Use of agents that modulate BBS-related proteins for treatment of fiberopathy

Abstract

The invention discloses application of a reagent for regulating and controlling BBS related protein to treatment of fiberopathy. The invention provides an application of a reagent for regulating and controlling protein related to Bardet-Biedl syndrome (BBS) in preparation of a medicine for preventing and / or treating fibrinopathy, and a preparation method of the reagent for regulating and controlling the protein related to the Bardet-Biedl syndrome (BBS). Wherein the fibrillopathy is a disorder associated with a gene mutation that does not encode a BBS-associated protein or encode a defective BBS-associated protein, resulting in abnormal cilia formation or function; wherein the reagent for regulating and controlling the BBS related protein comprises metformin or a pharmaceutically acceptable salt of the metformin, L-carnitine or a pharmaceutically acceptable salt of the L-carnitine, or a reagent for targeting a T10C6.10 gene or an expression product of the T10C6.10 gene.

Description

Technical Field

[0001] The present invention belongs to the field of molecular genetics and disease treatment technology, and relates to the use of reagents for regulating BBS-related proteins for treating ciliopathy. Specifically, it relates to a method for correcting ciliary dysfunction by targeted regulation of BBSome-related pathways, as well as the use of related gene mutants and pharmaceutical compositions in the treatment of Bardet-Biedl syndrome. Background Art

[0002] Cilia are organelles composed of microtubules and are widely present in a variety of eukaryotic cells. They undertake key functions such as signal perception, signal transduction, and developmental regulation. The structural construction, maintenance, and functional realization of cilia rely on a highly conserved protein transport system—intraflagellar transport (IFT). The IFT system is responsible for transporting structural proteins and signaling molecules synthesized in the cytoplasm to the ciliary assembly site or the ciliary tip, and transporting degraded or turnover proteins back to the cell body, thereby achieving bidirectional dynamic regulation of cilia. The IFT system is composed of motor proteins (such as the forward-moving kinesin type II (kinesin-II) and the reverse-moving IFT dynein) and two protein complexes, IFT-A and IFT-B. The former is mainly involved in reverse transport, while the latter mediates forward transport. In addition, the BBSome complex, as a type of carrier connector, is responsible for recognizing and transporting a variety of cargo proteins, assisting them in achieving efficient targeting through the IFT pathway.

[0003] The BBSome is a heteromeric complex composed of eight Bardet-Biedl syndrome-associated proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9, and BBS18). BBSome recognizes specific membrane proteins, such as G protein-coupled receptors (GPCRs), and facilitates their directional transport within primary cilia, serving as a crucial bridge between ciliary membrane cargo and the IFT transport machinery. BBSome assembly, entry, and exit regulation within cilia are coordinated with motor proteins. Its dysfunction leads to decreased IFT complex stability, ciliary structural disorder, and abnormal signaling.

[0004] Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder with ciliary dysfunction as its core etiology. At least 24 genes associated with BBS have been identified, with BBS1 to BBS21 being the classic BBS pathogenic genes. The clinical manifestations of BBS involve multiple organ systems and include early-onset obesity, retinitis pigmentosa, supernumerary digits, renal malformations, genital abnormalities, and cognitive impairment.

[0005] Currently, there is no cure for BBS, and clinical treatment primarily relies on symptomatic support. Although a collaborative relationship between the BBSome and IFT systems is known, its regulatory mechanisms and potential intervention targets remain unclear, and systemic genetic and pharmacological intervention strategies are lacking. Summary of the Invention

[0006] Problems to be solved by the invention

[0007] Due to its well-defined etiology and stable model system, BBS has become an important model disease for studying the function of cilia-related genes, signal transduction mechanisms, and therapeutic intervention strategies. To gain a deeper understanding of the BBSome regulatory network and explore possible therapeutic intervention pathways, this study focused on the BBSome core subunit BBS-7, using a forward genetic screening strategy to identify genetic suppressors that can alleviate the phenotype caused by its deletion. Combining in vivo imaging and mammalian cell models, this study further explored its mechanism of action and potential for drug reversibility.

[0008] Solutions for solving problems

[0009] [1] Use of an agent that regulates Bardet-Biedl syndrome (BBS)-related proteins in the preparation of a drug for preventing and / or treating ciliopathy; wherein the ciliopathy is a disease associated with a gene mutation that does not encode a BBS-related protein or encodes a defective BBS-related protein, resulting in abnormal cilia formation or function; wherein the agent that regulates BBS-related proteins comprises metformin or a pharmaceutically acceptable salt thereof, L-carnitine or a pharmaceutically acceptable salt thereof, or an agent that targets the T10C6.10 gene or its expression product.

[0010] [2] The use according to [1], wherein the ciliopathy is Bardet-Biedl syndrome (BBS).

[0011] [3] The use according to [1] or [2], wherein the BBS-related protein comprises at least one of: BBS1, BBS2, BBS3 / ARL6, BBS4, BBS5, BBS6 / MKKS, BBS7, BBS8 / TTC8, BBS9 / PTHB1, BBS10, BBS11 / TRIM32, BBS12, BBS13 / MKS1, BBS14 / CEP290, BBS15 / WDPCP, BBS16 / SDCCAG8, BBS17 / LZTFL1, BBS18 / BBIP1, BBS19 / IFT27, BBS20 / IFT74 and BBS21 / C8ORF37.

[0012] [4] The use according to any one of [1] to [3], wherein the BBS-related protein is selected from at least one of the proteins constituting the BBSome complex; optionally, the proteins constituting the BBSome complex include BBS1, BBS2, BBS4, BBS5, BBS7, BBS8 / TTC8, BBS9 / PTHB1 and BBS18 / BBIP1.

[0013] [5] The use according to any one of [1] to [4], wherein the BBS-related protein is selected from at least one of BBS1, BBS7 and BBS8 / TTC8.

[0014] [6]. The use according to any one of [1] to [5], wherein the sequence of the T10C6.10 gene is as shown in SEQ ID NO: 17.

[0015] [7]. The use according to any one of [1] to [6], wherein the expression product of the T10C6.10 gene is selected from the group consisting of: cDNA, mRNA, T10C6.10 precursor protein, mature T10C6.10 protein, and fragments thereof.

[0016] [8] The use according to any one of [1] to [7], wherein the agent targeting the T10C6.10 gene or its expression product is selected from: nucleic acid, polypeptide, ribonucleoprotein complex or small molecule drug.

[0017] [9]. The use according to any one of [1] to [8], wherein the ciliopathy is a ciliopathy in a subject.

[0018]

[10] The use according to any one of [1] to [9], wherein the subject comprises a human, a mouse or a nematode.

[0019] Effects of the Invention

[0020] The present invention systematically reveals the function of T10C6.10 as a novel BBS genetic suppressor, and proposes reagents for regulating BBS-related proteins, such as metformin and L-carnitine, and targeting the T10C6.10 gene as potential strategies for correcting the BBS phenotype, providing a new theoretical basis and therapeutic ideas for the study of the pathogenesis and targeted intervention of ciliopathies. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :The loss-of-function mutation of the sbbs-1 / T10C6.10 gene can partially alleviate the ciliary function defects of bbs-7(n1606) single mutant nematodes.

[0022] ( Figure 1A) Quantitative analysis of dye uptake defects in cilia of different genotypes (n=300 per group, data from three independent biological replicates). Statistical significance was assessed by Student's t-test, based on comparison of means of biological replicates: ****P<0.0001.

[0023] ( Figure 1 B) Schematic diagram of the nematode sbbs-1 / T10C6.10 gene structure and the mutation sites of the six genetic suppressor alleles, namely: G244D (cas10824), P287S (cas10821), G299* (cas10828) (* indicates nonsense mutation), K347N (cas10831) and D377K (cas10863).

[0024] ( Figure 1 C) Representative images show endogenous dual fluorescent signaling of IFT-A (DYF-2::mScarlet) and IFT-B (OSM-1::GFP) complexes in cilia of the head (top) and tail (bottom) sensilla. Images are shown for a bbs-7(n1606) single mutant, a bbs-7(n1606);sbbs-1(G299*) double mutant, and a worm overexpressing sbbs-1 (sbbs-1OE) in a bbs-7;sbbs-1(G299*) background. Arrowheads indicate the cilia base and transition zone, and arrowheads mark the junction between the mid and distal segments. Scale bar = 5 μm.

[0025] Figure 2 :L-Carnitine and metformin can partially alleviate the BBS phenotype of C. elegans.

[0026] ( Figure 2 A) Bar graph showing the ratio of ciliary dye uptake defects of different bbs mutants under treatment with 100 μM L-carnitine or 50 mM metformin (n=300 per group, data from three independent biological replicates). Statistical significance was assessed by Student's t-test, based on comparison of the means of biological replicates: ***P<0.001, ****P<0.0001.

[0027] ( Figure 2 B) Representative images show endogenous dual fluorescent signaling of IFT-A (DYF-2::mScarlet) and IFT-B (OSM-1::GFP) complexes in the cilia of the head (top) and tail (bottom) sensilla of different bbs mutants after treatment with 100 μM L-carnitine or 50 mM metformin. Arrowheads indicate the cilia base and transition zone, and arrowheads indicate the junction between the mid- and distal segments. Scale bar = 5 μm.

[0028] Figure 3 :L-Carnitine and metformin can partially alleviate the abnormal accumulation of smoothened protein (SMO) in primary cilia of human retinal pigment epithelial (RPE1) cells.

[0029] ( Figure 3 A) Schematic diagram of the human BBS7 gene structure and the gene deletion site of the first exon in BBS7 gene knockout RPE1 cells.

[0030] ( Figure 3 B) Representative immunofluorescence images showing the localization of endogenous SMO in primary cilia in wild-type RPE1 cells, RPE1 cells expressing shBBS1, shBBS7, or shBBS8, and RPE1 cells with BBS7 knockout, following treatment with 10 μM L-carnitine or metformin. ARL13B was used as a primary cilium marker. Scale bar = 5 μm.

[0031] ( Figure 3 C) Scatter plots showing the ratio of the SMO fluorescence signal intensity in the ciliary region to the surrounding background before and after drug treatment in the above cells (n>100). Statistical significance was assessed by Welth's t-test: **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Figure 4 :L-Carnitine and metformin can partially alleviate the abnormal accumulation of smoothin (SMO) in primary cilia of mouse embryonic fibroblast (NIH / 3T3) cells.

[0033] ( Figure 4 A) Representative immunofluorescence images showing the localization of endogenous SMO in primary cilia of wild-type NIH / 3T3 cells expressing shBBS1, shBBS7, or shBBS8, following treatment with 10 μM L-carnitine or metformin. ARL13B was used as a primary cilium marker. Scale bar = 5 μm.

[0034] ( Figure 4 B) Scatter plots show the ratio of the SMO fluorescence signal intensity in the ciliary region to the surrounding background before and after drug treatment in the above cells (n>100). Statistical significance was assessed by Welth's t test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns indicates not significant. DETAILED DESCRIPTION

[0035] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior or preferred over other embodiments.

[0036] In addition, numerous specific details are provided in the following detailed description to better illustrate the present invention. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other instances, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main points of the present invention.

[0037] Unless otherwise stated, the units used in this specification are international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0038] In this specification, the use of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0039] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the specific elements (e.g., features, structures, properties, and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0040] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints A and B.

[0041] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

[0042] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0043] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0044] As used herein, "administer," "give," and "treat" as applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer," "give," and "treat" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by a reagent, a diagnostic agent, a binding composition, or by another cell. "Treat" as applied to a human, veterinary, or research subject refers to treatment, prophylaxis, or preventative measures, research, and diagnostic applications.

[0045] As used herein, "treatment" means administering an internal or external therapeutic agent, such as a recombinant immune cell comprising the present invention, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the patient or population being treated, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be evaluated by any clinical test method commonly used by a physician or other health care professional to evaluate the severity or progression of the symptoms.

[0046] In this specification, the term "prevention" refers to preventive treatment of a subject who does not currently have a disease or has not had a disease in the past but is at risk of developing a disease or who has had a disease in the past and does not currently have a disease but is at risk of recurrence of the disease.

[0047] As used herein, an "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also refers to an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0048] As used herein, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall therapy; reduces or avoids symptoms, signs, or causes of a condition; and / or enhances the therapeutic efficacy of another therapeutic agent.

[0049] As used herein, a "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition or to prevent its recurrence. A prophylactically effective amount refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

[0050] In this specification, the term "pharmaceutically acceptable" (or "pharmacologically acceptable", "pharmaceutically usable") refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other untoward reactions when administered to animals or humans, as appropriate. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

[0051] The term "pharmaceutically acceptable salt" refers to a salt that possesses the potency of the parent agent and is not biologically or otherwise undesirable (e.g., is neither toxic nor otherwise harmful to the recipient thereof). Suitable salts include acid addition salts, which can be formed, for example, by mixing a solution of the parent compound with a solution of a pharmaceutically acceptable acid (e.g., hydrochloric acid, sulfuric acid, acetic acid, trifluoroacetic acid, or benzoic acid). If the drug has an acidic moiety (e.g., COOH or a phenolic group), pharmaceutically acceptable salts thereof can include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands (e.g., quaternary ammonium salts).

[0052] As used herein, "subject" or "host" refers to a human or non-human animal, including mammals. For example, primates (e.g., humans, monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice, and the like. "Subjects" or "hosts" include both therapeutic and non-therapeutic subjects. "Subjects" or "hosts" also include experimental animal models or animals used to produce biomolecules expressing therapeutic diseases, i.e., "non-therapeutic hosts" or "non-therapeutic subjects," such as nematodes, such as Caenorhabditis elegans. Detailed Description of the Invention

[0054] The primary cilium is a cellular organelle composed of microtubules that is widely involved in signal perception, transduction, and tissue development. Its structural construction and functional maintenance depend on the coordinated action of the intraflagellar transport (IFT) system and the BBSome complex. Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder closely associated with ciliary dysfunction. To date, there is no effective treatment strategy, and its pathogenic mechanisms remain under investigation.

[0055] Using the core BBSome subunit BBS-7 in the Caenorhabditis elegans nematode as a model, the present invention employed a forward genetic screening strategy to identify a genetic suppressor, sbbs-1, that alleviates the ciliary defects of the bbs-7(n1606) mutant. Loss-of-function mutations in this gene, particularly the nonsense mutation G299*, significantly reduced the ciliary dye uptake defect and restored the normal localization of the IFT-A marker DYF-2. Further overexpression of the wild-type sbbs-1 sequence reversed this inhibitory effect, confirming that the phenotypic correction depended on the functional loss of sbbs-1.

[0056] Given the potential role of sbbs-1 in lipid metabolism, the present study screened two metabolic modulators, metformin and L-carnitine, and found that they could alleviate ciliary staining defects and restore DYF-2 localization in various bbs nematode mutants. This effect was also significant in mammalian cell models: in human RPE1 cells and NIH / 3T3 cells, knockdown or deletion of the BBS gene led to abnormal accumulation of smoothened (Smo) in cilia, while drug treatment effectively reduced its signal intensity, suggesting that the ciliary signaling pathway was partially restored.

[0057] Agents that regulate BBS-related proteins for preventing and / or treating ciliopathy

[0058] In some aspects of the present invention, provided is the use of an agent that regulates a BBS-associated protein in the preparation of a medicament for preventing and / or treating a ciliary disorder.

[0059] In some aspects of the present invention, a method for preventing and / or treating ciliopathy is also provided, comprising administering to a subject in need thereof a preventively and / or therapeutically effective amount of an agent that regulates BBS-related proteins.

[0060] In some aspects of the present invention, agents for regulating BBS-related proteins are also provided, which are used to prevent and / or treat ciliary diseases.

[0061] In the above aspects, the ciliopathy is a disorder associated with a mutation in a gene that does not encode a Bardet-Biedl syndrome-associated protein (BBS-associated protein) or encodes a defective Bardet-Biedl syndrome-associated protein, resulting in abnormal cilia formation or function.

[0062] In the above aspects, the agent for regulating BBS-related proteins includes L-carnitine or a pharmaceutically acceptable salt thereof, metformin or a pharmaceutically acceptable salt thereof, or an agent targeting the T10C6.10 gene or its expression product.

[0063] (Ciliopathy)

[0064] Ciliopathies are a group of disorders associated with mutations in genes encoding defective proteins that lead to abnormal cilia formation or function.

[0065] In this specification, ciliopathy is defined as "a disorder associated with a mutation in a gene that does not encode a Bardet-Biedl syndrome-associated protein or encodes a defective Bardet-Biedl syndrome-associated protein, resulting in abnormal cilia formation or function."

[0066] In some specific embodiments, the ciliopathy is Bardet-Biedl syndrome.

[0067] In some embodiments, the BBS-associated proteins include: at least one of: BBS1, BBS2, BBS3 / ARL6, BBS4, BBS5, BBS6 / MKKS, BBS7, BBS8 / TTC8, BBS9 / PTHB1, BBS10, BBS11 / TRIM32, BBS12, BBS13 / MKS1, BBS14 / CEP290, BBS15 / WDPCP, BBS16 / SDCCAG8, BBS17 / LZTFL1, BBS18 / BBIP1, BBS19 / IFT27, BBS20 / IFT74 and BBS21 / C8ORF37.

[0068] BBS related protein information:

[0069]

[0070]

[0071] Where, “ / ” means none.

[0072] In some embodiments, the BBS-associated protein is selected from at least one of the proteins that constitute the BBSome complex.

[0073] In some specific embodiments, the proteins that constitute the BBSome complex include BBS1, BBS2, BBS4, BBS5, BBS7, BBS8 / TTC8, BBS9 / PTHB1 and BBS18 / BBIP1.

[0074] In some preferred embodiments, the BBS-related protein is selected from at least one of BBS1, BBS7 and BBS8 / TTC8.

[0075] (Agents for regulating BBS-related proteins)

[0076] In some embodiments, regulating Bardet-Biedl syndrome (BBS)-associated proteins includes rescuing ciliary staining defects caused by not encoding Bardet-Biedl syndrome-associated proteins (BBS-associated proteins) or encoding defective Bardet-Biedl syndrome-associated proteins. In some embodiments, regulating Bardet-Biedl syndrome (BBS)-associated proteins includes reducing abnormal accumulation of SMO in primary cilia caused by not encoding Bardet-Biedl syndrome-associated proteins (BBS-associated proteins) or encoding defective Bardet-Biedl syndrome-associated proteins. In some embodiments, regulating Bardet-Biedl syndrome (BBS)-associated proteins includes restoring dysregulation of Hedgehog signaling pathway activity caused by not encoding Bardet-Biedl syndrome-associated proteins (BBS-associated proteins) or encoding defective Bardet-Biedl syndrome-associated proteins.

[0077] In the present invention, the reagents for regulating BBS-related proteins include L-carnitine or a pharmaceutically acceptable salt thereof, metformin or a pharmaceutically acceptable salt thereof, or a reagent targeting the T10C6.10 gene or its expression product.

[0078] In some embodiments, L-carnitine or a pharmaceutically acceptable salt thereof, metformin or a pharmaceutically acceptable salt thereof, or an agent targeting the T10C6.10 gene or its expression product is used as an essential active ingredient for preventing and / or treating ciliopathy.

[0079] In some embodiments, L-carnitine or a pharmaceutically acceptable salt thereof, metformin or a pharmaceutically acceptable salt thereof, or an agent targeting the T10C6.10 gene or its expression product is used as the sole active ingredient for preventing and / or treating ciliopathy.

[0080] In some embodiments, L-carnitine or a pharmaceutically acceptable salt thereof or metformin or a pharmaceutically acceptable salt thereof alleviates the abnormal accumulation of smoothened protein (SMO) in primary cilia in subjects with ciliopathy, ie, significantly reduces the level of SMO in primary cilia.

[0081] In some embodiments, L-carnitine or a pharmaceutically acceptable salt thereof or metformin or a pharmaceutically acceptable salt thereof restores the dysregulated Hedgehog (Hh) signaling pathway in a subject suffering from a ciliopathy, eg, to a normal state.

[0082] L-Carnitine

[0083] In this invention, L-carnitine (CAS Registry No. 541-15-1), also known as L-carnitine or carnitine, is an amino acid that promotes the conversion of fat into energy. Red meat is the primary source of L-carnitine. It is non-toxic to the human body and highly absorbs moisture. L-carnitine's primary physiological function is to promote the conversion of fat into energy. Taking L-carnitine can reduce body fat and weight without sacrificing water or muscle mass.

[0084] The term "L-carnitine" as used herein refers to L-carnitine or a pharmaceutically acceptable salt thereof.

[0085] In the present invention, L-carnitine or a pharmaceutically acceptable salt thereof may be contained in any suitable dosage form.

[0086] In some exemplary embodiments, the pharmaceutically acceptable salt of L-carnitine is L-carnitine hydrochloride.

[0087] Metformin

[0088] In the present invention, metformin (CAS Reg. No. 657-24-9), also known as N,N-dimethyliminodicarboniminodiamide and 1,1-dimethylbiguanide, is disclosed in Werner, EA et al., J. Chem. Soc. (1922) 121: 1790-1794. This compound, its preparation, and use are also disclosed in, for example, US Pat. No. 3,174,901.

[0089] The term "metformin" as used herein refers to metformin or a pharmaceutically acceptable salt thereof, such as: hydrochloride; metformin (2:1) succinate, metformin (2:1) fumarate disclosed in U.S. application No. 09 / 262,526 filed on March 4, 1999; hydrobromide; p-chlorophenoxyacetate or embonate; and other known monocarboxylic acid metformin salts and dicarboxylic acid metformin salts including the salt disclosed in U.S. Patent No. 3,174,901; all of these salts are collectively referred to as metformin.

[0090] In some exemplary embodiments, the pharmaceutically acceptable salt of metformin is metformin hydrochloride.

[0091] Metformin can be included in any suitable dosage form. For example, metformin can be present in a powder, tablet, capsule, etc. Such dosage forms may also include a dedicated coating, matrix, etc. in some embodiments to achieve sustained release, controlled release, enteral release, etc.

[0092] Agents targeting the T10C6.10 gene or its expression product

[0093] In some embodiments, the expression product of the T10C6.10 gene refers to various forms of molecules of the T10C6.10 gene at various stages, such as but not limited to molecules produced during the amplification, replication, transcription, splicing, processing, translation, and modification of the T10C6.10 gene, such as cDNA, mRNA, precursor protein, mature protein, and fragments thereof.

[0094] In some embodiments, the sequence of the T10C6.10 gene is as shown in SEQ ID NO:17.

[0095] In some embodiments, the sequence of the expression product of the T10C6.10 gene (eg, mature T10C6.10 protein) is shown in SEQ ID NO:3.

[0096] In some embodiments, an agent targeting the T10C6.10 gene or its expression product can recognize and bind to the T10C6.10 gene or its expression product. In some embodiments, an agent targeting the T10C6.10 gene or its expression product can modulate the level or activity of the T10C6.10 gene or its expression product. In some specific embodiments, an agent targeting the T10C6.10 gene or its expression product can reduce the level or activity of the T10C6.10 gene or its expression product. In some specific embodiments, an agent targeting the T10C6.10 gene or its expression product can silence the T10C6.10 gene or its expression product.

[0097] In some embodiments, the agent targeting the T10C6.10 gene or its expression product is selected from the group consisting of: a nucleic acid, a polypeptide, a ribonucleoprotein complex (RNP), or a small molecule drug. In some embodiments, the nucleic acid is selected from the group consisting of: DNA, RNA, or DNA / RNA; the polypeptide is selected from the group consisting of: an antibody or its antigen-binding fragment; and the RNP is selected from the group consisting of: a CRISPR / cas system. In some specific embodiments, the agent targeting the T10C6.10 gene or its expression product is selected from the group consisting of: an antisense oligonucleotide, siRNA, dsRNA, a ribozyme, small interfering RNA (esiRNA) prepared by endoribonuclease III, a short hairpin RNA (shRNA), a CRISPR / cas system, or a small molecule drug.

[0098] In some specific embodiments, the agent targeting the T10C6.10 gene or its expression product comprises a small molecule drug that can reduce or silence the level or activity of the T10C6.10 gene or its expression product.

[0099] The term "small molecule" refers to a low molecular weight compound that can be produced synthetically or obtained from natural sources and has a molecular weight of less than 2000 Daltons (Da), less than 1500 Da, less than 1000 Da, less than 900 Da, less than 800 Da, less than 700 Da, less than 600 Da, or less than 500 Da.

[0100] In some embodiments, small molecule drugs can be organic compounds, inorganic compounds, or combinations of organic and / or inorganic compounds. In some specific embodiments, small molecule drugs are chemically prepared active substances or compounds. Typically, these compounds are synthesized in a classical manner by chemical reactions between different organic and / or inorganic compounds.

[0101] In some embodiments, a small molecule drug can exert its activity in the form in which it is administered, or the small molecule drug can be a prodrug. Thus, "small molecule drug" encompasses both the active form and the prodrug.

[0102] The term "prodrug" refers to a compound or substance that is converted into a therapeutically active agent under physiological conditions. In some embodiments, a prodrug is a compound or substance that is metabolized into a pharmaceutically active form in a subject after administration (e.g., by enzymatic activity in the subject).

[0103] The term "small molecule drug" also encompasses its pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to any salt form of a small molecule drug that is safe and effective for administration to a subject and that possesses the desired biological, pharmaceutical, and / or therapeutic activity.

[0104] (Subject)

[0105] In some embodiments, the ciliopathy is a ciliopathy in a subject.

[0106] In some preferred embodiments, the subject includes mammals, such as humans, mice, etc.

[0107] In some more preferred embodiments, the subject is a human.

[0108] In other embodiments, the subject comprises a nematode, such as Caenorhabditis elegans.

[0109] In some specific embodiments, the subject does not encode a Bardet-Biedl syndrome-associated protein or encodes a defective Bardet-Biedl syndrome-associated protein.

[0110] Example

[0111] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0112] 1. Experimental Materials

[0113] Caenorhabditis elegans: Caenorhabditis Genetics Center (CGC);

[0114] DiI: Sigma Cat#468495;

[0115] NGM medium / NGM plates:

[0116] NaCl 3.0g

[0117] Bacto-Agar 17.0g

[0118] Bacto-peptone 2.5g

[0119] ddH2O to 1L

[0120] Autoclave at 121°C for 30 minutes. After cooling to approximately 60°C, add the following reagents:

[0121] 1M CaCl2 1mL

[0122] 1M MgSO4 1mL

[0123] 1M KPO4 (pH=6) 25mL

[0124] After mixing, pour into a plastic petri dish and let solidify for use.

[0125] Ethyl methanesulfonate: Sigma M0880;

[0126] Metformin (1,1-Dimethylbiguanide hydrochloride): Sigma D150959;

[0127] L-Carnitine: Sangon A503002-0100; L-Carnitine hydrochloride;

[0128] RPE1 cells: Zhou Tianhua laboratory;

[0129] NIH / 3T3 cells: Purnosel;

[0130] Triton-X100: Beyotime P0096;

[0131] Mouse anti-ARL13B antibody: Proteintech 66739-1-Ig;

[0132] Rabbit anti-SMO antibody: Beyotime AF8010;

[0133] 488 Anti-rabbit antibody: Abbkine A23240;

[0134] 591 anti-mouse antibody: Abbkine A23210;

[0135] Opti-MEM:gibco 31985;

[0136] Lipo3000: invitrogen L3000-001.

[0137] 2. Experimental Methods

[0138] 1. Fluorescent Dye Filling Experiment of C. elegans Cilia

[0139] A dye uptake assay using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) was performed to assess the integrity of cilia in the head and tail sensilla. Adult worms were washed from NGM medium, soaked in a diluted DiI dye solution (20 μg / ml) at room temperature for 1 hour, and then transferred to a fresh NGM plate. After incubation at 20°C for approximately 5 hours in the dark, dye uptake in sensilla neurons was observed under a fluorescence microscope. Individuals exhibiting significant staining defects were defined as having ciliary staining defects. Three independent analyses were performed for each strain, with 100 worms counted each time.

[0140] 2. C. elegans strains and forward genetic screening

[0141] A forward genetic screen was performed using the bbs-7(n1606) single mutant nematode as the background.

[0142] The bbs-7 (n1606) single mutant nematodes were synchronized to the late L4 stage (P0) and treated with 0.05% ethyl methanesulfonate (EMS) for 4 hours at room temperature. After washing three times, they were transferred to NGM medium for culture. After 20 hours, the P0 generation nematodes were killed and the eggs were collected and further cultured to obtain mutant populations. The second generation offspring (F2 generation) individuals were screened for individuals with significantly alleviated staining defects by the dye filling experiment described in "1. Nematode cilia fluorescent dye filling experiment" above. A total of 6 candidate genetic suppressor mutants were obtained, numbered cas10821, cas10824, cas10828, cas10831, cas10834 and cas10863. The mutations of each strain were obtained by whole genome sequencing.

[0143] The bbs-7(n1606) single mutant nematodes were obtained from the CGC; the relevant references are as follows:

[0144] Mark Audeh et al. (2003)International Worm Meeting"Mutations in the worm orthologue of the Bardet-Biedl Syndrome gene BBS7 produce sensory neurondysfunction."

[0145] BBS-7 wild type WT (SEQ ID NO:1):

[0146] MQNYSRTDFAQVGTTNRGCMRVIPSDKEKEHDLIVVGGQNGSLICLSRKSNDTTIIFKTQPGYPVQSLALGGPASSKKKDKIFVASQNTVRGVNRKGKTFFSMETNMAEVANRMFVRGLDVVLTGRKSYSRYHDTVDSNSYLCTEDIHDVVSLVFEEAWGSREYTSILACGNSTLQIIEGNNFAYDVRLDSVPFTVSLFMGDGGHTKLLVLYGTKTGRLGLVSVPQGGGKILWEIDTTSGACVTTIVCFNVTGGQFPDIIVGKEDGLIEIYVIDETDHAHLFGTFSCDESITGISCGHVSSKSEIDIIICTFTGWLFSLAKTSRPMIENLPVAANFSVKMQQLRSEVEELQTKVNEERLRYEEITKRQGSGTGSTFFHSFQVHENFEYSAAHGAYNLTIELVIPIDFVVVQSQLPIRLMEVEKNASVVSEVRQDALNPWPLLASYRCQANVCRLELRVQANEGDSGVINLYVCPKVMPKCAQITTHYIKALSSHMRSHDFDLYRPMNTLQFTGNFSIAEAHAWLHNLLPNVPSKCPPADTITNNYQCSVNGGTQLQVVYSKGSAVFRSDCMTTICIIRDKVSEQTMKMQIRVEVACELNQDSVNHCLKLIDPKITSMLNIEKNKLYAAALKELESNNDDVFSFLSAANAKILKDHDAIYEKSEGVSIEDSGVLAILENLMVARGKLTGRSSKGRGDAIRDLISTDYSLENMQTLFKNAMND

[0147] BBS-7(n1606):W233*(SEQ ID NO:2)

[0148] MQNYSRTDFAQVGTTNRGCMRVIPSDKEKEHDLIVVGGQNGSLICLSRKSNDTTIIFKTQPGYPVQSLALGGPASSKKKDKIFVASQNTVRGVNRKGKTFFSMETNMAEVANRMFV RGLDVVLTGRKSYSRYHDTVDSNSYLCTEDIHDVVSLVFEEAWGSREYTSILACNGNSTLQIIEGNNFAYDVRLDSVPFTVSLFMGDGGHTKLLVLYGTKTGRLGLVSVPQGGGKIL*

[0149] 3. Sbbs-1 Functional Verification and Transgenic Manipulation

[0150] Wild-type sbbs-1 genomic DNA (gDNA) containing the promoter and 3'UTR regions was obtained from the nematode genome by PCR. Young adult nematodes were selected in the bbs-7(n1606); sbbs-1(G299*) double mutant background, and sbbs-1 gDNA (20ng / μl) was injected into the nematode gonads. Stably inherited sbbs-1 overexpression (sbbs-1OE) nematode strains were selected from the offspring to verify the causal relationship between sbbs-1 functional loss and phenotypic suppression.

[0151] The bbs-7(n1606), sbbs-1(G299*) double mutant was obtained from screening for EMS positive suppressors in nematodes with a bbs-7(n1606) background.

[0152] SBBS-1WT (SEQ ID NO: 3):

[0153] MGLIFSLCKPKNRRPPPELIPFSQFLKLPSKALRIVLHELDAHHIFELSQVSAELAALINPAHHTFDHIEVNISKEKKSISFYNNWQKDRPLTFNFTSKRAVFNRTVKFGDLRLPMKYDPKTRYHCSTDIYEECLQPCLDFFATIFSCRGGIAVVNMDECTMRVRQHTDHPIFKNNLVAKLSGNINPNEHVSLQSSIFPEYVLLEGIIVEFYFSIPLFDLTQIFDLNRVCIMDARWVTFGYVMGANFTRLMLSNTKWDVETVNQFISRWFRDGRWPHLERFELHFTPDSHGCFNPTILGSGSYPKKWNHRRSSLDSTRRRYYVMDCHRDPYLLDTKNDLDVTRGDGKIATIVNSPRAFLFLVWPDPPVEHKYRIETEPIIHNF[[ID=I]]

[0154] sbbs-1(G299*)(SEQ ID NO:4):

[0155] MGLIFSLCKPKNRRPPPELIPFSQFLKLPSKALRIVLHELDAHHIFELSQVSAELAALINPAHHTFDHIEVNISKEKKSISFYNNWQKDRPLTFNFTSKRAVFNRTVKFGDLRLPMKYDPKTRYHCSTDIYEECLQPCLDFFATIFSCRGGIAVVNMDECTMRVRQHTDHPIFKNNLVAKLSGNINPNEHVSLQSSIFPEYVLLEGIIVEFYFSIPLFDLTQIFDLNRVCIMDARWVTFGYVMGANFTRLMLSNTKWDVETVNQFISRWFRDGRWPHLERFELHFTPDSHGCFNPTIL*

[0156] 4. Ciliary Substructure Labeling and In Vivo Imaging

[0157] To analyze the localization of the IFT complex in different genetic backgrounds, an endogenous dual-fluorescence labeling system was constructed: DYF-2::mScarlet labels the IFT-A complex, and OSM-1::GFP labels the IFT-B complex. Cilia of the head and tail sensilla were imaged using intravital confocal microscopy to analyze the localization of DYF-2 in the middle and distal segments. Young adult individuals of each nematode strain were anesthetized with 0.1 mM / L levamisole and imaged using a Zeiss Axio Observer Z1 spinning disk laser confocal microscope.

[0158] Among them, the two endogenous fluorescent marker nematodes DYF-2::mScarlet and OSM-1::GFP were respectively inserted by Shangyuan Biosciences into the C-termini of the DYF-2 and OSM-1 genes in situ in the N2 wild-type nematode strain using CRISPR-Cas9 technology, and then the double-marked nematode strain was constructed by hybridization.

[0159] 5. Nematode Drug Treatment Experiment

[0160] Metformin (50 mM) and L-carnitine (100 μM), both FDA-approved lipid metabolism regulators, were added to NGM plates. L1-stage nematodes expressing bbs-1(ok1111), bbs-7(n1606), and bbs-8(nx77) mutants were transferred to the drug plates and treated for 48 hours. After drug treatment, changes in ciliary function were assessed by DiI staining (see "1. Fluorescent Dye Filling Assay for C. elegans Cilia") and DYF-2 localization (see "4. Cilia Substructure Labeling and Live Imaging").

[0161] The bbs-1(ok1111) and bbs-8(nx77) mutant nematodes were obtained from the Caenorhabditis elegans Genetics Center.

[0162] bbs-1(ok1111) Reference: The C. elegans Deletion Mutant Consortium et al. (2012) G3 (Bethesda) "large-scale screening for targeted knockouts in the Caenorhabditis elegans genome."

[0163] BBS-1 wild type WT (SEQ ID NO:5):

[0164]

[0165] bbs-1(ok1111)(SEQ ID NO:6):

[0166]

[0167] BBS-8 wild type WT (SEQ ID NO: 7):

[0168]

[0169] bbs-8(nx77)(SEQ ID NO:8):

[0170]

[0171] 6. Mammalian Cell Model Construction and Drug Treatment

[0172] Lentiviral vectors containing short hairpin RNA (shRNA) were packaged and transfected into human RPE1 cells and mouse NIH / 3T3 cells to knock down BBS1, BBS7, and BBS8, respectively. Serum starvation was used to induce ciliogenesis in cultured cells. After 24 hours of culture in serum-free medium, the cells were treated with 10 μM metformin or L-carnitine, respectively. After another 24 hours of culture, the localization of Smoothened (SMO) in primary cilia was observed using immunostaining.

[0173] Among them, shBBS1, shBBS7, and shBBS8 are from LentiElite whole genome shRNA library, the specific sequence is as follows

[0174] Human:

[0175] shBBS1 (SEQ ID NO: 9):

[0176] CCGGCCCAGGAAACTTCTCTGAAATCTCGAGATTTCAGAGAAGTTTCCTGGGTTTTTGshBBS7(SEQID NO:10):

[0177] CCGGCCCAAGGATCATGTAAGAGAACTCGAGTTCTCTTACATGATCCTTGGGTTTTTGshBBS8(SEQID NO:11):

[0178] CCGGCCTCATTTGAACGTGCCCTTTCTCGAGAAAGGGCACGTTCAAATGAGGTTTTTG

[0179] Mouse:

[0180] shBBS1 (SEQ ID NO: 12):

[0181] CCGGTTGACTGCTGCCTGCCGAAATCTCGAGATTTCGGCAGGCAGCAGTCAATTTTTGshBBS7(SEQID NO:13):

[0182] CCGGTTGCTGCAGGGTCCGAGATTACTCGAGTAATCTCGGACCCTGCAGCAATTTTTGshBBS8(SEQID NO:14):

[0183] CCGGCCGAACCTGTTGTCTGAACTTCTCGAGAAGTTCAGACAACAGGTTCGGTTTTTG

[0184] The lentiviral packaging method is as follows:

[0185] The shRNA-containing plasmid, along with the pSPAX2 and pMD2.G packaging plasmids, was packaged using the liposome reagent Lipo3000 and then transformed into 293T cells for viral packaging. After 72 hours, the supernatant containing mature virus was collected and used to transfect RPE1 or NIH / 3T3 cell lines.

[0186] The serum starvation method is as follows:

[0187] For adherent cells, remove the original complete medium and add serum-free Opti-MEM medium, and continue culturing for 48 hours.

[0188] 7. Immunofluorescence Staining of Mammalian Cells

[0189] After drug treatment, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with Triton-X100 for 15 minutes, and blocked with BSA for 2 hours. Primary cilia were labeled with mouse anti-ARL13B antibodies, and the localization of smoothened protein (SMO) in cilia was detected with goat anti-SMO antibodies. The cells were incubated overnight at 4°C. Fluorescent secondary antibodies, 488 anti-goat antibodies and 591 anti-mouse antibodies, were used for staining and incubated for 2 hours at room temperature. Images were acquired using a Zeiss Axio Observer Z1 spinning disk laser confocal microscope, and fluorescence intensity was quantified.

[0190] 8. Construction of BBS7 gene knockout RPE1 cells

[0191] CRISPR-Cas9 gene editing technology was used to knock out BBS7 in human RPE1 cells. A Cas9 and sgRNA expression vector was constructed in RPE1, where the sgRNA recognition position was located in the first exon of the BBS7 gene. The vector was packaged using a lentiviral vector and transfected into RPE1 cells, and the cell line was selected for sequencing. Sequencing revealed that one allele had an 8bp deletion in the promoter region and exon 1 (destroying the start codon), while the other allele had a 14bp deletion in exon 1, resulting in a frameshift mutation and loss of BBS7 function, indicating that the constructed cell line is a homozygous BBS7 knockout model.

[0192] sgRNA(SEQ ID NO:15):

[0193] ATCCATGATGACTACGCGGAGGG

[0194] Cas9(SEQ ID NO:16):

[0195]

[0196] Vector: lentiCRISPR V2 (Addgene, #52961).

[0197] 3. Experimental Results

[0198] 1. Forward genetic screening revealed that a loss-of-function mutation in sbbs-1 can suppress ciliary defects caused by bbs-7(n1606)

[0199] Using the method described above in "1. Fluorescent dye filling assay for nematode cilia", it was found that 100% of adult worms in the bbs-7(n1606) single mutant exhibited a dye-filling defect in their cilia. To identify genetic suppressors of this mutant, the inventors conducted a large-scale forward genetic screen in bbs-7(n1606) mutant worms (see "2. Nematode strains and forward genetic screen" described above). The screening strategy was as follows: bbs-7(n1606) adults were first treated with the chemical mutagen ethyl methanesulfonate (EMS), and their second-generation progeny (F2) were then analyzed for dye uptake (see "1. Fluorescent dye filling assay for nematode cilia" described above). By screening individuals with significantly alleviated staining defects, a total of 6 candidate suppressor mutants were finally obtained, numbered cas10821, cas10824, cas10828, cas10831, cas10834 and cas10863. Among these suppressor mutants, the proportion of ciliary staining defects decreased to 36%, 42%, 28%, 38%, 44% and 45%, respectively. Figure 1 A).

[0200] Whole genome sequencing results showed that these six genetic suppressor mutants all had mutations in the nematode gene T10C6.10. Given that it can alleviate the ciliary function defects of the bbs-7(n1606) mutant, T10C6.10 was named sbbs-1( s uppressor of bbs -7No. 1 These six suppressors carry the following sbbs-1 allele variants: P287S (cas10821), G244D (cas10824), G299* (cas10828), K347N (cas10831), and D377K (cas10863) ( Figure 1(B in Figure 1). G299* is a nonsense mutation, while the others are missense mutations. The sbbs-1(G299*) nonsense mutation (* represents a premature stop codon) showed the strongest ability to rescue the ciliary staining phenotype, reducing the staining defect from 100% to approximately 28%. Subsequent functional studies primarily used the bbs-7(n1606); sbbs-1(G299*) double mutant as an experimental model.

[0201] >T10C6.10 (SBBS-1), wild-type isoform A (SEQ ID NO: 3):

[0202] MGLIFSLCKPKNRRPPPELIPFSQFLKLPSKALRIVLHELDAHHIFELSQVSAELAALINPAHHTFDHIEVNISKEKKSISFYNNWQKDRPLTFNFTSKRAVFNRTVKFGDLRLPMKYDPKTRYHCSTDIYEECLQPCLDFFATIFSCRGGIAVVNMDECTMRVRQHTDHPIFKNNLVAKLSGNINPNEHV SLQSSIFPEYVLLEGIIVEFYFSIPLFDLTQIFDLNRVCIMDARWVTFGYVMGANFTRLMLSNTKWDVETVNQFISRWFRDGRWPHLERFELHFTPDSHGCFNPTILGSGSYPKKWNHRRSSLDSTRRRYYVMDCHRDPYLLDTKNDLDVTRGDGKIATIVNSPRAFLFLVWPDPPVEHKYRIETEPIIHNF

[0203]

[0204] >sbbs-1 (cas10824, G244D) mutant (SEQ ID NO:18):

[0205] gaaatttgtcattccttcctattcaccccgttcgactgtgcaATGGGTTTAATCTTTTCACTTTGTAAACCGAAGAATCGACGACCGCCGCCTGAATTGATACCGTTTAGTCAGTTTCTGAAGCTACCTTCCAAGGCATTGCGCATCGTTCTCCATGAATTAGACGCTCATCACATATTCGAGTTGTCCCAAGTGTCGGCGGAATTGGCCGCTTTGATCAACCCTGCTCACCACACATTTGACCATATTGAGGTCAACATTTCAAAAGAGAAAAAATCAATATCTTTCTACAATAATTGGCAAAAGGATCGACCACTCACTTTCAACTTCACCTCTAAACGTGCAGTTTTTAATAGGACTGTGAAATTCGGAGATCTAAGATTGCCGATGAAATACGACCCGAAAACCAGATATCACTGCTCCACGGACATCTATGAAGAGTGTCTACAACCCTGTCTCGATTTTTTTGCCACAATTTTCAGTTGCCGTGGTGGAATCGCAGTGGTCAATATGGATGAATGTACTATGAGAGTGAGACAACATACTGACCATCCAATTTTCAAAAATAACCTCGTGGCGAAATTGTCGGGAAATATAAACCCGAACGAACACGTCAGTCTGCAGTCATCAATTTTTCCGGAATATGTGCTTTTGGAGGGAATCATCGTTGAGTTTTACTTCTCCATACCTCTCTTCGATCTGACTCAGATCTTTGACCTGAATCGTGTGTGCATCATGGATGCAAGATGGGTCACATTTGGATATGTTATGG AACCAAATTTCACTCGGCTTATGTTGTCAAACACTAAGTGGGATGTCGAAACTGTGAACCAGTTCATCTCGCGTTGGTTCCGTGATGGTCGATGGCCACATTTGGAGAGATTTGAGCTTCACTTCACTCCCGATTCTCATGGCTGCTTCAATCCGACAATACTTGGATCGGGTTCGTATCCGAAGAAATGGAATCATCGACGCTCATCCCTGGATTCTACGAGAAGAAGATATTATGTAATGGACTGCCATCGGGACCCGTATCTCCTGGACACTAAAAACGATCTCGACGTGACAAGAGGTGACGGAAAAATCGCGACAATTGTGAATTCGCCTCGAGCATTTTTGTTCCTCGTCTGGCCCGATCCTCCCGTTGAGCATAAATATCGTATCGAGACTGAACCAATTATTCATAACTTTTGAattatttcgtttatatgtattttaactattttgaaaaatat

[0206] >sbbs-1 (cas10821, P287S) mutant (SEQ ID NO:19):

[0207] GaaatttgtcattccttcctattcaccccgttcgactgtgcaATGGGTTTAATCTTTTCACTTTGTAAACCGAAGAATCGACGACCGCCGCCTGAATTGATACCGTTTAGTCAGTTTCTGAAGCTACCTTCCAAGGCATTGCGCATCGTTCTCCATGAATTAGACGCTCATCACATATTCGAGTTGTCCCAAGTGTCGGCGGAATTGGCCGCTTTGATCAACCCTGCTCACCACACATTTGACCATATTGAGGTCAACATTTCAAAAGAGAAAAATTCAATATCTTTCTACAATAATTGGCAAAAGGATCGACCACTCACTTTCAACTTCACCTCTAAACGTGCAGTTTTTAATAGGACTGTGAAATTCGGAGATCTAAGATTGCCGATGAAATACGACCCGAAAACCAGATATCACTGCTCCACGGACATCTATGAAGAGTGTCTACAA CCCTGTCTCGATTTTTTTGCCACAATTTTCAGTTGCCGTGGTGGAATCGCAGTGGTCAATATGGATGAATGTACTATGAGAGTGAGACAACATACTGACCATCCAATTTTCAAAAATAACCTCGTGGCGAAATTGTCGGGAAATATAAACCCGAACGAACACGTCAGTCTGCAGTCATCAATTTTTCCGGAATATGTGCTTTTGGAGGGAATCATCGTTGAGTTTTACTTCTCCATACCTCTCTTCGATCTGACTCAGATCTTTGACCTGAATCGTGTGTGCATCATGGATGCAAGATGGGTCACATTTGGATATGTTATGGGACCAAATTTCACTCGGCTTATGTTGTCAAACACTAAGTGGGATGTCGAAACTGTGAACCAGTTCATCTCGCGTTGGTTCCGTGATGGTCGATGGCCACATTTGGAGAGATTTGAGCTTCACTTCACT TCCGATTCTCATGGCTGCTTCAATCCGACAATACTTGGATCGGGTTCGTATCCGAAGAAATGGAATCATCGACGCTCATCCCTGGATTCTACGAGAAGAAGATATTATGTAATGGACTGCCATCGGGACCCGTATCTCCTGGACACTAAAAACGATCTCGACGTGACAAGAGGTGACGGAAAAATCGCGACAATTGTGAATTCGCCTCGAGCATTTTTGTTCCTCGTCTGGCCCGATCCTCCCGTTGAGCATAAATATCGTATCGAGACTGAACCAATTATTCATAACTTTTGAattatttcgtttatatgtattttaactattttgaaaaatat

[0208] >Mutant of sbbs-1 (cas10828, G299*) (SEQ ID NO:20):

[0209] gaaatttgtcattccttcctattcaccccgttcgactgtgcaATGGGTTTAATCTTTTCACTTTGTAAACCGAAGAATCGACGACCGCCGCCTGAATTGATACCGTTTAGTCAGTTTCTGAAGCTACCTTCCAAGGCATTGCGCATCGTTCTCCATGAATTAGACGCTCATCACATATTCGAGTTGTCCCAAGTGTCGGCGGAATTGGCCGCTTTGATCAACCCTGCTCACCACACATTTGACCATATTGAGGTCAACATTTCAAAAGAGAAAAATTCAATATCTTTCTACAATAATTGGCAAAAGGATCGACCACTCACTTTCAACTTCACCTCTAAACGTGCAGTTTTTAATAGGACTGTGAAATTCGGAGATCTAAGATTGCCGATGAAATACGACCCGAAAACCAGATATCACTGCTCCACGGACAATCTATGAAGAGTGTCTACAACCCTGTCTCGATTTTTTT GCCACAATTTTCAGTTGCCGTGGTGGAATCGCAGTGGTCAATATGGATGAATGTACTATGAGAGTGAGACAACATACTGACCATCCAATTTTCAAAAATAACCTCGTGGCGAAATTGTCGGGAAATATAAACCCGAACGAACACGTCAGTCTGCAGTCATCAATTTTTCCGGAATATGTGCTTTTGGAGGGAATCATCGTTGAGTTTTACTTCTCCATACCTCTCTTCGATCTGACTCAGATCTTTGACCTGAATCGTGTGTGCATCATGGATGCAAGATGGGTCACATTTGGATATGTTATGGGACCAAATTTCACTCGGCTTATGTTGTCAAACACTAAGTGGGATGTCGAAACTGTGAACCAGTTCATCTCGCGTTGGTTCCGTGATGGTCGATGGCCACATTTGGAGAGATTTGAGCTTCACTTCACTCCCGATTCTCATGGCTGCTTCAATCCGACAATACTT TGATCGGGTTCGTATCCGAAGAAATGGAATCATCGACGCTCATCCCTGGATTCTACGAGAAGAAGATATTATGTAATGGACTGCCATCGGGACCCGTATCTCCTGGACACTAAAAACGATCTCGACGTGACAAGAGGTGACGGAAAAATCGCGACAATTGTGAATTCGCCTCGAGCATTTTTGTTCCTCGTCTGGCCCGATCCTCCCGTTGAGCATAAATATCGTATCGAGACTGAACCAATTATTCATAACTTTTGAattatttcgtttatatgtattttaactattttgaaaaatat

[0210] >sbbs-1 (cas10834, S312P) mutant (SEQ ID NO:21):

[0211] gaaatttgtcattccttcctattcaccccgttcgactgtgcaATGGGTTTAATCTTTTCACTTTGTAAACCGAAGAATCGACGACCGCCGCCTGAATTGATACCGTTTAGTCAGTTTCTGAAGCTACCTTCCAAGGCATTGCGCATCGTTCTCCATGAATTAGACGCTCATCACATATTCGAGTTGTCCCAAGTGTCGGCGGAATTGGCCGCTTTGATCAACCCTGCTCACCACACATTTGACCATATTGAGGTCAACATTTCAAAAGAGAAAAATTCAATATCTTTCTACAATAATTGGCAAAAGGATCGACCACTCACTTTCAACTTCACCTCTAAACGTGCAGTTTTTAATAGGACTGTGAAATTCGGAGATCTAAGATTGCCGATGAAATACGACCCGAAAACCAGATATCACTGCTCCACGGAATCTATGAAGAGTGTCTACAACCCTGTCTCGATTTTTTTGCCACAATTTTCAGTTGCC GTGGTGGAATCGCAGTGGTCAATATGGATGAATGTACTATGAGAGTGAGACAACATACTGACCATCCAATTTTCAAAAATAACCTCGTGGCGAAATTGTCGGGAAATATAAACCCGAACGAACACGTCAGTCTGCAGTCATCAATTTTTCCGGAATATGTGCTTTTGGAGGGAATCATCGTTGAGTTTTACTTCTCCATACCTCTCTTCGATCTGACTCAGATCTTTGACCCTGAATCGTGTGTGCATCATGGATGCAAGATGGGTCACATTTGGATATGTTATGGGACCAAATTTCACTCGGCTTATGTTGTCAAACACTAAGTGGGATGTCGAAACTGTGAACCAGTTCATCTCGCGTTGGTTCCGTGATGGTCGATGGCCACATTTGGAGAGATTTGAGCTTCACTTCACTCCCGATTCTCATGGCTGCTTCAATCCGACAATACTTGGATCGGGTTCGTATCCGAAGAAATGGAATCATCGACGC CCATCCCTGGATTCTACGAGAAGAAGATATTATGTAATGGACTGCCATCGGGACCCGTATCTCCTGGACACTAAAAACGATCTCGACGTGACAAGAGGTGACGGAAAAATCGCGACAATTGTGAATTCGCCTCGAGCATTTTTGTTCCTCGTCTGGCCCGATCCTCCCGTTGAGCATAAATATCGTATCGAGACTGAACCAATTATTCATAACTTTTGAattatttcgtttatatgtattttaactattttgaaaaatat

[0212] >Mutant of sbbs-1 (cas10831, K347N) (SEQ ID NO: 22):

[0213] C ATCGCGACAATTGTGAATTCGCCTCGAGCATTTTTGTTCCTCGTCTGGCCCGATCCTCCCGTTGAGCATAAATATCGTATCGAGACTGAACCAATTATTCATAACTTTTGAattatttcgtttatatgtattttaactattttgaaaaatat

[0214] >Mutant of sbbs-1 (cas10863, D377K) (SEQ ID NO: 23):

[0215] A AACCAATTATTCATAACTTTTGAattatttcgtttatatgtattttaactattttgaaaaatat

[0216] The underlines in the above SEQ ID NOs: 18-23 indicate mutation sites.

[0217] To confirm the causal relationship between sbbs-1 loss of function and phenotypic rescue, we overexpressed (OE) the sbbs-1 wild-type gene fragment in the bbs-7(n1606); sbbs-1(G299*) double mutant background by microinjection (see "3. sbbs-1 Functional Verification and Transgenic Manipulation" above). The results showed that sbbs-1 overexpression (OE) could significantly reverse the genetic compensation effect, increasing the ciliary staining defect rate from 28% to 88% (n=300) ( Figure 1 A), confirming that the inhibitory function of sbbs-1 depends on its loss-of-function mutation.

[0218] By constructing a dual fluorescence reporter system for ciliary transport complexes (red fluorescent marker DYF-2::mScarlet to mark endogenous IFT-A complex; green fluorescent marker OSM-1::GFP to mark endogenous IFT-B complex) and in vivo fluorescence imaging (see "4. Ciliary Substructure Labeling and In Vivo Imaging" above), the inventors analyzed the ultrastructure of cilia in different genetic backgrounds. In bbs-7(n1606) single mutant nematodes, the ciliary localization of the IFT-B complex (OSM-1::GFP) is basically normal, but the IFT-A complex (DYF-2::mScarlet) is significantly absent in the middle and distal segments of the cilia ( Figure 1 In the bbs-7(n1606); sbbs-1(G299*) double mutant, the localization of DYF-2 in cilia was significantly restored, especially in the tail sensor cilia ( Figure 1 However, after the introduction of sbbs-1 overexpression, the abnormal DYF-2 localization reappeared in bbs-7(n1606);sbbs-1(G299*);sbbs-1OE nematodes ( Figure 1 C), further confirming that loss of sbbs-1 function is necessary for the restoration of IFT-A complex localization, supporting sbbs-1(G299*) as a suppressor of the bbs-7(n1606) mutant.

[0219] 2. Screening for drug candidates that can alleviate the BBS phenotype in nematodes based on the mechanism of action of sbbs-1

[0220] The functional loss of sbbs-1 provides a potential intervention target for the treatment of Bardet-Biedl syndrome (BBS). The N-terminal end of the SBBS-1 protein contains an F-box domain. Although its biological function is still unclear, it was reported in a 2003 genome-wide RNAi screening that knocking down sbbs-1 can lead to a decrease in nematode fat content (Ashrafi K, Chang FY, Watts JL, et al. Genome-wide RNAi analysis of Caenorhabditis elegans fat regulatory genes. Nature. 2003; 421 (6920): 268-272. doi: 10.1038 / nature01279). Therefore, two FDA-approved drugs with the function of regulating fat metabolism were screened: metformin and L-carnitine.

[0221] Using the experimental method described in "5. Nematode drug treatment experiment" above, the above drugs were applied to bbs-1(ok1111), bbs-7(n1606), and bbs-8(nx77) nematode mutants, and each group was treated with 50mM metformin and 100μM L-carnitine. Similar to bbs-7(n1606), these three mutants all showed 100% dye uptake defects and DYF-2 localization was lost in the mid- and distal segments of the cilia. However, drug treatment significantly alleviated the dyeing defects and promoted the localization of DYF-2 in the cilia ( Figure 2 ), suggesting that these two drugs have the potential to correct the BBS phenotype.

[0222] 3. Metformin and L-carnitine can alleviate BBS-related ciliary phenotypes in mammalian cell BBS models.

[0223] To validate the drug's efficacy in mammalian systems, the experimental methods described in "6. Mammalian Cell Model Construction and Drug Treatment" and "7. Mammalian Cell Immunofluorescence Staining" above were used to knock down BBS1, BBS7, and BBS8 in two mammalian cell lines—human retinal pigment epithelial (RPE1) cells and mouse embryonic fibroblasts (NIH / 3T3)—using short hairpin RNA (shRNA). Primary cilia were labeled with a mouse anti-ARL13B antibody, and the localization of SMO within cilia was detected with a goat anti-SMO antibody. SMO is a key transduction protein in the Hedgehog (Hh) signaling pathway, acting as a "switch" in the Hh pathway. Its signal is typically weak in the primary cilia of normal cells.

[0224] This study observed that SMO was abnormally enriched in primary cilia in cells transfected with shBBS1, shBBS7, and shBBS8. However, after treatment with metformin or L-carnitine, the signal intensity of SMO in cilia decreased significantly ( Figure 3 BC and Figure 4 ), suggesting that these two drugs can partially restore the normal state of the ciliary signaling pathway.

[0225] To further verify the alleviating effect of the above-mentioned drugs on the BBS phenotype, the experimental method described above, "8. Construction of BBS7 gene knockout RPE1 cells", was used to construct a BBS7 gene knockout (KO) human retinal pigment epithelial cell line (RPE1) using CRISPR-Cas9 gene editing technology. Both BBS7 alleles in this cell line carry loss-of-function mutations: one allele has an 8bp deletion in the promoter region and the first exon (3bp in the promoter region and 5bp in exon 1), destroying the start codon; the other allele has a 14bp deletion in exon 1, causing a frameshift mutation, which is expected to lead to premature translation termination ( Figure 3 These biallelic mutations collectively result in a complete loss of BBS7 protein expression, indicating that this cell line is a homozygous BBS7 knockout model. Consistent with the shRNA knockdown results, SMO abnormally accumulates in primary cilia in BBS7 KO cells, suggesting dysregulated Hedgehog signaling. Treatment with metformin or L-carnitine significantly reduced SMO signaling in cilia ( Figure 3 This further supports the potential of these two drugs to alleviate BBS-related ciliary dysfunction in mammalian models.

[0226] It should be noted that, although the technical solutions of the present invention are described with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0227] While various embodiments of the present invention have been described above, the above descriptions are intended to be illustrative, non-exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is selected to best explain the principles of the embodiments, their practical applications, or technological improvements in the marketplace, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Use of an agent for regulating Bardet-Biedl syndrome (BBS)-related proteins in the preparation of a medicament for preventing and / or treating ciliopathy; in, The ciliopathy is a disorder associated with mutations in genes that do not encode BBS-related proteins or encode defective BBS-related proteins, leading to abnormal cilia formation or function; Wherein, the reagents for regulating BBS-related proteins include metformin or a pharmaceutically acceptable salt thereof, L-carnitine or a pharmaceutically acceptable salt thereof, or reagents targeting the T10C6.10 gene or its expression product.

2. The use according to claim 1, wherein The ciliopathy is Bardet-Biedl syndrome (BBS).

3. The use according to claim 1 or 2, wherein The BBS-related proteins include: at least one of BBS1, BBS2, BBS3 / ARL6, BBS4, BBS5, BBS6 / MKKS, BBS7, BBS8 / TTC8, BBS9 / PTHB1, BBS10, BBS11 / TRIM32, BBS12, BBS13 / MKS1, BBS14 / CEP290, BBS15 / WDPCP, BBS16 / SDCCAG8, BBS17 / LZTFL1, BBS18 / BBIP1, BBS19 / IFT27, BBS20 / IFT74 and BBS21 / C8ORF37.

4. The use according to any one of claims 1 to 3, wherein The BBS-associated protein is selected from at least one of the proteins constituting the BBSome complex; Optionally, the proteins constituting the BBSome complex include BBS1, BBS2, BBS4, BBS5, BBS7, BBS8 / TTC8, BBS9 / TTC8 and BBS18 / BBIP1.

5. The use according to any one of claims 1 to 4, wherein The BBS-related protein is selected from at least one of BBS1, BBS7 and BBS8 / TTC8.

6. The use according to any one of claims 1 to 5, wherein The sequence of the T10C6.10 gene is shown in SEQ ID NO:

17.

7. The use according to any one of claims 1 to 6, wherein The expression product of the T10C6.10 gene is selected from the group consisting of: cDNA, mRNA, T10C6.10 precursor protein, mature T10C6.10 protein, and fragments thereof.

8. The use according to any one of claims 1 to 7, wherein The agent targeting the T10C6.10 gene or its expression product is selected from: nucleic acid, polypeptide, ribonucleoprotein complex or small molecule drug.

9. The use according to any one of claims 1 to 8, wherein The ciliopathy is a ciliopathy in a subject.

10. The use according to any one of claims 1 to 9, wherein The subject includes a human, a mouse or a nematode.

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