, bupropion, naltrexone, and liraglutide.

In some embodiments, the metabolic disorder is hypertension, and the therapeutic agent is selected from the group consisting of chlorthalidone, chlorthiazide, hydrochlorothiazide, indapamide, mecopril, acebutolol, atenolol, betaxolol, bisoprolol fumarate, carboplatin hydrochloride, metoprolol tartrate, mecalol succinate, nadoprolol hydrochloride, benazepril, captopril maleate, fosinopril sodium, lisinopril, moexipril, perindopril, quinapril hydrochloride, ramipril, trandolapril, candesartan, eprosartan, irbesartan, losartan potassium, telmisartan, valsartan, amlodipine besylate, benazepril, diltiazem hydrochloride, felodipine, veradipine, nifedipine hydrochloride, doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, methyldopa, carvedilol hydrochloride, alpha methylgliclazide, guanamine hydrochloride, guanadine hydrochloride, guanadine, guanamine hydrochloride, and guanamine hydrochloride.

In some embodiments, the metabolic disorder is elevated triglycerides and the therapeutic agent is selected from rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil, fenofibrate, niacin, and omega-3 fatty acids.

In some embodiments, the metabolic disorder is lipodystrophy and the therapeutic agent is selected from the group consisting of(temorelin), ->(metformin),>(Poly-L-lactic acid), - (Y) and (B) in the formula>(calcium hydroxyapatite), polymethyl methacrylate (e.g. PMMA), -poly (methyl methacrylate) (PMMA)>(bovine collagen),(human collagen), silicone and hyaluronic acid. In some embodiments, therapeutic agents that treat or inhibit lipodystrophy include, but are not limited to: temorelin, metformin, poly-l-lactic acid, calcium hydroxyapatite, polymethyl methacrylate, bovine collagen, human collagen, silicone and hyaluronic acid.

In some embodiments, the metabolic disorder is liver inflammation and the therapeutic agent is selected from the group consisting of a hepatitis therapeutic agent and a hepatitis vaccine.

In some embodiments, the metabolic disorder is fatty liver disease and the therapeutic agent or treatment procedure is bariatric surgery and/or dietary intervention.

In some embodiments, the metabolic disorder is hypercholesterolemia and the therapeutic agent is selected from the group consisting of: the statin(s) (e.g., (atorvastatin), ->(fluvastatin), lovastatin, < - > and->(pitavastatin),(Pravastatin), ->(rosuvastatin calcium) and +.>(simvastatin)); bile acid sequestrants (e.g.)>(cholestyramine) and (B) Cryptophan>(colesevelam) and(colestipol)); PCSK9 inhibitors (e.g.)>(aliskiren)(avokuzumab); nicotinic acid (e.g., nisetum and nisetum); fibrates (e.g. fenofibrate and(gemfibrozil)); and ATP Citrate Lyase (ACL) inhibitors (e.g., +.>(Bei Peiduo)). In some embodiments, therapeutic agents that treat or inhibit hypercholesterolemia include, but are not limited to: statins (e.g., atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, and simvastatin); bile acid sequestrants (e.g., cholestyramine, colesevelam and colestipol); PCSK9 inhibitors (e.g., aliskiren and exekumumab; nicacid (e.g., nicarbazin and nicarbazin; fibrates (e.g., fenofibrate and gemfibrozil)), and ACL inhibitors (e.g., bei Peiduo). In some embodiments, the therapeutic agent that treats or inhibits hypercholesterolemia is aliskiren or exekumumab.

In some embodiments, the metabolic disorder is elevated liver enzymes (such as, for example, ALT and/or AST), and the therapeutic agent is selected from coffee, folic acid, potassium, vitamin B6, statin, and fiber, or any combination thereof.

In some embodiments of the present invention, in some embodiments, the metabolic disorder is NASH and the therapeutic agent is obeticholic acid, sec Long Se, ai Labu no, cinmivir, gr_md_02, mgl_3196, IMM124E, eicosanoyl-cholanic acid, GS0976, emlicarbazepine, wo Liba t, NGM282, GS9674, trapezil, mn_001, LMB763, bi_1467335, msdc_0602, pf_05221304, DF102, sha Luoge list bundle, BMS986036, lanuno, cord Ma Lutai, nitazoxanide, gri_0621, EYP001, VK2809, nalmefene, LIK066, mt_3995, enoxib Namod son, fu Lei Lushan anti, SAR425899, sogliflozin, EDP_305, exopat, jicabin, TERN_101, KBP_042, PF_06865571, DUR928, PF_06835919, NGM313, BMS_986171, na Ma Xizuo mab, CER_209, ND_L02_s0201, RTU_1096, DRX_065, IONIS_DGAT2Rx, INT_767, NC_001, seradpa, PXL770, TERN_201, NV556, AZD2693, SP_1373, VK0214, hepatstein, TGFTX4, RL112BN 7, GKT_137831, RYI _018, CB4209-CB4211 and JH_0920.

In some embodiments, the therapeutic agent that treats or inhibits a metabolic disorder is a melanocortin 4 receptor (MC 4R) agonist. In some embodiments, the MC4R agonist comprises a protein, peptide, nucleic acid molecule, or small molecule. In some embodiments, the protein is a peptide analog of MC 4R. In some embodiments, the peptide is semanteme. In some embodiments, the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp. In some embodiments, the small molecule is 1,2,3r, 4-tetrahydroisoquinoline-3-carboxylic acid. In some embodiments, the MC4R agonist is ALB-127158 (a).

The present disclosure also provides a therapeutic agent for treating or inhibiting a cardiovascular disease for treating a cardiovascular disease in a subject having: an INHBE variant genomic nucleic acid molecule encoding a predictive loss-of-function INHBE polypeptide; an INHBE variant mRNA molecule encoding a predictive loss-of-function INHBE polypeptide; or an INHBE variant cDNA molecule encoding a predictive loss-of-function INHBE polypeptide.

In some embodiments, the cardiovascular disease is hypertension, and the therapeutic agent is selected from the group consisting of chlorthalidone, chlorthiazide, hydrochlorothiazide, indapamide, mecopril, acebutolol, atenolol, betaxolol, bisoprolol fumarate, carboplatin hydrochloride, metoprolol tartrate, mecalol succinate, nadoprolol hydrochloride, benazepril, captopril maleate, fosinopril sodium, lisinopril, moexipril, perindopril, quinapril hydrochloride, ramipril, trandolapril, candesartan, eprosartan, irbesartan, losartan potassium, telmisartan, valsartan, amlodipine besylate, benazepril, diltiazem hydrochloride, felodipine, veradipine, nifedipine hydrochloride, doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, methyldopa, carvedilol hydrochloride, alpha methylgliclazide, guanamine hydrochloride, guanadine hydrochloride, guanadine, guanamine hydrochloride, and guanamine hydrochloride.

In some embodiments, the cardiovascular disease is cardiomyopathy and the therapeutic agent is selected from: 1) Antihypertensives such as ACE inhibitors, angiotensin II receptor blockers, beta blockers and calcium channel blockers; 2) Heart rate slowing agents such as beta blockers, calcium channel blockers, and digoxin; 3) Agents that maintain the heart beating at normal rhythms, such as antiarrhythmic agents; 4) Electrolyte balancing agents such as aldosterone blockers; 5) Agents that remove excess fluid and sodium from the body, such as diuretics; 6) Agents that prevent clot formation, such as anticoagulants or blood diluents; and 7) agents that reduce inflammation, such as corticosteroids.

In some embodiments, the cardiovascular disease is heart failure, and the therapeutic agent is selected from the group consisting of: ACE inhibitors, angiotensin 2 receptor blockers, beta blockers, mineralocorticoid receptor antagonists, diuretics, ivabradine, sarcandesartan, nitrate-containing hydralazine, and digoxin.

The present disclosure also provides an INHBE inhibitor for treating or inhibiting a metabolic disorder, for use in treating a metabolic disorder in a subject having: an INHBE variant genomic nucleic acid molecule encoding a predictive loss-of-function INHBE polypeptide; an INHBE variant mRNA molecule encoding a predictive loss-of-function INHBE polypeptide; or an INHBE variant cDNA molecule encoding a predictive loss-of-function INHBE polypeptide.

The present disclosure also provides an INHBE inhibitor for treating or inhibiting a cardiovascular disease for use in treating a cardiovascular disease in a subject having: an INHBE variant genomic nucleic acid molecule encoding a predictive loss-of-function INHBE polypeptide; an INHBE variant mRNA molecule encoding a predictive loss-of-function INHBE polypeptide; or an INHBE variant cDNA molecule encoding a predictive loss-of-function INHBE polypeptide.

In some embodiments, the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA. In some embodiments, the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule. In some embodiments, the Cas protein is Cas9 or Cpf1. In some embodiments, the gRNA recognition sequence is located within SEQ ID NO. 1. In some embodiments, the proscenium sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence adjacent to the motif (Protospacer Adjacent Motif, PAM) sequence. In some embodiments, the gRNA comprises about 17 to about 23 nucleotides. In some embodiments, the gRNA recognition sequence comprises a nucleotide sequence according to any of SEQ ID NOs 9-27.

All patent documents, websites, other publications, accession numbers and the like cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference. If different versions of a sequence are associated with an accession number at different times, then the version associated with the accession number at the date of the effective application of the present application is significant. The effective date of application means the date of application (if applicable) earlier than the actual date of application or priority application involving an accession number. Also, if different versions of publications, websites, etc. are published at different times, the most recently published version at the date of the effective application of the present application is significant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure may be used in combination with any other feature, step, element, embodiment, or aspect unless specifically indicated otherwise. Although the present disclosure has been described in 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.

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

Examples

Example 1: loss of function in INHBE is associated with more favourable fat distribution and protection against human type 2 diabetes

The fat distribution was subjected to a whole exome correlation analysis, which was measured by a waist-to-hip ratio (BMI-adjusted WHR) adjusted for body mass index. BMI-adjusted WHR is a measure of body fat distribution and is not related to global obesity. For each gene in the genome, rare predicted loss of function genetic variants (surrogate allele frequencies [ AAF ]<1% plofvariant) is associated with BMI-adjusted WHR. In this analysis, at the level of the whole exome with statistical significance (p<3.6x10 ^-7 Ponfronib (Bonferroni) correction corresponding to the number of tests), rare in INHBE (AAF<1%) the burden of predicting loss of function (pLOF) variants was associated with more favorable fat distribution (i.e., lower WHR for BMI adjustment; see fig. 1 and 2). Table 6 shows the results of correlation of pLOF variants in INHBE with fat distribution among 285,605 European blood system participants in UKB cohorts (vsCorrelation of BMI adjusted WHR; genetic exposure is AAF<1% of the burden of the plofvariant). INHBE plorf was strongly correlated with lower BMI-adjusted WHR (see table 6). This association of statistical significance was further replicated in meta-analysis of additional data, including the second set of UKB data (more than 140,000 participants of european descent) and more than 95,000 us participants who had mixed blood from MCPS studies (see fig. 1).

Table 6: INHBE gene burden-associated results for BMI-adjusted WHR in UKB

Abbreviations: UKB = uk biological sample library study population, AAF = plofallele frequency of plofvariants in genes, RR = count of individuals without heterozygous or homozygous observations of plofvariants in genes, RA = count of individuals with at least one heterozygous plofs and no homozygous plofvariants in genes, AA = count of individuals with at least one homozygous plofvariants in genes, CI = confidence interval, plofs = predictive function loss, SD = standard deviation.

Table 6 shows the correlation of INHBE pLOF with BMI-adjusted WHR in European blood individuals of a study population of the British biological sample library. The effect of INHBE pluf variants was estimated, in Standard Deviation (SD) units and WHR ratio units. Table 6 shows that INHBE pLOF carriers have lower BMI-adjusted WHR in the analysis adjusted for covariates, lineages and correlations compared to the average of individuals not carrying these genetic variants. Genotype counts demonstrate the number of individuals in a population study that do not carry a variant (RR) of plorf that results in an INHBE, carry one or more variants (RA) of plorf that result in a single INHBE allele, or carry one or more plorf variants (AA) of both INHBE alleles.

This association of INHBE ploflow variants with lower BMI-adjusted WHR was consistent in men and women from the british biological sample library cohort (see table 7; genetic exposure is the burden of AAF <1% ploflow variants).

Table 7: sex-stratified INHBE plofvariant association in UKB

Table 7 shows the correlation of INHBE pLOF with BMI-adjusted WHR in European blood individuals from British biological sample library studies by gender stratification. The effect of INHBE pluf variants was estimated, in Standard Deviation (SD) units and WHR ratio units. Genotype counts demonstrate the number of individuals in a population study that do not carry a variant (RR) of plorf that results in an INHBE, carry one or more variants (RA) of plorf that result in a single INHBE allele, or carry one or more plorf variants (AA) of both INHBE alleles. The association of INHBE ploflow variants with lower BMI-adjusted WHR was similar in intensity in men and women included in this analysis.

Among the pLOF variants in INHBE, the variant most strongly associated with BMI-regulated WHR was the c.299-1g > c (12:57456093:g: c according to the GRCh38/hg38 human genome assembly coordinates) mutation, predicted to affect the intron 1 receptor splice site, shortening exon 2 by 12 nucleotides at the 5' -end (see fig. 3 and table 8), and resulting in an in-frame deletion within the leader domain of INHBE protein (pro-domain) (see fig. 4).

Table 8: influence of the 12:57456093:G:C acceptor splice site variants on splicing as predicted by SpliceAI software.

Delta score: a value between 0 and 1 is interpreted as the probability of a variant having a splice variation effect on the INHBE gene.

Table 8 shows the predicted effect of variant 12:57456093:G:C on INHBE gene splicing.

In Chinese Hamster Ovary (CHO) cells, the c.299-1g > c splice variant was expressed and found to produce lower molecular weight proteins that were not secreted outside the cell, indicating loss of function (see fig. 5).

The plofvariant in INHBE correlates with larger hip size, higher arm and leg fat mass, indicating a greater ability to store calories in peripheral adipose tissue (see fig. 6 and table 9).

Table 9: correlation of plofgenetic variants in meta-analyzed INHBEs with obese phenotype in UKB, galsinge health system (Geisinger Health System, GHS) and MCPS studies

Abbreviations: UKB = uk biological sample library study population, GHS = galsinge health system study population, MCPS = mexico market prospective study population, AAF = plofallele frequency of plofvariant in gene, RR = count of individuals without heterozygous or homozygous observations of plofvariant in gene, RA = count of individuals with at least one heterozygous plofvariant in gene and no homozygous plofvariant, AA = count of individuals with at least one homozygous plofvariant in gene, CI = confidence interval, ploff = prediction function loss, SD = standard deviation, kg/m ² =kg per square meter, cm=cm. Genotype counts demonstrate that the population study does not carry variants (RR) of pLOF that result in INHBE, carries variants (RA) of pLOF that result in a single INHBE allele, or carries one or more pLOF of two INHBE allelesNumber of individuals of variant (AA).

Table 9 shows the relationship of INHBE pLOF to BMI, waist circumference and hip circumference. The effect of INHBE plofs was quantified in standard deviation units or in corresponding clinical units for each anthropometric variable.

Rare plofvariants in INHBE are also associated with protection against human type 2 diabetes. The INHBE plofvariants were also found to be associated with lower risk of type 2 diabetes (T2D) (see table 10; genetic exposure is the burden of AAF <1% plofvariants), which constitutes the first evidence linking LOFs in INHBE to human type 2 diabetes.

Table 10: correlation of plofgenetic variants in INHBE with T2D in UKB, GHS and MCPS studies

Abbreviations: meta-analysis = combined analysis of all listed study populations, AAF = plofallele frequency of plofvariants in genes, RR = count of individuals without heterozygous or homozygous observations of plofvariants in genes, RA = count of individuals with at least one heterozygous plofs and no homozygous plofvariants in genes, AA = count of individuals with at least one homozygous plofvariants in genes, CI = confidence interval, plofs = predictive loss of function, SD = standard deviation. Genotype counts demonstrate the number of individuals in a population study as cases of T2D or not in the T2D category that do not carry a variant (RR) of plorf that results in an INHBE, carry a variant (RA) of plorf that results in a single INHBE allele, or carry one or more plorf variants (AA) of both INHBE alleles.

Table 10 shows the correlation of ploflo variants with T2D in INHBEs resulting from analysis of the UKB biological sample library (UKB), the cover-singer health system (GHS) and the mexico market prospective study (MCPS) population. The results show that INHBE pLOF variants are associated with lower T2D risk in each study population, and this is demonstrated in meta-analysis combining the results of all three study populations.

Furthermore, in the analysis across multiple queues, INHBE pLOF variants were associated with favorable metabolic profiles (see Table 11; genetic exposure is the burden of an AAF <1% of INHBE pLOF variants) including lower HbA1C, ALT, triglycerides and LDL-C and higher HDL-C.

Table 11: correlation of pLOF genetic variants in meta-analyzed INHBE with metabolism in UKB, GHS and MCPS studies

Abbreviations: UKB = uk biological sample library study population, GHS = galsinge health system study population, MCPS = mexico market prospective study, AAF = plofallele frequency of plofvariants in genes, RR = count of individuals without heterozygous or homozygous observations of plofvariants in genes, RA = count of individuals with at least one heterozygous plofvariants in genes and no homozygous plofvariants, AA = count of individuals with at least one homozygous plofvariants in genes, CI = confidence interval, plofs = prediction function loss, SD = standard deviation, mg/dL = milligrams per deciliter, U/L = units per liter. Genotype counts demonstrate the number of individuals in a population study that do not carry a variant (RR) of plorf that results in an INHBE, carry one or more variants (RA) of plorf that result in a single INHBE allele, or carry one or more plorf variants (AA) of both INHBE alleles.

Table 11 shows the association of INHBE pLOF variants with a range of metabolic phenotypes as estimated in the meta-analysis of UKB, GHS and MCPS study groups. Results are shown in units of standard deviation and original clinical units of relevant metabolic phenotype.

Furthermore, INHBE pLOF variants are associated with a decrease in liver inflammation index in magnetic resonance imaging (see Table 12; genetic exposure is the burden of AAF <1% of INHBE pLOF variants).

Table 12: correlation of the plofr genetic variants in INHBE in UKB with liver imaging phenotype

^a The technological covariates including BMI, alcohol consumption and diabetes are regulated.

Abbreviations: PDFF = proton density fat fraction (defined as the ratio of the density of mobile protons from fat (triglyceride) to the total density of protons from mobile triglyceride and mobile water, and reflecting the fat concentration within the tissue), ECF = extracellular fluid, t1 = time constant for longitudinal magnetization recovery. This is a relaxation time that measures the speed at which the net magnetization returns to its ground state. It may vary significantly based on the strength of the magnetic field and the composition of the tissue. Furthermore, it increases with increasing magnetic field, whereas it decreases with the presence of fat and/or iron in the tissue, ct1=t 1 corrected for the effect of liver iron content (which leads to an underestimation of the T1 value), ukb=british biological sample library study population, aaf=the plofallele frequency of plofvariant in the gene, rr=the count of individuals without heterozygous or homozygous observations of plofvariant in the gene, ra=the count of individuals with at least one heterozygous plofvariant in the gene and without homozygous plofvariant, aa=the count of individuals with at least one homozygous plofvariant in the gene, ci=confidence interval, plof= predictive function loss, sd=standard deviation.

Table 12 shows the association of INHBE plofloc variants with a range of liver imaging phenotypes in european ancestor individuals from the british biological sample library study population. The results show that INHBE pLOF variants correlate with lower levels of ECF and cT1, which are measures of liver inflammation defined by magnetic resonance imaging.

In addition, it was investigated whether INHBE pLOF variants correlated with liver histopathological phenotype in 3,565 bariatric patients from GHS cohorts with exome sequencing and perioperative liver wedge biopsies. Only three carriers of the plorf variants of INHBE were in the pool, but the carrier status was correlated with a lower non-alcoholic fatty liver disease activity score (see table 13), which is a measure of liver disease severity at biopsy, which is the sum of steatosis, lobular inflammation and balloon-like grade (Kleiner et al, hepatology,2005,41,1313-21).

Table 13: correlation of rare plif variants in INHBE with lower non-alcoholic fatty liver disease activity scores

The association of rare plofvariants in INHBE with NAFLD activity scores (results) is reported. The association was estimated in 3,565 bariatric patients from GHS.

Finally, common variants near INHBE were found to correlate with higher liver expression levels of INHBE mRNA (12:57259799: A: C; rs7966846; AAF, 0.28) (0.3 SD of INHBE transcript abundance per allele, quantified by RNASeq in more than 2,000 GHS participants who underwent liver biopsies as part of bariatric surgery). The 12:57259799:A:C variant was also found to be associated with higher BMI-adjusted WHR, triglycerides and type 2 diabetes risk. Allele C causing expression and higher BMI-adjusted WHR (p-value=1.5x10 ^-4 ) Higher triglycerides (p-value=2.0x10 ^-11 ) Higher T2D risk (p-value=0.03) is relevant (see table 14). This suggests that genetically determined INHBE overexpression is associated with a higher risk of metabolic disease, whereas loss of function is associated with favorable metabolic characteristics and lower risk of diabetes (as indicated above by the plofvariant association).

Table 14: correlation of INHBE eQTL 12:57259799:a:c in UKB and GHS queues with various metabolic phenotypes

^a The estimate is in the form of a dominance ratio.

Abbreviations: AAF = allele frequency of the allele causing liver expression of INHBE (i.e., substitution allele), CI = confidence interval, SD = standard deviation, RR = reference-reference allele, RA = reference-substitution allele, AA = substitution-substitution allele, mg/dL = milligrams per deciliter. Genotype counts show the number of individuals in the population study who had no copy of the allele (RR) that caused liver expression of INHBE, only one copy of the allele (RA) that caused liver expression of INHBE, and 2 copies of the allele (AA) that caused liver expression of INHBE. Genotype counts were further stratified within individuals classified as T2D cases in the study population.

The association of 12:57259799:A:C with triglyceride levels, WHRadjBMI and T2D risk was studied in all European pedigree participants from the British biological sample library and the Gastrodia health study. The results show that 12:57259799:A:C are significantly associated with higher triglyceride levels and higher BMI adjusted WHR; furthermore, a higher T2D risk is also associated.

Example 2: INHBE is highly expressed in human hepatocytes and its expression is up-regulated in patients with steatosis and nonalcoholic steatohepatitis

INHBE mRNA expression in tissues from humans of the genotype tissue expression association (GTEx) was examined and found to be highest in liver among GTEx tissues (see fig. 7). mRNA expression of INHBE in each cell type was also examined in data from human protein profile (HPA) and found to be highest in hepatocytes (see FIG. 7). Levels of INHBE expression in the liver of over 2,000 bariatric surgery patients with liver RNASeq were also estimated in GHS. As a result, it was found that INHBE expression was up-regulated in liver steatosis patients compared to individuals with normal liver, INHBE expression was up-regulated in non-alcoholic steatohepatitis patients compared to individuals with normal liver, and INHBE expression was up-regulated in non-alcoholic steatohepatitis patients compared to steatohepatitis patients (see fig. 8).

Example 3: correlation of INHBE identified in BMI-adjusted WHR discovery analysis with visceral to gluteal femoral fat ratio measured by MRI

A subset of approximately 46,000 participants in UKB were subjected to two-point Dixon (Dixon, radiology,1984,153,189-194) using a Siemens (Siemens) MAGNETOM aero 1.5T clinical MRI scanner (Littlejohns et al, nat. Commun.,2020,11,2624) and divided into six different imaging series. The subset includes 38,880 people with available exome sequencing. Stitching of six different scan positions has been corrected for overlapping slices, partial scans, repeated scans, fat-water exchanges, misalignments between imaging series, bias fields, artificial dark slices, and local hot spots, similar to the previously performed operations (Basty et al Image Processing and Quality Control for Abdominal Magnetic Resonance Imaging in the UK Biobank,2020, arxiv abs/2007.01251). Whole-body Dixon MRI of a total of 52 subjects was manually labeled as six different fat categories: upper body fat, abdominal fat, visceral fat, mediastinal fat, gluteal thigh fat and lower leg fat. Particular attention is paid to adjusting the training dataset to attempt to span the expected phenotypic diversity, including specifically training subjects with genetic mutations that predispose the subjects to abnormal fat and muscle phenotypes such as ppavg (Ludtke et al, j.med.genet.,2007,44, e 88), PLIN1 (Gandotra et al, n.engl.j.med.,2011,364,740-748), LMNA (jelu et al, j.med.genet.,2017,54,413-416), LIPE (Zolotov et al, am.j.med.genet., a 173, 190-194) and MC4R (Akbari et al, science,2021,373). These annotations are then used to train a multi-class segmented deep neural network employing the UNet (Weng et al, IEEE Access,2021,9,16591-16603) architecture with the res net34 (He et al, in 2016IEEE Conference on Computer Vision and Pattern Recognition (CVPR), 2016, 770-778) skeleton, and a loss function of the sum of Jaccard (Jaccard) index and classification focus loss (categorical focal loss) (Lin et al, IEEE Transactions on Pattern Analysis and Machine Intelligence,2020,42,318-327). Fat volume phenotypes are calculated by summing the segmentation maps generated by the neural network for each respective fat category. The visceral to thigh fat ratio is then calculated as the ratio of visceral to thigh fat volume for a given individual.

Of the 38,880 subsets of people who performed whole-body MRI in UKB (i.e., -6% of the found samples), rare coding variants in INHBE associated with BMI-adjusted WHR showed a high correlation with visceral to gluteal-femoral fat ratio under MRI (an accurate fat distribution measurement method) (see table 15). In the UKB subset with MRI data, INHBE pLOF variants were nominally significantly associated with lower MRI-defined visceral to gluteal femoral fat ratios (β for each allele, in SD units of fat ratio, -0.24;95% CI, -0.45 to-0.02; p=0.03; see table 15).

TABLE 15

Each gene burden result in the table was analyzed in a model taking into account the gender specific effects of age, body mass index and height on visceral to gluteal femoral fat ratio.

Abbreviations: pluf, loss of predictive function; AAF, substitution allele frequency; CI, confidence interval; SD, standard deviation; BMI, body mass index; p, P value; RR, reference homozygous genotype; RA, reference-surrogate genotype; AA, replacing homozygous genotypes.

Example 4: correlation of INHBE predictive function loss with left ventricular ejection fraction increase and cardiomyopathy protection

The case in this example is any study participant without heart disease. The results are based on meta-analysis of UKB, GHS, SINAI, UPENN-PMBB, MDCS, indiana-Chalasani (Indiana-Chalasani). The predicted loss of function in INHBE associated with increased left ventricular ejection fraction and cardiomyopathy protection is shown in table 16 (burden of rare pluf variants of INHBE (M1.1)).

Table 16

Results 1 are left ventricular ejection fraction.

Results 2 are non-ischemic cardiomyopathy.

* Left ventricular ejection fraction obtained by cardiac MRI of participants of the british biological sample library.

* Cases of non-ischemic cardiomyopathy are defined as study participants with one or more of the following ICD10 codes: i420 (dilated cardiomyopathy), I425 (other restrictive cardiomyopathy), I428 (other cardiac densification incomplete cardiomyopathy (noncompaction cardiomyopathies)), I429 (primary cardiomyopathy |unspecified), and the absence of one or more of any ICD10 codes indicates myocardial infarction (i21|i22|i23|i252|i256) and hypertrophic cardiomyopathy (I421, I422).

The association of the plofvariant with lower blood pressure (see table 17; burden-M1.1 of rare plofvariants of inhbe) is consistent with a beneficial effect on hemodynamic properties.

TABLE 17

Trait 1 is diastolic blood pressure (corrected by treatment).

Trait 2 is systolic blood pressure (corrected by treatment).

Various modifications of the subject matter in addition to those described herein will be 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 disclosure (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 by reference in its entirety and for all purposes.

Sequence listing

<110> Lei Jiena Rong pharmaceutical Co., ltd (Regeneron Pharmaceuticals, inc.)

L.A. Luo tower (Lotta, luca Andrea)

P.Acarbari (Akbari, parsa)

O.Sosina (Olukayode)

M.A.R. Fei Leila (Ferriera, manuel Allen Revez)

A, barass (Aris)

<120> methods of treating metabolic disorders and cardiovascular diseases with inhibitors of inhibin subunit beta E (INHBE)

<130> 189238.05702 (3448) (10854WO01)

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<170> PatentIn version 3.5

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<212> DNA

<213> homo sapiens (homo sapiens)

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tggcagtggt gtctgctgtc actgtgccct cattggcccc cagcaatcag actcaacaga 180

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ctgatgtcca gctctggctg gtgctgctgt gggcactggt gcgagcacag gggacagggt 300

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ctcatcctcc accccaggca gcgctgacca gagccctccg gagactacag ccagggagtg 480

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tcccaccacc tgtaccatgc ccgcctgtgg ctgcacgtgc tccccaccct tcctggcact 900

ctttgcttga ggatcttccg atggggacca aggaggaggc gccaagggtc ccgcactctc 960

ctggctgagc accacatcac caacctgggc tggcatacct taactctgcc ctctagtggc 1020

ttgaggggtg agaagtctgg tgtcctgaaa ctgcaactag actgcagacc cctagaaggc 1080

aacagcacag ttactggaca accgaggcgg ctcttggaca cagcaggaca ccagcagccc 1140

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gaactgggat ggcgggactg gatactgcag cccgaggggt accagctgaa ttactgcagt 1320

gggcagtgcc ctccccacct ggctggcagc ccaggcattg ctgcctcttt ccattctgcc 1380

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gtgtactctg taggcttctg ggaggtccct tttcaggggt gtccacaaag tcaaagctat 2580

tttcataata atactaacat gttatttgcc ttttgaattc tcattatctt aaaattgtat 2640

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aatggaatgt gtgcttgta 2719

<210> 2

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<212> RNA

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ucugcuguca cugugcccuc auuggccccc agcaaucaga cucaacagac ggagcaacug 180

ccauccgagg cuccugaacc agggccauuc accaggagca ugcggcuccc ugauguccag 240

cucuggcugg ugcugcugug ggcacuggug cgagcacagg ggacaggguc uguguguccc 300

uccugugggg gcuccaaacu ggcaccccaa gcagaacgag cucuggugcu ggagcuagcc 360

aagcagcaaa uccuggaugg guugcaccug accagucguc ccagaauaac ucauccucca 420

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cucuuggaca cagcaggaca ccagcagccc uuccuagagc uuaagauccg agccaaugag 900

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cccgaggggu accagcugaa uuacugcagu gggcagugcc cuccccaccu ggcuggcagc 1080

ccaggcauug cugccucuuu ccauucugcc gucuucagcc uccucaaagc caacaauccu 1140

uggccugcca guaccuccug uugugucccu acugcccgaa ggccccucuc ucuccucuac 1200

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ggcugcagcu agcaagagga ccuggggcuu uggagugaag agaccaagau gaaguuuccc 1320

aggcacaggg caucugugac uggaggcauc agauuccuga uccacacccc aacccaacaa 1380

ccaccuggca auaugacuca cuugaccccu augggaccca aaugggcacu uucuugucug 1440

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aaggggucua cccagaaagc augauuuccu gcccuaaguc cugugagaag augucaggga 1560

cuagggaggg agggagggaa ggcagagaaa aauuacuuag ccucucccaa gaugag 1616

<210> 3

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<212> RNA

<213> homo sapiens (homo sapiens)

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agcugugagg gucaagcaca gcuauccauc agaugaucua cuuucagccu uccugagucc 60

cagacaauag aagacaggug gcuguacccu uggccaaggg uagguguggc aguggugucu 120

gcugucacug ugcccucauu ggcccccagc aaucagacuc aacagacgga gcaacugcca 180

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uggcuggugc ugcugugggc acuggugcga gcacagggga cagggucugu gugucccucc 300

ugugggggcu ccaaacuggc accccaagca gaacgagcuc uggugcugga gcuagccaag 360

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caggcagcgc ugaccagagc ccuccggaga cuacagccag ggaguguggc uccagggaau 480

ggggaggagg ucaucagcuu ugcuacuguc acagacucca cuucagccua cagcucccug 540

cucacuuuuc accuguccac uccucggucc caccaccugu accaugcccg ccuguggcug 600

cacgugcucc ccacccuucc uggcacucuu ugcuugagga ucuuccgaug gggaccaagg 660

aggaggcgcc aagggucccg cacucuccug gcugagcacc acaucaccaa ccugggcugg 720

cauaccuuaa cucugcccuc uaguggcuug aggggugaga agucuggugu ccugaaacug 780

caacuagacu gcagaccccu agaaggcaac agcacaguua cuggacaacc gaggcggcuc 840

uuggacacag caggacacca gcagcccuuc cuagagcuua agauccgagc caaugagccu 900

ggagcaggcc gggccaggag gaggaccccc accugugagc cugcgacccc cuuauguugc 960

aggcgagacc auuacguaga cuuccaggaa cugggauggc gggacuggau acugcagccc 1020

gagggguacc agcugaauua cugcaguggg cagugcccuc cccaccuggc uggcagccca 1080

ggcauugcug ccucuuucca uucugccguc uucagccucc ucaaagccaa caauccuugg 1140

ccugccagua ccuccuguug ugucccuacu gcccgaaggc cccucucucu ccucuaccug 1200

gaucauaaug gcaauguggu caagacggau gugccagaua ugguggugga ggccuguggc 1260

ugcagcuagc aagaggaccu ggggcuuugg agugaagaga ccaagaugaa guuucccagg 1320

cacagggcau cugugacugg aggcaucaga uuccugaucc acaccccaac ccaacaacca 1380

ccuggcaaua ugacucacuu gaccccuaug ggacccaaau gggcacuuuc uugucugaga 1440

cucuggcuua uuccagguug gcugaugugu ugggagaugg guaaagcguu ucuucuaaag 1500

gggucuaccc agaaagcaug auuuccugcc cuaaguccug ugagaagaug ucagggacua 1560

gggagggagg gagggaaggc agagaaaaau uacuuagccu cucccaagau gagaaagucc 1620

ucaagugagg ggaggaggaa gcagauagau gguccagcag gcuugaagca ggguaagcag 1680

gcuggcccag gguaagggcu guugagguac cuuaagggaa ggucaagagg gagaugggca 1740

aggcgcugag ggaggaugcu uaggggaccc ccagaaacag gagucaggaa aaugaggcac 1800

uaagccuaag aaguucccug guuuuuccca ggggacagga cccacuggga gacaagcauu 1860

uauacuuucu uucuucuuuu uuauuuuuuu gagaucgagu cucgcucugu caccaggcug 1920

gagugcagug acacgaucuu ggcucacugc aaccuccguc uccuggguuc aagugauucu 1980

ucugccucag ccucccgagc agcugggauu acaggcgccc acuaauuuuu guauucuuag 2040

uagaaacgag guuucaacau guuggccagg auggucucaa ucucuugacc ucuugaucca 2100

cccgacuugg ccucccgaag ugaugagauu auaggcguga gccaccgcgc cuggcuuaua 2160

cuuucuuaau aaaaaggaga aagaaaauca acaaauguga gucauaaaga aggguuaggg 2220

ugauggucca gagcaacagu ucuucaagug uacucuguag gcuucuggga ggucccuuuu 2280

cagggguguc cacaaaguca aagcuauuuu cauaauaaua cuaacauguu auuugccuuu 2340

ugaauucuca uuaucuuaaa auuguauugu ggaguuuucc agaggccgug ugacauguga 2400

uuacaucauc uuucugacau cauuguuaaa aaaaaaaaaa aaaaa 2445

<210> 4

<211> 2459

<212> RNA

<213> homo sapiens (homo sapiens)

<400> 4

gggucaagca cagcuaucca ucagaugauc uacuuucagc cuuccugagu cccagacaau 60

agaagacagg uggcuguacc cuuggccaag gguaggugug gcaguggugu cugcugucac 120

ugugcccuca uuggccccca gcaaucagac ucaacagacg gagcaacugc cauccgaggc 180

uccugaacca gggccauuca ccaggagcau gcggcucccu gauguccagc ucuggcuggu 240

gcugcugugg gcacuggugc gagcacaggg gacagggucu gugugucccu ccuguggggg 300

cuccaaacug gcaccccaag cagaacgagc ucuggugcug gagcuagcca agcagcaaau 360

ccuggauggg uugcaccuga ccagucgucc cagaauaacu cauccuccac cccaggcagc 420

gcugaccaga gcccuccgga gacuacagcc agggagugug gcuccaggga auggggagga 480

ggucaucagc uuugcuacug ucacagacuc cacuucagcc uacagcuccc ugcucacuuu 540

ucaccugucc acuccucggu cccaccaccu guaccaugcc cgccuguggc ugcacgugcu 600

ccccacccuu ccuggcacuc uuugcuugag gaucuuccga uggggaccaa ggaggaggcg 660

ccaagggucc cgcacucucc uggcugagca ccacaucacc aaccugggcu ggcauaccuu 720

aacucugccc ucuaguggcu ugagggguga gaagucuggu guccugaaac ugcaacuaga 780

cugcagaccc cuagaaggca acagcacagu uacuggacaa ccgaggcggc ucuuggacac 840

agcaggacac cagcagcccu uccuagagcu uaagauccga gccaaugagc cuggagcagg 900

ccgggccagg aggaggaccc ccaccuguga gccugcgacc cccuuauguu gcaggcgaga 960

ccauuacgua gacuuccagg aacugggaug gcgggacugg auacugcagc ccgaggggua 1020

ccagcugaau uacugcagug ggcagugccc uccccaccug gcuggcagcc caggcauugc 1080

ugccucuuuc cauucugccg ucuucagccu ccucaaagcc aacaauccuu ggccugccag 1140

uaccuccugu ugugucccua cugcccgaag gccccucucu cuccucuacc uggaucauaa 1200

uggcaaugug gucaagacgg augugccaga uaugguggug gaggccugug gcugcagcua 1260

gcaagcagga ccuggggcuu uggagugaag agaccaagau gaaguuuccc aggcacaggg 1320

caucugugac uggaggcauc agauuccuga uccacacccc aacccaacaa ccaccuggca 1380

auaugacuca cuugaccccu augggaccca aaugggcacu uucuugucug agacucuggc 1440

uuauuccagg uuggcugaug uguugggaga uggguaaagc guuucuucua aaggggucua 1500

cucagaaagc augauuuccu gcccuaaguc cugugagaag augucaggga cuagggaggg 1560

agggagggaa ggcagagaaa aauuacuuag ccucucccaa gaugagaaag uccucaagug 1620

aggggaggag gaagcagaua gaugguccag caggcuugaa gcaggguaag caggcuggcc 1680

caggguaagg gcuguugagg uaccuuaagg gaaggucaag agggagaugg gcaaggcgcu 1740

gagggaggau gcuuagggga cccccagaaa caggagucag gaaaaugagg cacuaagccu 1800

aagaaguucc cugguuuuuc ccaggggaca ggacccacug ggcgacaagc auuuauacuu 1860

ucuuucuucu uuuuuauuuu uuugagaucg agucucgcuc ugucaccagg cuggagugca 1920

gugacacgau cuuggcucac ugcaaccucc gucuccuggg uucaagugau ucuucugccu 1980

cagccucccg agcagcuggg auuacaggcg cccacuaauu uuuguauucu uaguagaaac 2040

gagguuucaa cauguuggcc aggauggucu caaucucuug accucuugau ccacccgacu 2100

uggccucccg aagugaugag auuauaggcg ugagccaccg cgccuggcuu auacuuucuu 2160

aauaaaaagg agaaagaaaa ucaacaaaug ugagucauaa agaaggguua gggugauggu 2220

ccagagcaac aguucuucaa guguacucug uaggcuucug ggaggucccu uuucaggggu 2280

guccacaaag ucaaagcuau uuucauaaua auacuaacau guuauuugcc uuuugaauuc 2340

ucauuaucuu aaaauuguau uguggaguuu uccaaaggcc gugugacaug ugauuacauc 2400

aucuuucuga caucaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2459

<210> 5

<211> 1616

<212> DNA

<213> homo sapiens (homo sapiens)

<400> 5

atgagctgtg agggtcaagc acagctatcc atcagatgat ctactttcag ccttcctgag 60

tcccagacaa tagaagacag gtggctgtac ccttggccaa gggtaggtgt ggcagtggtg 120

tctgctgtca ctgtgccctc attggccccc agcaatcaga ctcaacagac ggagcaactg 180

ccatccgagg ctcctgaacc agggccattc accaggagca tgcggctccc tgatgtccag 240

ctctggctgg tgctgctgtg ggcactggtg cgagcacagg ggacagggtc tgtgtgtccc 300

tcctgtgggg gctccaaact ggcaccccaa gcagaacgag ctctggtgct ggagctagcc 360

aagcagcaaa tcctggatgg gttgcacctg accagtcgtc ccagaataac tcatcctcca 420

ccccaggcag cgctgaccag agccctccgg agactacagc cagggagtgt ggctccaggg 480

aatggggagg aggtcatcag ctttgctact gtcacagact ccacttcagc ctacagctcc 540

ctgctcactt ttcacctgtc cactcctcgg tcccaccacc tgtaccatgc ccgcctgtgg 600

ctgcacgtgc tccccaccct tcctggcact ctttgcttga ggatcttccg atggggacca 660

aggaggaggc gccaagggtc ccgcactctc ctggctgagc accacatcac caacctgggc 720

tggcatacct taactctgcc ctctagtggc ttgaggggtg agaagtctgg tgtcctgaaa 780

ctgcaactag actgcagacc cctagaaggc aacagcacag ttactggaca accgaggcgg 840

ctcttggaca cagcaggaca ccagcagccc ttcctagagc ttaagatccg agccaatgag 900

cctggagcag gccgggccag gaggaggacc cccacctgtg agcctgcgac ccccttatgt 960

tgcaggcgag accattacgt agacttccag gaactgggat ggcgggactg gatactgcag 1020

cccgaggggt accagctgaa ttactgcagt gggcagtgcc ctccccacct ggctggcagc 1080

ccaggcattg ctgcctcttt ccattctgcc gtcttcagcc tcctcaaagc caacaatcct 1140

tggcctgcca gtacctcctg ttgtgtccct actgcccgaa ggcccctctc tctcctctac 1200

ctggatcata atggcaatgt ggtcaagacg gatgtgccag atatggtggt ggaggcctgt 1260

ggctgcagct agcaagagga cctggggctt tggagtgaag agaccaagat gaagtttccc 1320

aggcacaggg catctgtgac tggaggcatc agattcctga tccacacccc aacccaacaa 1380

ccacctggca atatgactca cttgacccct atgggaccca aatgggcact ttcttgtctg 1440

agactctggc ttattccagg ttggctgatg tgttgggaga tgggtaaagc gtttcttcta 1500

aaggggtcta cccagaaagc atgatttcct gccctaagtc ctgtgagaag atgtcaggga 1560

ctagggaggg agggagggaa ggcagagaaa aattacttag cctctcccaa gatgag 1616

<210> 6

<211> 2445

<212> DNA

<213> homo sapiens (homo sapiens)

<400> 6

agctgtgagg gtcaagcaca gctatccatc agatgatcta ctttcagcct tcctgagtcc 60

cagacaatag aagacaggtg gctgtaccct tggccaaggg taggtgtggc agtggtgtct 120

gctgtcactg tgccctcatt ggcccccagc aatcagactc aacagacgga gcaactgcca 180

tccgaggctc ctgaaccagg gccattcacc aggagcatgc ggctccctga tgtccagctc 240

tggctggtgc tgctgtgggc actggtgcga gcacagggga cagggtctgt gtgtccctcc 300

tgtgggggct ccaaactggc accccaagca gaacgagctc tggtgctgga gctagccaag 360

cagcaaatcc tggatgggtt gcacctgacc agtcgtccca gaataactca tcctccaccc 420

caggcagcgc tgaccagagc cctccggaga ctacagccag ggagtgtggc tccagggaat 480

ggggaggagg tcatcagctt tgctactgtc acagactcca cttcagccta cagctccctg 540

ctcacttttc acctgtccac tcctcggtcc caccacctgt accatgcccg cctgtggctg 600

cacgtgctcc ccacccttcc tggcactctt tgcttgagga tcttccgatg gggaccaagg 660

aggaggcgcc aagggtcccg cactctcctg gctgagcacc acatcaccaa cctgggctgg 720

cataccttaa ctctgccctc tagtggcttg aggggtgaga agtctggtgt cctgaaactg 780

caactagact gcagacccct agaaggcaac agcacagtta ctggacaacc gaggcggctc 840

ttggacacag caggacacca gcagcccttc ctagagctta agatccgagc caatgagcct 900

ggagcaggcc gggccaggag gaggaccccc acctgtgagc ctgcgacccc cttatgttgc 960

aggcgagacc attacgtaga cttccaggaa ctgggatggc gggactggat actgcagccc 1020

gaggggtacc agctgaatta ctgcagtggg cagtgccctc cccacctggc tggcagccca 1080

ggcattgctg cctctttcca ttctgccgtc ttcagcctcc tcaaagccaa caatccttgg 1140

cctgccagta cctcctgttg tgtccctact gcccgaaggc ccctctctct cctctacctg 1200

gatcataatg gcaatgtggt caagacggat gtgccagata tggtggtgga ggcctgtggc 1260

tgcagctagc aagaggacct ggggctttgg agtgaagaga ccaagatgaa gtttcccagg 1320

cacagggcat ctgtgactgg aggcatcaga ttcctgatcc acaccccaac ccaacaacca 1380

cctggcaata tgactcactt gacccctatg ggacccaaat gggcactttc ttgtctgaga 1440

ctctggctta ttccaggttg gctgatgtgt tgggagatgg gtaaagcgtt tcttctaaag 1500

gggtctaccc agaaagcatg atttcctgcc ctaagtcctg tgagaagatg tcagggacta 1560

gggagggagg gagggaaggc agagaaaaat tacttagcct ctcccaagat gagaaagtcc 1620

tcaagtgagg ggaggaggaa gcagatagat ggtccagcag gcttgaagca gggtaagcag 1680

gctggcccag ggtaagggct gttgaggtac cttaagggaa ggtcaagagg gagatgggca 1740

aggcgctgag ggaggatgct taggggaccc ccagaaacag gagtcaggaa aatgaggcac 1800

taagcctaag aagttccctg gtttttccca ggggacagga cccactggga gacaagcatt 1860

tatactttct ttcttctttt ttattttttt gagatcgagt ctcgctctgt caccaggctg 1920

gagtgcagtg acacgatctt ggctcactgc aacctccgtc tcctgggttc aagtgattct 1980

tctgcctcag cctcccgagc agctgggatt acaggcgccc actaattttt gtattcttag 2040

tagaaacgag gtttcaacat gttggccagg atggtctcaa tctcttgacc tcttgatcca 2100

cccgacttgg cctcccgaag tgatgagatt ataggcgtga gccaccgcgc ctggcttata 2160

ctttcttaat aaaaaggaga aagaaaatca acaaatgtga gtcataaaga agggttaggg 2220

tgatggtcca gagcaacagt tcttcaagtg tactctgtag gcttctggga ggtccctttt 2280

caggggtgtc cacaaagtca aagctatttt cataataata ctaacatgtt atttgccttt 2340

tgaattctca ttatcttaaa attgtattgt ggagttttcc agaggccgtg tgacatgtga 2400

ttacatcatc tttctgacat cattgttaaa aaaaaaaaaa aaaaa 2445

<210> 7

<211> 2459

<212> DNA

<213> homo sapiens (homo sapiens)

<400> 7

gggtcaagca cagctatcca tcagatgatc tactttcagc cttcctgagt cccagacaat 60

agaagacagg tggctgtacc cttggccaag ggtaggtgtg gcagtggtgt ctgctgtcac 120

tgtgccctca ttggccccca gcaatcagac tcaacagacg gagcaactgc catccgaggc 180

tcctgaacca gggccattca ccaggagcat gcggctccct gatgtccagc tctggctggt 240

gctgctgtgg gcactggtgc gagcacaggg gacagggtct gtgtgtccct cctgtggggg 300

ctccaaactg gcaccccaag cagaacgagc tctggtgctg gagctagcca agcagcaaat 360

cctggatggg ttgcacctga ccagtcgtcc cagaataact catcctccac cccaggcagc 420

gctgaccaga gccctccgga gactacagcc agggagtgtg gctccaggga atggggagga 480

ggtcatcagc tttgctactg tcacagactc cacttcagcc tacagctccc tgctcacttt 540

tcacctgtcc actcctcggt cccaccacct gtaccatgcc cgcctgtggc tgcacgtgct 600

ccccaccctt cctggcactc tttgcttgag gatcttccga tggggaccaa ggaggaggcg 660

ccaagggtcc cgcactctcc tggctgagca ccacatcacc aacctgggct ggcatacctt 720

aactctgccc tctagtggct tgaggggtga gaagtctggt gtcctgaaac tgcaactaga 780

ctgcagaccc ctagaaggca acagcacagt tactggacaa ccgaggcggc tcttggacac 840

agcaggacac cagcagccct tcctagagct taagatccga gccaatgagc ctggagcagg 900

ccgggccagg aggaggaccc ccacctgtga gcctgcgacc cccttatgtt gcaggcgaga 960

ccattacgta gacttccagg aactgggatg gcgggactgg atactgcagc ccgaggggta 1020

ccagctgaat tactgcagtg ggcagtgccc tccccacctg gctggcagcc caggcattgc 1080

tgcctctttc cattctgccg tcttcagcct cctcaaagcc aacaatcctt ggcctgccag 1140

tacctcctgt tgtgtcccta ctgcccgaag gcccctctct ctcctctacc tggatcataa 1200

tggcaatgtg gtcaagacgg atgtgccaga tatggtggtg gaggcctgtg gctgcagcta 1260

gcaagcagga cctggggctt tggagtgaag agaccaagat gaagtttccc aggcacaggg 1320

catctgtgac tggaggcatc agattcctga tccacacccc aacccaacaa ccacctggca 1380

atatgactca cttgacccct atgggaccca aatgggcact ttcttgtctg agactctggc 1440

ttattccagg ttggctgatg tgttgggaga tgggtaaagc gtttcttcta aaggggtcta 1500

ctcagaaagc atgatttcct gccctaagtc ctgtgagaag atgtcaggga ctagggaggg 1560

agggagggaa ggcagagaaa aattacttag cctctcccaa gatgagaaag tcctcaagtg 1620

aggggaggag gaagcagata gatggtccag caggcttgaa gcagggtaag caggctggcc 1680

cagggtaagg gctgttgagg taccttaagg gaaggtcaag agggagatgg gcaaggcgct 1740

gagggaggat gcttagggga cccccagaaa caggagtcag gaaaatgagg cactaagcct 1800

aagaagttcc ctggtttttc ccaggggaca ggacccactg ggcgacaagc atttatactt 1860

tctttcttct tttttatttt tttgagatcg agtctcgctc tgtcaccagg ctggagtgca 1920

gtgacacgat cttggctcac tgcaacctcc gtctcctggg ttcaagtgat tcttctgcct 1980

cagcctcccg agcagctggg attacaggcg cccactaatt tttgtattct tagtagaaac 2040

gaggtttcaa catgttggcc aggatggtct caatctcttg acctcttgat ccacccgact 2100

tggcctcccg aagtgatgag attataggcg tgagccaccg cgcctggctt atactttctt 2160

aataaaaagg agaaagaaaa tcaacaaatg tgagtcataa agaagggtta gggtgatggt 2220

ccagagcaac agttcttcaa gtgtactctg taggcttctg ggaggtccct tttcaggggt 2280

gtccacaaag tcaaagctat tttcataata atactaacat gttatttgcc ttttgaattc 2340

tcattatctt aaaattgtat tgtggagttt tccaaaggcc gtgtgacatg tgattacatc 2400

atctttctga catcaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 2459

<210> 8

<211> 350

<212> PRT

<213> homo sapiens (homo sapiens)

<400> 8

Met Arg Leu Pro Asp Val Gln Leu Trp Leu Val Leu Leu Trp Ala Leu

1 5 10 15

Val Arg Ala Gln Gly Thr Gly Ser Val Cys Pro Ser Cys Gly Gly Ser

20 25 30

Lys Leu Ala Pro Gln Ala Glu Arg Ala Leu Val Leu Glu Leu Ala Lys

35 40 45

Gln Gln Ile Leu Asp Gly Leu His Leu Thr Ser Arg Pro Arg Ile Thr

50 55 60

His Pro Pro Pro Gln Ala Ala Leu Thr Arg Ala Leu Arg Arg Leu Gln

65 70 75 80

Pro Gly Ser Val Ala Pro Gly Asn Gly Glu Glu Val Ile Ser Phe Ala

85 90 95

Thr Val Thr Asp Ser Thr Ser Ala Tyr Ser Ser Leu Leu Thr Phe His

100 105 110

Leu Ser Thr Pro Arg Ser His His Leu Tyr His Ala Arg Leu Trp Leu

115 120 125

His Val Leu Pro Thr Leu Pro Gly Thr Leu Cys Leu Arg Ile Phe Arg

130 135 140

Trp Gly Pro Arg Arg Arg Arg Gln Gly Ser Arg Thr Leu Leu Ala Glu

145 150 155 160

His His Ile Thr Asn Leu Gly Trp His Thr Leu Thr Leu Pro Ser Ser

165 170 175

Gly Leu Arg Gly Glu Lys Ser Gly Val Leu Lys Leu Gln Leu Asp Cys

180 185 190

Arg Pro Leu Glu Gly Asn Ser Thr Val Thr Gly Gln Pro Arg Arg Leu

195 200 205

Leu Asp Thr Ala Gly His Gln Gln Pro Phe Leu Glu Leu Lys Ile Arg

210 215 220

Ala Asn Glu Pro Gly Ala Gly Arg Ala Arg Arg Arg Thr Pro Thr Cys

225 230 235 240

Glu Pro Ala Thr Pro Leu Cys Cys Arg Arg Asp His Tyr Val Asp Phe

245 250 255

Gln Glu Leu Gly Trp Arg Asp Trp Ile Leu Gln Pro Glu Gly Tyr Gln

260 265 270

Leu Asn Tyr Cys Ser Gly Gln Cys Pro Pro His Leu Ala Gly Ser Pro

275 280 285

Gly Ile Ala Ala Ser Phe His Ser Ala Val Phe Ser Leu Leu Lys Ala

290 295 300

Asn Asn Pro Trp Pro Ala Ser Thr Ser Cys Cys Val Pro Thr Ala Arg

305 310 315 320

Arg Pro Leu Ser Leu Leu Tyr Leu Asp His Asn Gly Asn Val Val Lys

325 330 335

Thr Asp Val Pro Asp Met Val Val Glu Ala Cys Gly Cys Ser

340 345 350

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 9

cgtctgttga gtctgattgc 20

<210> 10

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 10

gacggagcaa ctgccatccg 20

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 11

atcagggagc cgcatgctcc 20

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 12

ctgaaccagg gccattcacc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 13

cctggttcag gagcctcgga 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 14

catccgaggc tcctgaacca 20

<210> 15

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 15

ccatccgagg ctcctgaacc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 16

gccacctgtc ttctattgtc 20

<210> 17

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 17

agccgcatgc tcctggtgaa 20

<210> 18

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 18

gtctgttgag tctgattgct 20

<210> 19

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 19

aagacaggtg gctgtaccct 20

<210> 20

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 20

ctgattgctg ggggccaatg 20

<210> 21

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 21

tgattgctgg gggccaatga 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 22

ccacctgtct tctattgtct 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 23

atgctcctgg tgaatggccc 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 24

ctgttgagtc tgattgctgg 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 25

ctggtgaatg gccctggttc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 26

accactgcca cacctaccct 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> gRNA recognition sequence

<400> 27

tctgttgagt ctgattgctg 20

<210> 28

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> original exon 2

<400> 28

agactccact tcagcctaca gct 23

<210> 29

<211> 23

<212> DNA

<213> Artificial sequence (artificial sequence)

<220>

<223> predictive exon 2

<400> 29

acactccact tcagcctaca gct 23

Claims

1. A method of treating a subject suffering from or at risk of developing a metabolic disorder, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

2. A method of treating a subject having or at risk of developing type 2 diabetes, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

3. A method of treating a subject suffering from or at risk of developing obesity, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

4. A method of treating a subject having or at risk of developing elevated triglyceride levels, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

5. A method of treating a subject suffering from or at risk of developing lipodystrophy, comprising administering to the subject a inhibin subunit βe (INHBE).

6. A method of treating a subject having or at risk of developing liver inflammation, the method comprising administering to the subject a inhibin subunit βe (INHBE).

7. A method of treating a subject suffering from or at risk of developing fatty liver disease, the method comprising administering to the subject a inhibin subunit βe (INHBE).

8. A method of treating a subject suffering from or at risk of developing hypercholesterolemia, the method comprising administering to the subject a inhibin subunit βe (INHBE).

9. A method of treating a subject having elevated liver enzymes or at risk of developing, the method comprising administering to the subject a inhibin subunit βe (INHBE).

10. A method of treating a subject having or at risk of developing non-alcoholic steatohepatitis (NASH), the method comprising administering to the subject a inhibin subunit βe (INHBE).

11. A method of treating a subject suffering from or at risk of developing a cardiovascular disease, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

12. A method of treating a subject suffering from or at risk of developing cardiomyopathy, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

13. A method of treating a subject suffering from or at risk of developing hypertension, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

14. A method of treating a subject suffering from heart failure or at risk of developing, the method comprising administering to the subject an inhibitor of inhibin subunit βe (INHBE).

15. The method of any one of claims 1 to 14, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA.

16. The method of any one of claims 1 to 14, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule.

17. The method of claim 16, wherein the Cas protein is Cas9 or Cpf1.

18. The method of claim 15 or claim 16, wherein the gRNA recognition sequence is located within SEQ ID No. 1.

19. The method of claim 15 or claim 16, wherein a pre-mid region sequence adjacent motif (PAM) sequence is about 2 to about 6 nucleotides downstream of the gRNA recognition sequence.

20. The method of any one of claims 16-19, wherein the gRNA comprises about 17 to about 23 nucleotides.

21. The method according to any one of claims 16 to 19, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 9-27.

22. The method of any one of claims 1 to 21, further comprising detecting the presence or absence of an INHBE variant nucleic acid molecule encoding an INHBE predicted loss of function polypeptide in a biological sample from said subject.

23. The method of claim 22, wherein when the subject is an INHBE reference, the subject is further administered a therapeutic agent that treats or inhibits a metabolic disorder or cardiovascular disease in a standard dose amount.

24. The method of claim 22, wherein when the subject is heterozygous or homozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predictive-function-lost polypeptide, a therapeutic agent that treats or inhibits a metabolic disorder or cardiovascular disease is also administered to the subject in a dose amount equal to or less than a standard dose amount.

25. The method of any one of claims 22 to 24, wherein the INHBE variant nucleic acid molecule is a missense variant, splice site variant, termination gain variant, start loss variant, termination loss variant, frameshift variant or in-frame insertion deletion variant, or a variant encoding a truncated INHBE polypeptide.

26. The method of claim 25, wherein the INHBE variant nucleic acid molecule encodes a truncated INHBE polypeptide.

27. The method of any one of claims 22 to 26, wherein the INHBE variant nucleic acid molecule is a genomic nucleic acid molecule.

28. The method of claim 27, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of the INHBE genomic nucleic acid molecule in the biological sample.

29. The method of any one of claims 22 to 26, wherein the INHBE variant nucleic acid molecule is an mRNA molecule.

30. The method of claim 29, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of the INHBE mRNA molecule in the biological sample.

31. The method of any one of claims 22 to 26, wherein the INHBE variant nucleic acid molecule is a cDNA molecule produced from an mRNA molecule.

32. The method of claim 31, wherein the detecting step comprises sequencing at least a portion of the nucleotide sequence of the INHBE cDNA molecule produced from an mRNA molecule in the biological sample.

33. The method of any one of claims 22 to 32, wherein the detecting step comprises sequencing the entire nucleic acid molecule.

34. A method of treating a subject with a therapeutic agent that treats or inhibits a metabolic disorder, wherein the subject has a metabolic disorder, the method comprising the steps of:

determining whether the subject has an inhibin subunit βe (INHBE) variant nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide by:

obtaining or having obtained a biological sample from the subject; and

genotyping or having been performed on the biological sample to determine whether the subject has a genotype comprising the INHBE variant nucleic acid molecule; and

When the subject is an INHBE reference, then administering or continuing to administer the therapeutic agent that treats or inhibits the metabolic disorder to the subject in a standard dose amount, and administering an INHBE inhibitor to the subject; and

when the subject is heterozygous for an INHBE variant nucleic acid molecule, then administering to the subject or continuing to administer the therapeutic agent that treats or inhibits the metabolic disorder in an amount equal to or less than a standard dose amount, and administering an INHBE inhibitor to the subject;

when the subject is homozygous for the INHBE variant nucleic acid molecule, then administering or continuing to administer the therapeutic agent to treat or inhibit the metabolic disorder to the subject in an amount equal to or less than the standard dose amount;

wherein the presence of a genotype having the INHBE variant nucleic acid molecule encoding an INHBE predictive function loss polypeptide is indicative of a reduced risk of the subject developing the metabolic disorder.

35. The method of claim 34, wherein the subject is an INHBE reference and the subject is administered or continues to be administered the therapeutic agent that treats or inhibits the metabolic disorder and an INHBE inhibitor at a standard dose amount.

36. The method of claim 34, wherein the subject is heterozygous for an INHBE variant nucleic acid molecule and is administered or continues to administer the therapeutic agent that treats or inhibits the metabolic disorder to the subject in an amount equal to or less than a standard dose amount, and an INHBE inhibitor is administered.

37. A method of treating a subject with a therapeutic agent that treats or inhibits a cardiovascular disease, wherein the subject has a cardiovascular disease, the method comprising the steps of:

obtaining or having obtained a biological sample from the subject; and

when the subject is an INHBE reference, then administering or continuing to administer the therapeutic agent that treats or inhibits the cardiovascular disease to the subject in a standard dose amount, and administering an INHBE inhibitor to the subject; and

when the subject is heterozygous for an INHBE variant nucleic acid molecule, then administering to the subject or continuing to administer the therapeutic agent that treats or inhibits the cardiovascular disease in an amount equal to or less than a standard dose amount, and administering an INHBE inhibitor to the subject;

When the subject is homozygous for the INHBE variant nucleic acid molecule, then administering or continuing administration of the therapeutic agent to treat or inhibit the cardiovascular disease to the subject in an amount equal to or less than the standard dose amount;

wherein the presence of a genotype having the INHBE variant nucleic acid molecule encoding an INHBE predictive function loss polypeptide is indicative of a reduced risk of the subject developing the cardiovascular disease.

38. The method of claim 37, wherein the subject is an INHBE reference and the subject is administered or continues to be administered the therapeutic agent that treats or inhibits the cardiovascular disease and an INHBE inhibitor at a standard dose amount.

39. The method of claim 37, wherein the subject is heterozygous for an INHBE variant nucleic acid molecule and is administered or continues to administer the therapeutic agent that treats or inhibits the cardiovascular disease to the subject in an amount equal to or less than a standard dose amount and an INHBE inhibitor.

40. The method of claim 34 or claim 37, wherein the INHBE variant nucleic acid molecule is a genomic nucleic acid molecule.

41. The method of claim 40, wherein said genotyping assay comprises sequencing at least a portion of the nucleotide sequence of said INHBE genomic nucleic acid molecule in said biological sample.

42. The method of claim 34 or claim 37, wherein the INHBE variant nucleic acid molecule is an mRNA molecule.

43. The method of claim 42, wherein said genotyping assay comprises sequencing at least a portion of the nucleotide sequence of said INHBE mRNA molecule in said biological sample.

44. The method of claim 34 or claim 37, wherein the INHBE variant nucleic acid molecule is a cDNA molecule produced from an mRNA molecule.

45. The method of claim 44, wherein said genotyping assay comprises sequencing at least a portion of the nucleotide sequence of said INHBE cDNA molecule produced from an mRNA molecule in said biological sample.

46. The method of claim 34 or claim 37, wherein the INHBE variant nucleic acid molecule is a missense variant, splice site variant, termination gain variant, initiation loss variant, termination loss variant, frameshift variant, or in-frame insertion deletion variant, or a variant encoding a truncated INHBE polypeptide.

47. The method of claim 34 or claim 37, wherein the INHBE variant nucleic acid molecule encodes a truncated INHBE polypeptide.

48. The method of any one of claims 34 to 47, wherein the genotyping assay comprises sequencing the entire nucleic acid molecule in the biological sample.

49. The method of any one of claims 34 to 48, wherein the genotyping assay comprises:

a) Amplifying at least a portion of the nucleic acid molecule encoding the INHBE polypeptide;

b) Labeling the amplified nucleic acid molecules with a detectable label;

c) Contacting the labeled nucleic acid molecules with a support comprising a probe that alters the specificity; and

d) Detecting the detectable label.

50. The method of claim 49, wherein the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed to cDNA prior to the amplifying step.

51. The method of any one of claims 34 to 48, wherein the genotyping assay comprises:

contacting the nucleic acid molecules in the biological sample with a change-specific probe comprising a detectable label; and

detecting the detectable label.

52. The method of any one of claims 34 to 51, wherein the nucleic acid molecule is present within a cell obtained from the subject.

53. The method of any one of claims 34 to 52, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA.

54. The method of any one of claims 34 to 52, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule.

55. The method of claim 54, wherein the Cas protein is Cas9 or Cpf1.

56. The method of claim 54 or claim 55, wherein the gRNA recognition sequence is located within SEQ ID NO. 1.

57. The method of claim 54 or claim 55, wherein a pre-mid region sequence adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence.

58. The method of any one of claims 54 to 57, wherein the gRNA comprises about 17 to about 23 nucleotides.

59. The method of any one of claims 54 to 57, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 9-27.

60. The method of any one of claims 34 to 36, wherein the metabolic disorder is type 2 diabetes and the therapeutic agent is selected from metformin, insulin, glibenclamide, glipizide, glimepiride, repaglinide, nateglinide, thiazolidinedione, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, cable Ma Lutai, canagliflozin, dapagliflozin, and engagliflozin, or any combination thereof.

61. The method of any one of claims 34 to 36, wherein the metabolic disorder is obesity and the therapeutic agent is selected from orlistat, phentermine, topiramate, bupropion, naltrexone, and liraglutide, or any combination thereof.

62. The method of any one of claims 34 to 36, wherein the metabolic disorder is elevated triglycerides and the therapeutic agent is selected from rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil, fenofibrate acid, niacin and omega-3 fatty acids, or any combination thereof.

63. The method of any one of claims 34 to 36, wherein the metabolic disorder is lipodystrophy and the therapeutic agent is selected from temorelin, metformin, poly-l-lactic acid, calcium hydroxyapatite, polymethyl methacrylate, bovine collagen, human collagen, silicone and hyaluronic acid, or any combination thereof.

64. The method of any one of claims 34 to 36, wherein the metabolic disorder is liver inflammation and the therapeutic agent is a hepatitis therapeutic agent or a hepatitis vaccine.

65. The method of any one of claims 34 to 36, wherein the metabolic disorder is fatty liver disease and bariatric surgery and/or dietary intervention is administered to the subject.

66. The method of any one of claims 34 to 36, wherein the metabolic disorder is hypercholesterolemia and the therapeutic agent is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, simvastatin, cholestyramine, colesevelam and colestipol, aliskirab, epothilone, nisalopa, nikk, fenofibrate, gemfibrozil Ji Hebei pegapped, or any combination thereof.

67. The method of any one of claims 34 to 36, wherein the metabolic disorder is elevated liver enzymes and the therapeutic agent is selected from coffee, folic acid, potassium, vitamin B6, statin, and fiber, or any combination thereof.

68. The method according to any one of claim 34 to 36, wherein the metabolic disorder is nonalcoholic steatohepatitis (NASH) and the therapeutic agent is obeticholic acid, se Long Se-t, ai Labu-no, cinnivirol, gr_md_02, mgl_3196, IMM124E, eicosanoyl-cholanic acid, GS0976, entikasheng, wo Liba-t, NGM282, GS9674, trapezil, mn_001, LMB763, bi_1467335, msdc_0602, pf_05221304, DF102, sha Luoge-column bundle, BMS986036, lanono, cord Ma Lutai, nitazoxanide, gri_0621, EYP001, VK2809, nalmefene, LIK066, mt_3995 eloxibat, namode son, fo Lei Lushan antibody, SAR425899, sogliflozin, edp_305, esobut, gemcabene, tert_101, kbp_042, pf_06865571, DUR928, pf_06835919, NGM313, bms_986171, nata23 mab, cer_209, nd_l02_s0201, rtu_1096, drx_065, ionis_dgat2rx, int_767, nc_001, serradpa, PXL770, tert_201, NV556, AZD2693, sp_1373, VK0214, hepatan, TGFTX4, rl112bn 7, gkt_137831, RYI _018, CB4209-CB4211, and jh_0920.

69. The method of any one of claims 34 to 36, wherein the therapeutic agent that treats or inhibits the metabolic disorder is a melanocortin 4 receptor (MC 4R) agonist.

70. The method of claim 69, wherein the MC4R agonist comprises a protein, peptide, nucleic acid molecule, or small molecule.

71. The method of claim 69, wherein the protein is a peptide analog of MC 4R.

72. The method of claim 71, wherein the peptide is semaphorin.

73. The method of claim 69, wherein the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp.

74. The method of claim 70, wherein the small molecule is 1,2,3R, 4-tetrahydroisoquinoline-3-carboxylic acid.

75. The method of claim 69, wherein the MC4R agonist is ALB-127158 (a).

76. The method of any one of claims 37-39, wherein the cardiovascular disease is hypertension and the therapeutic agent is selected from the group consisting of chlorthalidone, chlorthiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol, betaxolol fumarate, cartilalol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril maleate, enalapril, lisinopril, moxipril, perindopril, quinapril, ramipril, trandolapril, candesartan, eprosartan, irbesartan, losartan, telmisartan, valsartan, amlodipine besylate, benazepril, diltiazem hydrochloride, felodipine, iridipine, nicardipine, nifedipine, nisoldipine hydrochloride, verapamil, doxazosin hydrochloride, fluzazin, fluvoxazin, tolidine hydrochloride, fluvoxazin, tolidine hydrochloride, and any combination thereof.

77. The method of any one of claims 37-39, wherein the cardiovascular disease is cardiomyopathy and the therapeutic agent is an ACE inhibitor, an angiotensin II receptor blocker, a beta blocker, a calcium channel blocker, digoxin, an antiarrhythmic, an aldosterone blocker, a diuretic, an anticoagulant, a blood diluent, and a corticosteroid.

78. The method of any one of claims 37-39, wherein the cardiovascular disease is heart failure and the therapeutic agent is an ACE inhibitor, an angiotensin 2 receptor blocker, a beta blocker, a mineralocorticoid receptor antagonist, a diuretic, ivabradine, sarcandesartan, nitrate-containing hydralazine, and digoxin.

79. A method of identifying a subject at increased risk of developing a metabolic disorder, wherein the method comprises:

determining or having determined the presence or absence of an inhibin subunit beta E (INHBE) variant nucleic acid molecule encoding an INHBE predicted loss of function polypeptide in a biological sample obtained from said subject;

wherein:

when the subject is an INHBE reference, then the subject is at increased risk of developing the metabolic disorder; and is also provided with

When the subject is heterozygous or homozygous for the INHBE variant nucleic acid molecule, then the subject is at reduced risk of developing the metabolic disorder.

80. A method of identifying a subject at increased risk of developing a cardiovascular disease, wherein the method comprises:

wherein:

when the subject is an INHBE reference, then the subject is at increased risk of developing the cardiovascular disease; and is also provided with

When the subject is heterozygous or homozygous for the INHBE variant nucleic acid molecule, then the subject's risk of developing the cardiovascular disease is reduced.

81. The method of claim 79 or claim 80, wherein the INHBE variant nucleic acid molecule is a genomic nucleic acid molecule.

82. The method of claim 81, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the INHBE genomic nucleic acid molecule in the biological sample.

83. The method of claim 79 or claim 80, wherein the INHBE variant nucleic acid molecule is an mRNA molecule.

84. The method of claim 83, wherein the determining step comprises sequencing at least a portion of the nucleotide sequence of the INHBE mRNA molecule in the biological sample.

85. The method of claim 79 or claim 80, wherein the INHBE variant nucleic acid molecule is a cDNA molecule produced from an mRNA molecule.

86. The method of claim 85 wherein said determining step comprises sequencing at least a portion of the nucleotide sequence of said INHBE cDNA molecule produced from an mRNA molecule in said biological sample.

87. The method of claim 79 or claim 80, wherein the INHBE variant nucleic acid molecule is a missense variant, splice site variant, termination gain variant, initiation loss variant, termination loss variant, frameshift variant, or in-frame insertion deletion variant, or a variant encoding a truncated INHBE polypeptide.

88. The method of claim 79 or claim 80, wherein the INHBE variant nucleic acid molecule encodes a truncated INHBE polypeptide.

89. The method of any one of claims 79 to 88, wherein the determining step comprises sequencing the entire nucleic acid molecule in the biological sample.

90. The method of any one of claims 79 to 89, wherein the determining step comprises:

b) Labeling the amplified nucleic acid molecules with a detectable label;

d) Detecting the detectable label.

91. The method of claim 90, wherein the nucleic acid molecule in the sample is mRNA and the mRNA is reverse transcribed to cDNA prior to the amplifying step.

92. The method of any one of claims 79 to 89, wherein the determining step comprises:

detecting the detectable label.

93. The method of any one of claims 79 to 92, wherein the determining step is performed in vitro.

94. The method of any one of claims 79 to 93, wherein the subject is an INHBE reference and the subject is administered a therapeutic agent that treats or inhibits the metabolic disorder or cardiovascular disease and an INHBE inhibitor in a standard dose amount.

95. The method of any one of claims 79 to 93, wherein the subject is heterozygous for an INHBE variant nucleic acid molecule encoding an INHBE-predictive loss-of-function polypeptide, and a therapeutic agent that treats or inhibits the metabolic disorder or cardiovascular disease is administered to the subject in an amount equal to or less than a standard dose amount, and an INHBE inhibitor is administered.

96. The method of claim 94 or claim 95, wherein the INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA.

97. The method of claim 94 or claim 95, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule.

98. The method of claim 97, wherein the Cas protein is Cas9 or Cpf1.

99. The method of claim 97 or claim 98, wherein the gRNA recognition sequence is located within SEQ ID No. 1.

100. The method of claim 97 or claim 98, wherein a pre-mid region sequence adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence.

101. The method of any one of claims 97-100, wherein the gRNA comprises about 17 to about 23 nucleotides.

102. The method of any one of claims 97-100, wherein the gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 9-27.

103. The method of claim 94 or claim 95, wherein the metabolic disorder is type 2 diabetes and the therapeutic agent is selected from the group consisting of metformin, insulin, glibenclamide, glipizide, glimepiride, repaglinide, nateglinide, thiazolidinedione, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, cable Ma Lutai, canagliflozin, dapagliflozin, and engagliflozin, or any combination thereof.

104. The method of claim 94 or claim 95, wherein the metabolic disorder is obesity and the therapeutic agent is selected from the group consisting of orlistat, phentermine, topiramate, bupropion, naltrexone, and liraglutide, or any combination thereof.

105. The method of claim 94 or claim 95, wherein the metabolic disorder is elevated triglycerides and the therapeutic agent is selected from rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil, fenofibrate, niacin, and omega-3 fatty acids, or any combination thereof.

106. The method of claim 94 or claim 95, wherein the metabolic disorder is lipodystrophy and the therapeutic agent is selected from temorelin, metformin, poly-l-lactic acid, calcium hydroxyapatite, polymethyl methacrylate, bovine collagen, human collagen, silicone, and hyaluronic acid, or any combination thereof.

107. The method of claim 94 or claim 95, wherein the metabolic disorder is liver inflammation and the therapeutic agent is a hepatitis therapeutic agent or a hepatitis vaccine.

108. The method of claim 94 or claim 95, wherein the metabolic disorder is fatty liver disease and bariatric surgery and/or dietary intervention is administered to the subject.

109. The method of claim 94 or claim 95, wherein the metabolic disorder is hypercholesterolemia and the therapeutic agent is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, simvastatin, cholestyramine, colesevelam and colestipol, aliskirab, epothilone, nisalopa, nikk, fenofibrate, gemfibrozil Ji Hebei pegapped, or any combination thereof.

110. The method of claim 94 or claim 95, wherein the metabolic disorder is elevated liver enzymes and the therapeutic agent is selected from the group consisting of coffee, folic acid, potassium, vitamin B6, statin, and fiber, or any combination thereof.

111. The method of claim 94 or claim 95, wherein the metabolic disorder is nonalcoholic steatohepatitis (NASH) and the therapeutic agent is obeticholic acid, se Long Se-t, ai Labu-no, cinnivirol, gr_md_02, mgl_3196, IMM124E, eicosanoyl-cholanic acid, GS0976, entikasheng, wo Liba-t, NGM282, GS9674, trapezil, mn_001, LMB763, bi_1467335, msdc_0602, pf_05221304, DF102, sha Luoge-column bundle, BMS986036, lanono, cord Ma Lutai, nitazoxanide, gri_0621, EYP001, VK2809, nalmefene, LIK066, mt_3995 eloxibat, namode son, fo Lei Lushan antibody, SAR425899, sogliflozin, edp_305, esobut, gemcabene, tert_101, kbp_042, pf_06865571, DUR928, pf_06835919, NGM313, bms_986171, nata23 mab, cer_209, nd_l02_s0201, rtu_1096, drx_065, ionis_dgat2rx, int_767, nc_001, serradpa, PXL770, tert_201, NV556, AZD2693, sp_1373, VK0214, hepatan, TGFTX4, rl112bn 7, gkt_137831, RYI _018, CB4209-CB4211, and jh_0920.

112. The method of claim 94 or claim 95, wherein the therapeutic agent that treats or inhibits the metabolic disorder is a melanocortin 4 receptor (MC 4R) agonist.

113. The method of claim 112, wherein the MC4R agonist comprises a protein, peptide, nucleic acid molecule, or small molecule.

114. The method of claim 113, wherein the protein is a peptide analog of MC 4R.

115. The method of claim 113, wherein the peptide is semaphorin.

116. The method of claim 112, wherein the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp.

117. The method of claim 113, wherein the small molecule is 1,2,3r, 4-tetrahydroisoquinoline-3-carboxylic acid.

118. The method of claim 112, wherein the MC4R agonist is ALB-127158 (a).

119. The method of claim 94 or claim 95, wherein the cardiovascular disease is hypertension and the therapeutic agent is selected from the group consisting of chlorthalidone, chlorthiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol, betaxolol, bisoprolol fumarate, cartilalol hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, pegaptanol, quinapril hydrochloride, ramipril, trandolapril, candesartan, eprosartan, losartan, telmisartan, valsartan, amlodipine besylate, benazepril, diltiazepine hydrochloride, felodipine, verapamil hydrochloride, doxazosin hydrochloride, fluzazoxazine, methylxazoxazin, fluvoxaglide, fluzamide hydrochloride, fluvoxazin, fluvoxaglide, guanadine hydrochloride, and any combination thereof.

120. The method of claim 94 or claim 95, wherein the cardiovascular disease is cardiomyopathy and the therapeutic agents are ACE inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, digoxin, antiarrhythmic, aldosterone blockers, diuretics, anticoagulants, blood diluents, and corticosteroids.

121. The method of claim 94 or claim 95, wherein the cardiovascular disease is heart failure and the therapeutic agent is an ACE inhibitor, an angiotensin 2 receptor blocker, a beta blocker, a mineralocorticoid receptor antagonist, a diuretic, ivabradine, sarcobratic, sarcandesartan, nitrate-containing hydralazine, and digoxin.

122. A therapeutic agent for treating or inhibiting a metabolic disorder for treating the metabolic disorder in a subject having:

an INHBE variant genomic nucleic acid molecule encoding an inhibin subunit βe (INHBE) predicted loss-of-function polypeptide;

an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or (b)

An INHBE variant cDNA molecule encoding an INHBE predicted loss of function polypeptide.

123. The therapeutic agent of claim 122, wherein the metabolic disorder is type 2 diabetes and the therapeutic agent is selected from the group consisting of metformin, insulin, glibenclamide, glipizide, glimepiride, repaglinide, nateglinide, thiazolidinedione, rosiglitazone, pioglitazone, sitagliptin, saxagliptin, linagliptin, exenatide, liraglutide, so Ma Lutai, canagliptin, dapagliflozin, and engagliflozin, or any combination thereof.

124. The therapeutic agent of claim 122, wherein the metabolic disorder is obesity and the therapeutic agent is selected from orlistat, phentermine, topiramate, bupropion, naltrexone, and liraglutide, or any combination thereof.

125. The therapeutic agent of claim 122, wherein the metabolic disorder is elevated triglycerides and the therapeutic agent is selected from rosuvastatin, simvastatin, atorvastatin, fenofibrate, gemfibrozil, fenofibrate acid, nicotinic acid, and omega-3 fatty acids, or any combination thereof.

126. The method of claim 122, wherein the metabolic disorder is lipodystrophy and the therapeutic agent is selected from temorelin, metformin, poly-l-lactic acid, calcium hydroxyapatite, polymethyl methacrylate, bovine collagen, human collagen, silicone, and hyaluronic acid, or any combination thereof.

127. The method of claim 122, wherein the metabolic disorder is liver inflammation and the therapeutic agent is a hepatitis therapeutic agent or a hepatitis vaccine.

128. The method of claim 122, wherein the metabolic disorder is fatty liver disease and bariatric surgery and/or dietary intervention is administered to the subject.

129. The method of claim 122, wherein the metabolic disorder is hypercholesterolemia and the therapeutic agent is selected from the group consisting of: atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin calcium, simvastatin, cholestyramine, colesevelam and colestipol, aliskirab, epothilone, nisalopa, nikk, fenofibrate, gemfibrozil Ji Hebei pegapped, or any combination thereof.

130. The method of claim 122, wherein the metabolic disorder is elevated liver enzymes and the therapeutic agent is selected from coffee, folic acid, potassium, vitamin B6, statins, and fiber, or any combination thereof.

131. The method according to claim 122, wherein the metabolic disorder is nonalcoholic steatohepatitis (NASH) and the therapeutic agent is obeticholic acid, se Long Se-t, ai Labu-no, cinnivirol, gr_md_02, mgl_3196, IMM124E, eicosanoyl-cholanic acid, GS0976, entikasheng, wo Liba-t, NGM282, GS9674, trapezil, mn_001, LMB763, bi_1467335, msdc_0602, pf_05221304, DF102, sha Luoge-column bundle, BMS986036, lanono, cord Ma Lutai, nitazoxanide, gri_0621, EYP001, VK2809, nalmefene, LIK066, mt_3995 eloxibat, namode son, fo Lei Lushan antibody, SAR425899, sogliflozin, edp_305, esobut, gemcabene, tert_101, kbp_042, pf_06865571, DUR928, pf_06835919, NGM313, bms_986171, nata23 mab, cer_209, nd_l02_s0201, rtu_1096, drx_065, ionis_dgat2rx, int_767, nc_001, serradpa, PXL770, tert_201, NV556, AZD2693, sp_1373, VK0214, hepatan, TGFTX4, rl112bn 7, gkt_137831, RYI _018, CB4209-CB4211, and jh_0920.

132. The therapeutic agent according to claim 122, wherein the therapeutic agent that treats or inhibits the metabolic disorder is a melanocortin 4 receptor (MC 4R) agonist.

133. The therapeutic agent according to claim 132, wherein the MC4R agonist comprises a protein, peptide, nucleic acid molecule or small molecule.

134. The therapeutic of claim 133, wherein the protein is a peptide analog of MC 4R.

135. The therapeutic agent according to claim 133, wherein the peptide is semaphorin.

136. The therapeutic agent of claim 132, wherein the MC4R agonist is a peptide comprising the amino acid sequence His-Phe-Arg-Trp.

137. The therapeutic agent according to claim 133, wherein the small molecule is 1,2,3r, 4-tetrahydroisoquinoline-3-carboxylic acid.

138. The therapeutic agent of claim 132, wherein the MC4R agonist is ALB-127158 (a).

139. A therapeutic agent for treating or inhibiting a cardiovascular disease for treating the cardiovascular disease in a subject, the subject having:

140. The therapeutic agent of claim 139, wherein the cardiovascular disease is hypertension and the therapeutic agent is selected from the group consisting of chlorthalidone, chlorthiazide, hydrochlorothiazide, indapamide, metolazone, acebutolol, atenolol, betaxolol, bisoprolol fumarate, carboplatin hydrochloride, metoprolol tartrate, metoprolol succinate, nadolol, benazepril hydrochloride, captopril, enalapril maleate, fosinopril sodium, lisinopril, moexipril, pegapped, quinapril hydrochloride, ramipril, trandolapril, candesartan, eprosartan, losartan, telmisartan, valsartan, amlodipine besylate, benazepril, diltiazepine hydrochloride, felodipine hydrochloride, verapamil, doxazosin mesylate, terazosin, methylxazosin, fabazamate hydrochloride, favoxagliclazide, guanadine hydrochloride, guanafidamide hydrochloride, and any combination thereof.

141. The therapeutic agent of claim 139, wherein the cardiovascular disease is cardiomyopathy and the therapeutic agent is ACE inhibitors, angiotensin II receptor blockers, beta blockers, calcium channel blockers, digoxin, anti-arrhythmics, aldosterone blockers, diuretics, anticoagulants, blood diluents, and corticosteroids.

142. The therapeutic agent of claim 139, wherein the cardiovascular disease is heart failure and the therapeutic agent is an ACE inhibitor, an angiotensin 2 receptor blocker, a beta blocker, a mineralocorticoid receptor antagonist, a diuretic, ivabradine, sarabiraterone, nitrate-containing hydralazine, and digoxin.

143. A inhibin subunit βe (INHBE) inhibitor for use in treating or inhibiting a metabolic disorder, for treating the metabolic disorder in a subject having:

144. A inhibin subunit βe (INHBE) inhibitor for use in the treatment and/or prophylaxis of a metabolic disorder in a subject, said subject:

a) Is a reference to an INHBE genomic nucleic acid molecule, an INHBE mRNA molecule, or an INHBE cDNA molecule; or (b)

b) The following are heterozygous:

i) An INHBE variant genomic nucleic acid molecule encoding an INHBE predicted loss-of-function polypeptide;

ii) an INHBE variant mRNA molecule encoding an INHBE predicted loss-of-function polypeptide; or (b)

iii) An INHBE variant cDNA molecule encoding an INHBE predicted loss of function polypeptide.

145. The INHBE inhibitor according to claim 144, wherein said INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA.

146. The INHBE inhibitor of claim 144, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule.

147. The INHBE inhibitor of claim 146, wherein the Cas protein is Cas9 or Cpf1.

148. The INHBE inhibitor of claim 146 or claim 147, wherein said gRNA recognition sequence is located within SEQ ID No. 1.

149. The INHBE inhibitor of claim 146 or claim 147, wherein a prodomain sequence adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence.

150. The INHBE inhibitor of any one of claims 146-149, wherein the gRNA comprises about 17 to about 23 nucleotides.

151. The INHBE inhibitor according to any one of claims 146-149, wherein said gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 9-27.

152. A inhibin subunit βe (INHBE) inhibitor for use in the treatment or inhibition of a cardiovascular disease in a subject having:

153. A inhibin subunit βe (INHBE) inhibitor for use in the treatment and/or prophylaxis of cardiovascular disease in a subject, said subject:

b) The following are heterozygous:

154. The INHBE inhibitor of claim 153, wherein said INHBE inhibitor comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), or short hairpin RNA (shRNA) that hybridizes to INHBE mRNA.

155. The INHBE inhibitor of claim 153, wherein the INHBE inhibitor comprises a Cas protein and a guide RNA (gRNA) that hybridizes to a gRNA recognition sequence within an INHBE genomic nucleic acid molecule.

156. The INHBE inhibitor of claim 155, wherein the Cas protein is Cas9 or Cpf1.

157. The INHBE inhibitor according to claim 155 or claim 156 wherein said gRNA recognition sequence is located within SEQ ID No. 1.

158. The INHBE inhibitor according to claim 155 or claim 156 wherein a pre-mid region sequence adjacent motif (PAM) sequence is about 2 to 6 nucleotides downstream of the gRNA recognition sequence.

159. The INHBE inhibitor according to any one of claims 155-158, wherein said gRNA comprises about 17 to about 23 nucleotides.

160. The INHBE inhibitor according to any one of claims 155-158, wherein said gRNA recognition sequence comprises a nucleotide sequence according to any one of SEQ ID NOs 9-27.