Application of reagent for knocking down or inhibiting ANT1 in preparation of medicine for preventing and / or treating heterotopic ossification

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of a reagent for knocking down or inhibiting ANT1 to preparation of a medicine for preventing and / or treating heterotopic ossification. The application of the reagent for knocking down or inhibiting ANT1 in preparation of the medicine for preventing and / or treating heterotopic ossification is found for the first time. After ANT1 is inhibited, heterotopic ossification formed after soft tissue injury can be remarkably reduced; infiltration of osteoblasts, chondrocytes and inflammatory cells in injured tendons is reduced, formation of marrow cavities and blood vessels is reduced, and formation of bone tissues is reduced; the expression of osteogenesis related markers OCN, RUNX2 and ALP in the injured tendon is obviously inhibited; and ectopic ossification can be effectively prevented and / or treated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of an agent for knocking down or inhibiting ANT1 in the preparation of a drug for preventing and / or treating heterotopic ossification. Background Art

[0002] Heterotopic ossification (HO) refers to the formation of pathological bone tissue in non-skeletal tissue (including muscle, tendon or other soft tissue), which is mainly secondary to trauma, burns, nerve damage and joint replacement surgery, and is a serious complication in clinical practice. Its basic pathological changes are the formation of mature lamellar bone, cancellous bone, medullary cavity, nutrient vessels in fibrous connective tissue, with a small amount of hematopoietic function and muscle fiber membranes and other structures similar to normal bone. Its clinical symptoms are mainly inflammatory manifestations around soft tissues, namely swelling, pain, fever, skin erythema, and reduced range of motion of the affected area. Due to the high incidence of heterotopic ossification and the complexity of its pathogenesis, its prevention and treatment have always been valued by people, and the key to treatment is to relieve the patient's pain and improve motor function. However, the pathogenesis of heterotopic ossification is still unclear, and the most effective prevention plan, the choice of surgical timing, the necessity of physical therapy, and the clinical application of gene targeted therapy are still unclear.

[0003] At present, the treatment of heterotopic ossification mainly relies on surgical removal of heterotopic bone tissue, but this will inevitably cause additional trauma, which can easily lead to the recurrence of heterotopic ossification. At present, the most effective drug recognized for preventing the formation of heterotopic ossification is non-steroidal anti-inflammatory drugs, which can relieve pain and prevent the progression of heterotopic ossification by inhibiting cyclooxygenase to reduce the synthesis of inflammatory mediators prostaglandins, inhibit the differentiation of progenitor cells in heterotopic ossification into osteoblasts, and prevent the progression of heterotopic ossification. However, it has certain side effects, such as causing gastrointestinal damage and symptoms of the nervous system, urinary system, and cardiovascular system. In addition, radiotherapy, physical therapy, and molecular biological mechanism intervention also have certain therapeutic effects on heterotopic ossification, but these methods have different effects and have certain side effects. At present, there is still a lack of an effective means of targeted prevention and treatment of heterotopic ossification.

[0004] Adenine nucleotide translocase 1 (ANT1) is encoded by the SLC25A4 gene and is located in the inner membrane of mitochondria. It is responsible for the exchange of ADP / ATP in mitochondria and the regulation of mitochondrial function. The Gene ID in NCBI is 11739, also known as SLC25A4. Whether ANT1 plays a role in the formation of heterotopic ossification has not been reported. In recent years, adeno-associated virus (AAV) has become the preferred delivery vector for gene therapy due to its low immunogenicity, high safety, strong tissue-specific affinity and efficient gene delivery ability. Different serotypes of AAV recognize and bind to different cell surface receptors, which can achieve tissue-specific gene delivery and sustained and stable expression in vivo. It is widely used in tissues such as the heart, liver, eyes, nervous system, and motor system. However, in the field of heterotopic ossification research, there are few reports on the therapeutic application of AAV carrying related genes.

[0005] In view of this, the present invention provides an application of an agent for knocking down or inhibiting ANT1 in the preparation of a drug for preventing and / or treating heterotopic ossification, provides a new medical use of ANT1, solves the problem that the prior art is unclear about the exact role of ANT1 in heterotopic ossification and lacks corresponding drugs for treating heterotopic ossification, and opens up a new path for the research on gene therapy for heterotopic ossification. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes for the first time a solution for treating heterotopic ossification by knocking down or knocking out ANT1, which specifically includes the following aspects.

[0007] The first aspect of the present invention aims to provide use of an ANT1 inhibitor in the preparation of a drug for treating heterotopic ossification.

[0008] The second aspect of the present invention aims to provide a shRNA for inhibiting the expression of ANT1.

[0009] The third aspect of the present invention aims to provide a recombinant vector for inhibiting the expression of ANT1.

[0010] The fourth aspect of the present invention aims to provide a virus that inhibits the expression of ANT1.

[0011] The fifth aspect of the present invention aims to provide a medicine.

[0012] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is: The first aspect of the present invention provides a use of an ANT1 inhibitor in the preparation of a drug for treating heterotopic ossification.

[0013] The ANT1 inhibitor includes at least one of 1) and 2): 1) Substances that inhibit the ANT1 gene at the genetic level; 2) Substances that inhibit ANT1 protein at the protein level.

[0014] In some embodiments of the present invention, specifically, the ANT1 inhibitor includes at least one of a substance that reduces the content or expression level of ANT1 gene or protein, a substance that reduces the activity of ANT1 gene or protein, and a substance that promotes the degradation of ANT1 gene or protein.

[0015] In some embodiments of the present invention, the amino acid sequence of the ANT1 protein is as shown in a1) to a4): a1) a protein with an amino acid sequence as shown in SEQ ID NO: 5; a2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 5; a3) an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 5; a4) A derivative related to any one of the proteins described in a1), a2) and a3).

[0016] Preferably, common substitutions in proteins with the same function in a2) are considered conservative substitutions, such as substitutions among aliphatic amino acids Ala, Val, Leu and Ile, interchange of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe and Tyr.

[0017] Preferably, a3) means that the given amino acid sequence shares at least 85% identity with the reference sequence, for example, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. Alternatively, it means that the given amino acid sequence differs from the reference sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids. For polypeptides, such differences are preferably amino acid substitutions or deletions. The difference in identity here can come from conservative substitutions of amino acids, or from the introduction of tag proteins, or the introduction of nuclear import sequences, or the introduction and modification of signal peptide sequences. For example, adding His, GFP, HA or other tags to the C-terminus of SEQ ID NO: 4 results in a change in identity, or adding a signal peptide to the N-terminus of SEQ ID NO: 4, or adding a conventional nuclear import sequence to the N-terminus, thereby resulting in a change in identity.

[0018] Preferably, the protein-related derivatives described in a4) include ANT1 fusion proteins connected to tag proteins, pharmaceutically acceptable salts of ANT1 proteins, and pharmaceutically acceptable chemical modifications of ANT1 proteins.

[0019] Preferably, the tag protein includes but is not limited to at least one of His, Flag, Myc, GST, GFP, and HA.

[0020] In some embodiments of the present invention, the nucleic acid sequence encoding the ANT1 protein comprises: The nucleotide sequence shown in SEQ ID NO: 4; or The nucleotide sequence shown in SEQ ID NO: 4 is substituted, deleted and / or added with one or several nucleotides to encode a nucleotide sequence of the same active protein.

[0021] In some embodiments of the present invention, the substance that reduces the content or expression level of ANT1 gene or protein includes a small molecule inhibitor targeting ANT1 protein, or a nucleotide that reduces the content or expression level of ANT1 protein.

[0022] In some embodiments of the present invention, the nucleotides that reduce the content or expression level of ANT1 protein include at least one of a small guide RNA (sgRNA) that inhibits ANT1 expression, a short hairpin RNA (shRNA) that inhibits ANT1 expression, a Knock Out system for knocking out genes, dsRNA that inhibits ANT1 expression, dsRNA microRNA that inhibits ANT1 expression, and dsRNA siRNA that inhibits ANT1 expression.

[0023] In some embodiments of the present invention, the interference fragment sequence of the shRNA that inhibits ANT1 expression is shown in SEQ ID NO: 1.

[0024] In some embodiments of the present invention, the shRNA for inhibiting ANT1 expression further comprises other structures, such as a stem-loop structure. Specifically, the nucleotide sequence of the shRNA for inhibiting ANT1 expression is shown in SEQ ID NO:2.

[0025] The sgRNA can be applied to conventional CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology in the art to achieve inhibition of ANT1. The shRNA can be applied to conventional shRNA-mediated gene editing technology in the art to achieve inhibition of ANT1.

[0026] Preferably, the Knock Out system includes at least one of complete gene knockout and conditional gene knockout.

[0027] Preferably, the Knock Out system includes at least one of the Cre / LoxP system, the Gin / Gix system, the FLP / FRT system, the R / RS system, and the CRISPR-Cas9 system.

[0028] The editing of the ANT1 gene using the above-mentioned nucleotides (including but not limited to sgRNA, shRNA, siRNA, microRNA, dsRNA, Knock Out system) that reduce the content or expression level of ANT1 can be performed in vitro or in vivo. The above-mentioned nucleotides that reduce the content or expression level of ANT1 and the supporting related components can be delivered in vivo using a delivery system known in the prior art to achieve in vivo editing of the ANT1 gene in target cells, thereby inhibiting the expression of ANT1. Relevant delivery systems include but are not limited to liposome delivery systems, adenovirus delivery systems, adeno-associated virus delivery systems, lentivirus delivery systems, and nanoemulsion delivery systems.

[0029] The present invention uses an adeno-associated virus delivery system as an example for experiments, specifically an AAV9 delivery system, and the selection of the delivery system does not constitute a limitation of the present invention.

[0030] In a second aspect, the present invention provides a shRNA for inhibiting the expression of ANT1, wherein the interference sequence of the shRNA is shown in SEQ ID NO:1.

[0031] In some embodiments of the present invention, the nucleotide sequence of the shRNA that inhibits ANT1 expression is shown in SEQ ID NO:2.

[0032] The third aspect of the present invention provides a recombinant vector for inhibiting ANT1 expression, wherein the recombinant vector comprises the shRNA described in the second aspect of the present invention.

[0033] In some embodiments of the present invention, the recombinant vector is an adeno-associated virus vector; the adeno-associated virus vector can be applicable to various shuttle systems such as AdEasy, AdMAX and GateWay.

[0034] The present invention uses a plasmid with a vector structure of pAAV-U6-shSLC25A4-CMV-EGFP as an example for experimental testing, which does not mean that only the recombinant plasmid with this structure can achieve the expected technical effect. Those skilled in the art should know that other conventional AAV vector systems can be used with the shRNA described in the second aspect of the present invention to achieve the expected technical effect.

[0035] The fourth aspect of the present invention provides a virus for inhibiting the expression of ANT1, wherein the virus comprises the recombinant vector described in the third aspect of the present invention.

[0036] In some embodiments of the present invention, the virus is an adeno-associated virus, and the serotype of the adeno-associated virus is AAV9. Other adeno-associated virus types commonly found in the art can also achieve the technical effects of the present invention.

[0037] The fifth aspect of the present invention provides a drug, comprising the shRNA described in the second aspect of the present invention, the recombinant vector described in the third aspect of the present invention, or the virus described in the fourth aspect of the present invention.

[0038] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient. The excipient is generally recognized for this purpose and as an inactive ingredient of the medicament. A compilation of pharmaceutically acceptable excipients can be found in Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and PJ Weller; published by American Pharmaceutical Association, Washington and The Pharmaceutical Gess, London, 1994); Pharmacopoeia of the People's Republic of China - Catalogue of Pharmaceutical Excipients and other reference books.

[0039] Preferably, the drug is administered by one or more methods selected from the following groups: oral, injection, gold-coated gene gun bombardment, reproduction-defective bacteria carrying plasmid DNA, replication-defective adenovirus carrying target DNA or target gene encoded protein, electroporation, intravenous, pulmonary, mucosal, nasal, intraperitoneal, intracranial, intratumoral, sublingual, buccal, and transdermal administration.

[0040] Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsule preparations, lyophilized powder injection preparations, injections, suppositories, sprays, powders, soft capsules, and sustained-release preparations.

[0041] The beneficial effects of the present invention are: 1) The present invention discovers for the first time that intervening in ANT1 expression can significantly reduce heterotopic ossification formed after soft tissue injury; reduce the infiltration of osteoblasts, chondrocytes, and inflammatory cells in damaged tendons, reduce the formation of bone marrow cavities and blood vessels, and reduce bone tissue formation; significantly inhibit the expression of osteogenic-related markers OCN, RUNX2, and ALP in tendons after injury; and can effectively prevent and / or treat heterotopic ossification.

[0042] 2) The present invention provides an application of knocking down or inhibiting ANT1 in the preparation of a drug for preventing and / or treating heterotopic ossification, provides a new medical use of ANT1 inhibitors, solves the problem that the prior art is unclear about the exact role of ANT1 in heterotopic ossification and lacks corresponding drugs for treating heterotopic ossification, and provides a new target for the clinical development of drugs for preventing and / or treating heterotopic ossification.

[0043] 3) The present invention discovered that knocking down or inhibiting ANT1 can be used to treat heterotopic ossification, and provided shSLC25A4 and its nucleotide sequence for knocking down ANT1 gene expression, as well as an adeno-associated virus vector AAV9-shSLC25A4, which provides new ideas and strategies for preventing and / or treating post-traumatic soft tissue heterotopic ossification and has broad clinical application prospects.

[0044] 4) The present invention provides a reference for gene therapy research on other locomotor system diseases and can promote the development of the gene therapy field. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the adeno-associated virus vector pAAV-U6-shSLC25A4-CMV-EGFP vector map, and the vector name is AAV-0403.

[0046] Figure 2 It is the result of agarose gel electrophoresis of AAV target plasmid, among which, lane M is DNA Ladder map, lane 1 is AAV-0403 restriction map, and lane 2 is AAV-0403 plasmid map.

[0047] Figure 3 It is the 3D reconstruction image and parameter statistical analysis of micro-CT scanning.

[0048] Figure 4 The results of H&E staining (scale bar: 100 μm).

[0049] Figure 5 It is the result of qRT-PCR detection of osteogenesis-related genes. DETAILED DESCRIPTION

[0050] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0051] During the preliminary screening process, the applicant discovered that the ANT1 gene was associated with heterotopic ossification, and then verified whether it could be used as a relevant target for the treatment of heterotopic ossification.

[0052] The relevant experimental process, methods and results are as follows.

[0053] Example 1 Construction of AAV9-shSLC25A4 adeno-associated virus vector Adeno-associated virus vector construction, AAV virus packaging, and AAV control were commissioned to Beijing Qingke Biotechnology Co., Ltd. The adeno-associated virus vector serotype is AAV9.

[0054] The shRNA interference sequence fragment of ANT1 gene (SLC25A4) is 5'-GCACAUUAUCGUGAGCUGGAU-3' (SEQ ID NO: 1).

[0055] The nucleotide sequence of shSLC25A4 is 5′-GCACATTATCGTGAGCTGGATttcaagagaATCCAGCTCACGATAATGTGCttttt-3′ (SEQ ID NO: 2).

[0056] The preparation method of AAV9-shSLC25A4 is as follows: the shRNA that knocks down the expression of the SLC25A4 gene is recombined into AAV9 to obtain the recombinant adeno-associated virus AAV9-shSLC25A4. The AAV9-shSLC25A4 vector structure is pAAV-U6-shSLC25A4-CMV-EGFP. The vector map is as follows Figure 1 shown.

[0057] The sequence of shNC is 5'-GCGTGATCTTCACCGACAAGA-3' (SEQ ID NO: 3). The AAV9-shNC vector structure is pAAV-CMV-EGFP. The adeno-associated virus construction process was carried out by Beijing Qingke Biotechnology Co., Ltd., and the constructed AAV9-shSLC25A4 and AAV9-shNC were provided by Beijing Qingke Biotechnology Co., Ltd., with titers of 1×10 12 vgs / mL.

[0058]

[0059] The protein sequence of SLC25A4 / ANT1 is: MGDQALSFLKDFLAGGIAAAVSKTAVAPIERVK LLLQVQHASKQISAEKQYKGIIDCVVRIPKEQGFLSFWRGNLANVIRYFPTQALNFAFKDKYKQIFLGGVDRHKQFWRYFAGNLASGGAAGATSLCFVYPLDFARTRLAADVGKGSSQREFNGLGDCLTKIFKS DGLKGLYQGFSVSVQGIIIYRAAYFGVYDTAKGLMLPPKNVHIIVSWMIAQSVTAVAGLVSYPFDTVRRRMMMQSGRKGADIMYTGTLDCWRKIAKDEGANAFFKGAWSNVLRGMGGAFVLVLYDEIKKYV (SEQ ID NO: 5).

[0060] Example 2 Mouse Animal Experiment Model 1) Establishment of mouse heterotopic ossification model Seven-week-old female C57BL / 6 mice were selected for Achilles tendon tenotomy to induce heterotopic ossification: After successful anesthesia with tribromoethanol injection before surgery, the mice were placed supine on the operating table, the skin of the right heel was prepared and routinely disinfected, and then a skin incision of about 5 mm parallel to the long axis of the lower limb was made in the skin of the right heel to expose the fascia and Achilles tendon, and a complete transverse incision was made at the midpoint of the Achilles tendon, and the skin was sutured. Achilles tendon samples were collected 10 weeks later.

[0061] 2) Adeno-associated virus injection into mouse Achilles tendon Thirty-two 7-week-old female C57BL / 6 mice were randomly divided into two groups. Heterotopic ossification was induced by Achilles tendon transection. On the first day after surgery, 6 μL of the constructed virus was locally injected into the Achilles tendon rupture site using a microsyringe. Samples were collected for further testing 10 weeks later. The specific protocol is shown in Table 1.

[0062] Table 1 Experimental animal groups

[0063] 3) Micro-CT detection of mouse Achilles tendon Ten weeks after modeling, 10 mice were randomly selected from the AAV9-shNC group and the AAV9-shSLC25A4 group, and the right lower limbs of the mice were removed after being killed. The skin was removed, rinsed with PBS, and fixed with 4% paraformaldehyde solution for 24 hours and stored in a PBS test tube. The right lower limbs were placed parallel and fixed in the detection chamber of the micro-CT scanner (Skyscan 1176). After the scan, the size of heterotopic ossification was analyzed using CTAn software.

[0064] The experimental results are as follows Figure 3 As shown, AAV9-shSLC25A4 significantly reduced the volume of ectopic bone formed after soft tissue injury (*: p<0.05, with significant statistical difference).

[0065] 4) H&E staining of mouse Achilles tendon The mouse Achilles tendon samples after micro-CT examination were routinely decalcified, dehydrated, transparent, paraffin-embedded, and sliced. Appropriate slices were selected, routinely dewaxed, rehydrated, and then stained with hematoxylin and eosin (H&E) according to standard methods, that is, staining with hematoxylin staining solution for 5-7 minutes, rinsing with running water, differentiation with 0.2% hydrochloric acid ethanol for 2 seconds, rinsing with running water, staining with eosin staining solution for 30 seconds, dehydration, transparentization, and neutral resin sealing. Observe under a microscope and collect image analysis.

[0066] The experimental results are as follows Figure 4 As shown, AAV9-shSLC25A4 reduced the infiltration of osteoblasts, chondrocytes, and inflammatory cells in the tendon, reduced the formation of the bone marrow cavity and blood vessels, and reduced bone tissue formation.

[0067] 5) qRT-PCR detection of mouse Achilles tendon Ten weeks after modeling, 6 mice were randomly selected from the AAV9-shNC group and the AAV9-shSLC25A4 group, and the right Achilles tendons of the mice were taken after being killed. After being fully ground, the total RNA was extracted using the Trizol method. The Achilles tendon specimen was placed in a 1.5 ml RNase-free EP tube, and then 200 μL Trizol was added. The tissue was quickly and fully ground using a handheld electric grinder on ice, and then Trizol was added to a total volume of 1 ml. The Achilles tendon tissue was fully lysed by repeatedly blowing with a pipette gun, and then placed on ice for 5 min. Then 200 μL chloroform was added, and after vigorous shaking for about 15 seconds, it was placed on ice for 5 minutes to allow natural stratification. Low-temperature high-speed centrifugation (4°C, 12000g) was performed for 15 minutes. 400-500 μL of the supernatant was aspirated into a new 1.5 ml EP tube, and an equal volume of isopropanol was added. After being gently and fully mixed for about 10 seconds, it was placed on ice for 5 minutes. Centrifuge at low temperature and high speed (4℃, 12000g) for 15min, and remove the supernatant. Add 1ml75% ethanol and rotate the EP tube to rinse the precipitate. Centrifuge at low temperature and high speed (4℃, 12000g) for 15min, and remove 75% ethanol. Open the EP tube cap, dry the precipitate on ice in the clean bench, and then dissolve the RNA precipitate of each sample in an appropriate amount of RNase-free water. Measure the RNA concentration using a spectrophotometer and adjust the concentration of the RNA sample to 100ng / μL with RNase-free water.

[0068] Use the AG reverse transcription kit and perform reverse transcription according to the standard method in the manual to obtain cDNA. According to the 20μL system, add 4μL of RT Master Mix, 4μL of Total RNA, and 12μL of RNase-free water in sequence, and perform reverse transcription on the BioRed PCR amplification instrument. The reaction conditions are: 37℃: 15min, 85℃: 5s, 4℃: ∞, and obtain cDNA.

[0069] The AG qPCR kit was used to perform the reaction according to the standard method in the instruction manual. According to the 10 μL PCR reaction system, 5 μL SYBR Premix EX TaqⅡ, 3 μL RNAase Free water, 1 μL cDNA, 0.2 μL ROX, and 0.4 μL of upstream and downstream primers were added to the eight-tube strip in sequence. The mixture was centrifuged and the reaction was performed on the ABI PCR instrument to detect the expression of osteogenesis-related genes in the samples. The primer sequences used are shown in Table 2.

[0070] Table 2 Primer sequences of osteogenesis-related genes and internal reference genes

[0071] The experimental results are as follows Figure 5As shown: AAV9-shSLC25A4 significantly inhibited the expression of osteogenesis-related genes (OCN, RUNX2, ALP) in the Achilles tendon after injury (*: p<0.05, with significant statistical difference).

[0072] In summary, the present invention has discovered for the first time the use of reagents that knock down or inhibit ANT1 in the preparation of drugs for preventing and / or treating heterotopic ossification. Inhibiting ANT1 can significantly reduce heterotopic ossification formed after soft tissue injury; reduce the infiltration of osteoblasts, chondrocytes, and inflammatory cells in damaged tendons, reduce the formation of bone marrow cavities and blood vessels, and reduce bone tissue formation; significantly inhibit the expression of osteogenic-related markers OCN, RUNX2, and ALP in tendons after injury; and can effectively prevent and / or treat heterotopic ossification. It provides new ideas and strategies for preventing and / or treating heterotopic ossification of soft tissues after trauma, and has broad clinical application prospects.

Claims

1. Application of ANT1 inhibitors in the preparation of drugs for the treatment of heterotopic ossification.

2. The use according to claim 1, characterized in that: The ANT1 inhibitor includes at least one of 1) and 2): 1) Substances that inhibit the ANT1 gene at the genetic level; 2) Substances that inhibit ANT1 protein at the protein level. Preferably, the ANT1 inhibitor includes at least one of a substance that reduces the content or expression level of ANT1 gene or protein, a substance that reduces the activity of ANT1 gene or protein, and a substance that promotes the degradation of ANT1 gene or protein.

3. The use according to claim 2, characterized in that: The amino acid sequence of ANT1 is shown in a1) to a4): a1) a protein with an amino acid sequence as shown in SEQ ID NO: 5; a2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 5; a3) an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 5; a4) A derivative related to any one of the proteins described in a1), a2) and a3).

4. The use according to claim 2, characterized in that: The substance that reduces the content or expression level of ANT1 gene or protein includes a small molecule inhibitor targeting ANT1 gene or protein, or a nucleotide that reduces the content or expression level of ANT1 gene or protein; Preferably, the nucleotides that reduce the content or expression level of ANT1 protein include at least one of a small guide RNA that inhibits ANT1 protein expression, a short hairpin RNA that inhibits ANT1 protein expression, a Knock Out system for knocking out genes, a dsRNA that inhibits ANT1 expression, a dsRNA microRNA that inhibits ANT1 expression, and a dsRNA siRNA that inhibits ANT1 expression.

5. The use according to claim 4, characterized in that: The interference sequence of the shRNA for inhibiting ANT1 expression is shown in SEQ ID NO:

1.

6. A shRNA for inhibiting ANT1 expression, characterized in that: The interference sequence of the shRNA is shown in SEQ ID NO: 1; Preferably, the nucleotide sequence of the shRNA is as shown in SEQ ID NO:

2.

7. A recombinant vector for inhibiting ANT1 expression, characterized in that: The recombinant vector comprises the shRNA according to claim 6; Preferably, the recombinant vector comprises an AAV vector.

8. A virus for inhibiting ANT1 expression, characterized in that: The virus comprises the recombinant vector according to claim 7.

9. A drug comprising the shRNA according to claim 6, the recombinant vector according to claim 7, or the virus according to claim 8.

10. The drug according to claim 9, characterized in that: The drug also includes pharmaceutically acceptable excipients.

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