Gene regulation method and application thereof

Abstract

The invention provides a method for preparing a non-human animal, a GIGYF1 gene of the non-human animal is deleted, the non-human animal can be used for screening recombinant nucleic acid to treat specific diseases, and particularly, the invention further provides application of the recombinant nucleic acid to treat metabolic diseases and / or symptoms.

Description

A gene regulation method and its application Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a gene regulation method and its application. Background Art

[0002] Type 2 diabetes mellitus (T2DM) affects a large number of patients, and diabetes is also a major risk factor for coronary and peripheral artery disease and other complications. Despite the availability of numerous medications and other therapies, many oral T2DM medications are ineffective and have adverse effects in subjects.

[0003] Therefore, there is an urgent need in the art for a therapy that can be used for alleviating, preventing and / or treating metabolic diseases.

[0004] Summary of the Invention

[0005] The present application provides a recombinant nucleic acid that can be used for alleviating, preventing and / or treating metabolic diseases and / or symptoms.

[0006] On the one hand, the present application provides a preparation for intervening in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof, and use thereof in the preparation of a medicament for alleviating, preventing and / or treating specific diseases and / or symptoms.

[0007] In one aspect, the present application provides a preparation for intervening in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof, which is used to alleviate, prevent and / or treat specific diseases and / or symptoms.

[0008] In one aspect, the present application provides a method for alleviating, preventing and / or treating specific diseases and / or symptoms, comprising intervening in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof.

[0009] In one aspect, the present application provides a vector comprising a nucleic acid molecule encoding GIGYF1, GIGYF1 variants, truncations, and analogs thereof. In certain embodiments, the vector is an AAV vector. In certain embodiments, the vector is AAV9.

[0010] In another aspect, the present application provides a cell comprising an agent intervening in GIGYF1, GIGYF1 variants, truncations and analogs thereof.

[0011] On the other hand, the present application provides a kit comprising nucleic acid encoding GIGYF1, GIGYF1 variants, truncations and analogs thereof, a vector comprising the above nucleic acid and / or the cells described in the present application.

[0012] In certain embodiments, the GIGYF1 comprises the amino acid sequence shown in SEQ ID NO: 2.

[0013] In certain embodiments, the GIGYF1 comprises the amino acid sequence shown in SEQ ID NO: 1. In certain embodiments, the amino acid sequence of the GIGYF1 is shown in SEQ ID NO: 1.

[0014] In certain embodiments, the nucleotide sequence encoding the GIGYF1 protein is shown in SEQ ID NO: 5. In certain embodiments, the gene encoding the GIGYF1 protein comprises the nucleotide sequence shown in SEQ ID NO: 5.

[0015] In certain embodiments, the GIGYF1 is configured for delivery to a tissue or cell selected from the group consisting of liver, heart, muscle, pancreas, and brain.

[0016] In certain embodiments, the disease and / or condition is associated with GIGYF1 expression.

[0017] In certain embodiments, the GIGYF1 is used to alleviate, prevent and / or treat metabolic diseases and / or symptoms.

[0018] In certain embodiments, the GIGYF1 is used to alleviate, prevent and / or treat diabetes and / or its complications.

[0019] In certain embodiments, the disease and / or symptom is selected from the following group: developmental delay, hyperglycemia, hyperlipidemia, increased glycosylated hemoglobin ratio, insulin resistance, decreased glucose tolerance, increased fat, fatty liver, spleen hyperplasia, decreased insulin secretion, increased glucagon secretion, decreased reproductive ability, kidney damage, skin damage, swollen finger (toe) joints, heart damage, and cognitive impairment.

[0020] After administering the GIGYF1, GIGYF1 variants, truncations and analogs thereof described in the present application or nucleic acids encoding the same, the phenotype and / or symptoms of diabetes are improved.

[0021] In another aspect, the present application provides a modified non-human animal having reduced expression of the GIGYF1 gene compared to a wild-type non-human animal. In certain embodiments, the modified non-human animal has reduced expression of the GIGYF1 protein compared to a wild-type non-human animal. In certain embodiments, the GIGYF1 protein comprises the amino acid sequence set forth in SEQ ID NO. 1.

[0022] In certain embodiments, the GIGYF1 gene of the modified non-human animal is knocked out or knocked down. In certain embodiments, the GIGYF1 gene comprises the nucleic acid sequence shown in SEQ ID NO.5.

[0023] In certain embodiments, the modification comprises contacting the genome of a non-human animal cell, stem cell, or fertilized egg with: 1) a CRISPR-associated (Cas) protein; and 2) one or more ribonucleic acid (RNA) sequences. For example, the one or more ribonucleic acid (RNA) sequences comprise a sequence complementary to the GIGYF1 gene and a sequence that binds to the Cas protein.

[0024] In certain embodiments, the modified Cas protein is Cas9. In certain embodiments, the stem cell is selected from the group consisting of embryonic stem cells, adult stem cells, totipotent stem cells, unipotent stem cells, and induced pluripotent stem cells.

[0025] In certain embodiments, the sequence that binds to the Cas protein includes a tracrRNA sequence.

[0026] In certain embodiments, the one or more ribonucleic acid (RNA) sequences comprise the sequence shown in any one of SEQ ID NOs. 3-4.

[0027] In certain embodiments, the modified non-human animal displays phenotypes and / or symptoms of a metabolic disease.

[0028] In certain embodiments, the modified non-human animal displays a phenotype and / or symptoms of diabetes.

[0029] In certain embodiments, the phenotype and / or symptoms of diabetes are selected from any one or more of the following groups: (1) developmental delay; (2) increased blood lipids; (3) increased blood glucose concentration; (4) abnormal fat metabolism, for example, increased fat mass in females, decreased fat mass in males, fatty liver, lipomas, and spleen hyperplasia; (5) reproductive abnormalities, for example, decreased sperm count in male mice; (6) poor renal function, for example, abnormal urine color (yellow urine); (7) partial hair loss, skin ulcers, or swollen knuckles; (8) increased mortality; (9) abnormal heart condition; (10) cognitive abnormalities; (11) slower glucose clearance; (12) decreased sensitivity to insulin; (13) increased expression of tumor necrosis factor α; (14) decreased glucagon secretion; (15) decreased insulin secretion; and (16) decreased energy exchange.

[0030] In another aspect, the present application provides a modified living part of a non-human animal. In certain embodiments, the non-human animal comprises a mouse.

[0031] In another aspect, the present application provides a modified cell, tissue or organ of a non-human animal. In certain embodiments, the non-human animal comprises a mouse.

[0032] On the other hand, the present application provides the use of the modified non-human animals in screening drugs for alleviating, preventing and / or treating metabolic diseases and / or symptoms. In certain embodiments, the use includes administering one or more candidate agents to the modified non-human animals. If the metabolic diseases and / or symptoms of the modified non-human animals improve, the one or more candidate agents are considered to be drugs that can alleviate, prevent and / or treat metabolic diseases and / or symptoms; if the metabolic diseases and / or symptoms of the modified non-human animals do not improve or worsen, the one or more candidate agents are considered to be drugs that cannot alleviate, prevent and / or treat metabolic diseases and / or symptoms.

[0033] In certain embodiments, the medicament is used to alleviate, prevent and / or treat diabetes and / or its complications.

[0034] In certain embodiments, the non-human animal comprises a mouse.

[0035] On the other hand, the present application provides a method for screening drugs for alleviating, preventing and / or treating metabolic diseases and / or symptoms. If one or more candidate agents can reduce the expression level of the body's GIGYF1 gene or the expression amount of the GIGYF1 protein, the one or more candidate agents are identified as drugs that can alleviate, prevent and / or treat metabolic diseases and / or symptoms. If one or more candidate agents cannot reduce the expression level of the body's GIGYF1 gene or the expression amount of the GIGYF1 protein, the one or more candidate agents are identified as drugs that cannot alleviate, prevent and / or treat metabolic diseases and / or symptoms.

[0036] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0038] Figure 1 shows random blood glucose concentrations in Gigyf1 knockout mice;

[0039] Figure 2 shows the fasting blood glucose concentrations of Gigyf1 knockout mice;

[0040] Figure 3 shows the results of a glucose tolerance test in Gigyf1 knockout mice;

[0041] Figure 4 shows the results of the insulin sensitivity test in Gigyf1 knockout mice;

[0042] Figure 5 shows the body weight of Gigyf1 knockout mice;

[0043] Figure 6 shows the body fat percentage of Gigyf1 knockout mice;

[0044] Figure 7 shows the energy consumption of Gigyf1 knockout mice;

[0045] Figure 8 shows fasting insulin and glucagon levels in Gigyf1 knockout mice;

[0046] Figure 9 shows the survival curve of Gigyf1 knockout mice;

[0047] FIG10 shows the responses to glucose and insulin in mature-aged knockout mice after delivery of GIGYF1;

[0048] FIG11 shows the responses to glucose and insulin in aged knockout mice after delivery of GIGYF1. DETAILED DESCRIPTION

[0049] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0050] Definition of terms

[0051] In this application, the terms "GRB10 Interacting GYF Protein 1" and "GIGYF1 protein" generally have the same meaning and are used interchangeably herein. GIGYF1 is a member of the GIGYF family, and the protein it encodes contains a GYF motif. For example, the GIGYF1 can include unprocessed GIGYF1, any processed form of GIGYF1, GIGYF1 variants, or substances comprising functionally active fragments of GIGYF1. For more information on human GIGYF1 protein, see Uniport O75420; for more information on mouse GIGYF1 protein, see Uniport Q99MR1.

[0052] In this application, the term "GIGYF1 gene" generally refers to a gene encoding GIGYF1 protein, a member of the gyf adaptor protein family. For more information about the human GIGYF1 gene, see Ensembl: ENSG00000146830 or NCBI Gene ID: 64599.

[0053] In this application, the term "GIGYF1" or its various uppercase and lowercase forms can refer to a protein, gene or RNA. When not explicitly stated, a person skilled in the art can clearly know which substance the term "GIGYF1" or its uppercase and lowercase forms represents based on the context. In this application, the term "encoding" generally refers to the inherent properties of a specific nucleotide sequence in a polynucleotide such as a gene, DNA or mRNA, that is, the sequence can serve as a template to synthesize other polymers and macromolecules with a specified nucleotide sequence (such as rRNA, tRNA and mRNA) or a specified amino acid sequence in a biological process and the resulting biological properties. Therefore, when the transcription and translation of the mRNA corresponding to a gene in a cell or other biological system leads to the production of a protein, the gene, cDNA, or RNA can encode the protein. Both the coding strand (whose nucleotide sequence is the same as the mRNA sequence, usually provided in the sequence listing) and the non-coding strand (used as a template for transcription of the gene or cDNA) can be referred to as encoding a protein or other product of the gene or cDNA.

[0054] In this application, the term "vector" generally refers to an expression vector containing the recombinant nucleic acid or fusion nucleic acid DNA sequence of the present application and suitable transcription / translation control signals. The method of constructing the vector can include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The described DNA sequence can be effectively connected to the appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector can also include a ribosome binding site, regulatory sequence and transcription terminator for translation initiation. The regulatory sequence can include a promoter, enhancer, transcription termination signal, polyadenylation sequence, replication origin, nucleic acid restriction site and homologous recombination site that are operably connected to the nucleic acid sequence. The vector can also include a selective marker, for example, to determine the expression of the vector in a growth system (for example, bacterial cells) or in pancreas, liver, muscle target cells. For example, a vector comprising the above-mentioned appropriate DNA sequence and appropriate promoter or regulatory sequence can be used to transform a suitable host cell so that it can express a polypeptide.

[0055] In this application, the term "viral vector" generally refers to a gene therapy vector suitable for transduction and expression in target cells. For example, the target cells can be pancreatic, liver, or muscle target cells. Viral vectors include those derived from adenovirus, adeno-associated virus (AAV) including mutated forms, retroviruses, lentiviruses, herpes viruses, vaccinia viruses, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxviruses, and SV40. For example, viral vectors can generally maintain an extrachromosomal state without integrating into the genome of pancreatic, liver, or muscle target cells, or they can integrate into the genome of pancreatic, liver, or muscle target cells. The viral vector can be an AAV vector, such as a self-complementary adeno-associated virus (scAAV). Selective targeting can be achieved using specific AAV serotypes (AAV serotype 2 to AAV serotype 12) or modified versions of any of these serotypes (including AAV 4YF and AAV 7m8 vectors).

[0056] In this application, the term "adeno-associated virus" or "AAV" is also commonly referred to as adeno-associated virus, which belongs to the genus Dependinovirus of the family Parvoviridae. It is the simplest type of single-stranded DNA defective virus discovered so far and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in the inverted repeat sequences (ITR) at the two ends. The inverted repeat sequences at the ends play a decisive role in the replication and packaging of the virus. The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus. Adeno-associated viruses can infect a variety of cells. For example, adeno-associated viruses can be used as recombinant adeno-associated virus vectors to integrate into the genome of the cells they infect in a stable and site-specific manner. Adeno-associated virus vectors can be prepared using standard methods in the art, and any serotype of adeno-associated virus is suitable. Replication-deficient recombinant adeno-associated virus can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (e.g., adenovirus): a plasmid containing a nucleic acid sequence of interest flanked by two adeno-associated virus inverted terminal repeat (ITR) regions, and a plasmid carrying adeno-associated virus encapsidation genes (rep and cap genes). The resulting adeno-associated virus recombinant is then purified by standard techniques. For example, a recombinant adeno-associated virus vector can be encapsidated into virions (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16 AAV virions).

[0057] In this application, the term "serotype" generally refers to the detection of epitopes on the surface of the adeno-associated virus capsid by serological methods and the typing of the adeno-associated virus. Adeno-associated virus has a variety of common serotypes and more than 100 virus variants. In this application, AAV capsid, ITR and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, any known or mentioned AAV variants or AAVs yet to be discovered or their variants or mixtures.

[0058] In this application, the term "host cell" generally refers to prokaryotic cells, lower eukaryotic cells, or higher eukaryotic cells, such as mammalian cells (including human and non-human mammals). Representative examples include the following: animal cells such as CHO, NS0, COS7, or 293 cells. For example, neural cells can be selected as host cells. For example, the host cells are selected from the group consisting of pancreas, liver, muscle cells, or a combination thereof. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0059] In this application, the term "culture" generally refers to culturing host cells using conventional methods to express the protein encoded by the gene of the application. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for host cell growth. After the host cells grow to an appropriate cell density, the promoter of choice is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0060] In this application, the term "recombinant nucleic acid" generally refers to a recombined gene sequence. The recombinant nucleic acid of this application can be in the form of DNA or RNA. For example, the recombinant nucleic acid is DNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0061] In this application, the term "operably linked" may refer to a nucleic acid sequence that is functionally related to the sequence to which it is operably linked, being linked in such a way that they affect the expression or function of each other. For example, a nucleic acid sequence that is operably linked to a promoter will have an expression pattern affected by the promoter.

[0062] In the present application, the term "expression" may refer to the recombinant nucleic acid, fusion nucleic acid, or vector of the present application transcribing a target RNA and / or translating a target protein in a host cell.

[0063] In this application, the term "treatment" generally refers to an intervention that attempts to alter the natural course of the subject being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, curbing, reducing or inhibiting any direct or indirect pathological consequences of the disease, ameliorating or alleviating the disease state, and causing remission or improving prognosis.

[0064] As used herein, the term "pharmaceutically acceptable carrier" or "excipient" refers to one or more compatible solid or liquid fillers or gel substances suitable for human use, and which must be of sufficient purity and sufficiently low toxicity. Compatibility herein may refer to the ability of the components of the composition to be compatible with the active ingredient of the present invention, and with each other, without significantly reducing the efficacy of the active ingredient. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0065] In this application, the terms "subject" and "subject in need" generally refer to any mammal or non-mammal. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, rabbits, rats, mice, horses, dogs, cats, pigs, sheep, and goats.

[0066] In this application, the term "about" generally refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0067] In this application, the term "comprising" or "including (comprising)" generally refers to open, semi-closed and closed forms. In other words, the term also includes "essentially composed of..." or "composed of..."

[0068] In this application, sequence "identity" or "identity" is generally determined by comparing two aligned sequences along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence "identity" or "identity" of nucleotide sequences is a method well known to those skilled in the art. For example, "identity" can refer to the relatedness of two sequences based on a nucleotide-by-nucleotide comparison over a specific comparison window or segment. Thus, identity is defined as the degree of identity, correspondence or equivalence between the same strands (sense or antisense) of two DNA segments. "Percent sequence identity" is calculated as follows: two optimally aligned sequences are compared over a specific region; the number of positions at which the same base or amino acid occurs in the two sequences is determined to obtain the number of matched positions; the number of such positions is divided by the total number of positions in the compared segment, and the resulting quotient is multiplied by 100. Optimal alignment of sequences can be performed using established algorithms.

[0069] Detailed Description of the Invention

[0070] The present application provides a recombinant nucleic acid that can be used to alleviate, prevent, and / or treat metabolic diseases and / or symptoms. The association between the GIGYF1 target and disease has not yet been clearly elucidated; the present application provides a method and application for intervening in related diseases by intervening in GIGYF1. Preferably, by intervening in the expression of GIGYF1 in specific tissues, the applicant has discovered unexpected effects in intervening in diseases.

[0071] On the one hand, the present application provides a preparation for intervening in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof, and use thereof in the preparation of a medicament, which can be used to alleviate, prevent and / or treat specific diseases and / or symptoms.

[0072] In one aspect, the present application provides a preparation for intervening in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof, which can be used to alleviate, prevent and / or treat specific diseases and / or symptoms.

[0073] In one aspect, the present application provides a method for alleviating, preventing and / or treating specific diseases and / or symptoms, which may comprise intervening in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof.

[0074] For example, the GIGYF1 comprises a GYF motif, and the sequence of the GYF motif is shown in SEQ ID NO: 2. For example, the GIGYF1 of the present application may comprise a functionally active fragment of GIGYF1, for example, a truncated form having the functionally active fragment may retain the biological activity of the GYF motif.

[0075] For example, the GIGYF1 may comprise expression products of one or more exons of GIGYF1. For example, the GIGYF1 may comprise expression products of 27 exons of GIGYF1. For example, the GIGYF1 may comprise expression products of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 27 exons of GIGYF1.

[0076] For example, the GIGYF1 comprises human GIGYF1. For example, the GIGYF1 comprises mammalian GIGYF1. For example, the GIGYF1 comprises primate GIGYF1. For example, the GIGYF1 comprises human, monkey, rat, mouse, or rabbit GIGYF1.

[0077] For example, the amino acid sequence of GIGYF1 is shown in SEQ ID NO: 1. For example, the amino acid sequence of GIGYF1 has an amino acid sequence that is about at least 50% identical to SEQ ID NO: 1. For example, the amino acid sequence of GIGYF1 may have an amino acid sequence that is about at least 50%, about at least 60%, about at least 70%, about at least 80%, about at least 85%, about at least 90%, about at least 91%, about at least 92%, about at least 93%, about at least 94%, about at least 95%, about at least 96%, about at least 97%, about at least 98%, or about at least 99% identical to SEQ ID NO: 1.

[0078] For example, the nucleotide sequence encoding the GIGYF1 is shown in SEQ ID NO: 4. For example, the amino acid sequence of the GIGYF1 has a nucleotide sequence that is about at least 50% identical to SEQ ID NO: 4. For example, the nucleotide sequence of the GIGYF1 may have a nucleotide sequence that is about at least 50%, about at least 60%, about at least 70%, about at least 80%, about at least 85%, about at least 90%, about at least 91%, about at least 92%, about at least 93%, about at least 94%, about at least 95%, about at least 96%, about at least 97%, about at least 98%, or about at least 99% identical to SEQ ID NO: 4.

[0079] For example, the agent that intervenes in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof may also optionally contain a substance that reduces the expression of the gene. For example, the agent that intervenes in the expression of GIGYF1, GIGYF1 variants, truncations and analogs thereof may contain a CRISPR editing system, small inhibitory RNA (siRNA), microRNA (miRNA) or small hairpin RNA (shRNA).

[0080] For example, agents that interfere with the expression of GIGYF1, GIGYF1 variants, truncations, and analogs thereof can include substances that increase the expression of the gene. For example, the nucleic acid or nucleic acid sequence can be an oligonucleotide, a nucleotide, or a polynucleotide; for example, the nucleic acid or nucleic acid sequence can be a fragment of any of the above; for example, the nucleic acid or nucleic acid sequence can be DNA or RNA of genomic or synthetic origin, which can be single-stranded or double-stranded and can represent a sense strand or an antisense strand; for example, the nucleic acid or nucleic acid sequence can be a peptide nucleic acid (PNA); or for example, the nucleic acid or nucleic acid sequence can be any DNA-like or RNA-like substance of natural or synthetic origin. For example, the nucleic acid of the present application can include any fragment of an oligonucleotide, a nucleotide, a polynucleotide, or any of these; for example, the nucleic acid of the present application can include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin, which can be single-stranded or double-stranded; for example, the nucleic acid of the present application can include sense strand or antisense strand, or peptide nucleic acid (PNA), or any DNA-like or RNA-like material of natural or synthetic origin, including, for example, iRNA, ribonucleoprotein (e.g., double-stranded iRNA, e.g., iRNP). For example, the nucleic acid of the present application can include nucleic acids containing known analogs of natural nucleotides, i.e., modified oligonucleotides.

[0081] For example, the nucleic acid sequence of GIGYF1 is packaged by a non-viral vector and / or a viral vector. For example, the viral vector can be selected from the following group: a recombinant parvovirus, a recombinant lentivirus, a recombinant retrovirus, and a recombinant adenovirus. For example, the non-viral vector can be selected from the following group: naked DNA, naked RNA, chemically modified DNA, chemically modified RNA, an inorganic particle vector, a lipid particle vector, a liposome-based vector, a polymer-based vector, or a chitosan-based vector.

[0082] For example, the carrier of the present application also includes plasmid, expression vector, recombinant virus, any form of recombinant " naked DNA " carrier etc.For example, the carrier of the present application can comprise can infect, transfect, transiently or permanently transduce the nucleic acid of cell.For example, the carrier of the present application can be naked nucleic acid or with protein or lipid compound nucleic acid.For example, the carrier of the present application can comprise viral or bacterial nucleic acid and / or protein and / or membrane (for example, cell membrane, viral lipid envelope etc.).For example, the carrier of the present application can include but not limited to replicon (for example, RNA replicon, bacteriophage), and DNA fragment can be connected with this replicon and be replicated.For example, the carrier of the present application can include but not limited to RNA, autonomous self-replicating circular or linear DNA or RNA.

[0083] For example, the viral vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector and / or an adeno-associated viral vector. For example, the nucleic acid sequence of GIGYF1 is packaged by a viral vector selected from the following groups: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2N587A, AAV2E548A, AAV2N708A, AAVV708K, ovine AAV, bovine AAV, mouse AAV, AAV1 / AAV2 chimeric type, and rAAV2 / HBoV1 serotype. For example, the viral vector can be AAV9.

[0084] For example, the GIGYF1 gene can be operably linked to a regulatory element. For example, the regulatory element can be selected from the group consisting of a promoter, a polyadenylation sequence, an intron, an enhancer, and a polyA sequence. For example, the promoter can comprise a liver-specific promoter, a pancreas-specific promoter, and / or a muscle-specific promoter.

[0085] For example, the "promoter" of the present application can include all sequences that can drive the transcription of a coding sequence in a cell (e.g., a mammalian cell, such as a heart, lung, muscle, nerve or brain cell). For example, the "promoter" of the present application can include cis-acting transcription control elements and regulatory sequences that participate in regulating or controlling the timing and / or rate of gene transcription. For example, the "promoter" of the present application can include cis-acting transcription control elements, such as enhancers, promoters, transcription terminators, replication origins, chromosomal integration sequences, 5' and 3' untranslated regions or intron sequences, which participate in the regulation of transcription. For example, cis-acting sequences can interact with proteins or other biomolecules to implement (turn on / off, regulate, control, etc.) transcription.

[0086] For example, the present application may include a "constitutive" promoter, which can be those that continuously drive expression under most environmental conditions and developmental states or cell differentiation states. For example, the present application may include an "inducible" or "regulatable" promoter, which can direct expression of the nucleic acid of the present application under the influence of environmental conditions, applied chemicals, or developmental conditions.

[0087] For example, the GIGYF1 is configured to specifically target tissues or cells. For example, the GIGYF1 is configured to target tissues or cells in which gene editing is desired. For example, the GIGYF1 is configured to be delivered to tissues or cells selected from the group consisting of liver, heart, muscle, pancreas, and brain. For example, the GIGYF1 is configured to be delivered to liver cells, heart cells, pancreatic cells, brain cells, nerve cells, and / or reproductive cells. For example, the subject is female. For example, the subject is male.

[0088] For example, the formulations of the present application can be delivered by intramuscular (IM) injection, by intravenous (IV) injection, by subcutaneous injection, by inhalation, by a biolistic particle delivery system (e.g., a so-called "gene gun"), and the like.

[0089] For example, the disease and / or symptom is related to GIGYF1 expression. For example, the GIGYF1 is used to alleviate, prevent and / or treat metabolic diseases and / or conditions. For example, the GIGYF1 is used to alleviate, prevent and / or treat diabetes and / or its complications. For example, the disease and / or symptom is selected from the following group: developmental delay, hyperglycemia, hyperlipidemia, increased glycosylated hemoglobin ratio, insulin resistance, decreased glucose tolerance, increased fat, fatty liver, spleen hyperplasia, decreased insulin secretion, increased glucagon secretion, decreased reproductive ability, kidney damage, skin damage, swollen finger (toe) joints, heart damage, and cognitive impairment.

[0090] For example, the methods of the present application can be used to improve or treat any of a variety of diabetes-related heart diseases and cardiovascular diseases, for example, diabetes-related heart diseases and cardiovascular diseases, such as coronary artery disease; atherosclerosis; thrombosis; restenosis; vasculitis, including autoimmune vasculitis and viral vasculitis, such as polyarteritis nodosa, allergic granulomatosis with polyangiitis, and rickettsial vasculitis; atherosclerotic aneurysms; cardiac hypertrophy; congenital heart disease; ischemic heart disease and angina; acquired valvular disease / endocardial disease; primary cardiomyopathy, including myocarditis; arrhythmia; and transplant rejection; metabolic cardiomyopathy and cardiomyopathy, such as congestive cardiomyopathy, hypertrophic cardiomyopathy or restrictive cardiomyopathy; and / or heart transplantation. For example, the methods of the present application can be used to treat, improve or prevent (prevent) diabetes or prediabetes in a patient or individual; or to inhibit weight gain in a patient or individual, or to suppress appetite, or to stimulate or initiate weight loss; or to treat, improve or prevent (prevent) diabetes in a patient or individual.

[0091] In one aspect, the present application provides a cell comprising an agent that intervenes with GIGYF1, GIGYF1 variants, truncations, and analogs thereof. For example, the cells of the present application can be used to screen for candidate agents that can intervene with GIGYF1, GIGYF1 variants, truncations, and analogs thereof. For example, cells in which GIGYF1, GIGYF1 variants, truncations, and analogs thereof are intervened can be used for administration into a subject. For example, the cells can comprise a desired cell type, such as a mammalian cell. For example, the cells can comprise skeletal muscle cells or tissues, heart cells, liver cells, spleen cells, kidney cells, lung cells, pancreatic islet cells, bone cells, germ cells, neural cells, and the like.

[0092] For example, the present application provides products comprising the cells of the present application (e.g., cells modified to express a GIGYF1-encoded polypeptide to implement the methods of the present application) and uses of the cells prepared by the methods of the present application, including, for example, in vivo implantation of cells, implants, artificial organs, bioreactor systems, cell culture systems, cell culture plates, cell culture vessels, cell culture tubes, cell culture bottles, and cell culture flasks, wherein the above products comprise cells modified to express a GIGYF1-encoded polypeptide to implement the methods of the present application.

[0093] In one aspect, the present application provides a kit comprising nucleic acids encoding GIGYF1, GIGYF1 variants, truncations, and analogs thereof, vectors comprising the nucleic acids, and / or cells described herein. For example, the kit may further comprise instructions describing methods, such as in vitro or ex vivo methods for treating, preventing, or ameliorating diabetes or diabetic complications.

[0094] In another aspect, the present application provides a modified non-human animal, wherein the expression level of the GIGYF1 gene in the modified non-human animal is reduced compared to a wild-type non-human animal. For example, by at least 10%. For example, by at least 20%. For example, by at least 30%. For example, by at least 40%. For example, by at least 50%. For example, by at least 60%. For example, by at least 70%. For example, by at least 80%. For example, by at least 90%. For example, by at least 95%. For example, by at least 99%. For example, by at least 100%.

[0095] In certain embodiments, the expression level of the GIGYF1 protein in the modified non-human animal is reduced compared to a wild-type non-human animal. For example, by at least 10%. For example, by at least 20%. For example, by at least 30%. For example, by at least 40%. For example, by at least 50%. For example, by at least 60%. For example, by at least 70%. For example, by at least 80%. For example, by at least 90%. For example, by at least 95%. For example, by at least 100%.

[0096] In certain embodiments, the Gigyf1 protein comprises the amino acid sequence shown in SEQ ID NO. 1. In certain embodiments, the Gigyf1 protein comprises the amino acid sequence shown in SEQ ID NO. 1 or a functional fragment thereof.

[0097] In certain embodiments, the GIGYF1 gene of the modified non-human animal is knocked out or knocked down. In certain embodiments, the GIGYF1 gene comprises the nucleic acid sequence shown in SEQ ID NO.5.

[0098] For example, zinc-finger nuclease (ZFN) can be used to knock out or knock down the GIGYF1 gene of the modified non-human animal. For example, transcription activator-like effector nuclease (TALEN) can be used to knock out or knock down the GIGYF1 gene of the modified non-human animal. For example, CRISPR / Cas can be used to knock out or knock down the GIGYF1 gene of the modified non-human animal. For example, RNAi can be used to knock out or knock down the GIGYF1 gene of the modified non-human animal. For example, shRNA can be used to knock out or knock down the GIGYF1 gene of the modified non-human animal. For example, siRNA can be used to knock out or knock down the GIGYF1 gene of the modified non-human animal.

[0099] In certain embodiments, the modification comprises contacting the genome of a non-human animal cell, stem cell, or fertilized egg with: 1) a CRISPR-associated (Cas) protein; and 2) one or more ribonucleic acid (RNA) sequences. For example, the one or more ribonucleic acid (RNA) sequences comprise a sequence complementary to the GIGYF1 gene and a sequence that binds to the Cas protein.

[0100] In certain embodiments, the modified Cas protein is Cas9.

[0101] In certain embodiments, the stem cell is selected from the group consisting of embryonic stem cells, adult stem cells, totipotent stem cells, unipotent stem cells, and induced pluripotent stem cells.

[0102] In certain embodiments, the sequence that binds to the Cas protein includes a tracrRNA sequence.

[0103] In certain embodiments, the one or more ribonucleic acid (RNA) sequences comprise the sequence shown in any one of SEQ ID NOs. 3-4.

[0104] In certain embodiments, the modified non-human animal displays phenotypes and / or symptoms of a metabolic disease.

[0105] In certain embodiments, the modified non-human animal displays a phenotype and / or symptoms of diabetes.

[0106] In certain embodiments, the phenotype and / or symptoms of diabetes are selected from any one or more of the following groups: (1) developmental delay; (2) elevated blood lipids; (3) elevated blood glucose concentration; (4) abnormal fat metabolism, for example, increased fat mass in females, decreased fat mass in males, fatty liver, lipomas, and spleen hyperplasia; (5) reproductive abnormalities, for example, decreased sperm count in male mice; (6) renal dysfunction, for example, abnormal urine color (yellow urine); (7) partial hair loss, skin ulcers, or swollen knuckles; (8) increased mortality; (9) abnormal heart condition; (10) cognitive abnormalities; (11) slower glucose clearance; (12) decreased sensitivity to insulin; (13) increased expression of tumor necrosis factor α; (14) decreased glucagon secretion; (15) decreased insulin secretion; and (16) decreased energy exchange.

[0107] In certain embodiments, the modified non-human animal is developmentally delayed compared to a wild-type or unmodified non-human animal. For example, at least 10% delayed. For example, at least 20% delayed. For example, at least 30% delayed. For example, at least 40% delayed. For example, at least 50% delayed. For example, at least 60% delayed. For example, at least 70% delayed. For example, at least 80% delayed. For example, at least 90% delayed.

[0108] In certain embodiments, the modified non-human animal has elevated blood glucose, which may include random blood glucose and / or fasting blood glucose, compared to a wild-type or unmodified non-human animal. For example, an increase of at least 5%. For example, an increase of at least 10%. For example, an increase of at least 15%. For example, an increase of at least 20%. For example, an increase of at least 30%. For example, an increase of at least 40%. For example, an increase of at least 50%. For example, an increase of at least 60%. For example, an increase of at least 70%. For example, an increase of at least 80%. For example, an increase of at least 90%.

[0109] In certain embodiments, the modified non-human animal has an increase in body weight compared to a wild-type or unmodified non-human animal. For example, an increase of at least 5%. For example, an increase of at least 10%. For example, an increase of at least 15%. For example, an increase of at least 20%. For example, an increase of at least 30%. For example, an increase of at least 40%. For example, an increase of at least 50%. For example, an increase of at least 60%. For example, an increase of at least 70%. For example, an increase of at least 80%. For example, an increase of at least 90%.

[0110] In certain embodiments, the modified non-human animal has an increased body fat percentage compared to a wild-type or unmodified non-human animal. For example, an increase of at least 5%. For example, an increase of at least 10%. For example, an increase of at least 15%. For example, an increase of at least 20%. For example, an increase of at least 30%. For example, an increase of at least 40%. For example, an increase of at least 50%. For example, an increase of at least 60%. For example, an increase of at least 70%. For example, an increase of at least 80%. For example, an increase of at least 90%.

[0111] In certain embodiments, the modified non-human animal has a reduced lean mass percentage compared to a wild-type or unmodified non-human animal. For example, a reduction of at least 5%. For example, a reduction of at least 10%. For example, a reduction of at least 15%. For example, a reduction of at least 20%. For example, a reduction of at least 30%. For example, a reduction of at least 40%. For example, a reduction of at least 50%. For example, a reduction of at least 60%. For example, a reduction of at least 70%. For example, a reduction of at least 80%. For example, a reduction of at least 90%.

[0112] In certain embodiments, the modified non-human animal has reduced energy expenditure compared to a wild-type or unmodified non-human animal. For example, by at least 5%. For example, by at least 10%. For example, by at least 15%. For example, by at least 20%. For example, by at least 30%. For example, by at least 40%. For example, by at least 50%. For example, by at least 60%. For example, by at least 70%. For example, by at least 80%. For example, by at least 90%.

[0113] In certain embodiments, the modified non-human animal has reduced insulin secretion compared to a wild-type or unmodified non-human animal. For example, by at least 5%. For example, by at least 10%. For example, by at least 15%. For example, by at least 20%. For example, by at least 30%. For example, by at least 40%. For example, by at least 50%. For example, by at least 60%. For example, by at least 70%. For example, by at least 80%. For example, by at least 90%.

[0114] In certain embodiments, the modified non-human animal has reduced glucagon secretion compared to a wild-type or unmodified non-human animal. For example, by at least 5%. For example, by at least 10%. For example, by at least 15%. For example, by at least 20%. For example, by at least 30%. For example, by at least 40%. For example, by at least 50%. For example, by at least 60%. For example, by at least 70%. For example, by at least 80%. For example, by at least 90%.

[0115] In certain embodiments, the modified non-human animal has increased C-peptide expression compared to a wild-type or unmodified non-human animal. For example, an increase of at least 5%. For example, an increase of at least 10%. For example, an increase of at least 15%. For example, an increase of at least 20%. For example, an increase of at least 30%. For example, an increase of at least 40%. For example, an increase of at least 50%. For example, an increase of at least 60%. For example, an increase of at least 70%. For example, an increase of at least 80%. For example, an increase of at least 90%.

[0116] In certain embodiments, the modified non-human animal has increased expression of tumor necrosis factor compared to a wild-type or unmodified non-human animal. For example, an increase of at least 5%. For example, an increase of at least 10%. For example, an increase of at least 15%. For example, an increase of at least 20%. For example, an increase of at least 30%. For example, an increase of at least 40%. For example, an increase of at least 50%. For example, an increase of at least 60%. For example, an increase of at least 70%. For example, an increase of at least 80%. For example, an increase of at least 90%.

[0117] On the other hand, the present application provides the use of the modified non-human animals in screening drugs for alleviating, preventing and / or treating metabolic diseases and / or symptoms. In certain embodiments, the use includes administering one or more candidate agents to the modified non-human animals. If the metabolic diseases and / or symptoms of the modified non-human animals improve, the one or more candidate agents are considered to be drugs that can alleviate, prevent and / or treat metabolic diseases and / or symptoms; if the metabolic diseases and / or symptoms of the modified non-human animals do not improve or worsen, the one or more candidate agents are considered to be drugs that cannot alleviate, prevent and / or treat metabolic diseases and / or symptoms.

[0118] In certain embodiments, the medicament is used to alleviate, prevent and / or treat diabetes and / or its complications.

[0119] Technical Solution

[0120] 1. A preparation for intervening GIGYF1, GIGYF1 variants, truncations and analogs thereof, and use thereof in the preparation of a medicament for alleviating, preventing and / or treating a specific disease and / or symptom.

[0121] 2. The use as described in technical solution 1, wherein the GIGYF1 comprises a GYF motif, and the sequence of the GYF motif is shown in SEQ ID NO: 2.

[0122] 3. The use according to any one of technical solutions 1-2, wherein the GIGYF1 comprises the expression products of one or more exons of GIGYF1.

[0123] 4. The use according to any one of technical solutions 1 to 3, wherein the GIGYF1 comprises human GIGYF1.

[0124] 5. The use according to any one of technical aspects 1-4, wherein the amino acid sequence of GIGYF1 is shown in SEQ ID NO: 1.

[0125] 6. The use according to any one of technical solutions 1 to 5, wherein the nucleotide sequence encoding the GIGYF1 is shown in SEQ ID NO: 4.

[0126] 7. The use according to any one of technical solutions 1 to 6, wherein the nucleic acid sequence encoding the GIGYF1 is packaged by a non-viral vector and / or a viral vector.

[0127] 8. The use as described in Technical Solution 7, wherein the viral vector comprises a lentiviral vector, a retroviral vector, an adenoviral vector and / or an adeno-associated viral vector.

[0128] 9. The use as described in any one of technical solutions 1 to 8, wherein the nucleic acid sequence encoding the GIGYF1 is packaged by a viral vector selected from the following group: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2N587A, AAV2E548A, AAV2N708A, AAVV708K, sheep AAV, bovine AAV, mouse AAV, AAV1 / AAV2 chimeric type, and rAAV2 / HBoV1 serotype.

[0129] 10. The use according to any one of technical solutions 1 to 9, wherein the nucleic acid sequence encoding GIGYF1 is operably linked to a regulatory element.

[0130] 11. The use according to technical solution 10, wherein the regulatory element is selected from the following group: a promoter, a polyadenylation sequence, an intron, an enhancer and a polyA sequence.

[0131] 12. The use as described in technical solution 11, wherein the promoter comprises a liver-related specific promoter, a pancreas-related specific promoter and / or a muscle-related specific promoter.

[0132] 13. The use according to any one of technical solutions 1 to 12, wherein the GIGYF1 is configured to specifically target tissues or cells.

[0133] 14. The use according to any one of technical solutions 1 to 13, wherein the GIGYF1 is configured to be delivered to tissues or cells selected from the following group: liver, heart, muscle, pancreas and brain.

[0134] 15. The use according to any one of technical solutions 1 to 14, wherein the disease and / or symptom is related to GIGYF1 expression.

[0135] 16. The use according to any one of technical solutions 1 to 15, wherein the GIGYF1 is used to alleviate, prevent and / or treat metabolic diseases and / or symptoms.

[0136] 17. The use according to any one of technical solutions 1 to 16, wherein the GIGYF1 is used to alleviate, prevent and / or treat diabetes and / or its complications.

[0137] 18. The use according to any one of technical solutions 1 to 17, wherein the disease and / or symptom is selected from the following group: developmental delay, hyperglycemia, hyperlipidemia, increased glycosylated hemoglobin ratio, insulin resistance, decreased glucose tolerance, increased fat, fatty liver, spleen hyperplasia, decreased insulin secretion, increased glucagon secretion, decreased reproductive capacity, kidney damage, skin damage, swollen finger (toe) joints, heart damage, and cognitive impairment.

[0138] 19. A cell comprising an agent intervening GIGYF1, GIGYF1 variants, truncations and analogs thereof.

[0139] 20. A kit comprising nucleic acid encoding GIGYF1, GIGYF1 variants, truncations and analogs thereof, a vector comprising the above nucleic acid and / or the cell according to technical solution 19.

[0140] Without intending to be bound by any theory, the following embodiments are merely intended to illustrate the methods and uses of the present application and are not intended to limit the scope of the present invention.

[0141] Example

[0142] Example 1

[0143] The present application utilizes a wild-type gene sequence encoding the GIGYF1 protein, which may be optionally optimized, wherein the GIGYF1 protein sequence encoded by the GIGYF1 gene may be shown as SEQ ID NO: 1, and the sequence of the GIGYF1 gene may be shown as SEQ ID NO: 5.

[0144] Viral vector construction. The gene fragment encoding GIGYF1 described herein was ligated to a plasmid vector to produce a ligation product and a recombinant adeno-associated virus (RAV) expression vector. By transfecting the recombinant adeno-associated virus (RAV) expression vector into host cells, cells containing the viral vector were obtained. Testing confirmed that the titer and purity of the resulting virus met the requirements for subsequent applications.

[0145] Example 2

[0146] Animal model construction

[0147] A model animal is selected and the Gigyf1 gene is knocked out using gene editing tools. For example, a guide RNA as shown in SEQ ID NO: 3 or 4 can be provided.

[0148] PCR testing, sequencing analysis and growth observation showed that the constructed animal model had good growth ability and had the effect of preventing the expression of the Gigyf1 gene. Through phenotypic observation and testing, the Gigyf1 knockout animal model had at least the following effects: (1) developmental delay, such as similar conditions to early dwarfism; (2) abnormal blood indicators, such as high blood lipids and blood viscosity; (3) abnormal glucose metabolism, with diabetes and hyperglycemia phenotypes, such as impaired intraperitoneal glucose tolerance, resistance to insulin, and abnormal pancreatic islet morphology; (4) abnormal fat metabolism, such as increased fat mass in females, decreased fat mass in males, fatty liver, lipomas and spleen hyperplasia; (5) reproductive abnormalities, such as decreased sperm count in male mice; (6) poor renal function, such as abnormal urine color (yellow urine); (7) skin complications similar to diabetes, such as partial hair loss, skin ulcers, and swollen knuckles; (8) increased mortality; (9) abnormal heart condition; and (10) cognitive abnormalities.

[0149] As shown in Figures 1 and 2, blood glucose was measured in wild-type mice (n=40 males, 31 females) and Gigyf1 knockout mice (n=39 males, 18 females) at the same time each week using the glucose dehydrogenase method. The blood glucose concentrations of Gigyf1 knockout mice were abnormal. Compared with wild-type mice, the blood glucose concentrations of Gigyf1 knockout mice were higher in both fed and fasting conditions.

[0150] As shown in Figure 3, mice were fasted for 14-16 hours, but not water, and then blood was drawn from the tail vein. Basal blood glucose (i.e., blood glucose at 0 min) was measured using the glucose dehydrogenase assay. After weighing, mice were intraperitoneally injected with a glucose solution at a dose of 2 g glucose / kg body weight. Timer was immediately initiated after injection. Blood glucose levels were recorded at 15, 30, 60, 90, and 120 min. In the IPGTT test, Gigyf1 knockout mice (n = 30 males, 16 females) exhibited slower glucose clearance compared to wild-type mice (n = 37 males, 31 females), indicating that Gigyf1 knockout mice (both males and females) have reduced glucose utilization efficiency and a diabetic phenotype.

[0151] As shown in Figure 4, in the ITT test, blood was drawn from the tail vein, and basal blood glucose (i.e., blood glucose at 0 min) was measured using a glucometer. Body weight was then measured and recorded. A 0.1 U / mL insulin-saline solution was prepared and thoroughly mixed. Immediately after intraperitoneal injection at 1 U / kg body weight, a timer was started, and blood glucose levels were recorded at 15, 30, 60, and 120 minutes. The ITT curve was normalized to the 0 min blood glucose value as "1," and the area under the curve was calculated. Gigyf1 knockout mice (n = 20 males, 11 females) showed reduced insulin sensitivity compared to wild-type mice (n = 31 males, 18 females).

[0152] As shown in Figure 6, after measuring the body weight of wild-type mice (n = 20 males, 18 females) and Gigyf1 knockout mice (n = 10 males, 10 females), the fat and lean mass of each mouse was analyzed using a nuclear magnetic resonance biopsy analyzer. By comparing these components to body weight, body fat percentage and lean mass percentage were calculated. It can be seen that the fat percentage of Gigyf1 knockout female mice was higher than that of wild-type female mice and was increasing. However, the fat percentage of Gigyf1 knockout male mice was higher than that of wild-type male mice but was decreasing.

[0153] As shown in Figure 7, wild-type mice (n=8 males, 6 females) and Gigyf1 knockout mice (n=8 males, 6 females) were weighed at 9:00 AM and placed in metabolic cages. Oxygen consumption, carbon dioxide production, and activity levels were recorded over two days to calculate energy expenditure and respiratory exchange rates. The respiratory exchange rates of both male and female Gigyf1 knockout mice were significantly lower than those of wild-type mice, indicating that these mice prefer to use fat as a metabolic energy source compared to wild-type mice.

[0154] As shown in Figure 8, at 15 weeks of age, orbital blood was collected from wild-type mice (n=8 males, 6 females) and Gigyf1 knockout mice (n=7 males, 7 females) after 16 hours of starvation. Whole blood samples were incubated at room temperature for 0.5-1 hour and then centrifuged at 3500 rpm for 10 minutes at 4°C. Serum was retained and assayed using corresponding ELISA kits for C-peptide, insulin, and glucagon levels. Serum was centrifuged before use. There were no significant differences in serum insulin, glucagon, and C-peptide levels between KO mice and WT mice, indicating no significant changes in pancreatic islet function.

[0155] As shown in Figure 9, the survival of wild-type mice (n = 58 males, 59 females) and Gigyf1 knockout mice (n = 65 males, 52 females) was recorded for 50 weeks, and survival curves were plotted based on the results. It can be seen that the loss of the Gigyf1 gene leads to a high mortality rate, with mortality being most pronounced during the weaning period.

[0156] Example 3

[0157] The present application provides a recombinant nucleic acid encoding the GIGYF1 protein, which can be used for intervention, alleviation and treatment of diseases and / or conditions caused by abnormalities in the gene encoding the GIGYF1 protein.

[0158] Therapeutic effects of recombinant nucleic acids of GIGYF1

[0159] Fifteen-week-old wild-type mice (n=3 males) and Gigyf1 knockout mice (n=3 males) were intraorbitally injected with AAV9 virus at a titer of 1*10^14 vg / kg for systemic GIGYF1 complementation. Four weeks after injection, IPGTT and ITT tests were performed on the above two groups and on wild-type mice (n=3 males) and Gigyf1 knockout mice (n=3 males) that did not undergo complementation, as shown in Figure 10.

[0160] In the IPGTT test, mice are fasted for 14-16 hours but not water, then removed from the tail vein. Blood is drawn and basal blood glucose levels (i.e., blood glucose at 0 minutes) are measured using the glucose dehydrogenase method. After weighing, the mice are injected intraperitoneally with 2g of glucose / kg body weight, and timing begins immediately after injection. Blood glucose levels are recorded at 15, 30, 60, 90, and 120 minutes. After Gigyf1 repletion, the ability of Gigyf1 knockout mice to clear glucose from the blood returned to the level of wild-type mice, and zero-point blood glucose (fasting blood glucose) also decreased, indicating that the diabetic phenotype improved after repletion. Furthermore, overexpression of GIGYF1 in wild-type mice significantly improved their ability to clear glucose, suggesting that overexpression of this gene has a preventive effect on diabetes.

[0161] The mice in the ITT test were not starved. The test also adopted the tail vein blood sampling method. The basal blood glucose value (i.e., blood glucose at 0 min) was detected with a blood glucose meter, and the body weight was weighed and recorded. Prepare 0.1U / mL insulin-normal saline solution and make sure to mix it thoroughly. Immediately after intraperitoneal injection of 1U / kg body weight, the timing was started and the blood glucose values ​​were recorded at 15min, 30min, 60min, and 120min. The blood glucose value at 0min was normalized as "1" to obtain the ITT curve and calculate the area under the curve. The experimental results showed that the sensitivity of Gigyf1 gene knockout mice to insulin was greatly increased after gene therapy, and there was a trend of higher insulin efficiency compared with the WT results; overexpression of GIGYF1 in wild-type mice also showed that overexpression of this gene had a preventive effect on diabetes.

[0162] 40-week-old Gigyf1 knockout mice (n=4 males, 5 females) were injected intraorbitally with AAV9 virus at a titer of 1*10^14 vg / kg for systemic GIGYF1 complementation. Four weeks after injection, IPGTT and ITT tests were performed on the above presentation group, wild-type mice without complementation (n=10 males, 7 females), and Gigyf1 knockout mice (n=4 males). As shown in Figure 11, the traits of the Gigyf1 knockout mice that received gene presentation returned to the wild-type level, or even exceeded the wild-type level.

[0163] The results showed that gene delivery had the effect of treating and preventing diabetes in both young and old mice, and in both female and male mice.

[0164] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various changes to the embodiments listed in the present application are obvious to those skilled in the art and are intended to be within the scope of the appended claims and their equivalents.

Claims

1. A preparation for intervening GIGYF1, GIGYF1 variants, truncations and analogs thereof, and use thereof in the preparation of a medicament for alleviating, preventing and / or treating a specific disease and / or symptom.

2. The use according to claim 1, wherein the GIGYF1 comprises the amino acid sequence shown in SEQ ID NO:

2.

3. The use according to claim 1, wherein the GIGYF1 comprises the amino acid sequence shown in SEQ ID NO:

1.

4. The use according to any one of claims 1 to 3, wherein the nucleotide sequence encoding the GIGYF1 is shown in SEQ ID NO:

5.

5. The use according to any one of claims 1 to 4, wherein the GIGYF1 is configured to be delivered to tissues or cells selected from the group consisting of liver, heart, muscle, pancreas and brain.

6. The use according to any one of claims 1 to 5, wherein the disease and / or symptom is related to GIGYF1 expression.

7. The use according to any one of claims 1 to 6, wherein the GIGYF1 is used to alleviate, prevent and / or treat metabolic diseases and / or symptoms.

8. The use according to any one of claims 1 to 7, wherein the GIGYF1 is used to alleviate, prevent and / or treat diabetes and / or its complications.

9. The use according to any one of claims 1 to 8, wherein the disease and / or symptom is selected from the group consisting of developmental delay, hyperglycemia, hyperlipidemia, increased glycosylated hemoglobin ratio, insulin resistance, decreased glucose tolerance, increased fat, fatty liver, spleen hyperplasia, decreased insulin secretion, increased glucagon secretion, decreased reproductive capacity, kidney damage, skin damage, swollen finger (toe) joints, heart damage, and cognitive impairment.

10. A cell comprising an agent intervening with GIGYF1, GIGYF1 variants, truncations and analogs thereof.

11. A kit comprising a nucleic acid encoding GIGYF1, a GIGYF1 variant, a truncation and an analog thereof, a vector comprising the nucleic acid and / or the cell according to claim 10.

12. A modified non-human animal having reduced expression of the GIGYF1 gene compared to a wild-type non-human animal. The modified non-human animal according to claim 12 , wherein the GIGYF1 gene is knocked out or knocked down.

14. The modified non-human animal according to claim 12, wherein the GIGYF1 gene is knocked out by CRISPR / Cas technology.

15. The modified non-human animal according to claim 12, which displays phenotypes and / or symptoms of a metabolic disease.

16. Use of the non-human animal according to any one of claims 12 to 15 in screening drugs for alleviating, preventing and / or treating metabolic diseases and / or symptoms.

17. The use according to claim 16, wherein the medicament is used for alleviating, preventing and / or treating diabetes and / or its complications.

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