Use of an agent for knocking down or inhibiting ANT1 in the preparation of a drug for preventing and / or treating heterotopic ossification

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the use of reagents that knock down or inhibit ANT1 in the preparation of drugs for preventing and / or treating heterotopic ossification. The present invention is the first to discover 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 bone marrow cavity and blood vessel formation, and reduce bone tissue formation; significantly inhibit the expression of osteoblast-related markers OCN, RUNX2, and ALP in injured tendons; and can effectively prevent and / or treat heterotopic ossification.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly 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 pathological formation of bone tissue within non-skeletal tissue (including muscle, tendon, or other soft tissue). It primarily occurs secondary to trauma, burns, nerve damage, and joint replacement surgery and is a serious clinical complication. Its fundamental pathological changes involve the formation of mature lamellar bone, cancellous bone, a medullary cavity, and vasa vasorum within fibrous connective tissue, with minimal hematopoiesis and a myofibril membrane, resembling normal bone. Clinical symptoms primarily include inflammation of the surrounding soft tissues, including swelling, pain, fever, skin erythema, and decreased range of motion. Due to the high prevalence and complex pathogenesis of HO, its prevention and treatment have long been of great concern, with pain relief and improved motor function being key. However, the pathogenesis of HO remains unclear, and the most effective preventive measures, the timing of surgery, the necessity of physical therapy, and the clinical application of gene-targeted therapy remain uncertain.

[0003] Currently, the treatment of heterotopic ossification primarily relies on surgical resection of the heterotopic bone tissue, but this inevitably causes additional trauma, which can easily lead to recurrence of heterotopic ossification. Currently, the most recognized effective medication for preventing heterotopic ossification is nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs inhibit cyclooxygenase, reduce the synthesis of the inflammatory mediator prostaglandins, and inhibit the differentiation of progenitor cells in heterotopic ossification into osteoblasts, thereby relieving pain and preventing the progression of heterotopic ossification. However, these drugs have certain side effects, such as gastrointestinal damage and symptoms of the nervous system, urinary system, and cardiovascular system. In addition, radiotherapy, physical therapy, and molecular biological mechanism interventions also have some therapeutic effects on heterotopic ossification, but these methods have varying effectiveness and are associated with certain side effects. Currently, there is a lack of an effective targeted means of preventing and treating heterotopic ossification.

[0004] Adenine nucleotide translocase 1 (ANT1), encoded by the SLC25A4 gene, is located in the inner mitochondrial membrane and is responsible for intramitochondrial ADP / ATP exchange and regulation of mitochondrial function. Its NCBI Gene ID is 11739, and it is also known as SLC25A4. Whether ANT1 plays a role in 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 profile, strong tissue-specific affinity, and efficient gene delivery. Different AAV serotypes recognize and bind to different cell surface receptors, enabling tissue-specific gene delivery and sustained, stable expression in vivo. It has been widely used in tissues such as the heart, liver, eye, nervous system, and musculoskeletal system. However, few studies have reported on the therapeutic application of AAV-carrying genes for heterotopic ossification.

[0005] In view of this, the present invention provides a use of an agent for knocking down or inhibiting ANT1 in the preparation of a drug for preventing and / or treating heterotopic ossification, providing a new medical use of ANT1, solving the problem in the prior art that the exact role of ANT1 in heterotopic ossification is unclear and there is a lack of corresponding drugs to treat heterotopic ossification, and opening 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 that inhibits the expression of ANT1.

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

[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:

[0013] A first aspect of the present invention provides use of an ANT1 inhibitor in the preparation of a medicament for treating heterotopic ossification.

[0014] The ANT1 inhibitor includes at least one of 1) and 2) :

[0015] 1) Substances that inhibit the ANT1 gene at the genetic level;

[0016] 2) Substances that inhibit ANT1 protein at the protein level.

[0017] 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.

[0018] In some embodiments of the present invention, the amino acid sequence of the ANT1 protein is as shown in a1) to a4):

[0019] a1) a protein with the amino acid sequence shown in SEQ ID NO: 5;

[0020] a2) a protein having the same function as the amino acid sequence of SEQ ID NO: 5 obtained by substituting and / or deleting and / or adding one or more amino acid residues;

[0021] a3) an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 5;

[0022] a4) Derivatives related to the proteins described in any one of a1), a2), and a3).

[0023] Preferably, common substitutions in proteins with the same function in a2) are considered conservative substitutions, such as substitutions among the 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.

[0024] 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%, or 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, or 10 amino acids. For polypeptides, such differences preferably involve amino acid substitutions or deletions. Differences in identity can arise from conservative amino acid substitutions, the introduction of a tag protein, the introduction of a nuclear import sequence, or the introduction or modification of a signal peptide sequence. For example, the addition of a tag such as His, GFP, or HA to the C-terminus of SEQ ID NO:4 can result in a change in identity, or the addition of a signal peptide or a conventional nuclear import sequence to the N-terminus of SEQ ID NO:4 can result in a change in identity.

[0025] Preferably, the protein-related derivatives described in a4) include ANT1 fusion proteins linked to tag proteins, pharmaceutically acceptable salts of ANT1 proteins, and pharmaceutically acceptable chemical modifications of ANT1 proteins.

[0026] Preferably, the tag protein includes but is not limited to at least one of His, Flag, Myc, GST, GFP, and HA.

[0027] In some embodiments of the present invention, the nucleic acid sequence encoding the ANT1 protein comprises:

[0028] The nucleotide sequence shown in SEQ ID NO: 4; or

[0029] The nucleotide sequence shown in SEQ ID NO: 4 is substituted, deleted and / or added with one or more nucleotides to encode a nucleotide sequence of the same active protein.

[0030] 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.

[0031] In some embodiments of the present invention, the nucleotides that reduce the content or expression level of ANT1 protein include at least one of small guide RNA (sgRNA) that inhibits ANT1 expression, 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.

[0032] In some embodiments of the present invention, the interference fragment sequence of the shRNA that inhibits ANT1 expression is shown as SEQ ID NO: 1.

[0033] 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.

[0034] The sgRNA can be applied to conventional CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing technology in the art to inhibit ANT1. The shRNA can be applied to conventional shRNA-mediated gene editing technology in the art to inhibit ANT1.

[0035] Preferably, the Knock Out system includes at least one of complete gene knockout and conditional gene knockout.

[0036] 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.

[0037] Editing the ANT1 gene using the aforementioned nucleotides that reduce ANT1 content or expression (including but not limited to sgRNA, shRNA, siRNA, microRNA, dsRNA, and knock-out systems) can be performed in vitro or in vivo. These nucleotides and associated components can be delivered in vivo using known delivery systems to achieve in vivo editing of the ANT1 gene in target cells, thereby inhibiting ANT1 expression. These delivery systems include but are not limited to liposome delivery systems, adenoviral delivery systems, adeno-associated viral delivery systems, lentiviral delivery systems, and nanoemulsion delivery systems.

[0038] The present invention conducts experiments using an adeno-associated virus delivery system, specifically an AAV9 delivery system. The choice of this delivery system does not constitute a limitation of the present invention.

[0039] In a second aspect, the present invention provides a shRNA for inhibiting ANT1 expression, wherein the interference sequence of the shRNA is shown in SEQ ID NO: 1.

[0040] In some embodiments of the present invention, the nucleotide sequence of the shRNA that inhibits ANT1 expression is shown in SEQ ID NO: 2.

[0041] 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.

[0042] In some embodiments of the present invention, the recombinant vector is an adeno-associated viral vector; the adeno-associated viral vector can be applicable to various shuttle systems: AdEasy, AdMAX and GateWay.

[0043] The present invention uses a plasmid with a vector structure of pAAV-U6-shSLC25A4-CMV-EGFP as an example for experimental testing, but this does not mean that only recombinant plasmids with this structure can achieve the expected technical effects. Those skilled in the art will be aware 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 effects.

[0044] The fourth aspect of the present invention provides a virus for inhibiting ANT1 expression, wherein the virus comprises the recombinant vector according to the third aspect of the present invention.

[0045] 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.

[0046] 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.

[0047] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient. Such excipients are generally recognized for this purpose and serve as inactive ingredients in medicaments. A compilation of pharmaceutically acceptable excipients can be found in reference books such as the Handbook of Pharmaceutical Excipients (2nd edition, edited by A. Wade and P.J. Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Gess, London, 1994) and the Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients.

[0048] Preferably, the drug is administered by one or more methods selected from the following groups: oral administration, injection, gold-coated gene gun bombardment, replication-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.

[0049] Preferably, the dosage form of the drug comprises at least one of capsules, tablets, microcapsules, freeze-dried powder injections, injections, suppositories, sprays, powders, soft capsules, and sustained-release preparations.

[0050] The beneficial effects of the present invention are:

[0051] 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 injured tendons, reduce the formation of bone marrow cavities and blood vessels, and reduce bone tissue formation; significantly inhibit the expression of osteoblast-related markers OCN, RUNX2, and ALP in tendons after injury; and can effectively prevent and / or treat heterotopic ossification.

[0052] 2) The present invention provides the use of knocking down or inhibiting ANT1 in the preparation of drugs for preventing and / or treating heterotopic ossification, providing new medical uses of ANT1 inhibitors, solving the problems in the prior art of unclear exact role of ANT1 in heterotopic ossification and lack of corresponding drugs for treating heterotopic ossification, and providing new targets for the clinical development of drugs for preventing and / or treating heterotopic ossification.

[0053] 3) The present invention discovered that knocking down or inhibiting ANT1 can be used to treat heterotopic ossification, and provides 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.

[0054] 4) This invention provides a reference for gene therapy research on other musculoskeletal diseases and can promote the development of the gene therapy field. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is the map of the adeno-associated virus vector pAAV-U6-shSLC25A4-CMV-EGFP vector, and the vector name is AAV-0403.

[0056] Figure 2 This is the result of agarose gel electrophoresis of the AAV target plasmid, where lane M is the DNA ladder map, lane 1 is the AAV-0403 enzyme digestion map, and lane 2 is the AAV-0403 plasmid map.

[0057] Figure 3 It is the 3D reconstruction image and parameter statistical analysis of micro-CT scanning.

[0058] Figure 4 The results of H&E staining are shown (scale bar: 100 μm).

[0059] Figure 5 This is the result of qRT-PCR detection of osteogenesis-related genes. DETAILED DESCRIPTION

[0060] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described 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.

[0061] 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.

[0062] The relevant experimental process, methods and results are as follows.

[0063] Example 1 Construction of AAV9-shSLC25A4 adeno-associated virus vector

[0064] AAV vector construction, AAV virus packaging, and AAV control were commissioned to Beijing Qingke Biotechnology Co., Ltd. The adeno-associated virus vector serotype was AAV9.

[0065] The shRNA interference sequence fragment of the ANT1 gene (SLC25A4) is 5'-GCACAUUAUCGUGAGCUGGAU-3' (SEQ ID NO: 1).

[0066] The nucleotide sequence of shSLC25A4 is 5′-GCACATTATCGTGAGCTGGATttcaagagaATCCAGCTCACGATAATGTGCttttt-3′ (SEQ ID NO: 2).

[0067] 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 1shown.

[0068] 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. The constructed AAV9-shSLC25A4 and AAV9-shNC were provided by Beijing Qingke Biotechnology Co., Ltd., and the titer of each was 1×10 12 vgs / mL.

[0069]

[0070] The protein sequence of SLC25A4 / ANT1 is: MGDQALSFLKDFLAGGIAAAVSKTAVAPIERVK LLLQVQHASKQISAEKQYKGIIDCVVRIPKEQGFLSFWRGNLANVIRYFPTQALNFAFKDKYKQIFLGGVDRHKQFWRYFAGNLASGGAAGATSLCFVYPLDFARTRLAADVGKGSSQREFNGLGDCLTKIFKS DGLKGLYQGFSVSVQGIIIYRAAYFGVYDTAKGLMLPPKNVHIIVSWMIAQSVTAVAGLVSYPFDTVRRRMMMQSGRKGADIMYTGTLDCWRKIAKDEGANAFFKGAWSNVLRGMGGAFVLVLYDEIKKYV (SEQ ID NO: 5).

[0071] Example 2 Mouse Animal Experiment Model

[0072] 1) Establishment of a mouse heterotopic ossification model

[0073] Seven-week-old female C57BL / 6 mice underwent Achilles tenotomy to induce heterotopic ossification. After successful anesthesia with an injection of tribromoethanol, the mice were placed supine on the operating table. The skin of the right heel was prepared and routinely disinfected. A 5-mm incision parallel to the long axis of the lower limb was made at the right heel to expose the fascia and Achilles tendon. A complete transverse incision was performed at the midpoint of the Achilles tendon, and the skin was sutured. Tendon samples were collected 10 weeks later.

[0074] 2) Adeno-associated virus injection into mouse Achilles tendon

[0075] Thirty-two 7-week-old female C57BL / 6 mice were randomly divided into two groups. Heterotopic ossification was induced by Achilles tenotomy. 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 10 weeks later for further testing. The specific protocol is shown in Table 1.

[0076] Table 1 Experimental animal groups

[0077]

[0078] 3) Micro-CT detection of mouse Achilles tendon

[0079] Ten weeks after model establishment, 10 mice were randomly selected from each of the AAV9-shNC and AAV9-shSLC25A4 groups. After sacrifice, the right lower limbs of the mice were removed, the skin removed, rinsed with PBS, fixed in 4% paraformaldehyde for 24 hours, and stored in PBS tubes. The right lower limbs were positioned parallel to each other and fixed in the examination chamber of a micro-CT scanner (Skyscan 1176). After scanning, the size of the heterotopic ossification was analyzed using CTAn software.

[0080] The experimental results are as follows Figure 3 As shown in the results, AAV9-shSLC25A4 significantly reduced the volume of ectopic bone formed after soft tissue injury (*: p < 0.05, statistically significant).

[0081] 4) H&E staining of mouse Achilles tendon

[0082] Mouse Achilles tendon specimens following micro-CT examination were routinely decalcified, dehydrated, cleared, paraffin-embedded, and sectioned. Appropriate sections were routinely dewaxed and rehydrated, followed by hematoxylin and eosin (H&E) staining according to standard procedures: staining with hematoxylin 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 for 30 seconds, dehydration, clearing, and mounting with neutral resin. Microscopic observation and image analysis were performed.

[0083] The experimental results are as follows Figure 4 As shown, AAV9-shSLC25A4 reduced the infiltration of osteoblasts, chondrocytes, and inflammatory cells in tendons, reduced the formation of bone marrow cavities and blood vessels, and reduced bone tissue formation.

[0084] 5) qRT-PCR detection of mouse Achilles tendon

[0085] Ten weeks after model establishment, six mice were randomly selected from each of the AAV9-shNC and AAV9-shSLC25A4 groups. The right Achilles tendons of the mice were harvested and thoroughly ground, followed by total RNA extraction using the Trizol method. The Achilles tendon specimens were placed in 1.5 ml RNase-free EP tubes, and 200 μL of Trizol was added. The tissue was quickly and thoroughly ground using a handheld electric grinder on ice. Trizol was then added to a total volume of 1 ml. The tissue was thoroughly lysed by repeated pipetting with a pipette, and the tissue was allowed to stand on ice for 5 minutes. Then, 200 μL of chloroform was added, and the mixture was vigorously shaken for approximately 15 seconds. The mixture was then allowed to stand on ice for 5 minutes to allow for natural separation. The mixture was then centrifuged at low temperature (4°C, 12,000 g) for 15 minutes. 400-500 μL of the supernatant was transferred to a new 1.5 ml EP tube, and an equal volume of isopropanol was added. The mixture was gently mixed for approximately 10 seconds and allowed to stand on ice for 5 minutes. Centrifuge at low temperature (4°C, 12,000 g) for 15 minutes and remove the supernatant. Add 1 ml of 75% ethanol and rotate the EP tube to rinse the pellet. Centrifuge at low temperature (4°C, 12,000 g) for 15 minutes and remove the 75% ethanol. Open the EP tube cap and dry the pellet on ice in a clean hood. Dissolve the RNA pellet from each sample in an appropriate amount of RNase-free water. Measure the RNA concentration using a spectrophotometer and adjust the RNA sample concentration to 100 ng / μL using RNase-free water.

[0086] cDNA was obtained by reverse transcription using the AG reverse transcription kit according to the standard protocol in the manufacturer's instructions. To a 20 μL volume, 4 μL of RT Master Mix, 4 μL of Total RNA, and 12 μL of RNase-free water were added, and reverse transcription was performed on a BioRed PCR amplifier using the following reaction conditions: 37°C for 15 minutes, 85°C for 5 seconds, and 4°C for ∞.

[0087] The AG qPCR kit was used according to the standard protocol in the manufacturer's instructions. To a 10-μL PCR reaction system, the following were added, in order, to eight tube strips: 5 μL SYBR Premix EX Taq II, 3 μL RNAse-free water, 1 μL cDNA, 0.2 μL ROX, and 0.4 μL each of upstream and downstream primers. The mixture was centrifuged and then reacted on an ABI PCR instrument to detect the expression of osteogenesis-related genes in the samples. The primer sequences used are shown in Table 2.

[0088] Table 2 Primer sequences of osteogenesis-related genes and internal reference genes

[0089]

[0090] 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, statistically significant difference).

[0091] In summary, the present invention demonstrates for the first time the use of reagents that knock down or inhibit ANT1 in the preparation of drugs for the prevention and / or treatment of heterotopic ossification. ANT1 inhibition can significantly reduce heterotopic ossification following soft tissue injury; reduce infiltration of osteoblasts, chondrocytes, and inflammatory cells in injured tendons, decrease bone marrow cavities and blood vessel formation, and reduce bone tissue formation; and significantly inhibit the expression of osteoblast-related markers OCN, RUNX2, and ALP in injured tendons, effectively preventing and / or treating heterotopic ossification. This provides new insights and strategies for the prevention and / or treatment of post-traumatic soft tissue heterotopic ossification and has broad clinical application prospects.

Claims

1. Application of ANT1 inhibitors in the preparation of drugs for the treatment of heterotopic ossification; The ANT1 inhibitor is a nucleotide that reduces the expression level of the ANT1 gene or protein; The nucleotide that reduces the expression level of ANT1 gene or protein is shRNA; The interference fragment sequence of the shRNA is shown in SEQ ID NO: 1; The heterotopic ossification is traumatic heterotopic ossification.

2. The use according to claim 1, characterized in that: The sequence of the shRNA is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that: The amino acid sequence of ANT1 is shown in a1) to a2): a1) a protein with an amino acid sequence as shown in SEQ ID NO: 5; a2) A protein having the same function as the amino acid sequence shown in SEQ ID NO: 5 obtained by substituting and / or deleting and / or adding one or more amino acid residues.

4. The use according to claim 2, characterized in that: The shRNA delivery system is the AAV9 delivery system.

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