WO2023130973A1 - 一种用于骨修复的药物组合物 - Google Patents

一种用于骨修复的药物组合物 Download PDF

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WO2023130973A1
WO2023130973A1 PCT/CN2022/140988 CN2022140988W WO2023130973A1 WO 2023130973 A1 WO2023130973 A1 WO 2023130973A1 CN 2022140988 W CN2022140988 W CN 2022140988W WO 2023130973 A1 WO2023130973 A1 WO 2023130973A1
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self
pharmaceutical composition
mrna
gene
replicating
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French (fr)
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李亘
杨太华
王蕾
崔文国
阮慧瞳
杨仁豪
徐艺冬
张尹
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上海唯可生物科技有限公司
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Publication of WO2023130973A1 publication Critical patent/WO2023130973A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

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  • the invention relates to the field of biomedicine, in particular to a pharmaceutical composition for bone repair.
  • Bone tissue defects caused by trauma and disease are common and difficult clinical problems. How to promote the repair of bone tissue defects has always been the research focus of scholars in related fields.
  • the methods for clinical treatment of full-thickness bone-cartilage defects include microcracks, cartilage scraping, joint replacement, mosaic plasty, subchondral bone drilling, and pseudoarthrosis replacement.
  • these treatment modalities have limitations such as limited donor sources, lesions at the treatment site, loosening of grafts and limited prostheses, and poor durability.
  • bone growth Factors are prone to failure, and when used, the effective time is short, and continuous injections are required to take effect.
  • tissue engineering technology for bone repair through the combination of mesenchymal stem cells and scaffold materials to induce osteogenic differentiation, but the use of mesenchymal stem cells for repair is often regulated by the local microenvironment, inflammatory cytokines such as tumor Necrosis factors and interleukin 1 often inhibit the differentiation of mesenchymal stem cells into osteoblasts, affecting bone regeneration and repair, and the proliferation of stem cells is likely to cause tumors.
  • a specific embodiment of the present invention provides a pharmaceutical composition for bone repair that overcomes the above-mentioned problems, and the specific scheme is as follows:
  • a pharmaceutical composition for bone repair comprising self-replicating mRNA, wherein said self-replicating mRNA comprises a bone repair gene.
  • the bone repair gene is BMP gene or VEGFA gene.
  • the self-replicating mRNA is BMP2 gene self-replicating mRNA, VEGFA gene self-replicating mRNA or BMP2 and VEGFA dual gene self-replicating mRNA.
  • composition further includes liposomes, and the self-replicating mRNA is dispersed in the liposomes.
  • the liposome is composed of DOTAP, HSPC, Chol and PEG-DSPE.
  • the liposome comprises a cationic penetrating peptide.
  • the composition further includes a hydrogel, on which the self-replicating mRNA and liposomes are loaded.
  • the hydrogel is methacrylic anhydride gelatin hydrogel.
  • the weight percentage of methacrylic anhydride gelatin is 3wt%-9wt%.
  • sequence of the self-replicating mRNA of the BMP2 gene is shown in SEQ ID NO.1
  • sequence of the self-replicating mRNA of the VEGFA gene is shown in SEQ ID NO.2
  • the self-replicating mRNA of the BMP2 and VEGFA double genes The sequence is shown in SEQ ID NO.3.
  • the pharmaceutical composition is an orthotopic injection preparation for bone injury.
  • composition for bone repair comprises self-replicating mRNA, wherein said self-replicating mRNA comprises bone repair gene of BMP gene or VEGFA gene, said pharmaceutical composition is conducive to maintaining Bone-repair-promoting genes are highly expressed for a long time, and a small amount of small batches of injections to the bone trauma site can be effectively repaired.
  • Fig. 1 is 3wt%, 5wt%, 7wt%, and the microscopic observation of the hydrogel sphere prepared by the GelMA of 9wt% concentration in the embodiment and the internal characterization result of tissue section;
  • Fig. 2 is liposome, short-chain BMP2 gene mRNA and liposome complex in the embodiment, the SEM observation form figure of the mixture of self-replication BMP2 gene mRNA and liposome complex;
  • Fig. 3 is the self-replicating mRNA of BMP4 gene, the self-replicating mRNA of TGFB gene, the self-replicating mRNA of BMP2 gene, the self-replicating mRNA of VEGFA gene and the in vitro cell experiment results of BMP2 and VEGFA double-gene self-replicating mRNA in the embodiment;
  • Fig. 4 is an experimental effect diagram of bone repair in implementation.
  • a specific embodiment of the present invention provides a pharmaceutical composition for bone repair, the composition includes self-replicating mRNA, wherein the self-replicating mRNA includes a bone repair gene.
  • the inventors of the present invention have found that after in situ administration of the pharmaceutical composition at the bone trauma site, the bone repair-promoting gene will be continuously and significantly expressed for a long time, and the bone damage can be effectively repaired by a small amount of injection in a small number of batches .
  • the self-replicating mRNA includes a self-replicating sequence and a bone repair gene sequence.
  • the self-replicating sequence is derived from a positive-strand ssRNA virus, such as Venezuelan horse cerebrospinal cord TC83 Venezuelan Equine Encephalitis Virus, VEEV, Sin-dbis virus, Chikungunya virus, Eastern equine encephalitis virus, Western equine encephalitis virus (Western equine encephalitis virus), Mayarovirus (Mayarovirus), Semliki forest virus (Semliki forest virus), Venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus), etc., especially Venezuelan equine encephalitis virus (TC83 Venezuelan Equine Encephalitis Virus, VEEV).
  • a positive-strand ssRNA virus such as Venezuelan horse cerebrospinal cord TC83 Venezuelan Equine Encephalitis Virus, VEEV,
  • described self-replicating mRNA comprises BMP gene or VEGFA gene
  • described self-replicating mRNA is BMP2 gene self-replicating mRNA
  • the self-replicating mRNA is the self-replicating mRNA of the VEGFA gene.
  • the self-replicating mRNA is the self-replicating mRNA of BMP2 and VEGFA.
  • the self-replicating mRNA of the embodiment especially when When the self-replicating mRNA is BMP2 and VEGFA double-gene self-replicating mRNA, it has more significant expression of genes promoting bone repair.
  • the sequence of described BMP2 gene self-replication mRNA is as shown in SEQ ID NO.1
  • the sequence of described VEGFA gene self-replication mRNA is as SEQ ID NO.2
  • the sequence of the self-replicating mRNA of the BMP2 and VEGFA double genes is shown in SEQ ID NO.3.
  • the composition in order to improve the biological activity of the self-replicating mRNA, in some specific embodiments, the composition also includes liposomes, and the self-replicating mRNA is dispersed in the liposomes, The liposome is used as the delivery carrier of the pharmaceutical composition.
  • the lipid of the liposome is a phospholipid, such as hydrogenated soybean lecithin (HSPC), lecithin, phosphatidylethanolamine, sphingomyelin, cephalin, cardiolipin, and in some specific embodiments
  • the lipids also include cationic lipids such as 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxy-3-(trimethylamino)propane ( DOTAP), N-[1-(2,3-di(tetradecyloxy))propyl]-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N-[1 -(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxyethyl-ammonium bromide (DORIE), N-[1-(2,3-diolene Oxy)propyl]-N,N,
  • the liposome is composed of DOTAP, HSPC, Chol and PEG-DSPE.
  • the liposome Plastids also contain cationic penetrating peptides (CPPs).
  • the composition in order to realize the loading and slow release of the drug, in some specific embodiments, also includes a hydrogel, and the self-replicating mRNA and liposome are loaded on the water On the gel, the hydrogel has a hydrophilic polymer chain.
  • the hydrogel is compatible with cells, such as gelatin, methacrylic anhydride gelatin, alginic acid Sodium, silk fibroin, chitosan and collagen, etc.
  • the hydrogel is methacrylic anhydride gelatin hydrogel, in order to take into account the small collapse rate of the hydrogel ball and the large The liposome adsorption ratio, in some specific embodiments, in the hydrogel, the weight percentage of methacrylic anhydride gelatin is 3wt% ⁇ 9wt%.
  • the pharmaceutical composition according to the specific embodiment of the present invention is suitable for bone tissue defects caused by various traumas or diseases, such as fractures, bone defects or bone non-union sites and the like.
  • the bone repair includes regeneration or formation of bone.
  • the pharmaceutical composition in some specific embodiments, is an in situ injection preparation for a bone injury, and when used, the pharmaceutical composition is injected in situ into the bone injury.
  • GelMA methacrylic anhydride gelatin
  • MWCO 3500 dialysis bags
  • centrifuged at 7000rpm 15 minutes to remove insoluble matter
  • the supernatant was taken to continue dialysis for 2 to 3 days.
  • the suction-filtered GelMA was divided into multiple 10cm plates, and placed in a minus 80 refrigerator for frozen storage.
  • the product GelMA was obtained by lyophilization at low temperature.
  • Breaker adjust parameter power to 20-40%, work for 3 minutes and stop for 1 second, clean the ultrasonic head, submerge the ultrasonic head over the liquid level at the bottom of the flask, and perform hydration, pay attention to observe the hydration of the lipid film at the bottom, and use a 1mL needle to extract
  • the solutions were filtered through 0.45 ⁇ m and 0.22 ⁇ m filters into 2 mL EP tubes, sealed with parafilm, and stored at 4 °C.
  • RNA plasmid templates expressing BMP4 gene, TGFB gene, BMP2 gene, VEGFA gene and BMP2 and VEGFA genes were respectively constructed.
  • the constructed plasmid template was sequenced and verified (using a next-generation sequencer for gene sequence sequencing), and the verified plasmid template was amplified using the QRT-PCR method, and 60ug of BMP4 and TGFB were respectively amplified using restriction endonucleases.
  • BMP2, VEGFA, BMP2 and VEGFA gene plasmids were linearized, and the linearized BMP4, TGFB, BMP2, VEGFA, BMP2 and VEGFA gene templates obtained by precipitation and purification were 30-50ug.
  • the self-replicating mRNA solution 500ul (concentration 1ug/ul) prepared above is continued to be dissolved in 500ul serum-free medium, 500ul liposome solution (concentration 10mg/ml) is dissolved in 500ul serum-free medium, self-replicating mRNA and The liposome mixture was co-incubated at a ratio of 1:1 for 10 min to obtain a self-replicating mRNA/liposome mixture.
  • the mixture of short-chain BMP2 gene mRNA and liposome complexed was prepared in the above manner.
  • Liposome the mixture of short-chain BMP2 gene mRNA and liposome complex, the mixture of self-replication BMP2 gene mRNA and liposome complex are carried out SEM observation, and its shape is as shown in Figure 2, as shown in Figure 2, using the described
  • the size of the mixture of the liposome and the self-replicating BMP2 gene mRNA is basically the same as that of the liposome before mixing, indicating that the liposome has an excellent loading effect on the self-replicating BMP2 gene mRNA.
  • BMSC Bone marrow mesenchymal stem cells
  • GFP mRNA was set as the control group
  • BMP4 gene self-replicating mRNA (Sa-BMP4) and TGFB gene self-replicating mRNA obtained above were loaded on the hydrogel.
  • Sa-mRNA/Lip@GelMA the hydrogel-loaded BMP2 and VEGFA double-gene self-replicating mRNA and liposome complex prepared by the hydrogel-loaded preparation method of the self-replicating mRNA and liposome complex;
  • Lip@GelMA according to the above hydrogel loading preparation method of self-replicating mRNA and liposome complex, wherein the liposome is not mixed with self-replicating mRNA, the prepared hydrogel-loaded liposome;
  • mRNA/Lip@GelMA The hydrogel-loaded short-chain mRNA and liposome complex prepared according to the above hydrogel-loaded preparation method of self-replicating mRNA and liposome complexes, in which the self-replicating mRNA was replaced by short-chain BMP2 Gene mRNA was mixed with short-chain VEGFA gene mRNA (1mol:1mol).
  • mRNA/Lip(-CPPs)@GelMA The self-replicating mRNA and liposome complexes loaded with BMP2 and VEGFA were prepared in the hydrogel-loaded self-replicating mRNA and liposome complexes above, but Wherein the liposome does not contain cationic penetrating peptides (CPPs).
  • CPPs cationic penetrating peptides
  • step 2 Take more than 150 ul of the injection sample in step 2), and perform in situ injection at the joint injury site on the rat distal femoral bone defect model in step 1), and perform the second injection after an interval of 2 weeks, and after 2 weeks and 4 weeks All points were obtained from mouse joints for tissue fixation, and microCT samples were taken for analysis. The results are shown in Figure 4.

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Abstract

本发明提供一种用于骨修复的药物组合物，所述组合物包括自复制mRNA，其中，所述自复制mRNA包含骨修复基因，所述药物组合物有利于维持骨修复基因表达，对骨损伤位置少量少批次注射进行修复。

Description

一种用于骨修复的药物组合物 技术领域
本发明涉及生物医药领域，特别涉及一种用于骨修复的药物组合物。
背景技术
创伤和疾病等原因导致的骨组织缺损是临床常见且较难处理的问题，如何促进骨组织缺损的修复一直是相关领域学者的研究重点。临床治疗骨-软骨全层缺损的方法有：微裂纹、软骨刮削、关节置换、马赛克成形术、软骨下骨钻孔和假关节置换等。然而，这些治疗方式具有供体来源有限、治疗部位的病变、移植体的松脱和假体有限以及耐久性差等局限性。
随着生物技术的发展，新得的生物技术不断应用于骨修复，例如有研究通过经皮注射骨生长因子，直接将高效的骨诱导剂输送至骨折、骨缺损及骨不连接部位，具有创伤小，适应症广等优点，针对临床中大段粉碎性骨折患者，在闭合复位后向骨折部位注入骨生长因子能够大大提高骨折愈合率；而对于骨不连患者，在内固定或外固定完善的情况下，采取局部注射骨生长因子，可进一步扩大了非手术治疗的适应范围，但是该方法需要对骨生长因子进行提取纯化，纯化工艺复杂，同时，在纯化保存以及使用过程中，骨生长因子容易失效，并且使用时，有效时间短，需要持续给予注射才能起效。还有研究利用组织工程技术为骨修复，通过间充质干细胞与支架材料复合后进行成骨诱导分化，但利用间充质干细胞进行修复，常常受到局部微环境的调节，炎性细胞因子如肿瘤坏死因子和白细胞介素1等常常抑制间充质干细胞向成骨细胞分化，影响骨再生修复，同时干细胞增生容易引发肿瘤。
发明内容
本发明的具体实施方式提供一种克服以上所述问题的用于骨修复的药物组合物，具体方案如下：
一种用于骨修复的药物组合物，所述组合物包括自复制mRNA,其中，所述自复制mRNA包含骨修复基因。
可选的，所述骨修复基因为BMP基因或VEGFA基因。
可选的，所述自复制mRNA为BMP2基因自复制mRNA、VEGFA基因自复制mRNA或BMP2与VEGFA双基因自复制mRNA。
可选的，所述组合物还包括脂质体，所述自复制mRNA分散于脂质体中。
可选的，所述脂质体由DOTAP、HSPC、Chol和PEG-DSPE组成。
可选的，所述脂质体包含阳离子穿膜肽。
可选的，所述组合物还包括水凝胶，所述自复制mRNA与脂质体负载于所述水凝胶上。
可选的，所述水凝胶为甲基丙烯酸酐化明胶水凝胶。
可选的，所述水凝胶中，甲基丙烯酸酐化明胶的重量百分比为3wt％～9wt％。
可选的，所述BMP2基因自复制mRNA的序列如SEQ ID NO.1所示，所述VEGFA基因自复制mRNA的序列如SEQ ID NO.2所示，所述BMP2与VEGFA双基因自复制mRNA的序列如SEQ ID NO.3所示。
可选的，所述药物组合物为骨损伤部原位注射制剂。
本发明具体实施方式的用于骨修复的药物组合物，所述组合物包括自复制mRNA,其中，所述自复制mRNA包含BMP基因或VEGFA基因的骨修复基因，所述药物组合物有利于维持促骨修复基因长时间内高表达，对骨创伤位置少量少批次注射便可进行有效修复。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍。
图1为实施例中3wt％,5wt％,7wt％,和9wt％浓度的GelMA制备的水凝胶球的显微镜观察及组织切片内部表征结果；
图2为实施例中脂质体、短链BMP2基因mRNA与脂质体复合,自复制BMP2基因mRNA与脂质体复合的混合物的SEM观察形态图；
图3为实施例中BMP4基因自复制mRNA、TGFB基因自复制mRNA、BMP2基因自复制mRNA、VEGFA基因自复制mRNA、以及BMP2与VEGFA双基因自复制mRNA体外细胞实验结果；
图4为实施中骨修复的实验效果图。
具体实施方式
本发明的具体实施方式提供一种用于骨修复的药物组合物，所述组合物包括自复制mRNA,其中，所述自复制mRNA包含骨修复基因。本发明的发明人研究发现，当骨创伤位置原位施用所述药物组合物后，促骨修复基因会在长的时间内得到持续显著表达，少量少批次注射便可对骨损伤进行有效修复。
本发明具体实施方式的药物组合物，所述自复制mRNA包括自复制序列以及骨修复基因序列，在一些具体实施方式种，所述自复制序列来源于正链ssRNA病毒，具体例如委内瑞拉马脑脊髓炎病毒(TC83 Venezuelan Equine Encephalitis Virus，VEEV)，辛德毕斯病毒(Sin-dbis virus)、屈曲病毒(Chikungunya virus)、东方马脑脊髓炎病毒(Eastern equine encephali-tis virus)、西方马脑脊髓炎病毒(Western equineencephalitis virus)、马雅鲁病毒(Mayarovirus)、生里基森林病毒(Semliki forest virus)、委内瑞拉马脑脊髓炎病毒(Venezuelan equine encephalitisvirus)等，特别的为用委内瑞拉马脑脊髓炎病毒(TC83 Venezuelan Equine Encephalitis Virus，VEEV)。
本发明具体实施方式的药物组合物，在一些具体实施方式中，所述自复制mRNA包含BMP基因或VEGFA基因，在一些具体实施方式中，所述自复制mRNA为BMP2基因自复制mRNA，在一些具体实施方式中，所述自复制mRNA为VEGFA基因自复制mRNA，在一些具体实施方式中所述自复制mRNA为BMP2与VEGFA双基因自复制mRNA，所述实施方式的自复制mRNA，特别是当自复制mRNA为BMP2与VEGFA双基因自复制mRNA时，具有更为显著的促骨修复基因表达。
本发明具体实施方式的药物组合物，在一些具体实施方式中，所述BMP2基因自复制mRNA的序列如SEQ ID NO.1所示，所述VEGFA基因自复制mRNA的序列如SEQ ID NO.2所示，所述BMP2与VEGFA双基因自复制mRNA的序列如SEQ ID NO.3所示。
本发明具体实施方式的药物组合物，为提高所述自复制mRNA的生物活性，在一些具体实施方式中，所述组合物还包括脂质体，所述自复制mRNA 分散于脂质体中，通过所述脂质体作为所述药物组合物的递送载体。在一些具体实施方式中，所述脂质体的脂质为磷脂，例如氢化大豆卵磷脂(HSPC)，卵磷脂、磷脂酰乙醇胺、鞘磷脂、脑磷脂、心磷脂，在一些具体实施方式中所述脂质还包括阳离子脂质，例如1，2-二肉豆蔻酰-3-三甲基铵丙烷(DMTAP)、1，2-二油烯氧基-3-(三甲基氨基)丙烷(DOTAP)、N-[1-(2，3-二(十四烷氧基))丙基]-N，N-二甲基-N-羟乙基溴化铵(DMRIE)、N-[1-(2，3-二油烯氧基)丙基]-N，N-二甲基-N-羟乙基-溴化铵(DORIE)、N-[1-(2，3-二油烯氧基)丙基]-N，N，N-三甲基氯化铵(DOTMA)、3β[N-(N′，N′-二甲基氨基乙烷)氨基甲酰基]胆固醇(DC-Chol)和二甲基-二(十八烷基)溴化铵(DDAB)，在一些具体实施方式中，所述脂质还包括固醇，例如胆固醇(Chol)、胆固醇半琥珀酸酯、胆固醇硫酸酯或胆固醇的任何其他的衍生物，在一些具体实施方式中，所述脂质体还包括脂质聚合物，例如，PEG-DSPE(二硬脂酰磷脂酰乙醇胺)。作为更为有效递送的脂质体，在一具体实施方式中，所述脂质体由DOTAP、HSPC、Chol和PEG-DSPE组成，为进一步提高递送效率，在一些具体实施方式中，所述脂质体中还包含阳离子穿膜肽(CPPs)。
本发明具体实施方式的药物组合物，为实现药物的负载和缓慢释放，在一些具体实施方式中，所述组合物还包括水凝胶，所述自复制mRNA与脂质体负载于所述水凝胶上，所述水凝胶具有亲水性聚合物链，在一些具体实施方式中，所述水凝胶与细胞具有相容性，例如可以是明胶、甲基丙烯酸酐化明胶、海藻酸钠、丝素、壳聚糖和胶原蛋白等等，在一些具体实施方式中，所述水凝胶为甲基丙烯酸酐化明胶水凝胶，为兼顾既水凝胶球小的塌陷率以及大的脂质体吸附比例，在一些具体实施方式中，所述水凝胶中，甲基丙烯酸酐化明胶的重量百分比为3wt％～9wt％。
本发明具体实施方式的药物组合物，所述组合物适用于各种创伤或疾病等原因导致的骨组织缺损，例如骨折、骨缺损或骨不连接部位等等。
本发明具体实施方式的药物组合物，所述骨修复包括骨的再生或者形成。
本发明具体实施方式的药物组合物，在一些具体实施方式中，所述药物组合物为骨损伤部原位注射制剂，使用时，将所述药物组合物原位注射至所述骨损伤部位。
以下通过具体实施例对本发明做进一步说明。
实施例
水凝胶制备
称取20g明胶分散于200mlPBS(磷酸缓冲盐溶液)(0.01M)中，500mL烧瓶或烧杯中，置于水浴锅中加热搅拌，使明胶温度达到60℃，溶解呈澄清状态(约30min)。用注射器抽取16mlMA(甲基丙烯酸酐)，去除气泡，用微量注射泵以0.25ml/min的速度将MA缓慢加入明胶中，注意避光。加完MA后，水浴锅中继续反应2小时，后加200mlPBS终止反应。反应终止15min后，将GelMA(甲基丙烯酸酐化明胶)分装入透析袋中(MWCO 3500)，38℃透析过夜，7000rpm 15min离心除去不溶物，取上清继续透析2～3天。用真空泵抽滤，滤掉超过滤膜(0.22微米)孔径的大分子。将抽滤好的GelMA分装到多个10cm的平皿中，并置于负80冰箱中冷冻保存。低温冻干得到产物GelMA。
将3wt％,5wt％,7wt％,和9wt％浓度的GelMA溶于双蒸水后37℃加热，使用微流控装置制备微球，-30℃冰冻后蓝光照射5min。，如图1显微镜观察可以看出甲基丙烯酸酐化明胶的重量百分比为3wt％～9wt％时，可以保证既水凝胶球小的塌陷率以及大的脂质体吸附比例，特别时为5wt％时可以保证水凝胶球在最小的塌陷率的情况下实现最大的脂质体吸附比例。
脂质体制备
用电子分析天平精密量取DOTAP(SigmaAldrich)、HSPC(SigmaAldrich)、Cholesterol(SigmaAldrich)和mPEG2000-DSPE(上海子起生物科技有限公司)，289w(阳离子穿膜肽)(参考文献Acta BIomaterialia 63(2017)123-134 Discerning the composition of penetratin for safe penetration from cornea to retina合成)分别使用三氯甲烷溶解定容，各配制10mg/mL的溶液1.5mL，按以下量：DOTAP(MW＝698.5):0.706mg/70.6uL，HSPC(MW＝78.3):10mg/mL,7.92mg/792uL，Chol(MW＝386):3.52mg/352uL，DSPE.PEG2000(MW＝2750):1.667mg/166.7uL，289w 2.701mg/270.1uL分别加入上述溶液进入25mL烧瓶中，接旋转蒸发仪，使用标口夹夹紧，水浴38℃，打开旋转开关，打开真空泵，调整真空阀，缓慢抽真空。待烧瓶底部形成脂质膜后计20min，关闭旋转开关，调整烧瓶位置，擦干瓶底外部水，打开真空阀，关闭真空泵，移液器于瓶底缓慢滴入1.5mL纯水，打开超声细胞破碎仪，调整参数 功率20-40％，3min工作2s停1s，洗净超声头，将超声头没过烧瓶底部液面，进行水化，注意观察底部脂质膜水化情况，使用1mL针管抽取溶液分别经过0.45μm和0.22μm滤器滤入2mL EP管中，封口膜封闭，4℃保存。
自复制mRNA制备
利用SimpliconTM RNA质粒，分别构建了表达BMP4基因、TGFB基因、BMP2基因、VEGFA基因以及BMP2与VEGFA双基因的质粒模板。对构建好的质粒模板进行测序验证(利用二代测序仪进行基因序列测序)，并对验证好的质粒模板利用QRT-PCR方法进行扩增,利用限制性核酸内切酶分别对60ug BMP4、TGFB、BMP2、VEGFA、BMP2与VEGFA基因质粒线性化处理，并沉淀纯化得到的线性化BMP4、TGFB、BMP2、VEGFA、BMP2与VEGFA基因模板为30-50ug。利用线性化模板，进行T7启动子体外转录试剂盒体外转录，在37℃恒温条件下转录1-2小时，然后在37℃恒温条件下进行5’端加帽和3’端加尾修饰，修饰后的mRNA用转录纯化试剂盒进行纯化，最后用去离子水进行洗脱，得到BMP4、TGFB、BMP2、VEGFA、BMP2及VEGFA双基因自复制mRNA(Self-amplifying mRNA)溶液。
自复制mRNA和脂质体复合制备
将以上制备获得的自复制mRNA溶液500ul(浓度1ug/ul)继续溶解在500ul无血清培养基，将500ul脂质体溶液(浓度10mg/ml)溶解在500ul无血清培养液中，自复制mRNA与脂质体混合液以1：1比例共孵育10min得到自复制mRNA/脂质体混合物。
作为对比，按以上方式，制备短链BMP2基因mRNA与脂质体复合后的混合物。
将脂质体、短链BMP2基因mRNA与脂质体复合后的混合物、自复制BMP2基因mRNA与脂质体复合的混合物进行SEM观察，其形体如图2所示，如图2，采用所述脂质体与自复制BMP2基因mRNA复合后的混合物大小与混合前的脂质体基本一致的形貌，表明所述脂质体对自复制BMP2基因mRNA具有优异的负载作用。
自复制mRNA和脂质体复合物的水凝胶负载制备
将2000ul以上自复制mRNA与脂质体复合后的混合物放在15ml离心管A中，将以上制备的5wt％的GelMA水凝胶产物2000ul置于15ml离心管B 中，将离心管A中的自复制mRNA与脂质体复合后的混合物加入离心管B中，冰上孵育30min。
细胞实验
在六孔培养皿中体外培养骨髓间充质干细胞(BMSC)，设置GFP mRNA处理为对照组，将上述获得的水凝胶负载的BMP4基因自复制mRNA(Sa-BMP4)、TGFB基因自复制mRNA(Sa-TGFB)、BMP2基因自复制mRNA(Sa-BMP2)、VEGFA基因自复制mRNA(Sa-VEGF)、BMP2与VEGFA双基因自复制mRNA(Sa-BMP2+VEGF)作为实验组，每组设置三个重复实验孔，每孔转入1ug对应mRNA，通过qRT-PCR观察骨修复主要指标Runx2、OPN和OCN的表达情况，选取第五天的表达情况结果如图3所示，根据图3的结果显示，相对于空白样，BMP4以及TGFB基因自复制mRNA在第五天的Runx2、OPN和OCN表达略有提升，而BMP2基因自复制mRNA、VEGFA基因自复制mRNA、BMP2与VEGFA双基因自复制mRNA在第五天均具有显著的Runx2、OPN和OCN表达，特别是BMP2与VEGFA双基因自复制mRNA具有最为显著的表达。
骨修复实验
1)建立大鼠股骨远端骨缺损模型
根据每只实验动物体重，使用戊巴比妥钠+吸入异氟烷复合麻醉。大鼠仰卧固定于手术操作台，右侧后肢膝关节内侧周围备皮，常规消毒，铺巾。取膝关节内侧纵行切口，逐层进入，暴露股骨远端，使用直径3mm克氏针由股骨远端内侧面水平进针，构建直径3mm的骨隧道，随后根据分组注入生理盐水或不同材料，使用骨蜡封闭隧道口。反复冲洗后逐层关闭切口。术后放回单笼饲养。
2)注射样准备
sham：生理盐水
Sa-mRNA/Lip@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式制备的水凝胶负载BMP2与VEGFA双基因自复制mRNA和脂质体复合物；
Lip@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式,其中脂质体中不混合有自复制mRNA，制备的水凝胶负载脂质体；
mRNA/Lip@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式制备的水凝胶负载短链mRNA和脂质体复合物,其中的自复制mRNA替换为短链BMP2基因mRNA与短链VEGFA基因mRNA(1mol:1mol)混合。
mRNA/Lip(-CPPs)@GelMA:按以上自复制mRNA和脂质体复合物的水凝胶负载制备方式制备的水凝胶负载BMP2与VEGFA双基因自复制mRNA和脂质体复合物,但其中脂质体中不含阳离子穿膜肽(CPPs)。
3)骨修复分析
抽取150ul以上步骤2)中的注射样，对步骤1)中的大鼠股骨远端骨缺损模型进行关节损伤部位原位注射，间隔2周后进行第二次注射，2周和4周后时间点均获取小鼠关节进行组织固定，取样microCT分析，结果如图4所示。根据图4的结果可知，sham样与Lip@GelMA样无骨修复作用，mRNA/Lip(-CPPs)@GelMA样与mRNA/Lip@GelMA样仍存在有空洞，而Sa-mRNA/Lip@GelMA样的骨骺处骨质愈合最好，骨质增生紧致，表明BMP2与VEGFA双基因自复制mRNA由包含有阳离子穿膜肽的脂质体负载具有十分优异的骨修复作用。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (11)

  1. 一种用于骨修复的药物组合物，其特征在于，所述组合物包括自复制mRNA,其中，所述自复制mRNA包含骨修复基因。
  2. 根据权利要求1所述的药物组合物，其特征在于，所述骨修复基因为BMP基因或VEGFA基因。
  3. 根据权利要求2所述的药物组合物，其特征在于，所述自复制mRNA为BMP2基因自复制mRNA、VEGFA基因自复制mRNA或BMP2与VEGFA双基因自复制mRNA。
  4. 根据权利要求1所述的药物组合物，其特征在于，所述组合物还包括脂质体，所述自复制mRNA分散于脂质体中。
  5. 根据权利要求1所述的药物组合物，其特征在于，所述脂质体由DOTAP、HSPC、Chol和PEG-DSPE组成。
  6. 根据权利要求5所述的药物组合物，其特征在于，所述脂质体包含阳离子穿膜肽。
  7. 根据权利要求4所述的药物组合物，其特征在于，所述组合物还包括水凝胶，所述自复制mRNA与脂质体负载于所述水凝胶上。
  8. 根据权利要求7所述的药物组合物，其特征在于，所述水凝胶为甲基丙烯酸酐化明胶水凝胶。
  9. 根据权利要求6所述的药物组合物，其特征在于，所述水凝胶中，甲基丙烯酸酐化明胶的重量百分比为3wt％～9wt％。
  10. 根据权利要求3所述的药物组合物，其特征在于，所述BMP2基因自复制mRNA的序列如SEQ ID NO.1所示，所述VEGFA基因自复制mRNA的序列如SEQ ID NO.2所示，所述BMP2与VEGFA双基因自复制mRNA的序列如SEQ ID NO.3所示。
  11. 根据权利要求1-10任意一项所述药物组合物，其特征在于，所述药物组合物为骨损伤部原位注射制剂。
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