Treatment of muscle disorders with follicin interacting protein 1 (fnIP1) inhibitors and / or follicin (FLCN) inhibitors

Abstract

The present disclosure is generally directed to treating a subject having a muscle disorder or at risk of developing a muscle disorder by administering a follicin interacting protein 1 (FNIP1) inhibitor and / or a follicin (FLCN) inhibitor to the subject.

Description

[0001] References to sequence lists This application includes a sequence list submitted electronically as an XML file, named 381204286SEQ, created on August 21, 2024, and measuring 106,382 bytes. This sequence list is incorporated herein by reference. Technical Field

[0002] This disclosure generally relates to treating subjects with muscular dystrophy or at risk of developing muscular dystrophy by administering follicle-interacting protein 1 (FNIP1) inhibitors and / or follicle-coated protein (FLCN) inhibitors, and to methods for identifying subjects at increased risk of developing muscular dystrophy. Background Technology

[0003] Causes of muscle disorders include: injury or overuse, such as sprains or strains, spasms, or tendinitis; hereditary conditions, such as muscular dystrophy; certain cancers; inflammation, such as myositis, nerve diseases affecting muscles; infections; and certain medications. Muscle disorders include sarcopenia, Duchenne muscular dystrophy, and Pompe disease.

[0004] Follicle-stimulating hormone (FLCN) is encoded by a 25 kb gene located at 17p11.2. FLCN, a 579-amino acid long, is a 64 kDa multifunctional protein involved in cellular responses to amino acid availability and the regulation of glycolysis. Specifically, FLCN regulates the mTORC1 signaling cascade, controls the MiT / TFE factors TFEB and TFE3 in the cellular response to amino acid availability, and regulates glycolysis by binding to lactate dehydrogenase LDHA, acting as a non-competitive inhibitor. Furthermore, FLCN activates mTORC1 by stimulating the hydrolysis of GTP by RRAGC / RagC or RRAGC / RagD, acting as a GTPase activator, promoting the conversion of RRAGC / RagC or RRAGC / RagD to the GDP-bound state, and thus activating the kinase activity of mTORC1.

[0005] Follicle-interacting protein 1 (FNIP1) is encoded by a 155 kb gene located at 5q31.1. FNIP1 is a 130 kDa protein of 1166 amino acids that participates in the regulation of cellular metabolism and nutrient sensing by targeting AMP-activated protein kinase (AMPK) and the rapamycin signaling pathway. Furthermore, FNIP1 binds to the tumor suppressor protein follicle-90 and associates with the molecular chaperone heat shock protein-90 (Hsp90), negatively regulating its ATPase activity by promoting its association with follicle-90. Summary of the Invention

[0006] This disclosure provides a method for treating a subject with or at risk of developing a muscle disorder, the method comprising administering an FNIP1 inhibitor and / or an FLCN inhibitor to the subject.

[0007] This disclosure also provides a method for treating a subject with or at risk of developing myopathy by administering a myopathy treatment agent, the method comprising: determining, or having determined, whether the subject possesses an FNIP1 variant nucleic acid molecule and / or an FLCN variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject; performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype containing an FNIP1 variant nucleic acid molecule and / or an FLCN variant nucleic acid molecule; administering or continuing to administer a standard dose of the myopathy treatment agent, and / or administering it to the subject as an FNIP1 reference and / or FLCN reference. Administering FNIP1 inhibitors and / or FLCN inhibitors; administering or continuing to administer a myopathy treatment agent at the same or less than the standard dose; and / or administering FNIP1 inhibitors and / or FLCN inhibitors to subjects who are heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules; or administering or continuing to administer a myopathy treatment agent at the same or less than the standard dose to subjects who are homozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules; wherein the presence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules indicates a reduced risk of myopathy in the subject.

[0008] This disclosure also provides a method for identifying subjects with an increased risk of developing myopathy, the method comprising: determining, or having determined, the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules in a biological sample obtained from the subject; wherein: when the subject is an FNIP1 reference and / or FLCN reference, the subject has an increased risk of developing myopathy; and when the subject is heterozygous or homozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, the subject has a decreased risk of developing myopathy.

[0009] This disclosure also provides a muscle disease treatment agent for treating or preventing muscle diseases in subjects with FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules.

[0010] This disclosure also provides an FNIP1 inhibitor for treating or preventing muscle disorders in a subject who is FNIP1 reference or heterozygous for an FNIP1 variant nucleic acid molecule.

[0011] This disclosure also provides FLCN inhibitors for treating or preventing muscle disorders in subjects who are FLCN references or heterozygous for FLCN variant nucleic acid molecules. Attached Figure Description

[0012] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the Patent Office will provide a published copy of this patent or application with the color drawing.

[0013] Figure 1 This study demonstrates the identification of rare FNIP1 coding variants in ExWAS associated with blood lipids and liver enzymes; the maximum effect was observed at lower triglycerides and ALT.

[0014] Figure 2 The results showed that genome-wide association analysis identified a common missense variant (5:131672501:T:C--Gln648Arg) in FNIP1 that was associated with the TGL:HDL ratio.

[0015] Figure 3 The study showed an association between rare coding variants of FNIP1 and higher HDLC and lower triglyceride and LDLC levels; FLCN showed a similar association pattern.

[0016] Figure 4 Rare coding variants of FNIP1 and FLCN were shown to be associated with lower ALT levels; FLCN pLoF was associated with lower MRI-derived liver fat and inflammation.

[0017] Figure 5 The effects of rare coding variants of FNIP1 and FLCN on ALT and PDFF are shown.

[0018] Figure 6 This shows the association between rare FNIP1 coding variants and lower NALD odds.

[0019] Figure 7 The association between common FNIP1 missense variants and liver enzymes was shown.

[0020] Figure 8 The study showed that rare coding variants of FNIP1 and FLCN were associated with lower waist-to-hip ratio and body fat percentage.

[0021] Figure 9 The effects of rare variants of FNIP1 on body fat distribution were shown.

[0022] Figure 10 The study showed an association between rare coding variants of FNIP1 and FLCN and lower HbA1c levels and a lower chance of developing type 2 diabetes.

[0023] Figure 11 The association between rare coding variants of FNIP1 and FLCN and lower eGFR is shown.

[0024] Figure 12 The results showed that FNIP1 pLoF has a general protective cardiovascular metabolic association.

[0025] Figure 13 The results showed that FNIP1 pLoF has a general protective cardiovascular metabolic association.

[0026] Figure 14 The association between common missense variants of FNIP1 and the rare coding of FNIP1 is shown to be similar.

[0027] Figure 15 The study showed that common missense variants of FNIP1 were most strongly associated with lower T2D probability.

[0028] Figure 16 The study showed that inhibition of FNIP1 or FLCN in skeletal muscle induced an increase in gene expression in oxidative muscle fibers and mitochondria; AAVmyo2A was purchased from SignaGen Laboratories (catalog number SL100862).

[0029] Figure 17 The study showed that inhibition of FNIP1 or FLCN in skeletal muscle enhanced vascularization and muscle glycogen content.

[0030] Figure 18 The study showed that FLCN inhibition in skeletal muscle induced an increase in oxidized muscle fibers, enhanced vascular distribution, and increased glycogen content. Detailed Implementation

[0031] Various terms relating to aspects of this disclosure are used throughout the specification and claims. Unless otherwise specified, such terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0032] Unless otherwise expressly stated, it is never intended that any method or aspect stated herein require its steps to be performed in a particular order. Therefore, in the claims or description, where a method claim does not explicitly state that the steps are limited to a particular order, it is never intended in any way to infer the order. This applies to any possible non-expressive basis of interpretation, including logical matters relative to the arrangement of steps or the flow of operations, the general meaning derived from grammatical organization or punctuation, or the numbering or type of aspects described in the description.

[0033] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural referents.

[0034] As used herein, the term "about" means that the listed values ​​are approximate and that small variations will not significantly affect the practice of the disclosed embodiments. When using numerical values, unless the context otherwise indicates, the term "about" means that the values ​​may vary by ±10% and remain within the range of the disclosed embodiments.

[0035] As used herein, in certain embodiments, the term "comprising" may be replaced with "consisting of" or "substantially composed of" as needed.

[0036] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid sequence,” “polynucleotide,” or “oligonucleotide” can include polymeric forms of nucleotides of any length, can include DNA and / or RNA, and can be single-stranded, double-stranded, or multi-stranded. One strand of a nucleic acid also refers to its complementary sequence.

[0037] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cattle, and pigs), companion animals (e.g., dogs and cats), laboratory animals (e.g., mice, rats, and rabbits), and non-human primates. In some embodiments, the subject is a human. In some embodiments, the human is a patient under the care of a physician.

[0038] According to this disclosure, rare FNIP1 variant nucleic acid molecules and rare FLCN variant nucleic acid molecules (whether these variants are homozygous or heterozygous in a particular subject) have been observed to be associated with a reduced risk of developing myopathy. It is believed that FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules are not associated with myopathy in humans. Therefore, subjects who are heterozygous for FNIP1 and / or FLCN as a reference or for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules can be treated with FNIP1 inhibitors and / or FLCN inhibitors to suppress or prevent myopathy, alleviate or prevent its symptoms, and / or suppress or prevent the development of symptoms. It is also believed that such subjects with myopathy can be further treated with one or more myopathy therapeutic agents. Additionally, this disclosure provides methods for identifying or stratifying the risk of such subjects having myopathy using the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules in subjects, or methods for diagnosing subjects as having an increased risk of developing myopathy.

[0039] For the purposes of this disclosure, any particular subject, such as a human, may be classified as having one of three PARP1 genotypes: i) FNIP1 reference; ii) heterozygous for an FNIP1 variant nucleic acid molecule; or iii) homozygous for an FNIP1 variant nucleic acid molecule. A subject is an FNIP1 reference when it has no copy of an FNIP1 variant nucleic acid molecule. A subject is heterozygous for an FNIP1 variant nucleic acid molecule when it has a single copy. A subject is homozygous for an FNIP1 variant nucleic acid molecule when it has two copies.

[0040] For the purposes of this disclosure, any particular subject, such as a human, may be classified as having one of three FLCN genotypes: i) FLCN reference; ii) heterozygous for the FLCN variant nucleic acid molecule; or iii) homozygous for the FLCN variant nucleic acid molecule. A subject is FLCN reference when it has no copy of the FLCN variant nucleic acid molecule. A subject is heterozygous for the FLCN variant nucleic acid molecule when it has a single copy. A subject is homozygous for the FLCN variant nucleic acid molecule when it has two copies.

[0041] In any of the embodiments described herein, the FNIP1 variant nucleic acid molecule can be any nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, or cDNA molecule derived from an mRNA molecule) encoding an FNIP1 variant polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. The subject having a partially (or predictedly partially) lost-function (FNIP1) FNIP1 polypeptide is a meta-allele for FNIP1. In some embodiments, the FNIP1 variant nucleic acid molecule results in reduced or abnormal expression or activity of FNIP1 mRNA or polypeptide. In some embodiments, the FNIP1 variant nucleic acid molecule is associated with a weakened in vitro response to FNIP1 ligands compared to reference FNIP1. In some embodiments, the FNIP1 variant nucleic acid molecule is a splice site variant, a termination gain variant, a start loss variant, a termination loss variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated FNIP1 variant polypeptide. In some embodiments, the FNIP1 variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the FNIP1 variant nucleic acid molecule contains a single nucleotide polymorphism (SNP). In some embodiments, the FNIP1 variant nucleic acid molecule contains a variation in the coding region. In some embodiments, the FNIP1 variant nucleic acid molecule does not contain a variation in the non-coding region, but contains a variation in the splice acceptor region (two bases before the start of any exon except the first exon). In some embodiments, the FNIP1 variant nucleic acid molecule causes or is predicted to cause premature truncation of the FNIP1 polypeptide compared to reference FNIP1. In some embodiments, the FNIP1 predicted loss-of-function or missense variant nucleic acid molecule is a variant that, as predicted by an in vitro prediction algorithm (such as Polyphen, SIFT, or similar algorithms), causes impairment of protein function (therefore, in this case, protection is formed in humans). In some embodiments, the FNIP1 predicted loss-of-function or missense variant nucleic acid molecule is a variant that causes or is predicted to cause a non-synonymous amino acid substitution in the FNIP1 nucleic acid molecule and whose allele frequency is less than 1 / 100 alleles in the population from which subjects are selected. In some implementations, the FNIP1 variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%; or 1 / 1,000 alleles), or any splice site, termination gain, initiation loss, termination loss, frameshift or in-frame insertion / deletion variant, or other frameshift FNIP1 variant.

[0042] In any of the embodiments described herein, the FLCN variant nucleic acid molecule can be any nucleic acid molecule (such as a genomic nucleic acid molecule, mRNA molecule, or cDNA molecule derived from an mRNA molecule) encoding an FLCN variant polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function. The subject having a partially (or predictedly partially) lost-function FLCN polypeptide is a meta-allele for FLCN. In some embodiments, the FLCN variant nucleic acid molecule results in reduced or abnormal expression or activity of FLCN mRNA or polypeptide. In some embodiments, the FLCN variant nucleic acid molecule is associated with a weakened in vitro response to FLCN ligands compared to a reference FLCN. In some embodiments, the FLCN variant nucleic acid molecule is a splice site variant, a termination gain variant, a start loss variant, a termination loss variant, a frameshift variant, an in-frame insertion / deletion variant, or a variant encoding a truncated FLCN variant polypeptide. In some embodiments, the FLCN variant nucleic acid molecule is a missense variant nucleic acid molecule. In some embodiments, the FLCN variant nucleic acid molecule contains a single nucleotide polymorphism (SNP). In some embodiments, the FLCN variant nucleic acid molecule contains variations in the coding region. In some embodiments, the FLCN variant nucleic acid molecule does not contain variations in the non-coding region but contains variations in the splice acceptor region (two bases before the start of any exon except the first exon). In some embodiments, the FLCN variant nucleic acid molecule causes or is predicted to cause premature truncation of the FLCN polypeptide compared to the reference FLCN. In some embodiments, the FLCN-predicted loss-of-function or missense variant nucleic acid molecule is a variant that, as predicted by an in vitro prediction algorithm (such as Polyphen, SIFT, or similar algorithms), causes impairment of protein function (therefore, in this case, protection is formed in humans). In some embodiments, the FLCN-predicted loss-of-function or missense variant nucleic acid molecule is a variant that causes or is predicted to cause non-synonymous amino acid substitutions in the FLCN nucleic acid molecule and whose allele frequency is less than 1 / 100 alleles in the population from which subjects are selected. In some implementations, the FLCN variant nucleic acid molecule is any rare missense variant (allele frequency <0.1%; or 1 / 1,000 alleles), or any splice site, termination gain, initiation loss, termination loss, frameshift or in-frame insertion / deletion variant, or other frameshift FLCN variant.

[0043] In any of the embodiments described herein, the nucleotide sequence of the FNIP1 reference genomic nucleic acid molecule in the GRCh38 / hg38 human genome assembly is used as a reference sequence. The FNIP1 variant genomic nucleic acid molecule may include one or more variants at any position on chromosome 5 (i.e., positions 131,641,714-131,797,017) (see ENSG00000217128.13 and ENST00000510461.6 annotated in the Ensembl database (URL: "http: / / useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000217128;r=5:131641714-131797017;transcript=ENST00000510461.6" on the World Wide Web)). The sequences of the FNIP1 genomic nucleic acid molecules provided in these transcripts are merely exemplary sequences. Other sequences of the FNIP1 genomic nucleic acid molecule are also possible.

[0044] In any of the embodiments described herein, the FNIP1 variant nucleic acid molecule may contain one or more of the following genetic variations in the genomic nucleic acid molecule (referring to the chromosomal positions described in the GRCh38 / hg38 human genome assembly): 5:131644687:A:G, 5:131644689:A:T, 5:131644701:G:A, 5:131644702:C:T, 5:131644707:T:C, 5:131644707:T:G, 5:131644708:A:C, 5:131644717:G:A, 5:131644717:G:T, 5:131644720:T:C, 5:1316447 21:G:C、5:131644725:G:A、5:131644726:C:T、5:131644728:A:G、5:13164 4734:G:A、5:131644735:C:A、5:131644735:C:T、5:131644738:G:C、5:131 644744:G:A、5:131644759:C:T、5:131644761:A:G、5:131647089:C:T、5:1 31647090:C:A、5:131647090:C:T、5:131647091:C:A、5:131647093:A:G、5 :131647093:A:T、5:131647100:C:A、5:131647106:G:C、5:131647107:C:A , 5:131647111:T:C, 5:131647112:T:C, 5:131647117:T:G, 5:131647118:G :A, 5:131647123:C:CG, 5:131647123:C:G, 5:131647123:C:T, 5:13164712 4:G:A, 5:131647124:G:C, 5:131647125:C:T, 5:131647129:T:C, 5:131647 132:C:G、5:131647135:C:G、5:131647145:C:T、5:131647151:G:C、5:1316 47154:T:G、5:131647162:T:C、5:131647165:A:T、5:131647166:A:G、5:13 1647174:T:C、5:131647175:C:T、5:131647183:C:A、5:131647183:C:T、5: 131647184:G:A、5:131647187:C:G、5:131647188:T:G、5:131647190:C:T、5:131647195:T:C、5:131647195:T:G、5:131647196:G:GCATT、5:131647199:T:A、5:131647199:T:C、5:131647202:C:T、5:131647206:C:CTG、5:131651807:T:C、5:131651810:G:A、5:131651810:G:T、5:131651814:C:G、5:131651814:CA:C、5:131651817:GT:G、5:131651818:T:A、5:131651819:T:G、5:131651821:T:C、5:131651824:T:A、5:131651824:T:C、5:131651825:T:C、5:131651828:A:G、5:131651831:G:C、5:131651834:G:T、5:131651834:GAA:G、5:131651837:G:C、5:131651839:G:A、5:131651840:T:C、5:131651842:G:A、5:131651842:G:T、5:131651844:A:T、5:131651849:G:A、5:131651854:T:C、5:131651863:A:G、5:131651866:C:A、5:131651875:A:G、5:131651878:A:G、5:131651879:C:T、5:131651880:T:A、5:131651885:T:C、5:131651887:C:G、5:131651896:T:C、5:131651908:C:T、5:131651909:G:A、5:131651909:G:C、5:131651911:C:G、5:131651914:T:C、5:131651921:T:C、5:131651927:C:T、5:131651930:G:A、5:131651930:G:C、5:131651930:GA:G、5:131651933:C:T、5:131651935:G:C、5:131651944:T:C、5:131651946:C:T、5:131651947:A:C、5:131651947:A:G、5:131651948:T:C、5:131651956:A:G、5:131651963:A:G、5:131651963:A:T、5:131651966:C:G、5:131651966:C:T、5:131651968:G:C、5:131651969:C:T、5:131651972:C:T、5:131651978:T:C、5:131651993:C:G、5:131651995:G:A、5:131651999:G:T、5:131670467:A:G、5:131670468:C:T、5:131670471:C:T、5:131670472:A:T、5:131670473:T:C、5:131670473:T:G、5:131670474:G:T、5:131670476:G:A、5:131670476:G:C、5:131670477:A:C、5:131670482:T:A、5:131670483:C:G、5:131670483:C:T、5:131670487:C:T、5:131670493:A:T、5:131670500:C:A、5:131670500:C:T、5:131670501:G:A、5:131670502:G:C、5:131670503:A:T、5:131670505:C:G、5:131670506:C:T、5:131670510:C:T、5:131670511:A:C、5:131670514:A:C、5:131670519:C:A、5:131670521:A:G、5:131670540:C:G、5:131670543:G:T、5:131670546:C:T、5:131670550:A:AT、5:131670551:G:A、5:131670552:A:G、5:131670554:G:A、5:131670554:G:T、5:131670560:T:C、5:131670561:A:G、5:131670579:T:A、5:131670582:C:T、5:131670585:A:G、5:131670593:A:G、5:131670594:T:C、5:131670596:T:A、5:131670605:A:T、5:131670606:C:G、5:131670606:C:T、5:131670610:A:C、5:131670611:C:A、5:131670611:C:T、5:131670612:T:C、5:131670614:A:G、5:131670618:C:T、5:131670621:T:C、5:131670626:T:C、5:131670632:C:T、5:131671506:C:A、5:131671509:G:A、5:131671509:G:C、5:131671514:G:C、5:131671516:T:C、5:131671522:A:C、5:131671522:A:T、5:131671524:C:T、5:131671527:C:T、5:131671533:C:A、5:131671533:C:G、5:131671533:C:T、5:131671534:C:A、5:131671538:T:A、5:131671539:C:G、5:131671543:T:G、5:131671545:G:C、5:131671548:C:G、5:131671551:C:T、5:131671554:C:T、5:131671557:A:C、5:131671559:T:C、5:131671562:C:T、5:131671569:C:T、5:131671572:G:C、5:131671572:G:T、5:131671574:C:T、5:131671575:T:C、5:131671584:C:T、5:131671586:T:C、5:131671589:C:A、5:131671592:G:A、5:131671595:T:C、5:131671595:T:G、5:131671601:C:T、5:131671604:T:C、5:131671604:T:G、5:131671610:T:C、5:131671610:T:G、5:131671613:C:T、5:131671614:C:T、5:131671617:G:A、5:131671628:G:A、5:131671632:T:C、5:131671634:T:C、5:131671634:T:G、5:131671639:T:G、5:131671646:A:T、5:131671647:T:A、5:131671647:T:C、5:131671647:T:G、5:131671658:G:A、5:131671658:G:C、5:131671659:T:C、5:131671661:C:T、5:131671664:A:C、5:131671665:C:T、5:131671668:C:A、5:131671669:A:C、5:131671670:A:AT、5:131671670:A:T、5:131671673:T:C、5:131671677:T:A、5:131671680:C:T、5:131671683:A: C、5:131671685:C:A、5:131671686:T:C、5:131671691:T:C、5:131671692: T:C、5:131671694:T:A、5:131671697:C:A、5:131671700:T:C、5:13167170 0:TG:T、5:131671701:G:A、5:131671703:G:A、5:131671703:G:C、5:131671 704:G:A、5:131671705:GA:G、5:131671706:A:T、5:131671710:G:T、5:131 671712:A:C、5:131671713:T:C、5:131671713:TG:T、5:131671718:A:G、5: 131671718:A:T、5:131671726:T:A、5:131671732:G:C、5:131671734:C:T、 5:131671740:G:C、5:131671742:G:A、5:131671742:G:T、5:131671745:A:G 、5:131671751:G:A、5:131671751:G:T、5:131671754:T:C、5:131671755:T :A、5:131671758:A:G、5:131671760:G:A、5:131671761:A:T、5:131671762 :A:C、5:131671763:T:C、5:131671764:C:G、5:131671764:C:T、5:1316717 67:G:C、5:131671769:G:A、5:131671770:G:A、5:131671770:G:T、5:131671 772:A:C、5:131671772:A:G、5:131671773:C:A、5:131671775:G:A、5:1316 71776:T:C、5:131671778:T:C、5:131671779:C:G、5:131671779:C:T、5:13 1671780:T:C、5:131671782:T:C、5:131671784:C:T、5:131671788:T:G、5: 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C、5:131698921:A:G、5:131698930:G:C、5:131698932:G:A、5:131698932: G:C、5:131698932:G:T、5:131698933:C:A、5:131698936:C:A、5:13169893 6:C:T、5:131698942:T:C、5:131698944:C:A、5:131698944:C:G、5:131698 944:C:T、5:131698945:G:A、5:131698947:T:A、5:131698950:T:A、5:1316 98950:T:C、5:131698951:A:G、5:131698953:G:A、5:131698959:C:G、5:13 1698963:T:C、5:131698967:A:T、5:131698968:C:A、5:131698968:C:G、5: 131698969:T:C、5:131698972:C:T、5:131698974:T:C、5:131698978:C:G、5 :131698979:TG:T、5:131698981:A:C、5:131698981:A:G、5:131698983:C: T、5:131698984:T:C、5:131698986:C:T、5:131698987:G:A、5:131698991: C:T、5:131698993:T:C、5:131698997:C:G、5:131698998:A:G、5:13169899 9:T:C、5:131699001:G:A、5:131699001:G:T、5:131704062:TA:T、5:131704 072:A:G、5:131704073:T:C、5:131704075:G:A、5:131704079:T:A、5:1317 04081:T:C、5:131704090:T:C、5:131704094:T:C、5:131704096:T:A、5:13 1704099:C:T、5:131704108:A:T、5:131704109:G:C、5:131704111:G:A、5: 131704112:G:A、5:131704114:A:C、5:131704120:G:GA、5:131704124:A:G、5:131704127:A:G、5:131704128:G:T、5:131704130:A:C、5:131704131:T:A、5:131704132:T:A、5:131704135:T:C、5:131704137:A:C、5:131704138:A:G、5:131704138:A:T、5:131704140:T:G、5:131704141:T:G、5:131704142:T:C、5:131704144:T:C、5:131704147:T:C、5:131704153:T:A、5:131704157:C:T、5:131704159:T:A、5:131704160:CT:C、5:131704162:T:C、5:131704165:G:C、5:131704166:A:G、5:131704167:C:G、5:131704171:G:A、5:131704178:T:A、5:131704178:T:C、5:131704181:C:A、5:131704186:A:G、5:131704187:T:C、5:131704193:T:C、5:131704202:T:G、5:131704204:C:T、5:131704205:G:A、5:131704207:A:C、5:131704207:A:G、5:131704208:C:T、5:131704211:T:C、5:131704214:C:T、5:131704216:G:A、5:131704220:T:G、5:131704222:G:A、5:131704222:G:T、5:131704223:G:A、5:131704228:C:G、5:131704228:C:T、5:131704229:A:G、5:131704230:G:C、5:131704231:C:T、5:131704232:T:G、5:131704236:A:T、5:131704237:T:A、5:131704237:T:C、5:131704240:G:A、5:131704250:A:G、5:131704251:G:C、5:131704252:C:T、5:131704259:C:T、5:131704261:A:G、5:131704262:T:C、5:131706409:A:G、5:131706412:A:G、5:131706414:C:T、5:131706418:G:A、5:131706424:C:T、5:131706426:C:G、5:131706426:C:T、5:131706427:C:T、5:131706432:T:C、5:131706438:C:G、5:131706441:G:C、5:131706442:T:C、5:131706445:T:C、5:131706448:G:C、5:131706453:C:T、5:131706454:G:A、5:131706456:C:T、5:131706457:G:A、5:131706462:C:T、5:131706463:G:A、5:131706463:G:T、5:131706465:C:T、5:131706466:G:A、5:131706471:T:C、5:131706480:G:A、5:131706486:C:T、5:131706487:T:A、5:131706489:C:T、5:131706490:G:A、5:131706490:G:C、5:131706491:GGTAA:G、5:131706501:G:A、5:131706502:A:C、5:131706504:T:G、5:131706507:G:C、5:131706508:G:C、5:131706516:A:C、5:131706519:G:A、5:131706519:G:C、5:131706520:G:C、5:131706523:T:A、5:131706523:T:C、5:131706524:G:C、5:131706525:A:G、5:131706526:T:C、5:131706541:G:A、5:131706548:T:C、5:131709206:C:T、5:131709207:G:A、5:131709210:C:A、5:131709210:C:G、5:131709210:C:T、5:131709213:T:C、5:131709215:C:T、5:131709216:C:T、5:131709218:C:G、5:131709219:T:A、5:131709219:T:C、5:131709226:G:C、5:131709227:T:C、5:131709228:C:A、5:131709233:T:C、5:131709234:T:C、5:131709234:T:G、5:131709237:G:A、5:131709239:T:C、5:131709240:C:G、5:131709243:T:C、5:131709245:C:T、5:131709248:G: A、5:131709248:G:C、5:131709252:T:C、5:131709258:T:C、5:131709258: T:G、5:131709260:G:C、5:131709269:T:C、5:131709270:G:A、5:13171058 1:C:A、5:131710581:C:T、5:131710582:A:C、5:131710584:A:C、5:131710 593:C:T、5:131710594:G:T、5:131710595:C:T、5:131710600:G:C、5:1317 10604:A:G、5:131710605:G:C、5:131710607:C:T、5:131710608:G:A、5:13 1710610:A:C、5:131710613:G:A、5:131710613:G:T、5:131710614:G:C、5: 131710617:C:T、5:131710618:C:G、5:131710628:A:G、5:131710632:C:G、5 :131710632:C:T、5:131710634:C:T、5:131710635:G:A、5:131710637:C:A 、5:131710637:C:G、5:131710638:T:C、5:131710640:G:A、5:131710640:G :C、5:131710643:C:T、5:131710644:T:C、5:131710647:A:C、5:131710649 :A:T、5:131710653:G:A、5:131710655:G:A、5:131710656:A:G、5:1317165 64:C:G、5:131716566:T:C、5:131716567:A:G、5:131716570:T:C、5:13171 6573:C:A、5:131716582:C:A、5:131716582:C:T、5:131716589:T:C、5:131 716592:C:A, 5:131716592:C:T, 5:131716592:CTG:C, 5:131716600:T:A, 5:131716600:T:C, 5:131716601:T:C, 5:131716606:G:C, 5:131716607:C:A,5:131716607:C:T、5:131716613:A:C、5:131716615:G:T、5:131716615:GT:G、5:131716616:T:C、5:131716617:A:C、5:131716618:T:C、5:131716621:T:C、5:131716622:T:A、5:131716627:T:C、5:131716630:T:A、5:131716630:T:C、5:131716634:T:C、5:131716634:T:G、5:131716638:T:A、5:131716648:T:C、5:131716651:T:G、5:131716652:G:A、5:131716655:G:T、5:131718984:A:T、5:131718985:C:T、5:131718986:G:A、5:131718986:G:C、5:131718986:G:T、5:131718989:T:A、5:131718989:T:C、5:131718989:T:G、5:131718993:G:A、5:131718995:C:T、5:131718998:C:T、5:131719001:C:A、5:131719013:C:T、5:131719014:C:T、5:131719016:G:A、5:131719016:G:C、5:131719019:C:T、5:131719020:G:A、5:131719020:G:C、5:131719025:G:T、5:131719026:T:C、5:131719027:A:AAACACTTTGC、5:131719027:A:C、5:131719031:A:G、5:131719032:C:T、5:131719035:T:G、5:131719041:G:A、5:131719042:C:T、5:131719043:A:C、5:131719044:T:G、5:131719046:A:G、5:131719047:G:C、5:131719048:C:G、5:131719049:T:C、5:131719050:G:A、5:131719052:G:A、5:131719056:G:A、5:131719058:G:A、5:131719058:G:T、5:131719317:C:T、5:131719318:G:A、5:131719318:G:C、5:131719320:A:C、5:131719332:T:C、5:131719339:T:A、5:131719339:T:C、5:131719342:A:G、5:131719348:T:C、5:131719350:T:G、5:131719353:C:A、5:131719356:A:C、5:131719357:T:C、5:131719359:G:A、5:131719363:C:T、5:131719366:A:G、5:131719368:C:A、5:131719368:C:G、5:131719374:A:G、5:131719375:T:C、5:131719388:C:T、5:131719390:T:C、5:131719392:T:C、5:131719393:T:C、5:131719396:C:T、5:131719402:A:G、5:131719404:G:A、5:131719405:A:G、5:131719407:C:A、5:131719410:C:T、5:131719411:G:A、5:131719415:AC:A、5:131719416:C:T、5:131719417:C:G、5:131730903:C:G、5:131730906:G:T、5:131730908:T:C、5:131730909:A:G、5:131730915:G:C、5:131730917:C:T、5:131730921:G:C、5:131730921:G:T、5:131730922:G:C、5:131730922:G:T、5:131730924:C:G、5:131730926:T:A、5:131730927:T:C、5:131730929:A:G、5:131730932:T:C、5:131730933:C:A、5:131730933:C:G、5:131730935:G:C、5:131730936:A:C、5:131730938:G:T、5:131730939:A:G、5:131730945:T:C、5:131730948:C:A、5:131730948:C:G、5:131730948:C:T、5:131730950:G:C、5:131730950:G:T、5:131730950:GAACT:G、5:131730952:A:C、5:131730952:A:T、5:131730953:C:A、5:131730956:T:C、5:131730957:C:A、5:131730959:A:G、5:131730960:A:C、5:131730965:G:C、5:131730968:G:T、5:131730969:A:T、5:131730971:C:G、5:131730971:C:T、5:131730972:T:C、5:131730972:T:G、5:131730974:T:C、5:131730975:C:T、5:131730978:C:A、5:131730978:C:T、5:131730980:C:T、5:131730981:C:T、5:131730983:G:A、5:131730984:G:C、5:131730986:T:G、5:131730987:T:G、5:131730989:A:G、5:131730991:T:G、5:131730992:T:C、5:131730993:G:A、5:131730995:C:T、5:131730999:A:G、5:131731001:T:C、5:131731004:C:A、5:131731004:C:G、5:131731007:A:C、5:131731010:A:C、5:131731011:C:A、5:131731011:C:T、5:131731014:T:C、5:131731016:A:T、5:131731019:T:G、5:131731020:G:A、5:131731022:G:C、5:131731023:C:T、5:131731026:C:T、5:131731028:C:T、5:131731029:T:C、5:131731031:C:A、5:131731035:G:C、5:131731039:C:G、5:131731040:T:C、5:131744563:C:A、5:131744565:G:A、5:131744565:G:T、5:131744568:A:T、5:131744569:C:T、5:131744571:G:A、5:131744572:A:AT、5:131744572:A:G、5:131744577:T:A、5:131744577:T:C、5:131744578:C:A、5:131744578:C:T、5:131744590:T:C、5:131744596:C:T、5:131744598:C:A、5:131744599:T:C、5:131744601:G:C、5:131744605:C:G、5:131744613:A:G、5:131744616:T:C、5:131744617:T:G、5:131744623:C:A、5:131744623:C:G、5:131744623:C:T、5:131744623:CT:C、5:131744625:C:T、5:131744626:G:A、5:131744626:G:C、5:131744629:T:C、5:131744632:C:T、5:131744634:C:G、5:131744634:C:T、5:131744635:A:G、5:131744642:A:C、5:131744644:A:AT、5:131744646:A:G、5:131744647:C:T、5:131744648:A:C、5:131744650:T:C、5:131744655:C:T、5:131744656:G:A、5:131744658:A:G、5:131744659:T:C、5:131744660:C:G、5:131744662:G:A、5:131744662:G:C、5:131744668:G:T、5:131744670:T:G、5:131744671:C:G、5:131744671:C:T、5:131744673:A:G、5:131744675:C:A、5:131744676:T:C、5:131744676:T:G、5:131744677:C:G、5:131744679:G:A、5:131744680:G:A、5:131796828:A:G、5:131796829:C:T、5:131796830:C:G、5:131796834:A:G、5:131796838:G:C、5:131796843:C:A、5:131796843:C:G、5:131796845:G:A、5:131796849:C:A、5:131796851:C:G、5:131796851:C:T、5:131796852:G:A、5:131796852:G:C、5:131796858:C:A、5:131796860:C:T、5:131796863:C:A、5:131796863:C:T、5:131796866:G:C、5:131796867:G:A、5:131796872:C:A、5:131796872:C:G、5:131796875:A:G、5:131796878:C:T、5:131796879:C:T、5:131796883:C:G、5:131796884: C:G、5:131796887:T:C、5:131796889:G:C、5:131796891:T:A、5:131796897:G:C、5:131796899:T:C、5:1317969 00:T:C, 5:131796901:C:G, 5:131796904:G:C, 5:131796906:A:G, 5:131796911:G:A, 5:131796911:G:T, 5:131796912:T:A, 5:131796914:G:C, 5:131796915:G:A or 5:131796917:G:A, or mRNA molecules generated therefrom, or cDNA molecules generated from said mRNA molecules.

[0045] Subjects who are genotyped or identified as FNIP1 references have an increased risk of developing muscle disorders. Subjects who are genotyped or identified as FNIP1 references or who are heterozygous for FNIP1 variant nucleic acid molecules can be treated with ITGA1 inhibitors.

[0046] In any of the embodiments described herein, the nucleotide sequence of the FLCN reference genomic nucleic acid molecule in the GRCh38 / hg38 human genome assembly is used as a reference sequence. FLCN variant genomic nucleic acid molecules may include one or more variants at any position on chromosome 17 (i.e., positions 17,212,212-17,237,188) (see ENSG00000154803.13, ENST00000285071.9 annotated in the Ensembl database (URL: "http: / / useast.ensembl.org / Homo_sapiens / Gene / Summary?g=ENSG00000154803;r=17:17212212-17237188;transcript=ENST00000285071.9" on the World Wide Web)). The sequences of the FLCN genomic nucleic acid molecules provided in these transcripts are merely exemplary sequences. Other sequences of FLCN genomic nucleic acid molecules are also possible.

[0047] In any of the embodiments described herein, the FLCN variant nucleic acid molecule may contain one or more of the following genetic variations (referring to the chromosomal positions specified in the GRCh38 / hg38 human genome assembly): 17:17213659:TTCCGAGAC:T, 17:17213680:G:T, 17:17213686:C:G, 17:17213686:C:T, 17:17213686:CG:C, 17:17213687:G:A, 17:17213695:G:A, 17:17213703:G:C, 17:17213705:G:A, 17:17213705:GT:G , 17:17213711:T:C, 17:17213712:G:C, 17:17213712:G:T, 17:17213737: C:T、17:17213738:A:G、17:17213747:G:C、17:17213749:A:G、17:1721375 8:T:C、17:17213789:G:T、17:17213796:C:G、17:17213796:CTG:C、17:17 213798:G:A、17:17213805:C:G、17:17213815:C:CT、17:17213815:C:T、17 :17213816:G:A、17:17213823:C:CA、17:17213834:A:C、17:17213836:T:C , 17:17213848:T:C, 17:17213849:T:C, 17:17213852:C:T, 17:17213855:T :C、17:17214984:C:G、17:17214984:C:T、17:17214993:C:G、17:17214995 :C:T、17:17214996:C:A、17:17215001:T:A、17:17215001:T:G、17:172150 04:G:A、17:17215006:C:T、17:17215013:G:A、17:17215015:C:T、17:1721 5016:A:G、17:17215022:C:A、17:17215024:A:C、17:17215036:G:A、17:17 215036:G:C、17:17215037:A:C、17:17215048:T:C、17:17215057:G:C、17: 17215070:T:C、17:17215073:T:G、17:17215082:TG:T、17:17215087:C:T、17:17215184:C:G、17:17215184:C:T、17:17215188:G:A、17:17215190:T:TC、17:17215224:A:AC、17:17215228:G:C、17:17215236:TGA:T、17:17215256:CA:C、17:17215257:AC:A、17:17215263:C:CAGGGTGG、17:17215264:AGGGTGGAGGGTGGAACGTGC:A、17:17215282:T:TGCGGCTGCGTGGACCTC、17:17215302:C:T、17:17215311:CA:C、17:17215313:A:C、17:17215317:CTG:C、17:17216382:G:A、17:17216394:T:TG、17:17216394:TG:T、17:17216395:G:T、17:17216400:G:A、17:17216400:G:C、17:17216400:G:T、17:17216401:G:A、17:17216401:G:T、17:17216418:C:A、17:17216418:C:CT、17:17216424:C:A、17:17216424:C:T、17:17216426:C:CA、17:17216427:AG:A、17:17216433:T:C、17:17216435:G:T、17:17216440:G:A、17:17216443:A:T、17:17216453:G:C、17:17216454:T:C、17:17216458:G:A、17:17216464:T:C、17:17216466:T:C、17:17216466:T:TA、17:17216476:T:TG、17:17216478:C:A、17:17216479:G:C、17:17216482:C:A、17:17216484:C:A、17:17216484:C:T、17:17216485:A:G、17:17216487:C:G、17:17216491:C:CG、17:17217068:C:G、17:17217068:CCCGAAGTACTTCAAAAGCTGACT:C、17:17217074:G:T、17:17217086:C:G、17:17217092:G:A、17:17217112:C:CT、17:17217116:TC:T、17:17217124:A:T、17:17217127:TG:T、17:17217128:G:A、17:17217133:C:T、17:17217147:C:T、17:17217161:G:A、17:17217163:A:G、17:17217168:AG:A、17:17217174:AC:A、17:17217175:C:A、17:17217183:C:A、17:17217184:T:A、17:17217184:T:C、17:17219017:A:C、17:17219018:C:T、17:17219030:G:A、17:17219032:C:T、17:17219033:G:A、17:17219038:G:A、17:17219038:G:T、17:17219051:G:GCAGATTCCGGGGCTGC、17:17219082:TGAGA:T、17:17219126:C:CTTCTGTACTCTCTGGCAACACAGGGGCT、17:17219133:ACT:A、17:17219147:CAG:C、17:17219148:A:AG、17:17219174:C:CA、17:17219176:G:C、17:17219177:A:G、17:17219183:C:G、17:17219187:GCTTT:G、17:17219190:TTC:T、17:17219194:G:C、17:17219194:G:T、17:17219201:C:G、17:17219206:A:C、17:17221535:A:G、17:17221549:C:T、17:17221555:G:A、17:17221567:C:A、17:17221570:C:T、17:17221575:G:A、17:17221576:G:C、17:17221597:C:T、17:17221599:G:A、17:17221605:C:T、17:17221612:C:T、17:17221614:C:T、17:17221617:G:A、17:17221617:G:C、17:17221618:C:A、17:17221618:C:G、17:17221627:G:A、17:17221628:C:T、17:17221630:T:C、17:17222500:C:A、17:17222501:C:G、17:17222516:T:G、17:17222517:G:A、17:17222519:A:G、17:17222525:G:GC、17:17222532:G:T、17:17222540:T:C、17:17222541:CACTT:C、17:17222546:G:T、17:17222558:A:C、17:17222558:A:G、17:17222559:G:C、17:17222561:G:A、17:17222564:C:T、17:17222565:G:A、17:17222565:G:C、17:17222582:C:T、17:17222590:T:TA、17:17222615:A:C、17:17222618:C:A、17:17222628:G:A、17:17222632:TG:T、17:17222636:C:T、17:17222649:C:T、17:17222657:A:C、17:17222662:C:T、17:17222663:T:G、17:17223929:G:A、17:17223930:C:T、17:17223937:C:A、17:17223937:C:G、17:17223942:G:A、17:17223951:T:A、17:17223955:TC:T、17:17223959:C:A、17:17223960:G:A、17:17223966:T:A、17:17223975:G:C、17:17223978:A:T、17:17223980:G:A、17:17223981:G:T、17:17223983:C:A、17:17223986:G:A、17:17223989:T:G、17:17223995:A:G、17:17223996:G:A、17:17223998:T:C、17:17224001:A:G、17:17224001:A:T、17:17224002:T:A、17:17224004:C:T、17:17224005:G:A、17:17224008:C:A、17:17224012:CATGATGG:C、17:17224017:T:A、17:17224021:G:C、17:17224030:G:T、17:17224032:A:G、17:17224032:A:T、17:17224035:A:G、17:17224037:C:T、17:17224038:G:A、17:17224039:C:G、17:17224041:G:A、17:17224053:C:A、17:17224062:C:A、17:17224073:A:G、17:17224076:G:T、17:17224077:T:A、17:17224083:T:C、17:17224088:A:G、17:17224089:C:T、17:17224091:A:G、17:17224092:A:C、17:17224095:C:T、17:17224099:C:CTG、17:17224099:CT:C、17:17224104:C:T、17:17224107:C:T、17:17224110:C:T、17:17224111:G:C、17:17224111:G:T、17:17224115:A:C、17:17224121:G:A、17:17224121:G:C、17:17224124:C:A、17:17224125:C:T、17:17224128:C:T、17:17224130:C:A、17:17224130:C:T、17:17224131:G:A、17:17224136:G:A、17:17224136:G:C、17:17224137:G:T、17:17224143:C:A、17:17224143:C:T、17:17226174:A:C、17:17226186:A:C、17:17226187:G:C、17:17226192:C:T、17:17226193:G:A、17:17226193:G:C、17:17226202:C:T、17:17226207:C:G、17:17226208:G:A、17:17226210:A:G、17:17226220:A:C、17:17226224:C:CT、17:17226224:C:G、17:17226243:T:A、17:17226245:G:GT、17:17226247:G:A、17:17226250:T:TG、17:17226252:A:T、17:17226262:T:C、17:17226270:T:C、17:17226274:T:A、17:17226276:T:C、17:17226277:C:G、17:17226282:C:T、17:17226291:C:G、17:17226294:G:A、17:17226294:G:C、17:17226295:G:C、17:17226297:T:G、17:17226298:G:A, 17:17226301:C:T, 17:17226315:C:T, 17:17226316:G:A, 17:17226318:C:T, 17:172263 21:C:A、17:17226324:T:C、17:17227887:G:A、17:17227887:G:T、17:17227893:C:T、17:17227899:TCCGA: T, 17:17227904:CT:C, 17:17227923:CT:C, 17:17227930:C:A, 17:17227948:CG:C, 17:17227950:G:T, 17:1 7227979:CT:C、17:17228025:C:CT、17:17228040:TC:T、17:17228073:G:A、17:17228077:A:G、17:17228079 :A:T、17:17228082:A:G、17:17228085:G:T、17:17228088:C:A、17:17228088:C:T、17:17228089:G:A、17:1 7228091:G:A、17:17228095:C:T、17:17228104:C:G、17:17228104:C:T、17:17228105:G:C、17:17228105:G: T, 17:17228109:A:T, 17:17228112:T:C, 17:17228114:G:T, 17:17228115:C:T, 17:17228119:G:C, 17:17228124:A:C, 17:17228133:T:C, 17:17228136:A:G, or 17:17228137:T:C, or mRNA molecules derived therefrom, or cDNA molecules derived from said mRNA molecules.

[0048] Subjects who are genotyped or identified as FLCN references have an increased risk of developing muscle disorders. Subjects who are genotyped or identified as FLCN references or who are heterozygous for FLCN variant nucleic acid molecules can be treated with FLCN inhibitors.

[0049] In any of the embodiments described herein, the subject who prevents myopathy by administering an FNIP1 inhibitor and / or an FLCN inhibitor can be any subject at risk of developing myopathy, including but not limited to subjects with a genetic predisposition to myopathy. In some embodiments, an FNIP1 inhibitor and / or an FLCN inhibitor may be administered to a subject with myopathy to prevent recurrence of myopathy in a subject who already has myopathy. In any of the embodiments described herein, the method may be used to improve myopathy.

[0050] In any of the embodiments described herein, the FNIP1 predicted loss-of-function peptide can be any FNIP1 peptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.

[0051] In any of the embodiments described herein, the FLCN-predicted loss-of-function polypeptide can be any FLCN polypeptide having partial loss of function, complete loss of function, predicted partial loss of function, or predicted complete loss of function.

[0052] Any one or more of the FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules described herein (i.e., any combination thereof) can be used in any of the methods described herein to determine whether a subject has an increased or decreased risk of developing myopathy. Combinations of specific variants can form a mask for statistical analysis of a specific association between FNIP1 and / or FLCN and an increased or decreased risk of developing myopathy. In some embodiments, the mask used for statistical analysis of a specific association between FNIP1 and / or FLCN and an increased or decreased risk of developing myopathy may exclude any one or more of these FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules described herein.

[0053] In any of the embodiments described herein, the subject may have a muscle disease. In any of the embodiments described herein, the subject may be at risk of developing a muscle disease. In some embodiments, muscle diseases include sarcopenia, Duchenne muscular dystrophy, and Pompe disease. In some embodiments, muscle diseases include sarcopenia. In some embodiments, muscle diseases include Duchenne muscular dystrophy. In some embodiments, muscle diseases include Pompe disease.

[0054] This disclosure provides a method for treating a subject with or at risk of developing a muscle disorder, the method comprising administering an FNIP1 inhibitor and / or an FLCN inhibitor to the subject.

[0055] This disclosure provides a method for treating a subject with or at risk of developing muscular dystrophy complications, the method comprising administering an FNIP1 inhibitor and / or an FLCN inhibitor to the subject.

[0056] In some embodiments, the FNIP1 inhibitor comprises an inhibitory nucleic acid molecule. Examples of inhibitory nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such inhibitory nucleic acid molecules can be programmed to target any region of the FNIP1 nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within the FNIP1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the FNIP1 peptide in the subject's cells. In some embodiments, the FNIP1 inhibitor comprises an antisense molecule that hybridizes to the FNIP1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the FNIP1 peptide in the subject's cells. In some embodiments, the FNIP1 inhibitor comprises siRNA that hybridizes to the FNIP1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the FNIP1 peptide in the subject's cells. In some embodiments, the FNIP1 inhibitor comprises shRNA that hybridizes to the FNIP1 genomic nucleic acid molecule or mRNA molecule and reduces the expression of the FNIP1 peptide in the subject's cells.

[0057] In some embodiments, FLCN inhibitors comprise repressive nucleic acid molecules. Examples of repressive nucleic acid molecules include, but are not limited to, antisense nucleic acid molecules, small interfering RNA (siRNA), and short hairpin RNA (shRNA). Such repressive nucleic acid molecules may be programmed to target any region of the FLCN nucleic acid molecule. In some embodiments, the antisense RNA, siRNA, or shRNA hybridizes to a sequence within the FLCN genomic nucleic acid molecule or mRNA molecule and reduces the expression of the FLCN peptide in the subject's cells. In some embodiments, FLCN inhibitors comprise antisense molecules that hybridize to the FLCN genomic nucleic acid molecule or mRNA molecule and reduce the expression of the FLCN peptide in the subject's cells. In some embodiments, FLCN inhibitors comprise siRNA that hybridizes to the FLCN genomic nucleic acid molecule or mRNA molecule and reduces the expression of the FLCN peptide in the subject's cells. In some embodiments, FLCN inhibitors comprise shRNA that hybridizes to the FLCN genomic nucleic acid molecule or mRNA molecule and reduces the expression of the FLCN peptide in the subject's cells. For example, WO 2022 / 178411 discloses representative FLCN siRNA molecules.

[0058] Repressive nucleic acid molecules may include RNA, DNA, or both. Repressive nucleic acid molecules may also be linked or fused with heterologous nucleic acid sequences (such as heterologous nucleic acid sequences in a vector) or heterologous markers. For example, repressive nucleic acid molecules may be contained within a vector containing both the repressive nucleic acid molecule and the heterologous nucleic acid sequence, or as an exogenous donor sequence containing both the repressive nucleic acid molecule and the heterologous nucleic acid sequence. Repressive nucleic acid molecules may also be linked or fused with heterologous markers. Markers may be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such markers can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical methods. Such markers include, for example, radioactive markers, pigments, dyes, chromogens, spin markers, and fluorescent markers. Markers may also be, for example, chemiluminescent substances; metal-containing substances; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term "marker" may also refer to a "tag" or hapten that selectively binds 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 can be used as a tag, along with avidin or streptavidin conjugates of horseradish peroxidase (HRP), to bind to the tag and be checked using a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorescent substrate to detect the presence of HRP. Exemplary tags that can be used to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6XHis or polyhistidine, glutathione S-transferase (GST), maltose-binding proteins, epitope tags, or the Fc portion of immunoglobulins. Many tags include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other tags.

[0059] Inhibitory nucleic acid molecules may include, for example, nucleotides or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides containing modified bases, sugars, or phosphate groups, or nucleotides incorporating non-natural portions into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated, amination-modified, deamination-modified, alkylated, benzylated, and fluorescently labeled nucleotides.

[0060] Inhibitory nucleic acid molecules may also contain one or more nucleotide analogs or substitutions. Nucleotide analogs are nucleotides containing modifications to the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications to A, C, G, and T / U, as well as to various purine or pyrimidine bases (e.g., 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-halogenated, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deadenine, 7-deadenine, 3-deadenine and 3-deadenine.

[0061] Nucleotide analogs may also include modifications to the sugar moiety. Modifications to the sugar moiety include, but are not limited to, natural and synthetic modifications of ribose and deoxyribose. Sugar modifications include, but are not limited to, the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C- groups. 1-10 Alkyl or C 2-10 alkenyl and C 2-10 Alkyne group. Exemplary 2' sugar modifications also include, but are not limited to, -O[(CH2)]. n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON[(CH2) n CH3)]2, where n and m are independently 1 to approximately 10. Other modifications at the 2' position include, but are not limited to, C 1-10Alkyl groups, substituted lower alkyl groups, alkylaryl groups, aralkyl groups, O-alkylaryl or O-aralkyl groups, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocyclic alkyl groups, heterocyclic alkylaryl groups, aminoalkylamino groups, polyalkylamino groups, substituted silyl groups, RNA cleaving groups, reporter groups, intercalating agents, groups used to improve the pharmacokinetic properties of oligonucleotides, or groups used to improve the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, specifically at the 3' position of the sugar on the 3' terminal nucleotide or at the 3' position of the sugar and the 5' position of the 5' terminal nucleotide in 2'-5' linked oligonucleotides. Modified sugars can also include those sugars containing modifications (e.g., CH2 and S) at the bridging epoxy. Nucleotide sugar analogs can also have sugar mimics that replace furanopentoses, such as a cyclobutyl moiety.

[0062] Nucleotide analogs may also be modified at the phosphate ester moiety. Modified phosphate ester moieties include, but are not limited to, those that can be modified such that the link between two nucleotides contains the following modified phosphate ester moieties: thiophosphate, chiral thiophosphate, dithiophosphate, phosphate triester, aminoalkyl phosphate triester, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), hypophosphonates, aminophosphates (including 3'-aminoaminophosphate and aminoalkylaminophosphate), thiocarbonylaminophosphate, thiocarbonylalkylphosphonate, thiocarbonylalkyl phosphate triester, and boron phosphate. These phosphate ester or modified phosphate ester links between two nucleotides can be 3'-5' or 2'-5' linked, and said links may contain reversed polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0063] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first one to seven nucleotides at the 5' and 3' ends are each modified with 2'-methoxyethyl (2'-MOE). In some embodiments, the first five nucleotides at the 5' and 3' ends are each modified with 2'-MOE. In some embodiments, the first one to seven nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, each backbone link between nucleotides is a phosphate thioester linker.

[0064] 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, a non-hydrolyzable 5'-phosphate analogue, such as 5'-(E)-vinylphosphonate, is used.

[0065] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribonucleosides have been shown to enhance siRNA stability and in vivo bioavailability. The non-ester groups (-OH, =O) of the phosphodiester bond can be replaced with sulfur, boron, or acetate esters to obtain thiophosphate, borate phosphate, and phosphonoacetate bonds. Additionally, replacing the phosphodiester group with a phosphate triester 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 (a reaction catalyzed by exonucleases and endonucleases), thereby allowing the 2'-hydroxyl group to 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.

[0066] In some embodiments, the siRNA molecule has base modifications. In some embodiments, the bases may be substituted with modified bases such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0067] In some implementations, siRNA molecules are conjugated to lipids. Lipids may conjugate to the 5' or 3' end of the siRNA to enhance its bioavailability in vivo by allowing it 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.

[0068] In some implementations, a representative siRNA has the following formula: Meaningful: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antonym: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N Wherein: “N” is a base; “2F” is 2'-F modification; “m” is 2'-O-methyl modification; “I” is an internal base; and “*” is a thiophosphate skeleton bond.

[0069] In any of the embodiments described herein, the inhibitory nucleic acid molecule may be administered, for example, as an intravenous infusion or subcutaneous injection over one to two hours. In any of the embodiments described herein, the inhibitory nucleic acid molecule may range from about 50 mg to about 900 mg, about 100 mg to about 800 mg, about 150 mg to about 700 mg, or about 175 mg to about 640 mg (2.5 mg / kg to 9.14 mg / kg; 92.5 to 338 mg / m²). 2 –Based on the assumption of a body weight of 70 kg and the multiplier of the dose for humans at 37 mg / kg, the mg / kg dose level is shifted to mg / m 2 (Dose level conversion) dosage level administration.

[0070] This disclosure also provides vectors comprising one or more of the inhibitory nucleic acid molecules. In some embodiments, the vector comprises one or more of the inhibitory nucleic acid molecules and a heterologous nucleic acid. The vector can be a viral or non-viral vector capable of transporting nucleic acid molecules. In some embodiments, the vector is a plasmid or granule (e.g., a circular double-stranded DNA in which an additional DNA segment can be linked). In some embodiments, the vector is a viral vector in which an additional DNA segment can be linked to a viral genome. Expression vectors include, but are not limited to, plasmids, granules, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), episomes derived from Epstein-Barr virus (EBV), and other expression vectors known in the art.

[0071] This disclosure also provides compositions comprising one or more of the inhibitory nucleic acid molecules. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or an excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic acid-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid helices, and lipid microtubules. Carriers may include buffered saline solutions, such as PBS, HBSS, etc.

[0072] In some embodiments, FNIP1 inhibitors or FLCN inhibitors comprise nucleases that induce one or more nicks or double-strand breaks at one or more recognition sequences, or DNA-binding proteins that bind to recognition sequences within the ITGA1 genomic nucleic acid molecule. The recognition sequence may be located within the coding region of the FNIP1 or FLCN gene, or within a regulatory region affecting gene expression. The recognition sequence of the DNA-binding protein or nuclease may be located in an intron, exon, promoter, enhancer, regulatory region, or any non-protein coding region. The recognition sequence may include or be close to the start codon of the FNIP1 or FLCN gene. For example, the recognition sequence may be located approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 100, approximately 200, approximately 300, approximately 400, approximately 500, or approximately 1,000 nucleotides from the start codon. As another example, two or more nucleases may be used, each targeting a nuclease recognition sequence containing or close to the start codon. As another example, two nuclease agents can be used: one targeting a nuclease recognition sequence containing or near the start codon, and the other targeting a nuclease recognition sequence containing or near the stop codon, wherein cleavage by the nuclease agent results in the deletion of the coding region between the two nuclease recognition sequences. Any nuclease agent that induces a nick or double-strand break to the desired recognition sequence can be used in the methods and compositions disclosed herein. Any DNA-binding protein that binds to the desired recognition sequence can be used in the methods and compositions disclosed herein.

[0073] Suitable nucleases and DNA-binding proteins used in this study include, but are not limited to, zinc finger proteins or zinc finger nucleases (ZFN) pairs, transcription activator-like effector (TALE) proteins or transcription activator-like effector nucleases (TALENs), or clustered, regularly distributed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems. The recognition sequence length can vary and includes, for example, recognition sequences of approximately 30-36 bp for zinc finger proteins or ZFN pairs, approximately 15-18 bp for each ZFN, approximately 36 bp for TALE proteins or TALENs, and approximately 20 bp for CRISPR / Cas guide RNA.

[0074] In some embodiments, the CRISPR / Cas system can be used to modify intracellular FNIP1 or FLCN genomic nucleic acid molecules. The methods and compositions disclosed herein can utilize the CRISPR-Cas system by employing a CRISPR complex (containing a guide RNA (gRNA) complexed with a Cas protein) for site-specific cleavage of FNIP1 or FLCN nucleic acid molecules.

[0075] Cas proteins typically contain at least one RNA recognition or binding domain that interacts with gRNA. Cas proteins may also contain nuclease domains (e.g., DNase or RNase domains), DNA-binding domains, helicase domains, protein-protein interaction domains, dimerization domains, and other domains. Suitable Cas proteins include, for example, wild-type Cas9 and wild-type Cpf1 proteins (e.g., FnCpf1). Cas proteins may have full cleavage activity to produce double-strand breaks in FNIP1 or FLCN genomic nucleic acid molecules, or they may be cleavage enzymes that produce single-strand breaks in FNIP1 or FLCN genomic nucleic acid molecules. Other examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), and Cse4. CasC, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, as well as their homologs or modified forms. In some embodiments, the Cas system, such as Cas12a, may have multiple gRNAs encoding a single crRNA. Cas proteins may also be operatively linked as fusion proteins to heterologous peptides. For example, Cas proteins may be fused to cleavage domains, epigenetic modification domains, transcriptional activation domains, or transcriptional repression domains. Cas proteins may be provided in any form. For example, Cas proteins can be provided in the form of proteins (such as Cas proteins complexed with gRNA). Alternatively, Cas proteins can be provided in the form of nucleic acid molecules encoding Cas proteins, such as RNA or DNA.

[0076] In some embodiments, targeted genetic modification of the FNIP1 or FLCN genomic nucleic acid molecule can be achieved by contacting a cell with a Cas protein and one or more gRNAs, said gRNAs hybridizing to one or more gRNA recognition sequences within a target genomic locus in the FNIP1 or FLCN genomic nucleic acid molecule. The gRNA recognition sequence may include or be close to the start codon or stop codon of the FNIP1 or FLCN genomic nucleic acid molecule. For example, the gRNA recognition sequence may be located approximately 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start or stop codon.

[0077] In FNIP1 or FLCN genomic nucleic acid molecules, the gRNA recognition sequence within the target genomic locus is located near a protospacer adjacent motif (PAM) sequence, which is a DNA sequence of 2-6 base pairs immediately following the DNA sequence targeted by the Cas9 nuclease. A typical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two guanine ("G") nucleotides. The gRNA can transport Cas9 to any location in the genome for gene editing, but editing will not occur at any site other than the Cas9-recognized PAM site. Additionally, 5'-NGA-3' can serve as a highly efficient atypical PAM for human cells. Generally, the PAM is located approximately 2-6 nucleotides downstream of the gRNA-targeted DNA sequence. The PAM can be side-attached to the gRNA recognition sequence. In some embodiments, the gRNA recognition sequence may be side-attached to the 3' end by the PAM. In some embodiments, the gRNA recognition sequence may be side-attached to the 5' end by the PAM. For example, the cleavage site of the Cas protein can be approximately 1 to 10 base pairs, approximately 2 to 5 base pairs, or three base pairs upstream or downstream of the PAM sequence. In some embodiments (such as when using Streptococcus pyogenes...), S. pyogenes When N is a Cas9 or closely related Cas9, the PAM sequence of the non-complementary strand can be 5'-NGG-3', where N is any DNA nucleotide and is the 3' of the gRNA recognition sequence of the non-complementary strand of the target DNA. Therefore, the PAM sequence of the complementary strand will be 5'-CCN-3', where N is any DNA nucleotide and is the 5' of the gRNA recognition sequence of the complementary strand of the target DNA.

[0078] gRNA is an RNA molecule that binds to the Cas protein and targets the Cas protein to a specific location within the FNIP1 or FLCN genomic nucleic acid molecule. An exemplary gRNA is one that effectively guides the Cas enzyme to bind to or cleave the FNIP1 or FLCN genomic nucleic acid molecule, wherein the gRNA contains a DNA targeting region that hybridizes to a gRNA recognition sequence within the FNIP1 or FLCN genomic nucleic acid molecule. An exemplary gRNA contains a DNA targeting region that hybridizes to a gRNA recognition sequence present within the FNIP1 or FLCN genomic nucleic acid molecule, said gRNA recognition sequence containing or near a start codon or stop codon. For example, the gRNA can be selected to hybridize with a gRNA recognition sequence located at approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the start codon or approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, or 1,000 nucleotides from the stop codon. Suitable gRNAs may contain approximately 17 to approximately 25 nucleotides, approximately 17 to approximately 23 nucleotides, approximately 18 to approximately 22 nucleotides, or approximately 19 to approximately 21 nucleotides. In some embodiments, the gRNA may contain 20 nucleotides.

[0079] The Cas protein and gRNA form a complex, and the Cas protein cleaves the target FNIP1 or FLCN genomic nucleic acid molecule. The Cas protein can cleave the nucleic acid molecule at sites within or outside the DNA target segment of the target FNIP1 or FLCN genomic nucleic acid molecule that bind to the gRNA. For example, the formation of a CRISPR complex (containing gRNA that hybridizes to the gRNA recognition sequence and complexes with the Cas protein) can cause one or both strands to be cleaved in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) of the DNA target segment of the gRNA present in the FNIP1 or FLCN genomic nucleic acid molecule.

[0080] Such methods can produce FNIP1 or FLCN genomic nucleic acid molecules in which, for example, regions are disrupted, start codons are disrupted, stop codons are disrupted, or coding sequences are disrupted or deleted. Optionally, the cell may be further contacted with one or more additional gRNAs that hybridize to additional gRNA recognition sequences within target genomic loci in the FNIP1 or FLCN genomic nucleic acid molecule. By contacting the cell with one or more additional gRNAs (e.g., a second gRNA that hybridizes to a second gRNA recognition sequence), Cas protein cleavage can produce two or more double-strand breaks or two or more single-strand breaks.

[0081] In some implementations, FNIP1 and / or FLCN gRNA molecules may be combined. For example, FNIP1 gRNA molecules may be combined with 15 gRNAs per gene and packaged into multiple (e.g., 4, 5, or 6) gRNAs per vector (e.g., AAV). Similarly, FLCN gRNA molecules may be combined with 15 gRNAs per gene and packaged into multiple (e.g., 4, 5, or 6) gRNAs per vector (e.g., AAV).

[0082] In any treatment or prevention method described herein, the subject receiving treatment may contain FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules. In any treatment or prevention method described herein, the subject receiving treatment is heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules. In any treatment or prevention method described herein, the subject receiving treatment is homozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules. In some embodiments, the subject receiving treatment is an FNIP1 and / or FLCN reference. FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules can be any FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule disclosed herein. In some embodiments, FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules are FNIP1 or FLCN variant genomic nucleic acid molecules containing any one or more of the genetic variations described herein, or mRNA molecules derived from them, or cDNA molecules derived from mRNA molecules.

[0083] In some embodiments, the treatment or prevention method further includes detecting the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules in a biological sample from a subject. In some embodiments, the FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules can be any FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules disclosed herein. In some embodiments, the FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules are FNIP1 or FLCN variant genomic nucleic acid molecules containing any or more of the genetic variations described herein, or mRNA molecules derived therefrom, or cDNA molecules derived from mRNA molecules.

[0084] This disclosure also provides a method for treating a subject with a myopathy treatment agent, wherein the subject has or is at risk of developing myopathy. The method includes determining whether the subject possesses an FNIP1 variant nucleic acid molecule and / or an FLCN variant nucleic acid molecule by obtaining or having obtained a biological sample from the subject, and performing or having performed sequence analysis on the biological sample to determine whether the subject has a genotype containing the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule. In an embodiment where the subject is an FNIP1 and / or FLCN reference, the method further includes administering or continuing to administer the myopathy treatment agent to the subject at a standard dose, and / or administering an FNIP1 inhibitor and / or an FLCN inhibitor to the subject. In an embodiment where the subject is heterozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule, the method further includes administering or continuing to administer the myopathy treatment agent in an amount equal to or less than the standard dose for the subject, and / or administering an FNIP1 inhibitor and / or an FLCN inhibitor to the subject. In embodiments where the subject is homozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, the method further includes administering or continuing to administer an amount of a myopathy treatment agent that is the same as or less than a standard dose. The presence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules indicates a reduced risk of the subject developing myopathy. In some embodiments, the subject is an FNIP1 and / or FLCN reference. In some embodiments, the subject is heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules. In some embodiments, the subject is homozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules. In any of the embodiments described herein, an FNIP1 inhibitor or FLCN inhibitor is an example of a myopathy treatment agent. In some embodiments, the FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules are FNIP1 or FLCN variant genomic nucleic acid molecules containing any one or more of the genetic variations described herein, or mRNA molecules derived therefrom, or cDNA molecules derived from mRNA molecules.

[0085] For subjects whose genotype is identified as FNIP1 and / or FLCN reference or who are heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, FNIP1 inhibitors and / or FLCN inhibitors may be administered to such subjects as described herein.

[0086] Detecting the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules in biological samples from a subject and / or determining whether a subject possesses FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecules may be present within cells obtained from the subject.

[0087] In some implementations, when the subject is an FNIP1 reference, the subject is administered a myopathy treatment agent and / or an FNIP1 inhibitor at the same or less than the standard dose. In some implementations, when the subject is heterozygous for an FNIP1 variant nucleic acid molecule, the subject is administered a myopathy treatment agent and / or an FNIP1 inhibitor at the same or less than the standard dose.

[0088] In some implementations, when the subject is an FLCN reference, the subject is administered a myopathy treatment agent and / or an FLCN inhibitor at the same or less than the standard dose. In some implementations, when the subject is heterozygous for an FLCN variant nucleic acid molecule, the subject is administered a myopathy treatment agent and / or an FLCN inhibitor at the same or less than the standard dose.

[0089] In some embodiments, the treatment or prevention method includes detecting the presence or absence of reduced expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide in a biological sample from the subject. In some embodiments, when the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide in the subject is not reduced, the subject is administered a standard dose of a myopathy treatment agent, and / or an FNIP1 inhibitor and / or an FLCN inhibitor. In some embodiments, when the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide in the subject is reduced, the subject is administered an amount of a myopathy treatment agent that is the same as or less than a standard dose.

[0090] This disclosure also provides a method for treating a subject with a myopathy treatment agent, wherein the subject has or is at risk of developing myopathy. The method includes determining whether the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide is reduced by obtaining or having obtained a biological sample from the subject, and performing or having performed assays on the biological sample to determine whether the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide is reduced. In embodiments where the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide in the subject is not reduced, the method further includes administering or continuing to administer a standard dose of the myopathy treatment agent to the subject, and / or administering an FNIP1 inhibitor and / or an FLCN inhibitor to the subject. In embodiments where the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide in the subject is reduced, the method further includes administering or continuing to administer an amount of the myopathy treatment agent to the subject that is the same as or less than the standard dose. Decreased expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide indicates a reduced risk of myopathy in the subject. In some embodiments, the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide in the subject is decreased. In some embodiments, the expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide in the subject is not decreased. In any of the embodiments described herein, an FNIP1 inhibitor or FLCN inhibitor is an example of a myopathy treatment agent. In some embodiments, the FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule is a FNIP1 variant genomic nucleic acid molecule or FLCN variant genomic nucleic acid molecule containing any one or more of the genetic variations described herein, or an mRNA molecule derived therefrom, or a cDNA molecule derived from an mRNA molecule.

[0091] The reduced expression of FNIP1 variant mRNA or peptide and / or FLCN variant mRNA or peptide can be detected by a variety of known methods. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the mRNA or peptide may be present in cells obtained from the subject.

[0092] In some embodiments, the treatment or prevention method includes detecting the presence or absence of FNIP1 variant peptides and / or FLCN variant peptides in a biological sample from the subject. In some embodiments, when the subject does not have FNIP1 variant peptides and / or FLCN variant peptides, the subject is administered a myopathy treatment agent, and / or an FNIP1 inhibitor and / or an FLCN inhibitor, at a standard dose. In some embodiments, when the subject has FNIP1 variant peptides and / or FLCN variant peptides, the subject is administered a standard dose of a myopathy treatment agent.

[0093] This disclosure also provides a method of treating a subject with a myopathy treatment agent, wherein the subject has or is at risk of developing myopathy. The method includes determining whether the subject has an FNIP1 variant peptide and / or an FLCN variant peptide by obtaining or having obtained a biological sample from the subject, and performing or having performed a assay on the biological sample to determine whether the subject has an FNIP1 variant peptide and / or an FLCN variant peptide. When the subject does not have an FNIP1 variant peptide and / or an FLCN variant peptide, the subject is administered a myopathy treatment agent, and / or an FNIP1 inhibitor and / or an FLCN inhibitor, at a standard dose or less. When the subject has an FNIP1 variant peptide and / or an FLCN variant peptide, the subject is administered a standard dose of the myopathy treatment agent. The presence of the FNIP1 variant peptide and / or the FLCN variant peptide indicates a reduced risk of the subject developing myopathy. In some embodiments, the subject has an FNIP1 variant peptide and / or an FLCN variant peptide. In some embodiments, the subject does not have an FNIP1 variant peptide and / or an FLCN variant peptide.

[0094] This disclosure also provides a method for preventing a subject from developing myopathy by administering a myopathy treatment agent. In some embodiments, the method includes determining whether the subject has an FNIP1 variant peptide and / or an FLCN variant peptide by obtaining or having obtained a biological sample from the subject, and performing or having performed a assay on the biological sample to determine whether the subject has an FNIP1 variant peptide and / or an FLCN variant peptide. When the subject does not have an FNIP1 variant peptide and / or an FLCN variant peptide, administering to the subject an amount of a myopathy treatment agent, and / or an FNIP1 inhibitor and / or an FLCN inhibitor, equal to or less than a standard dose. When the subject has an FNIP1 variant peptide and / or an FLCN variant peptide, administering to the subject an amount of a myopathy treatment agent, equal to or less than a standard dose. The presence of the FNIP1 variant peptide and / or the FLCN variant peptide indicates a reduced risk of the subject developing myopathy. In some embodiments, the subject has an FNIP1 variant peptide and / or an FLCN variant peptide. In some embodiments, the subject does not have an FNIP1 variant peptide and / or an FLCN variant peptide.

[0095] Detecting the presence or absence of FNIP1 variant peptides and / or FLCN variant peptides in biological samples from a subject and / or determining whether a subject possesses FNIP1 variant peptides and / or FLCN variant peptides can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the peptides may be present within cells obtained from the subject.

[0096] In some embodiments, the FNIP1 inhibitor and / or FLCN inhibitor are small molecules. In some embodiments, the small molecule is a low molecular weight (<900 Daltons) organic compound.

[0097] In some embodiments, the FNIP1 inhibitor and / or FLCN inhibitor comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or its antigen-binding fragment specifically binds to human FNIP1 or FLCN. In some embodiments, the antibody is a fully human monoclonal antibody (mAb) or its antigen-binding fragment that specifically binds to and neutralizes, inhibits, blocks, eliminates, reduces, or interferes with at least one activity of FNIP1 or FLCN, particularly human FNIP1 or FLCN. In some embodiments, the antibody or its fragment can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of FNIP1 or FLCN by binding to an epitope of FNIP1 or FLCN that is directly involved in the targeting activity of FNIP1 or FLCN. In some embodiments, antibodies or fragments thereof can neutralize, inhibit, block, eliminate, reduce, or interfere with the activity of FNIP1 or FLCN by binding to an epitope of FNIP1 or FLCN that is not directly involved in the targeting activity of FNIP1 or FLCN, but the antibody or fragment bound to the epitope spatially or conformally inhibits, blocks, eliminates, reduces, or interferes with the targeting activity of FNIP1 or FLCN. In some embodiments, antibodies or fragments thereof bind to an epitope of FNIP1 or FLCN that is not directly involved in the targeting activity of FNIP1 or FLCN (i.e., non-blocking antibodies), but the antibody or fragment bound to the epitope results in enhanced clearance of FNIP1 or FLCN from circulation compared to the clearance of FNIP1 or FLCN in the absence of the antibody or fragment thereof, thereby indirectly inhibiting, blocking, eliminating, reducing, or interfering with the activity of FNIP1 or FLCN. The clearance of FNIP1 or FLCN from circulation can be particularly enhanced by a combination of two or more different non-blocking antibodies that do not compete with each other for specific binding to FNIP1 or FLCN. Antibodies can be full-length (e.g., IgG1 or IgG4 antibodies) or can contain only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragments), and can be modified to affect function, e.g., to eliminate residual effector function (Reddy et al., J. Immunol., 2000, 164, 1925-1933).

[0098] In some implementations, the equilibrium dissociation constant (Ki) for the specific binding of the antibody or its antigen-binding fragment to FNIP1 or FLCN is specified. D The Km is approximately 7 nM or less, approximately 6 nM or less, approximately 5 nM or less, approximately 4 nM or less, approximately 3 nM or less, approximately 2 nM or less, or approximately 1 nM or less, as measured by surface plasmon resonance assays (e.g., BIACORE™). In some embodiments, the antibody exhibits Km DThe values ​​are approximately 800 pM or less, approximately 700 pM or less, approximately 600 pM or less, approximately 500 pM or less, approximately 400 pM or less, approximately 300 pM or less, approximately 200 pM or less, approximately 100 pM or less, or approximately 50 pM or less.

[0099] In some implementations, anti-FNIP1 antibodies and / or anti-FLCN antibodies have modified glycosylation patterns. In some applications, modifications that remove undesirable glycosylation sites, or, for example, remove the fucose moiety to enhance antibody-dependent cytotoxicity (ADCC) function, may be useful (see Shield et al., J. Biol. Chem., 2002, 277, 26733). In other applications, removal of N-glycosylation sites may reduce undesirable immune responses against therapeutic antibodies or increase antibody affinity. In other applications, galactosylation may be modified to alter complement-dependent cytotoxicity (CDC).

[0100] In some implementations, the muscle disease treatment agents include, but are not limited to, glucocorticoids (e.g., prednisone, prednisolone, deflazacort, etc.), testosterone and other androgens or analogues, or androgen receptor modulators (e.g., ennosam, ataluren), oligonucleotides (e.g., eteplirsen, golodirsen, viltolarsen). (and casimersen), losmapimod, zetomipzomib, venous globulin-IH, activin II receptor antagonists (e.g., bimagrumab), myostatin inhibitors (e.g., trevogrumab and domagrozumab), activin A antagonists (e.g., garetosmab), and reldesemtiv.

[0101] This disclosure also provides compositions comprising a combination of an antibody or an antigen-binding fragment thereof with a treatment agent for muscle disorders.

[0102] In some implementations, the muscle disease treatment agent may be combined with an FNIP1 inhibitor and / or an FLCN inhibitor.

[0103] In some embodiments, the dose of the myopathy treatment agent may be reduced by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (i.e., less than the standard dose) for subjects homozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules or FNIP1 reference and / or FLCN reference. In some embodiments, the dose of the myopathy treatment agent may be reduced by approximately 10%, 20%, 30%, 40%, or 50%. In some embodiments, the dose of the myopathy treatment agent may be reduced by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% for subjects heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, compared to subjects using FNIP1 reference and / or FLCN reference. In addition, compared with subjects who are heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, subjects who are heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules or FNIP1 reference and / or FLCN reference can be administered muscle disease treatment agents at a lower frequency.

[0104] For example, the treatment agent for muscle disorders, and / or an FNIP1 inhibitor, and / or an FLCN inhibitor may be repeated after one, two, three, five, one week, two, three weeks, one month, five, six, seven, eight, two months, or three months. Repeated administration may be at the same dose or at different doses. Administration may be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more. For example, depending on certain dosage regimens, subjects may receive treatment for extended periods, such as six months, one year, or longer.

[0105] The administration of myopathy treatment agents and / or FNIP1 inhibitors and / or FLCN inhibitors may be carried out via any suitable route, including but not limited to parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular routes. The pharmaceutical composition for administration is ideally sterile and substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., a single-dose dose). The pharmaceutical composition may be formulated using one or more physiologically and pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. The term "pharmaceutically acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other components of the formulation and is substantially harmless to the recipient.

[0106] As used herein, the terms “treat,” “treating,” and “treatment,” as well as “prevent,” “preventing,” and “prevention,” refer to eliciting a desired biological response, such as therapeutic and preventative effects, respectively. In some embodiments, therapeutic effects include one or more of the following: reduction / alleviation of myopathy, reduction / alleviation of the severity of myopathy (e.g., reduction or inhibition of myopathy development), reduction / alleviation of symptoms and disease-related effects, delay of the onset of symptoms and disease-related effects, reduction of the severity of symptoms of disease-related effects, reduction of the number of symptoms and disease-related effects, shortening of the latency period of symptoms and disease-related effects, improvement of symptoms and disease-related effects, alleviation of secondary symptoms, alleviation of secondary infections, prevention of recurrence of myopathy, reduction of the number or frequency of recurrences, prolongation of the latency period between symptom onsets, increase of time to sustained progression, accelerated remission, or increased efficacy of alternative therapies or reduced resistance to alternative therapies and / or prolonged survival of affected host animals. Preventive effects may include complete or partial avoidance / inhibition or delay of the development / progression of myopathy after the administration of a treatment regimen (such as, for example, complete or partial avoidance / inhibition or delay), and increased survival time in affected host animals. Treatment of myopathy covers treating subjects diagnosed with any form of myopathy at any clinical stage or presentation, delaying the onset or evolution or aggravation or worsening of symptoms or signs of myopathy, and / or preventing and / or reducing the severity of myopathy.

[0107] In some embodiments, an FNIP1 inhibitor and / or FLCN inhibitor, along with a myopathy treatment agent, are disposed within the pharmaceutical composition. In some embodiments, an FNIP1 inhibitor and / or FLCN inhibitor are disposed within a first pharmaceutical composition, and a myopathy treatment agent is disposed within a second pharmaceutical composition. In some embodiments, the first and second pharmaceutical compositions are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0108] In any of the embodiments described herein, administration of the FNIP1 inhibitor and / or FLCN inhibitor may target a specific tissue. For example, in some embodiments, the FNIP1 inhibitor and / or FLCN inhibitor may target muscle tissue, muscle cells, skeletal muscle tissue, or skeletal muscle cells. In some embodiments described herein, administration of the FNIP1 inhibitor and / or FLCN inhibitor may target muscle tissue, muscle cells, skeletal muscle tissue, or skeletal muscle cells. For example, the FNIP1 inhibitor and / or FLCN inhibitor may be linked to a CACNG1 antibody or its antigen-binding fragment. In some embodiments, muscle-specific FNIP1 siRNA (CACNG1 mAB-siRNA) is used to promote oxidative metabolism and lysosomal biosynthesis in muscle fibers.

[0109] In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises a heavy chain CDR1 (HCDR1), said HCDR1 comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises a heavy chain CDR2 (HCDR2), said HCDR2 comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises a heavy chain CDR3 (HCDR3), said HCDR3 comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises a light chain CDR1 (LCDR1), said LCDR1 comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises a light chain CDR2 (LCDR2), said LCDR2 comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises a light chain CDR3 (LCDR3), said LCDR3 comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0110] In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises the HCVR amino acid sequence listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises the LCVR amino acid sequence listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0111] In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises an HC amino acid sequence selected from at least one of SEQ ID NO: 97 to SEQ ID NO: 108, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the CACNG1 antibody or its antigen-binding fragment comprises an LC amino acid sequence selected from at least one of SEQ ID NO: 109 to SEQ ID NO: 120, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0112] Table 1: Anti-CACNG1 antibodies

[0113] HCVR amino acid sequence: QVQLVESGGGVVQPGRSLRLSCTASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISGDNSKVYLQMNSLRAEDTAVYYCARRGTIRTAAPFDYWGQGTLVTVSS (SEQ ID NO: 1); QVQLQQWGAGLLKPSATLSRTCAVYGGSFSGYYWNWIRQSPGKGLEWIGEILHSGRTNYNPSLKSRVTISSVDTSKNQFSLKLTSVTAADTAVYYCAGRIAARHGWFDPWGQGTLVTVSS (SEQ ID NO: 2); QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDKYYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS (SEQ ID NO: 3); QVQLQESGPGLVKPSETLSLTCTVSGDSINNYYWTWLRQPPGKGLEWIGYIYYSGSANYNPSLKSRVTISSVDTSKNQFSLKLNSVTAADTAVYYCARGAVKYFRHWGQGTLVTVSS (SEQ ID NO: 4); QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWIDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGGIVVAAPFDYWGQGTLVTVSS (SEQ ID NO: 5); QVQLVESGGGVVQPGRSLRLSCEASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARRGHIATAAPFDYWGQGTLVTVSS (SEQ ID NO: 6); QVQLVESGGGVVQPGRSLRLSCTASGFTFRSYGMHWVRQAPGKGLEWVSVIWIDGNNIYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARRLAITSAAPFDYWGQGTLVTVSS (SEQ ID NO: 7); QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSS (SEQ ID NO: 8); EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWGQGTLVTVSS (SEQ ID NO: 9); EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMDVWGQGTTVTVSS (SEQ ID NO: 10); EVQLVESGGNLVQPGGSLRLSCAASGFTFTSHAMNWVRQAPGKGLEWVSVITGRGFDTHYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYCAKGLYDSGNYYIDYWGQGTLVTVSS (SEQ ID NO: 11); QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSS (SEQ ID NO: 12)。

[0114] HCDR1 amino acid sequence: GITFRNYG (SEQ ID NO: 13);GGSFSGYY (SEQ ID NO: 14);GFTFSTYG (SEQ ID NO: 15);GDSINNYY (SEQ ID NO: 16);GFTFSSYG (SEQ ID NO: 17);GITFRNYG (SEQ ID NO: 18);GFTFRSYG (SEQ ID NO: 19);GYAFTTYG (SEQ ID NO: 20);GLTLDSSA (SEQ ID NO: 21);GFTFNNYG (SEQ ID NO: 22);GFTFTSHA (SEQ ID NO: 23);GYAFTTYG (SEQ ID NO: 24)。

[0115] HCDR2 amino acid sequence: MWYDGSNK (SEQ ID NO: 25);ILHSGRT (SEQ ID NO: 26);IWHDGSDK (SEQ ID NO:27);IYYSGSA (SEQ ID NO: 28);IWIDGSNK (SEQ ID NO: 29);MWYDGSN (SEQ ID NO: 30);IWIDGNNI (SEQ ID NO: 31);ISAYNGN (SEQ ID NO: 32);IRNKANRYAT (SEQ ID NO: 33);SGSGGT (SEQ ID NO: 34);ITGRGFDT (SEQ ID NO: 35);ISAYNGNT (SEQ ID NO: 36)。

[0116] HCDR3 amino acid sequence: ARRGTIRTAAPFDY (SEQ ID NO: 37); AGRIAARHGWFDP (SEQ ID NO: 38); ARRGIRGTVFDH (SEQ ID NO: 39); ARGAVKYFRH (SEQ ID NO: 40); NO: 43); CARKGHYGSGTYYNPFGFD (SEQ ID NO: 44); TRNWKIFLFDY (SEQ ID NO: 45); CGKGGYCSSSGCRH (SEQ ID NO: 46); AKGLYDSGNYYIDY (SEQ ID NO: 47); ARKGHYGSGTYYNPFGFDF (SEQ ID NO: 48).

[0117] HC amino acid sequence: QVQLVESGGGVVQPGRSLRLSCTASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISGDNSKVYLQMNSLRAEDTAVYYCARRGTIRTAAPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPGGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF N STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 97) *The underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation via transglutaminase; QVQLQQWGAGLLKPSATLSRTCAVYGGSFSGYYWNWIRQSPGKGLEWIGEILHSGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAGRIAARHGWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF N STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 98) *The underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase; QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDKYYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 99) *The underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation via transglutaminase; QVQLQESGPGLVKPSETLSLTCTVSGDSINNYYWTWLRQPPGKGLEWIGYIYYSGSANYNPSLKSRVTISSVDTSKNQFSLKLNSVTAADTAVYYCARGAVKYFRHWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF N STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 100) *The underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation via transglutaminase; QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWIDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGGIVVAAPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF N STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 101) *The underlined and bold asparagine (N) can be mutated to glutamine (Q) for conjugation by transglutaminase; QVQLVESGGGVVQPGRSLRLSCTASGFTFRSYGMHWVRQAPGKGLEWVSVIWIDGNNIYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARRLAITSAAPFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 102) *The underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation via transglutaminase; EVQLVESGGNLVQPGGSLRLSCAASGFTFTSHAMNWVRQAPGKGLEWVSVITGRGFDTHYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAKGLYDSGNYYIDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF N STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 103) *The underlined and bolded asparagine (N) can be mutated to glutamine (Q) for conjugation via transglutaminase; QVQLVESGGGVVQPGRSLRLSCEASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARRGHIATAAPFDYWGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 104); QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 105); EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO: 106); EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMDVWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 107); QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 108)。

[0118] LCVR amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 49); DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIK (SEQ ID NO: 50); DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK (SEQ ID NO: 51); EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEIK (SEQ ID NO: 52); DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 53); DIQMTQSPSSLSASVGDRVTISCRASQSISSYLNWYQQKPGKAPKVLMYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 54); DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK (SEQ ID NO: 55); EIMLMQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYYGSPWTFGQGTKVEIK (SEQ ID NO: 56); EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK (SEQ ID NO: 57); QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYIYWYQRLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDTLSGYVFGTGTKVTVL (SEQ ID NO: 58); QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDLSLSFNWVFGGGTKLTVL (SEQ ID NO: 59); EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFALYFCQQYYGSPWTFGQGTKVEIK (SEQ ID NO: 60).

[0119] LCDR1 amino acid sequence: QSISSY (SEQ ID NO: 61); QDIRKW (SEQ ID NO: 62); QSISNK (SEQ ID NO: 63); QTINHNN (SEQ ID NO: 64); QSISSY (SEQ ID NO: 65); QSISSY (SEQ ID NO: 66); QSISSY(SEQ ID NO: 67); QSVSSSY (SEQ ID NO: 68); QSVGSKY (SEQ ID NO: 69); SSNIGNNY (SEQID NO: 70); SSNIGNNY (SEQ ID NO: 71); QSVSSSY (SEQ ID NO: 72).

[0120] Amino acid sequence of LCDR2: AAS (SEQ ID NO: 73); ATS (SEQ ID NO: 74); KAS (SEQ ID NO: 75); GAS (SEQID NO: 76); AAS (SEQ ID NO: 77); AAS (SEQ ID NO: 78); AAS (SEQ ID NO: 79); GA(SEQ ID NO: 80); GAS (SEQ ID NO: 81); RN (SEQ ID NO: 82); DNN (SEQ ID NO: 83); GAS (SEQ ID NO: 84).

[0121] Amino acid sequence of LCDR3: QQSYSTPPIT (SEQ ID NO: 85);QQANSFPFT (SEQ ID NO: 86);QQYNSYSWT (SEQID NO: 87);QQYGSLPLT (SEQ ID NO: 88);QQSYSTPPIT (SEQ ID NO: 89);QSYSTPPIT(SEQ ID NO: 90);QSYSTPPIT (SEQ ID NO: 91);CQQYYGSPW (SEQ ID NO: 92);QQYGSSPWT(SEQ ID NO: 93);CAAWDDTLSGY (SEQ ID NO: 94);GTWDLSLSFNWV (SEQ ID NO: 95);QQYYGSPWT (SEQ ID NO: 96)。

[0122] LC amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 109); DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 110); DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 111); EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 112); DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 113); DIQMTQSPSSLSASVGDRVTISCRASQSISSYLNWYQQKPGKAPKVLMYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC (SEQID NO: 114); DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 115); EIMLMQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYYGSPWTFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC (SEQID NO: 116); EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 117); QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYIYWYQRLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDTLSGYVFGTGTKVTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC(SEQ ID NO: 118); QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDLSLSFNWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS(SEQ ID NO: 119); EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFALYFCQQYYGSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQID NO: 120)。

[0123] In some embodiments described herein, administration of FNIP1 inhibitors and / or FLCN inhibitors can target adipose tissue or adipocytes. In some embodiments, fat-specific FNIP1 siRNA can be used to promote lipid oxidation in white adipose tissue (WAT) for therapeutic purposes. In some embodiments, FNIP1 inhibitors and / or FLCN inhibitors can be injected into adipose tissue. In some embodiments, according to the reverse transfection protocol reported by Isidor et al., Adipocyte, 2016, 5, 175-185, fat-specific FNIP1 siRNA and / or FLCN siRNA can be delivered to adipocytes via siRNA transfection of mature white adipocytes using a lipid-based transfection reagent. Essentially, the lipid-based reverse siRNA transfection reported herein can effectively silence FNIP1 and / or FLCN in mature adipocytes from various sources. In some implementations, fat-specific FNIP1 siRNA and / or FLCN siRNA can be delivered to adipose tissue by adhering to the “gold standard” proposed in Romanelli et al., Diabetes, 2020, 69, 2581-2588, with appropriate modifications. siRNA targeting FNIP1 or FLCN can be transferred via viral or non-viral vectors. Fat-cell-specific antibodies targeting fat-cell selective targets (e.g., amino acid transporter ASC-1 or brown cell surface markers) are expected to improve tropism.

[0124] Antigen-binding fragments include, but are not limited to: monovalent Fab', bivalent Fab2, F(ab)'3 fragments, single-chain variable region fragments (scFv), biscFv, (scFv)2, biantibodies, mini antibodies, nanobodies, triantibodies, tetraantibodies, disulfide-stabilized Fv proteins (dsFv), single-domain antibodies (sdAb), Ig NAR, bispecific antibodies or their binding fragments, bispecific T-cell conjugates (BiTE), trispecific antibodies and their chemically modified derivatives.

[0125] Bifunctional linkers, such as M3463 (available from Broadpharm, BP-22617), can be used as linkers to attach siRNA or antisense molecules to antibodies. The modified single-stranded linker can be synthesized using standard automated oligonucleotide synthesis. During bead-on synthesis, a 5' sense chain modification of a six-carbon chain with a primary amino terminus can be attached to a basic group, providing a handle for the linker. The chain is then subjected to standard deprotection and cleavage, followed by purification by reversed-phase high-performance liquid chromatography (HPLC) to obtain a chain that can be annealed to form double-stranded siRNA. An excess of the bifunctional linker (e.g., M3463) can be coupled to the terminal amino group of the 5' sense chain modification. The mixture can then be purified by reversed-phase HPLC to obtain a functionalized double-stranded siRNA ready for antibody conjugation. To bind the siRNA to the antibody, the antibody can be treated with 1 mM dithiothreitol or TCEP (tris(2-carboxyethyl)phosphine) at 37°C for 30 minutes. After gel filtration (G-25, pH 4.5 sodium acetate), bismaleimide linker siRNA was added to the reducing antibody, and the mixture was adjusted to pH 7.0 with 1 M HEPES (pH 7.4). After 1 hour, the conjugate was purified by size exclusion chromatography and sterile filtered. The effective loading concentrations of protein and linker were determined by UV spectroscopy. Size exclusion HPLC was used to determine that the conjugate used was >95% monomeric, and reversed-phase high-performance liquid chromatography (RP-HPLC) was used to determine the effective loading of <0.5% unconjugated linkers. UV (Hamblett et al., Cancer Res., 2004, 10, 7063) and hydrophobic interaction chromatography (HIC) were used to determine the loading of 1–1.6 siRNA / antibody.

[0126] This disclosure also provides methods for identifying subjects with an increased risk of developing metabolic disorders. In some embodiments, the method includes determining, or has been determined, the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules) in a biological sample obtained from the subject. When a subject lacks FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules (i.e., the subject is genotyped as an FNIP1 reference and / or FLCN reference), the subject has an increased risk of developing a muscle disorder. When a subject has FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules (i.e., the subject is heterozygous or homozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules), the subject has a decreased risk of developing a muscle disorder. In some embodiments, the FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules are FNIP1 variant genomic nucleic acid molecules or FLCN variant genomic nucleic acid molecules containing any one or more of the genetic variations described herein, or mRNA molecules derived therefrom, or cDNA molecules derived from mRNA molecules.

[0127] Having a single copy of the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule provides better protection against myopathy than having no copy. Without being limited to any particular theory or mechanism of action, it is believed that a single copy of the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule (i.e., heterozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule) protects a subject from myopathy, and it is also believed that having two copies of the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule (i.e., homozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule) may provide better protection against myopathy than having a single copy. Therefore, in some embodiments, a single copy of the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule may not be completely protective, but may provide partial or incomplete protection against myopathy. While we do not wish to be bound by any particular theory, there may be other factors or molecules involved in myopathy that are present in subjects with a single copy of the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule, thus resulting in less complete protection against myopathy.

[0128] Determining whether a biological sample from a subject contains FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules and / or whether a subject possesses FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecules may be present within cells obtained from the subject.

[0129] In some embodiments, when a subject is determined to have an increased risk of developing myopathy, a myopathy treatment agent and / or an FNIP1 inhibitor and / or an FLCN inhibitor are administered to the subject, as described herein. For example, when a subject is an FNIP1 reference and / or an FLCN reference, and therefore has an increased risk of developing myopathy, the subject is administered a myopathy treatment agent at a dose equal to or less than the standard dose, and / or an FNIP1 inhibitor and / or an FLCN inhibitor. In some embodiments, when a subject is heterozygous for an FNIP1 variant nucleic acid molecule and / or an FLCN variant nucleic acid molecule, the subject is administered a myopathy treatment agent at a dose equal to or less than the standard dose, and / or an FNIP1 inhibitor and / or an FLCN inhibitor. In some embodiments, when a subject is homozygous for an FNIP1 variant nucleic acid molecule and / or an FLCN variant nucleic acid molecule, the subject is administered a myopathy treatment agent at a dose equal to or less than the standard dose. In some embodiments, the subject is an FNIP1 reference and / or an FLCN reference. In some implementations, the subject is heterozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule. In some implementations, the subject is homozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule.

[0130] This disclosure also provides a method for determining the total burden or risk score of a subject having two or more FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, and / or two or more FNIP1 variant peptides or two or more FLCN variant peptides associated with a reduced risk of developing myopathy. Total burden is the sum of two or more genetic variants that can be performed in an association analysis with myopathy. In some embodiments, the subject is homozygous for one or more FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules associated with a reduced risk of developing myopathy. In some embodiments, the subject is heterozygous for one or more FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules associated with a reduced risk of developing myopathy. When a subject has a low total burden, the subject has an increased risk of developing myopathy, and the subject is given or continues to be given an amount of myopathy treatment agent, and / or an FNIP1 inhibitor and / or an FLCN inhibitor, equal to or less than the standard dose. When a subject has a high total burden, the subject has a reduced risk of developing myopathy, and the subject is given or continues to be given an amount of myopathy treatment agent, equal to or less than the standard dose. The higher the total load, the lower the risk of developing muscle disorders.

[0131] In some implementations, the total load of a subject having any two or more FNIP1 variant nucleic acid molecules and / or any two or more FLCN variant nucleic acid molecules represents a weighted sum of multiple any FNIP1 variant nucleic acid molecules and / or any FLCN variant nucleic acid molecules. In some implementations, the total burden is calculated using at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 100, at least about 120, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, at least about 500, at least about 1,000, at least about 10,000, at least about 100,000, or at least about or greater than 1,000,000 genetic variants present in or around the FNIP1 or FLCN gene (up to 10 Mb), where the genetic burden is the number of alleles multiplied by an association estimate with myopathy or a result associated with each allele (e.g., a weighted polygenic burden score). In some implementations, when a subject's total burden exceeds a desired threshold score, the subject's risk of developing myopathy is reduced. In some implementations, the risk of developing muscle disorders increases when a subject's total load falls below a desired threshold score.

[0132] In some implementations, the total burden may be divided into quintiles, such as the highest quintile, second quintile, middle quintile, fourth quintile, and lowest quintile, wherein the highest quintile of the total burden corresponds to the lowest risk group, and the lowest quintile of the total burden corresponds to the highest risk group. In some implementations, subjects with higher total burdens include those with the highest weighted total burden, including but not limited to the top 10%, top 20%, top 30%, top 40%, or top 50% of the total burden of the subject population. In some implementations, the genetic variants include those associated with myopathy that are among the top 10%, top 20%, top 30%, top 40%, or top 50% of the relevant p-value range. In some implementations, each of the identified genetic variants includes a myopathy-associated genetic variant with a p-value no greater than about 10. -2 Approximately 10 -3 Approximately 10 -4 Approximately 10 -5 Approximately 10 -6 Approximately 10 -7 Approximately 10 -8 Approximately 10 -9 Approximately 10 -10 Approximately 10 -11 Approximately 10 -12 Approximately 10 -13 Approximately 10 -14 Or about 10 -15 In some implementations, the identified genetic variants include those with p-values ​​less than 5 × 10⁻⁶. -8 Genetic variants associated with myopathy. In some implementations, the identified genetic variants include those associated with myopathy in high-risk subjects, having the following odds ratios (ORs) compared to the remainder of the reference population: approximately 1.5 or greater, approximately 1.75 or greater, approximately 2.0 or greater, or approximately 2.25 or greater for the top 20% of the distribution; or approximately 1.5 or greater, approximately 1.75 or greater, approximately 2.0 or greater, approximately 2.25 or greater, approximately 2.5 or greater, or approximately 2.75 or greater. In some implementations, the odds ratio (OR) may range from about 1.0 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, about 4.5 to about 5.0, about 5.0 to about 5.5, about 5.5 to about 6.0, about 6.0 to about 6.5, about 6.5 to about 7.0, or greater than 7.0. In some implementations, high-risk subjects have the lowest decile, quintile, or tertiary total load in the reference population. The threshold for total load may be determined based on the nature of the intended practical application and the risk difference that would be considered meaningful for said practical application.

[0133] In embodiments for determining the total burden of FNIP1 and / or FLCN genetic variants associated with myopathy, the total burden represents a risk score for the subject to have myopathy. In some embodiments, the total burden or risk score includes FNIP1 variant genomic nucleic acid molecules and / or FLCN variant genomic nucleic acid molecules containing any one or more of the genetic variants described herein, or mRNA molecules derived therefrom, or cDNA molecules derived from mRNA molecules. In some embodiments, the total burden of the subject may be determined for myopathy-associated FNIP1 and / or FLCN genetic variants in combination with other genetic variants of other genes associated with myopathy to generate a polygenic risk score (PRS) for myopathy. In some embodiments, the PRS includes FNIP1 variant genomic nucleic acid molecules and / or FLCN variant genomic nucleic acid molecules containing any one or more of the genetic variants described herein, or mRNA molecules derived therefrom, or cDNA molecules derived from mRNA molecules.

[0134] This disclosure also provides methods for detecting the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules (i.e., genomic nucleic acid molecules, mRNA molecules, or cDNA molecules derived from mRNA molecules) in biological samples from subjects. It should be understood that gene sequences and mRNA molecules encoded by such genes within a population can vary due to polymorphisms, such as single nucleotide polymorphisms.

[0135] Biological samples can be derived from any cells, tissues, or biological fluids from a subject. Biological samples may include any clinically relevant tissue, such as bone marrow samples, tumor biopsies, fine-needle aspiration, or bodily fluid samples, such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cystic fluid, or urine. In some cases, samples include oral swabs. The biological samples used in the methods disclosed herein can vary based on the assay format, the nature of the assay 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 FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, preliminary processing designed to isolate or enrich the genomic DNA of the biological sample may be employed. Various techniques can be used for this purpose. When detecting the level of any FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules, different techniques can be used to enrich the biological sample with mRNA molecules. Various methods can be used to detect the presence or level of mRNA molecules or the presence of specific variant genomic DNA sites.

[0136] In some embodiments, detecting FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules in a subject includes sequence analysis of a biological sample obtained from the subject to determine the presence of FNIP1 genomic nucleic acid molecules and / or FLCN genomic nucleic acid molecules, and / or FNIP1 mRNA molecules and / or FLCN mRNA molecules, and / or FNIP1 cDNA molecules and / or FLCN cDNA molecules generated from mRNA molecules in the biological sample. In some embodiments, the method detects FNIP1 variant genomic nucleic acid molecules and / or FLCN variant genomic nucleic acid molecules that contain any one or more of the genetic variations described herein, or mRNA molecules generated therefrom, or cDNA molecules generated from mRNA molecules.

[0137] In some embodiments, methods for detecting the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules (e.g., genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules derived from mRNA molecules) in a subject include assaying a biological sample obtained from the subject. The assay determines whether the nucleic acid molecules in the biological sample contain a specific nucleotide sequence.

[0138] In some embodiments, the biological sample comprises cells or cell lysates. Such methods may also include, for example, obtaining from a subject a biological sample containing FNIP1 genomic nucleic acid molecules or mRNA molecules and / or FLCN genomic nucleic acid molecules or mRNA molecules, and if mRNA is contained, optionally reverse transcribing the mRNA into cDNA. Such assays may include, for example, determining the identity of these sites in a specific FNIP1 nucleic acid molecule and / or FLCN nucleic acid molecule. In some embodiments, the method is an in vitro method.

[0139] In some embodiments, the determining step, detection step, or sequence analysis includes sequencing at least a portion of the nucleotide sequence of an FNIP1 genomic nucleic acid molecule and / or an FLCN genomic nucleic acid molecule, an FNIP1 mRNA molecule and / or an FLCN mRNA molecule, or an FNIP1 cDNA molecule and / or an FLCN cDNA molecule in a biological sample, said nucleotide sequence containing at least a portion of genetic variation compared to a corresponding FNIP1 reference molecule and / or an FLCN reference molecule. In some embodiments, the sequencing portion includes one or more variations that cause or are predicted to cause loss of function (partial or complete).

[0140] In some embodiments, the assay includes sequencing the entire nucleic acid molecule. In some embodiments, only the FNIP1 genomic nucleic acid molecule and / or only the FNIP1 mRNA and / or FLCN mRNA are analyzed. Only FNIP1 cDNA obtained from FNIP1 mRNA and / or FLCN cDNA obtained from FLCN mRNA are analyzed.

[0141] Modified-specific polymerase chain reaction (MCR) techniques can be used to detect mutations in nucleic acid sequences, such as SNPs. Modified-specific primers can be used because DNA polymerase will not extend when a mismatch with the template exists.

[0142] In some embodiments, the nucleic acid molecule in the sample is mRNA, and the mRNA is reverse transcribed into cDNA prior to the amplification step. In some embodiments, the nucleic acid molecule is present within cells obtained from a subject.

[0143] In some embodiments, the assay includes: under stringent conditions, contacting a biological sample with primers or probes (e.g., altering the specific primers or probes) that specifically hybridize with a variant FNIP1 genomic sequence, variant mRNA sequence, or variant cDNA sequence instead of the corresponding FNIP1 reference sequence, and determining whether hybridization has occurred; and / or contacting a biological sample with primers or probes (e.g., altering the specific primers or probes) that specifically hybridize with a variant FLCN genomic sequence, variant mRNA sequence, or variant cDNA sequence instead of the corresponding FLCN reference sequence, and determining whether hybridization has occurred.

[0144] In some implementations, the determining step, detection step, or sequence analysis includes: a) amplifying at least a portion of an FNIP1 nucleic acid molecule encoding an FNIP1 polypeptide and / or amplifying at least a portion of an FLCN nucleic acid molecule encoding an FLCN polypeptide; b) labeling the amplified nucleic acid molecule with a detectable label; c) contacting the labeled nucleic acid molecule with a vector containing a probe that modifies specificity; and d) detecting the detectable label.

[0145] In some embodiments, the assay includes RNA sequencing (RNA-Seq). In some embodiments, the assay also includes, for example, reverse transcription of mRNA into cDNA by reverse transcriptase polymerase chain reaction (RT-PCR).

[0146] In some embodiments, the method utilizes probes and primers of sufficient nucleotide length to bind to a target nucleotide sequence and specifically detect and / or identify polynucleotides containing FNIP1 variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules, or FLCN variant genomic nucleic acid molecules, variant mRNA molecules, or variant cDNA molecules. Hybridization or reaction conditions can be determined by the operator to achieve this result. The nucleotide length can be any length sufficient for the selected detection method (including any assay described or exemplified herein). Such probes and primers can specifically hybridize to the target nucleotide sequence under highly stringent hybridization conditions. Probes and primers can have complete nucleotide sequence identity with adjacent nucleotides within the target nucleotide sequence, but probes can be designed using conventional methods to differ from the target nucleotide sequence while retaining the ability to specifically detect and / or identify the target nucleotide sequence. The probes and primers can have approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or complementarity with the nucleotide sequence of the target nucleic acid molecule.

[0147] Illustrative examples of nucleic acid sequencing technologies include, but are not limited to, Sanger sequencing and dye-terminated sequencing. Other methods involve nucleic acid hybridization methods other than sequencing, including the use of labeled primers or probes for purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, target nucleic acid molecules can be amplified before or simultaneously with detection. Illustrative examples of nucleic acid amplification technologies 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).

[0148] In hybridization techniques, stringent conditions can be employed to ensure that a probe or primer specifically hybridizes with its target. In some embodiments, under stringent conditions, the polynucleotide primer or probe will hybridize with its target sequence to a detectable degree greater than, for example, at least 2, 3, 4, or more times (relative to background), including more than 10 times (relative to background). In some embodiments, under stringent conditions, the polynucleotide primer or probe will hybridize with its target nucleotide sequence to a detectable degree at least 2 times greater than with other nucleotide sequences. In some embodiments, under stringent conditions, the polynucleotide primer or probe will hybridize with its target nucleotide sequence to a detectable degree at least 3 times greater than with other nucleotide sequences. In some embodiments, under stringent conditions, the polynucleotide primer or probe will hybridize with its target nucleotide sequence to a detectable degree at least 4 times greater than with other nucleotide sequences. In some embodiments, under stringent conditions, the polynucleotide primer or probe will hybridize with its target nucleotide sequence to a detectable degree more than 10 times greater than with other nucleotide sequences (relative to background). The strict condition is sequence-dependent and will be different in different environments.

[0149] Suitable stringent conditions for promoting DNA hybridization (e.g., 6X sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by washing with 2X SSC at 50°C) are known and available in [the context of DNA hybridization]. Current Protocols in Molecular Biology John Wiley & Sons, NY (1989), 6.3.1–6.3.6. Typically, the stringent conditions used for hybridization and detection will be those described below: a salt concentration of less than approximately 1.5 M Na at pH 7.0 to 8.3. + Ions, typically about 0.01 to 1.0 M Na + The ion concentration (or other salts) and temperature are at least about 30°C for short probes (e.g., like 10 to 50 nucleotides) and at least about 60°C for longer probes (e.g., like more than 50 nucleotides). Tight conditions can also be achieved by adding a destabilizing agent (e.g., formamide). Optionally, the wash buffer may contain about 0.1% to about 1% SDS. The duration of hybridization is typically less than about 24 hours, usually about 4 to about 12 hours. The washing time will be at least long enough to reach equilibration.

[0150] In some implementations, the isolated nucleic acid molecules contain 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 5 5, 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 or composed thereof. In some embodiments, the isolated nucleic acid molecule comprises or consists 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.

[0151] In some embodiments, such isolated nucleic acid molecules are hybridized under stringent conditions to FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules (such as genomic nucleic acid molecules, mRNA molecules, and / or cDNA molecules). These nucleic acid molecules can be used as probes, primers, modified-specific probes, or modified-specific primers, as described or illustrated herein, and include, but are not limited to, primers, probes, antisense RNA, shRNA, and siRNA, each described in more detail elsewhere herein, and can be used in any of the methods described herein.

[0152] In some embodiments, the isolated nucleic acid molecule hybridizes with 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 with an FNIP1 variant nucleic acid molecule or an FLCN variant nucleic acid 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.

[0153] In some embodiments, the specific probe and specific primer are modified to contain DNA. In some embodiments, the specific probe and specific primer are modified to contain RNA.

[0154] In some embodiments, the probes and primers described herein (including modified-specific probes and modified-specific primers) have nucleotide sequences that specifically hybridize with any nucleic acid molecule disclosed herein or its complementary sequence. In some embodiments, the probes and primers specifically hybridize with any nucleic acid molecule disclosed herein under stringent conditions.

[0155] In some implementations, primers (including modified-specific primers) can be used in next-generation sequencing or high-throughput sequencing. In some cases, primers can be modified, including modified-specific primers. In particular, primers can contain various modifications used in different steps of, for example, massively parallel signature sequencing (MPSS), polymerase cloning sequencing (Polony sequencing), and 454 pyrosequencing. Modified primers can be used in several steps of the process, including the use of biotinylated primers in the cloning step and fluorescently labeled primers in the bead loading and detection steps. Polymerase cloning sequencing is typically performed using paired-end tagged libraries, where each DNA template molecule is approximately 135 bp in length. Biotinylated primers are used in the bead loading step and emulsion PCR. Fluorescently labeled degenerate nonameric oligonucleotides are used in the detection step. The adaptor may contain a 5'-biotin tag for immobilizing the DNA library onto streptavidin-coated beads.

[0156] The probes and primers described herein can be used to detect nucleotide variations in any of the FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules disclosed herein. The primers described herein can be used to amplify any FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule, or fragments thereof.

[0157] In the context of this disclosure, "specific hybridization" means that the probe or primer (e.g., by altering the specific probe or primer) does not hybridize with the nucleic acid sequence encoding the FNIP1 reference genomic nucleic acid molecule, the FNIP1 reference mRNA molecule and / or the FNIP1 reference cDNA molecule, and / or does not hybridize with the nucleic acid sequence encoding the FLCN reference genomic nucleic acid molecule, the FLCN reference mRNA molecule and / or the FLCN reference cDNA molecule.

[0158] In some embodiments, the probe (e.g., a modified specific probe) contains a label. In some embodiments, the label is a fluorescent label, a radioactive label, or biotin.

[0159] This disclosure also provides supports for substrates to which any one or more of the probes disclosed herein are attached. A solid support is a solid substrate or support to which molecules (such as any of the probes disclosed herein) can associate. One form of a solid support is an array. Another form of a solid support is an array of detectors. An array of detectors is a solid support to which multiple different probes are coupled in an array, grid, or other organized pattern. One form of a solid substrate is a microtiter dish, such as a standard 96-well type. In some embodiments, a porous glass slide, typically containing one array per well, may be used.

[0160] Genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can originate from any organism. For example, genomic nucleic acid molecules, mRNA molecules, and cDNA molecules can be human or orthologs of another organism (e.g., non-human mammals, rodents, mice, or rats). It should be understood that gene sequences within a population can vary due to polymorphisms such as single nucleotide polymorphisms.

[0161] This article also provides functional polynucleotides that can interact with the disclosed nucleic acid molecules. Examples of functional polynucleotides include, but are not limited to, antisense molecules, aptamers, ribozymes, triplet-forming molecules, and external guide sequences. Functional polynucleotides can act as influencers, inhibitors, regulators, and stimulators of the specific activities of target molecules, or they can possess novel activities independent of any other molecule.

[0162] The isolated nucleic acid molecules disclosed herein may include RNA, DNA, or both RNA and DNA. The isolated nucleic acid molecules may also be ligated or fused to heterologous nucleic acid sequences (such as in a vector) or heterologous tags. For example, the isolated nucleic acid molecules disclosed herein may be in a vector or as an exogenous donor sequence comprising the isolated nucleic acid molecule and a heterologous nucleic acid sequence. The isolated nucleic acid molecules may also be ligated or fused to heterologous tags. Tags may be directly detectable (e.g., fluorophores) or indirectly detectable (e.g., haptens, enzymes, or fluorophore quenchers). Such tags can be detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such tags include, for example, radioactive tags, pigments, dyes, chromogens, spin tags, and fluorescent tags. Tags may also be, for example, chemiluminescent substances; metal-containing substances; or enzymes, wherein enzyme-dependent secondary signal generation occurs. The term “tag” may also refer to a “label” or hapten that selectively binds 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 can be used as a tag, along with avidin or streptavidin conjugates of horseradish peroxidase (HRP), to bind to the tag and be checked using a calorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorescent substrate to detect the presence of HRP. Exemplary tags that can be used to facilitate purification include, but are not limited to, myc, HA, FLAG or 3XFLAG, 6Xhis or polyhistidine, glutathione S-transferase (GST), maltose-binding proteins, epitope tags, or the Fc portion of immunoglobulins. Many tags include, for example, particles, fluorophores, haptens, enzymes and their calorimetric, fluorescent, and chemiluminescent substrates, and other tags.

[0163] The percentage of identity (or complementarity) between specific elongations of nucleotide sequences within nucleic acid molecules or amino acid sequences within polypeptides can be determined using the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or routinely using the Gap program (Wisconsin Sequence Analysis Package for Unix, version 8, Genetics Computer Group, University Research Park, Madison Wis.) with default settings (which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489)). In this document, a higher percentage of sequence identity is preferred over a lower percentage when referring to sequence identity percentages.

[0164] This disclosure also provides myopathic therapeutic agents for treating, preventing, or inhibiting myopathic symptoms, for treating or preventing myopathic symptoms in subjects having FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules. Any myopathic therapeutic agent for treating, preventing, or inhibiting myopathic symptoms described herein is applicable hereto. Any FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules disclosed herein are applicable hereto. In some embodiments, the FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules are FNIP1 variant genomic nucleic acid molecules or FLCN variant genomic nucleic acid molecules containing any one or more of the genetic variations described herein, or mRNA molecules derived therefrom, or cDNA molecules derived from mRNA molecules.

[0165] This disclosure also provides the use of a myopathy therapeutic agent for treating, preventing, or inhibiting myopathy, for preparing a medicament for treating or preventing myopathy in a subject having an FNIP1 variant nucleic acid molecule and / or an FLCN variant nucleic acid molecule. Any myopathy therapeutic agent described herein for treating, preventing, or inhibiting myopathy may be used herein. Any FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule disclosed herein may be used herein. In some embodiments, the FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule is an FNIP1 variant genomic nucleic acid molecule or an FLCN variant genomic nucleic acid molecule containing any one or more of the genetic variations described herein, or an mRNA molecule derived therefrom, or a cDNA molecule derived from an mRNA molecule.

[0166] This disclosure also provides FNIP1 inhibitors for treating or preventing muscle disorders in subjects who are FNIP1 references or heterozygous for FNIP1 variant nucleic acid molecules. Any FNIP1 inhibitors described herein are applicable here. Any FNIP1 variant nucleic acid molecules disclosed herein are applicable here. In some embodiments, the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule is an FNIP1 variant genomic nucleic acid molecule or an FLCN variant genomic nucleic acid molecule containing any one or more of the genetic variations described herein, or an mRNA molecule derived therefrom, or a cDNA molecule derived from an mRNA molecule.

[0167] This disclosure also provides FNIP1 inhibitors for the preparation of medicaments for the treatment or prevention of muscle disorders in subjects who are FNIP1 references or heterozygous for FNIP1 variant nucleic acid molecules. Any FNIP1 inhibitors described herein are applicable here. Any FNIP1 variant nucleic acid molecules disclosed herein are applicable here. In some embodiments, the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule is an FNIP1 variant genomic nucleic acid molecule or an FLCN variant genomic nucleic acid molecule containing any one or more of the genetic variations described herein, or an mRNA molecule derived therefrom, or a cDNA molecule derived from an mRNA molecule.

[0168] This disclosure also provides FLCN inhibitors for treating or preventing muscle disorders in subjects who are FLCN references or heterozygous for FLCN variant nucleic acid molecules. Any FLCN inhibitors described herein are applicable here. Any FLCN variant nucleic acid molecules disclosed herein are applicable here. In some embodiments, the FLCN variant nucleic acid molecule is an FLCN variant genomic nucleic acid molecule containing any one or more of the genetic variations described herein, or an mRNA molecule derived therefrom, or a cDNA molecule derived from an mRNA molecule.

[0169] This disclosure also provides FLCN inhibitors for the preparation of medicaments for the treatment or prevention of muscle disorders in subjects who are FLCN references or heterozygous for FLCN variant nucleic acid molecules. Any FLCN inhibitors described herein are applicable here. Any FLCN variant nucleic acid molecules disclosed herein are applicable here. In some embodiments, the FLCN variant nucleic acid molecule is an FLCN variant genomic nucleic acid molecule containing any one or more of the genetic variations described herein, or an mRNA molecule derived therefrom, or a cDNA molecule derived from an mRNA molecule.

[0170] In some embodiments, an FNIP1 inhibitor and / or FLCN inhibitor, along with a myopathy treatment agent, are disposed within the pharmaceutical composition. In some embodiments, an FNIP1 inhibitor and / or FLCN inhibitor are disposed within a first pharmaceutical composition, and a myopathy treatment agent is disposed within a second pharmaceutical composition. In some embodiments, the first and second pharmaceutical compositions are administered simultaneously. In some embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition. In some embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition.

[0171] All patent documents, websites, other publications, registration numbers, etc., cited above or below are incorporated herein by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated as being incorporated by reference. If different versions of a sequence are associated with registration numbers at different times, the version associated with the registration number on the effective filing date of this application is indicated. The effective filing date is the earlier of the actual filing date or the filing date of the priority application referencing the registration number (if applicable). Similarly, if different versions of publications, websites, etc., are published at different times, the version most recently published on the effective filing date of this application is indicated, unless otherwise stated. Unless otherwise specifically stated, any feature, step, element, embodiment, or aspect of this disclosure may be used in combination with any other feature, step, element, embodiment, or aspect. Although this disclosure has been described in detail by illustration and example for clarity and understanding purposes, it will be apparent that certain changes and modifications may be made within the scope of the appended claims.

[0172] The following examples are provided to describe the embodiments in more detail. They are intended to illustrate, but not limit, the claimed embodiments. The following examples provide a disclosure and description, for those skilled in the art, of how the compounds, compositions, articles, apparatuses, and / or methods described herein can be prepared and evaluated, and are intended to be exemplary only and not to limit the scope of any claim. Efforts have been made to ensure accuracy regarding figures (e.g., amounts, temperatures, etc.), but some errors and deviations may be expected. Unless otherwise stated, parts are parts by weight, temperatures are in °C or at ambient temperature, and pressures are at or near atmospheric pressure.

[0173] Example Example 1: General Method Participation queue Genetic association studies were conducted on the following individuals: those of African or mixed-race African, mixed-race American, European, and East and South Asian descent from the UK Biobank (UKB) cohort (Bycroft et al., Nature, 2018, 562, 203-09; and Van Hout et al., Nature, 2020, 586, 749-56); the MyCode Community Health Initiative cohort from the Gaessinger Health System (GHS) (Carey et al., Genet. Med., 2016, 18, 906-13); the University of Pennsylvania Medical Biobank (UPENN-PMBB); the Malmö Diet and Cancer Study (MDCS) (Berglund et al., J. Int. Med., 1993, 233, 45-51); and the Mount Sinai BioMe Personalized Medicine Cohort (SINAI) (Gottesman et al., Genet. Med., 2013, 15). 761-71), Indiana Biobank (INDIANA), Colorado Biobank and Prospective Collection Project (COLORADO), UCLA Biobank (UCLA), Mayo Clinic Biobank (MAYO-CLINIC), Pakistani high-bloodline cohort recruited by the University of Pennsylvania Center for Noncommunicable Diseases (CNCD), Pima Indian Sequencing (NIDDK) of the National Institute for Diabetes and Digestive and Kidney Diseases (Knowler et al., Am. J. Epidemiol., 1978, 108, 497-505), Mexico City Prospective Study (MCPS) (Tapia-Conyer et al., Int. J. Epidemiol., 2006, 35, 243-9), and the University of Texas Southwestern Medical Center Dallas Heart Extension Study-Dallas Biobank (UTSW-COHEN).

[0174] The UKB is a population-based cohort study that recruited individuals aged 40 to 69 years from 22 testing centers in the UK between 2006 and 2010. Exome sequencing and phenotypic data covered up to 432,015 individuals. The GHS MyCode study is a health system-based cohort that recruited patients from Central and Eastern Pennsylvania (USA) between 2007 and 2019. A total of 134,731 participants from GHS were included, along with available whole-exome sequencing and phenotypic data. SINAI is a clinical care cohort associated with electronic health records, with 21,673 individuals having available phenotypic and exome sequencing data. MDCS is a population-based prospective cohort that included 5,215 individuals with available phenotypic and exome sequencing data. UPENN-PMBB included a total of 26,022 individuals with available phenotypic and exome sequencing data. The INDIANA Biobank included 5,389 participants with both exome sequencing and phenotypic data. MCPS was a population-based prospective study including 33,935 individuals with both phenotypic and exome sequencing data. CNCD was a population-based study of individuals in Pakistan including 34,831 individuals with both phenotypic and exome sequencing data. NIDDK included 5,175 individuals with both phenotypic and exome sequencing data. COLORADO, UCLA, and MAYO-CLINIC provided genotypic and phenotypic data for 35,320, 27,932, and 81,132 individuals, respectively, based on hospital electronic health records. UTSW-COHEN was a population-based African American and Hispanic cohort in Dallas, including 13,888 individuals.

[0175] Phenotype definition For population-based studies like UKB and MDCS, lipid levels are measured from samples collected upon initial visit to the research center. For cohorts using electronic health record data, lipid levels are obtained as median measurements. Generally, LDL cholesterol levels are estimated using the Friedewald equation, except at UKB, where LDL is measured directly. Individuals known to be taking lipid-lowering medications have their pre-treatment lipid levels estimated using a correction factor.

[0176] Liver MRI phenotype A subset of UKB participants underwent liver magnetic resonance imaging (MRI) (Littlejohns et al., Nat. Commun., 2020, 11, 2624). For liver fat imaging acquisition, approximately 10,000 subjects were imaged using the Dixon gradient echo protocol, while the remaining individuals who underwent imaging from 2016 onwards were imaged using the IDEAL sequence protocol (which utilizes echo asymmetry and least-squares estimation to iteratively decompose water and fat). Data from this acquisition are provided as a series of complex-valued 2D images for each subject. The in-plane pixel size is 2.5 × 2.5 mm; the slice thickness is 6 mm. All images were acquired on a Siemens MAGNETOM clinical MRI scanner.

[0177] The measurement of liver fat percentage (proton density liver fat fraction; PDFF), a measure of the proportion of fat content in the liver, is obtained by segmenting the liver on liver MRI images and applying a predefined mathematical model (Hernando et al., Magn. Reason. Med., 2012, 67, 638-44). This implementation was validated using the publicly available Phantom dataset, which contains vials with varying concentrations of fat (Hernando et al., Magn. Reason. Med., 2017, 77, 1516-24). PDFF is estimated as the proportion of fat signal relative to total fat plus water signal. Pixels belonging to the liver in the PDFF image were segmented using the Li thresholding method to identify liver tissue. To obtain a summary measurement for each trait for each subject, the average value of all pixels within the liver in each parametric image was taken. The PDFF was then used as the analysis result. Further details regarding the derivation of these phenotypes have been published (O'Dushlaine et al., Genome-wide association study of liver fat, iron, and extracellular fluid fraction in the UK Biobank, medRxiv: 2021.10.25.21265127).

[0178] Phenotype definition Clinical laboratory measurements of ALT, total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were extracted from the electronic health records (EHRs) of participants in the GHS, UPENN-PMBB, SINAI, INDIANA, UCLA, COLORADO, and MAYO-CLINIC biobanks, or measured at recruitment time in UKB, MDCS, CNCD, NIDDK, and UTSW-COHEN. For GHS, UPENN-PMBB, SINAI, INDIANA, UCLA, COLORADO, and MAYO-CLINIC, the median values ​​for all participants who underwent two or more measurements were calculated. In UKB, ALT, AST, total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were measured by analysis performed by the IFCC (International Federation for Clinical Chemistry) on a Beckman Coulter AU5800 at the baseline visit of the study, and the average was taken from multiple measurements. Prior to genetic association analysis, continuous phenotypic values ​​were transformed using an inverse standard normal function and applied to each ancestral group, separately for males and females.

[0179] Disease outcomes were defined according to the International Classification of Diseases, Ninth and Tenth Revisions (ICD-9 and ICD-10) and read codes stored in the EHR, with self-reports used where available; all of these were combined into a single variable to classify individuals as cases or controls. The previously described algorithm was used to identify individuals with type 2 diabetes (Eastwood et al., PLoS One, 2016, 11, e0162388). Individuals with coronary artery disease or liver disease were identified by combining EHR records, self-reports, and ALT measurements, as described in Table 6.

[0180] EHR records and self-reports are used to identify individuals with or without a disease. The OPCS4 code (operational procedures), f.20002 (self-reported disease), and f.20004 (self-reported operation procedures) variables are specific to the UKB. Within each queue, EHR records with ICD-9 or read codes are converted to ICD-10 codes.

[0181] Table 2

[0182] In each cohort, cases of type 2 diabetes were defined based on one or more of the following criteria: 1) Type 2 diabetes was recorded in an electronic health record (using ICD-10 diagnostic code E11 or O24.1 or the corresponding ICD-9 code), with at least one hospitalization or at least two outpatient visits, or as a recorded cause of death; 2) Blood glucose biomarker values ​​(HbA1c, random or fasting blood glucose) were within the diabetic range (Diabetes Care, 2021, 44(Supplement 1), S15-S33); 3) Antidiabetic medication use prescription records; 4) A self-reported physician diagnosis of type 2 diabetes; 5) Registration as a type 2 diabetes case in the diabetes registry. Where possible, individuals with a potential diagnosis of type 1 diabetes (using ICD-10 code E10 or O24.0, and whose prescription records only included insulin if no other diabetes medications were prescribed) were excluded from the case database. Individuals who did not meet any of the criteria for diabetes case status were used as controls. In addition, individuals were excluded from the control group if any of the following criteria were met: 1) an electronic health record diagnosis associated with any potential type of diabetes or a family history of diabetes; 2) blood glucose biomarker values ​​within the prediabetes range; or 3) any other cohort-specific phenotypes that might indicate a diagnosis of diabetes (e.g., disease registry entries or self-reported diagnoses of nonspecific diabetes).

[0183] Genotype data High-coverage whole-exome sequencing was performed as previously described (Science, 2016, 354:aaf6814; and Nature, 2020, 586, 749-756) and as summarized below. NimbleGen probes (VCRome; for a portion of the GHS cohort) or modified versions of the xGen design provided by Integrated DNA Technologies (IDT; for the remainder of the GHS and other cohorts) were used for exome target sequence capture. A unique 6-base-pair (bp) barcode (VCRome) or 10 bp barcode (IDT) was added to each DNA fragment during library preparation to facilitate multiplex exome capture and sequencing. Equal volumes of samples were pooled prior to exome capture. Sequencing was performed using 75 bp paired-end reads on an Illumina v4 HiSeq 2500 (for a portion of the GHS cohort) or NovaSeq (for the remainder of the GHS and other cohorts). The sequencing coverage depth (i.e., the number of sequence reads covering each nucleotide in the genomic target region) was sufficient to provide greater than 20x coverage of 85% of the target bases in 96% of VCRome samples and 20x coverage of 90% of the target bases in 99% of IDT samples. Data processing steps included sample splitting using Illumina software, alignment with the GRCh38 human genome reference sequence, including generating binary alignment and mapping files (BAMs), and processing the BAM files (e.g., marking repetitive reads and other read mapping evaluations). Variant calls were performed using the GLNexus system. Variant mapping and annotation were based on the GRCh38 human genome reference sequence and Ensembl v85 gene definitions using snpEff software. SnpEff predictions involving the annotated initiation and termination of protein-coding transcripts were then combined into single functional effect predictions by selecting the most harmful functional effect category for each gene. The levels of these annotations (from most harmful to least harmful) are frameshift, termination gain, termination loss, splice acceptor, splice donor, termination loss, in-frame insertion / deletion, missense, and other annotations. Predicted LOF genetic variants include: a) insertions or deletions resulting in frameshifts; b) insertions, deletions, or single nucleotide variants resulting in the introduction of premature stop codons or loss of transcription start or stop sites; and c) variants of donor or acceptor splice sites.Potential functional impacts were predicted using SIFT (Adzhubei et al., Nat. Methods, 2010, 7, 248-9), Polyphen2_HVAR (Adzhubei et al., Nat. Methods, 2010, 7, 248-9), LRT (Chun et al., Genome Res., 2009, 19, 1553-61), and MutationTaster (Schwarz et al., Nat. Methods, 2010, 7, 575-6). computer The prediction algorithm categorizes missense variants. For each gene, the alternative allele frequency (AAF) and functional annotation of each variant determine the inclusion of the following seven gene load exposures: 1) pLOF variants where AAF < 1%; 2) pLOF or missense variants predicted as harmful by all five of the five algorithms, where AAF < 1%; 3) pLOF or missense variants predicted as harmful by all five of the five algorithms, where AAF < 0.1%; 4) pLOF or missense variants predicted as harmful by at least one of the five algorithms, where AAF < 1%; 5) pLOF or missense variants predicted as harmful by at least one of the five algorithms, where AAF < 0.1%; 6) pLOF or any missense variant, where AAF < 1%; 7) pLOF or any missense variant, where AAF < 0.1%.

[0184] Gene load association analysis of rare loss-of-function variants Using REGENIE v1.0 software, the association between the negative loading of rare loss-of-function or missense variants in a given gene and the phenotype was examined by fitting linear regression models (for quantitative traits) or first-bias-corrected logistic regression models (for binary traits) and adjusting for polygenic scores based on an approximate genomic kinship matrix. Analysis was stratified by ancestry and based on age and chronological age. 2 Gender, Age × Gender and Age 2 × The sex interaction term, experimental batch-related covariates, principal components derived from 10 common variants, and principal components derived from 20 rare variants were adjusted. A fixed-effects inverse variance-weighted meta-analysis was used to combine cross-cohort results for each variant-phenotype association. In the gene load test, all individuals were labeled heterozygous if they carried one or more eligible rare variants (based on frequency and functional annotation, as described above), and homozygous if they carried any eligible variant in a homozygous state. This “composite genotype” was then used to test associations.

[0185] Example 2: Loss of function of genes encoding FNIP1 and FLCN is associated with decreased levels of triglycerides, alanine aminotransferase, liver fat as measured by MRI, HbA1c, and waist-to-hip ratio, as well as protection against type 2 diabetes. We analyzed exome sequencing data from participants from the following organizations: the Geisinger Health System MyCode Community Health Initiative Study (GHS), the Malmö Diet and Cancer Study (MDCS), the Mount Sinai BioMe Personalized Medicine Cohort (SINAI), the UK Biobank (UKB), the University of Pennsylvania Penn Medical Biobank (UPENN-PMBB), the Indiana University Biobank (INDIANA), the Colorado Biobank and Prospective Collection Project (COLORADO), the UCLA Biobank (UCLA), the Mayo Clinic Biobank (MAYO-CLINIC), the University of Pennsylvania Highly Closed Relatives Cohort (CNCD), the National Institute for Diabetes, Digestive and Kidney Disease Pima Indian Sequencing (NIDDK), the Mexico City Prospective Study (MCPS), and the University of Texas Southwestern Medical Center Dallas Heart Extension Study-Dallas Biobank (UTSW-COHEN). In a large-scale whole-exome association analysis of rare coding variants with blood lipids, loss-of-function variants of FNIP1 and FLCN were identified as being associated with decreased levels of triglycerides, LDL cholesterol, and alanine aminotransferase (ALT).

[0186] Compared to non-carriers, carriers of rare predictive loss-of-function variants in FNIP1 and FLCN had lower levels of triglycerides (-35 mg / dL and -21 mg / dL, respectively), LDL cholesterol (-7 mg / dL and -4 mg / dL, respectively), and apolipoprotein B (-7 mg / dL and -5 mg / dL, respectively) (see Table 3). Carriers of loss-of-function variants in both genes had lower alanine aminotransferase levels, while carriers of the FLCN loss-of-function variant had significantly lower levels of liver fat and inflammation, as measured by MRI (see Table 4). Rare predictive loss-of-function and harmful missense variants of FNIP1 were associated with a lower probability of non-alcoholic liver disease (see Table 5). Carriers of loss-of-function or harmful missense variants in both genes had favorable fat distribution, as indicated by a lower waist-to-hip ratio, lower body fat percentage, lower HbA1c, and a lower probability of type 2 diabetes (see Tables 6 and 7).

[0187] Table 3: Rare predictive loss-of-function variants in FNIP1 and FLCN are associated with lower levels of LDL cholesterol, triglycerides, and apolipoproteins.

[0188] Effect sizes are expressed as standard deviation (SD) and clinical units. RR: Count of homozygous individuals carrying the reference allele; RA: Count of heterozygous individuals carrying one pLOF variant; AA: Count of homozygous individuals carrying the pLOF variant in both alleles.

[0189] Table 4: FNIP1 and FLCN Rare variants of liver disease with loss of predictive function were associated with lower levels of liver enzymes, liver fat, and inflammation as measured by MRI.

[0190] Effect sizes are expressed as standard deviation (SD) and clinical units. RR: Count of homozygous individuals carrying the reference allele; RA: Count of heterozygous individuals carrying one pLOF variant; AA: Count of homozygous individuals carrying the pLOF variant in both alleles.

[0191] Table 5: FNIP1 Rare predictive loss-of-function and harmful missense variants are associated with the likelihood of non-alcoholic liver disease.

[0192] RR: Count of homozygous individuals with reference allele; RA: Count of heterozygous individuals carrying one pLOF or pLOF+ harmful missense variant; AA: Count of homozygous individuals with both alleles carrying pLOF+ harmful missense variant.

[0193] Table 6: Rare variants of FNIP1 and FLCN with loss of predictive function are associated with lower waist-to-hip ratio, body fat percentage, and HbA1c levels.

[0194] Effect sizes are expressed as standard deviation (SD) and clinical units. RR: Count of homozygous individuals carrying the reference allele; RA: Count of heterozygous individuals carrying one pLOF variant; AA: Count of homozygous individuals carrying the pLOF variant in both alleles.

[0195] Table 7: FNIP1 and FLCN Rare predictive loss-of-function and harmful missense variants were associated with a lower probability of type 2 diabetes.

[0196] RR: Count of homozygous individuals with reference allele; RA: Count of heterozygous individuals carrying one pLOF or pLOF+ harmful missense variant; AA: Count of homozygous individuals with both alleles carrying pLOF+ harmful missense variant.

[0197] Table 8: Predicted loss-of-function or harmful missense variants of FNIP1 identified by whole-exome sequencing

[0198] Table 9: Loss-of-function or harmful missense variants in FLCN identified by whole-exome sequencing

[0199] Other data are shown in Figure 1-18 middle.

[0200] Figure 1 This study demonstrates the identification of rare FNIP1 coding variants in ExWAS associated with blood lipids and liver enzymes; the maximum effect was observed at lower triglycerides and ALT.

[0201] Figure 2 The results showed that genome-wide association analysis identified a common missense variant (5:131672501:T:C--Gln648Arg) in FNIP1 that was associated with the TGL:HDL ratio.

[0202] Figure 3 The study showed an association between rare coding variants of FNIP1 and higher HDLC and lower triglyceride and LDLC levels; FLCN showed a similar association pattern.

[0203] Figure 4Rare coding variants of FNIP1 and FLCN were shown to be associated with lower ALT levels; FLCN pLoF was associated with lower MRI-derived liver fat and inflammation.

[0204] Figure 5 The effects of rare coding variants of FNIP1 and FLCN on ALT and PDFF are shown.

[0205] Figure 6 This shows the association between rare FNIP1 coding variants and lower NALD odds.

[0206] Figure 7 The association between common FNIP1 missense variants and liver enzymes was shown.

[0207] Figure 8 The study showed that rare coding variants of FNIP1 and FLCN were associated with lower waist-to-hip ratio and body fat percentage.

[0208] Figure 9 The effects of rare variants of FNIP1 on body fat distribution were shown.

[0209] Figure 10 The study showed an association between rare coding variants of FNIP1 and FLCN and lower HbA1c levels and a lower chance of developing type 2 diabetes.

[0210] Figure 11 The association between rare coding variants of FNIP1 and FLCN and lower eGFR is shown.

[0211] Figure 12 The results showed that FNIP1 pLoF has a general protective cardiovascular metabolic association.

[0212] Figure 13 The results showed that FNIP1 pLoF has a general protective cardiovascular metabolic association.

[0213] Figure 14 The association between common missense variants of FNIP1 and the rare coding of FNIP1 is shown to be similar.

[0214] Figure 15 The study showed that common missense variants of FNIP1 were most strongly associated with lower T2D probability.

[0215] Example 3: Effects of FNIP1 or FLCN knockdown on skeletal muscle In this embodiment, multiple gRNAs were simultaneously mixed using the AAV-myo2A vector to achieve intramuscular delivery of FNIP1 or FLCN gRNA, thereby achieving effective gene perturbation. Subsequently, its knockdown effect on muscle fiber transition and various mitochondrial gene expression patterns was observed.

[0216] Figure 16 This study demonstrated that FNIP1 inhibition in skeletal muscle induced increased expression of oxidative myofibrils and mitochondrial genes. The FNIP1 gRNA pool was co-packaged into AAVmyo2A, purchased from SignaGen Laboratories (catalog number SL100862). The AAV-gRNA pool was then delivered intramuscularly to male Cas9 mice at a dose of 1.5E11 vg per tibialis anterior (TA) muscle, resulting in effective gene perturbation in both the TA and extensor digitorum longus (EDL) muscles. Six weeks after AAV administration, the following phenotypes were observed: conversion of glycolytic myofibrils to oxidative myofibrils in both TA and EDL muscles, and increased mitochondrial gene expression. These data are consistent with literature on muscle-specific FNIP1 cKO mice.

[0217] Figure 17 The results showed that inhibition of FNIP1 in skeletal muscle enhanced vascularization and muscle glycogen content, and increased the expression of genes related to glucose and fatty acid uptake and metabolism. These results indicate that: a) FNIP1 gRNA increases muscle vascular distribution and enhances muscle nutrient uptake, consistent with the oxidative myofiber phenotype; b) in adults, skeletal muscle-specific targeting of FNIP1 induces exercise-mimicking effects, which may partially explain the beneficial human genetic associations of FNIP1 pLOF variants: reduced HbA1c and T2D risk, reduced plasma lipids, and reduced obesity.

[0218] Figure 18 This study demonstrated that FLCN inhibition in skeletal muscle induced an increase in oxidative muscle fibers, enhanced vascular distribution, and increased glycogen content. The FLCN gRNA pool was co-packaged into AAVmyo2A, purchased from SignaGenLaboratories (catalog number SL100862). The AAV-gRNA pool was then delivered intramuscularly to male Cas9 mice at a dose of 1.5E11 vg per tibialis anterior (TA) muscle. Six weeks after AAV administration, the following phenotypes were observed: conversion of glycolytic muscle fibers to oxidative muscle fibers in the TA muscle, increased capillary density, and increased glycogen storage. These data are consistent with literature on muscle-specific FLCN cKO mice and may partially explain the beneficial human genetic associations of the FLCN pLOF variant: reduced HbA1c and T2D risk, reduced plasma lipids, and reduced obesity.

[0219] In addition to the modifications described herein, various modifications to the subject matter 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. Every reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank accession numbers, etc.) is incorporated herein by reference in its entirety and for all purposes.

Claims

1. A method for treating a subject with or at risk of developing myopathy, the method comprising administering to the subject an inhibitor of follicle-interacting protein 1 (FNIP1) and / or an inhibitor of follicle-coated protein (FLCN).

2. The method of claim 1, wherein the muscle disease includes sarcopenia, Duchenne muscular dystrophy, or Pompe disease.

3. The method of claim 1 or claim 2, wherein the FNIP1 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FNIP1 nucleic acid molecule.

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

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

6. The method of claim 4, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

7. The method of any one of claims 1 to 6, wherein the FLCN inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FLCN nucleic acid molecule.

8. The method of claim 7, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

9. The method of claim 8, wherein the inhibitory nucleic acid molecule comprises siRNA.

10. The method of claim 8, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

11. The method of any one of claims 1 to 10, wherein the subject is further administered a muscle disease treatment agent.

12. The method of any one of claims 1 to 11, further comprising detecting the presence or absence of FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules in a biological sample from the subject.

13. The method of claim 12, further comprising administering a standard dose of a muscle disease treatment agent to the subject when the FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule are not present in the biological sample.

14. The method of claim 12, further comprising administering to the subject an amount of the muscle disease treatment agent that is the same as or less than the standard dose when the subject is heterozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule.

15. The method of any one of claims 12 to 14, wherein the FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule comprises splice site variants, termination gain variants, initiation loss variants, termination loss variants, frameshift variants, missense variants, in-frame insertion / deletion variants and / or variants encoding truncated FNIP1 variant peptides or truncated FLCN variant peptides.

16. The method of any one of claims 12 to 14, wherein the FNIP1 variant nucleic acid molecule comprises any one or more gene variants in Table 8.

17. The method of any one of claims 12 to 14, wherein the FLCN variant nucleic acid molecule comprises any one or more gene variants in Table 9.

18. A method of treating a subject suffering from or at risk of developing a myopathy by administering a myopathy treatment agent, the method comprising: Whether the subject possesses follicle-interacting protein 1 (FNIP1) variant nucleic acid molecules and / or follicle-coated protein (FLCN) variant nucleic acid molecules has been determined or has been determined by the following methods: Biological samples obtained from or already obtained from the subject; as well as The biological sample has been or is being sequenced to determine whether the subject has a genotype containing FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules; as well as The muscle disease treatment agent, and / or FNIP1 inhibitor and / or FLCN inhibitor, are administered or continued to be administered to subjects who are FNIP1 references and / or FLCN references in amounts equal to or less than the standard dose. Administer or continue administration of the muscle disease treatment agent, and / or FNIP1 inhibitors and / or FLCN inhibitors, to subjects who are heterozygous for the FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule; or Administer or continue to administer the standard dose of the muscle disease treatment agent to subjects who are homozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule; The presence of the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule indicates a reduced risk of the subject developing muscle disease.

19. The method of claim 18, wherein the muscle disease includes sarcopenia, Duchenne muscular dystrophy, or Pompe disease.

20. The method of claim 18 or claim 19, wherein the FNIP1 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FNIP1 nucleic acid molecule.

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

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

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

24. The method of any one of claims 18 to 23, wherein the FLCN inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FLCN nucleic acid molecule.

25. The method of claim 24, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

26. The method of claim 25, wherein the inhibitory nucleic acid molecule comprises siRNA.

27. The method of claim 25, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

28. The method of any one of claims 18 to 27, wherein the subject is heterozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule, and the subject is given or continues to be given an amount of the myopathy treatment agent, and the FNIP1 inhibitor and / or FLCN inhibitor, that is the same as or less than the standard dose.

29. The method of any one of claims 18 to 27, wherein the subject is an FNIP1 reference and / or FLCN reference, and the subject is given or continues to be given an amount of the myopathy treatment agent, and the FNIP1 inhibitor and / or FLCN inhibitor, that is the same as or less than the standard dose.

30. The method of any one of claims 18 to 29, wherein the FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule comprises splice site variants, termination gain variants, initiation loss variants, termination loss variants, frameshift variants, missense variants, in-frame insertion / deletion variants and / or variants encoding truncated FNIP1 variant peptides or truncated FLCN variant peptides.

31. The method of any one of claims 18 to 29, wherein the FNIP1 variant nucleic acid molecule comprises any one or more gene variants in Table 8.

32. The method of any one of claims 18 to 29, wherein the FLCN variant nucleic acid molecule comprises any one or more gene variants in Table 9.

33. A method for identifying subjects with an increased risk of developing a muscle disorder, the method comprising: The presence or absence of follicle-interacting protein 1 (FNIP1) variant nucleic acid molecules and / or follicle-coated protein (FLCN) variant nucleic acid molecules in biological samples obtained from the subject has been determined or has been determined. in: When the subject is an FNIP1 reference and / or FLCN reference, the subject has an increased risk of developing the aforementioned muscle condition; and When the subject is heterozygous or homozygous for the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule, the subject's risk of developing the muscle disease is reduced.

34. The method of claim 33, wherein the muscle disorder includes sarcopenia, Duchenne muscular dystrophy, or Pompe disease.

35. The method of claim 34 or claim 35, wherein the FNIP1 variant nucleic acid molecule and / or FLCN variant nucleic acid molecule comprises splice site variants, termination gain variants, initiation loss variants, termination loss variants, frameshift variants, missense variants, in-frame insertion / deletion variants and / or variants encoding truncated FNIP1 variant peptides or truncated FLCN variant peptides.

36. The method of any one of claims 33 to 35, wherein the FNIP1 variant nucleic acid molecule comprises any one or more gene variants in Table 8.

37. The method of any one of claims 33 to 35, wherein the FLCN variant nucleic acid molecule comprises any one or more gene variants in Table 9.

38. The method of any one of claims 33 to 37, further comprising administering to a subject, as a reference for FNIP1 and / or FLCN, an amount of a myopathy treatment agent equal to or less than a standard dose, and / or an FNIP1 inhibitor and / or an FLCN inhibitor.

39. The method of claim 38, further comprising administering to a subject, as a reference for FNIP1 and / or FLCN, an amount of a myopathy treatment agent equal to or less than a standard dose, and an FNIP1 inhibitor and / or an FLCN inhibitor.

40. The method of any one of claims 33 to 37, further comprising administering to a subject heterozygous for FNIP1 variant nucleic acid molecules and / or FLCN variant nucleic acid molecules an amount of a muscle disease treatment agent equal to or less than a standard dose, and / or an FNIP1 inhibitor and / or an FLCN inhibitor.

41. The method of claim 40, comprising administering to a subject heterozygous for a muscle disease treatment agent in an amount equal to or less than a standard dose, and an FNIP1 inhibitor and / or an FLCN inhibitor.

42. The method of any one of claims 33 to 41, wherein the FNIP1 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FNIP1 nucleic acid molecule.

43. The method of claim 42, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), and / or a short hairpin RNA (shRNA).

44. The method of claim 43, wherein the inhibitory nucleic acid molecule comprises siRNA.

45. The method of claim 43, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

46. ​​The method of any one of claims 33 to 45, wherein the FLCN inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FLCN nucleic acid molecule.

47. The method of claim 46, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

48. The method of claim 47, wherein the inhibitory nucleic acid molecule comprises siRNA.

49. The method of claim 47, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

50. A muscle disease treatment agent for treating or preventing muscle disease in a subject having an FNIP1 variant nucleic acid molecule and / or an FLCN variant nucleic acid molecule.

51. The muscle disease treatment agent of claim 50, wherein the FNIP1 variant nucleic acid molecule and / or the FLCN variant nucleic acid molecule is a splice site variant, a termination gain variant, a start loss variant, a stop loss variant, a frameshift variant, a missense variant, an in-frame insertion / deletion variant, or a variant encoding a truncated FNIP1 or FLCN variant polypeptide.

52. The muscle disease treatment agent of claim 50 or claim 51, wherein the FNIP1 variant nucleic acid molecule comprises any one or more gene variants in Table 8.

53. The muscle disease treatment agent of claim 50 or claim 51, wherein the FLCN variant nucleic acid molecule comprises any one or more gene variants in Table 9.

54. A follicle-interacting protein 1 (FNIP1) inhibitor for the treatment or prevention of muscle disease in a subject who is a FNIP1 reference or heterozygous for an FNIP1 variant nucleic acid molecule.

55. The FNIP1 inhibitor of claim 54, wherein the FNIP1 variant nucleic acid molecule is a splice site variant, a termination gain variant, a start loss variant, a stop loss variant, a frameshift variant, a missense variant, an in-frame insertion / deletion variant, or a variant encoding a truncated FNIP1 variant polypeptide.

56. The FNIP1 inhibitor of claim 54 or claim 55, wherein the FNIP1 variant nucleic acid molecule comprises any one or more gene variants in Table 7.

57. The FNIP1 inhibitor of any one of claims 54 to 56, wherein the FNIP1 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FNIP1 nucleic acid molecule.

58. The FNIP1 inhibitor of claim 57, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

59. The FNIP1 inhibitor of claim 58, wherein the inhibitory nucleic acid molecule comprises siRNA.

60. The FNIP1 inhibitor of claim 58, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

61. A follicle-nucleotide (FNIP) inhibitor for treating or preventing muscle disease in a subject, said subject being a FLCN reference or heterozygous for a FLCN variant nucleic acid molecule.

62. The FLCN inhibitor of claim 61, wherein the FLCN variant nucleic acid molecule is a splice site variant, a termination gain variant, a start loss variant, a stop loss variant, a frameshift variant, a missense variant, an in-frame insertion / deletion variant, or a variant encoding a truncated FLCN variant polypeptide.

63. The FLCN inhibitor of claim 61 or claim 62, wherein the FLCN variant nucleic acid molecule comprises any one or more gene variants in Table 9.

64. The FLCN inhibitor of any one of claims 61 to 63, wherein the FNIP1 inhibitor comprises an inhibitory nucleic acid molecule that hybridizes with an FNIP1 nucleic acid molecule.

65. The FLCN inhibitor of claim 64, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, small interfering RNA (siRNA), and / or short hairpin RNA (shRNA).

66. The FLCN inhibitor of claim 64, wherein the inhibitory nucleic acid molecule comprises siRNA.

67. The FLCN inhibitor of claim 64, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule.

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