CN117412776A - 用于mRNA递送的药学上可接受的含水凝胶组合物 - Google Patents
用于mRNA递送的药学上可接受的含水凝胶组合物 Download PDFInfo
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Abstract
本发明涉及一种药学上可接受的含水凝胶组合物,其包含胶凝剂、镁和/或锰或其二价离子,和编码目的蛋白质的mRNA分子。本公开还涉及用于诱导或促进人或动物受试者中组织的修复、再生或产生的治疗方法,所述方法包括将所述组合物施用至待修复、再生或产生的组织的部位。
Description
技术领域
本公开涉及用于递送mRNA的药物组合物,特别是药学上可接受的含水凝胶组合物,并涉及用于将mRNA原位靶向递送至特定位点的方法。
背景技术
1989年首次提出了使用mRNA作为疫苗开发平台的建议(Malone等,1989)。后来的出版物提出了不同的方法来增强细胞对外部mRNA的摄取,例如Ca和基于脂质的纳米颗粒(Schlake等,2012)。2020年,Pfizer/Biontech生产了第一种疫苗,然后由其它制药公司生产。
例如在WO9522611中已经提出了用于刺激骨生长的包含亲骨基因和骨相容性基质例如羟基磷灰石基质的组合物。
还建议通过基于羟基磷灰石的纳米载体,将长度通常比mRNA短得多的RNA分子,即siRNA,递送至小鼠胚胎干细胞(Zantye等,Mol.Pharmaceutics 2021,18,796-806)。
用于药物和siRNA递送的生物可降解羟基磷灰石粒子的合成方案也已公开(Liu等,Journal ofColloid and Interface Science,570(2020)402-410)。
发明内容
本发明基于这样的想法,即施用包含编码目的蛋白质的mRNA和钙磷灰石形成的催化剂的组合物,以诱导含mRNA的羟基磷灰石纳米颗粒的形成,用于将mRNA原位递送至细胞。每当需要表达新蛋白、修饰蛋白或增加已表达蛋白的量时,这种递送是有用的。这可能是所期望的,例如在细胞功能的修饰中。
本发明利用金属和金属氧化物的性质,来催化磷酸钙如羟基磷灰石的形成,其中mRNA作为磷酸盐源加入,导致mRNA-羟基磷灰石纳米颗粒的形成,其被靶细胞吸收。由于是这种颗粒的组成部分,mRNA分子被保护免于被血清酶分解,并且其还通过靶细胞的清除受体,促进mRNA的摄取以及对羟基磷灰石的摄取。
已经发现,在还包含mRNA分子的含水凝胶组合物中提供金属镁和/或锰或其二价离子,诱导了掺入mRNA分子的羟基磷灰石颗粒的原位形成。随后羟基磷灰石颗粒容易被细胞吸收,随后在细胞内降解以释放mRNA分子,mRNA分子被翻译成相应的蛋白质。
因此,根据本发明的第一方面,提供了一种药学上可接受的含水凝胶组合物,其包含胶凝剂、镁和/或锰或其二价离子,和编码目的蛋白质的mRNA分子。
根据一些实施方式,镁和/或锰或其二价离子,以在生理条件下足以诱导羟基磷灰石形成的量存在。
根据一些实施方式,胶凝剂选自淀粉或其衍生物;琼脂/琼脂糖或琼脂/琼脂糖的衍生物;透明质酸或透明质酸的衍生物;壳聚糖;明胶;和葡聚糖。在一些实施方式中,胶凝剂的浓度在0.5-10%w/w的范围内。
根据一些实施方式,目的蛋白质是参与组织修复、组织再生或组织生成的蛋白。
根据一些实施方式,所述蛋白质选自磷酸调节性中性内肽酶,X-连接的(由PHEX编码),热休克蛋白90-α(由HSP90AA1编码),腱蛋白样2(由CHRDL2编码),短瞬时受体电位通道4(由TRPC4编码),泛连接蛋白3(由PANX3编码),胶原XXIV型α1(由COL24A1编码),ATP283的基因产物,含有普列克底物蛋白同源结构域的B1(由PLEKHB1编码),白细胞免疫球蛋白样受体亚家族B成员4(由LILRB4编码),茴香胺5(由ANO5编码),ChaC谷胱甘肽特异性-谷氨基环转移酶1(由CHAC1编码),轴丝中链动力蛋白(由DNAI2编码),聚糖(由ACAN编码),整合素α10(由ITGA10编码),纤调蛋白(由FMOD编码),分泌型磷蛋白1(又称骨桥蛋白,由SPP1编码),载脂蛋白C1(由APOC1编码),负调控蛋白2(由PTCH2编码)、载脂蛋白E(由APOE编码)、髓系细胞上触发受体2(由TREM2编码)、平足蛋白(由PDPN编码)、溶质载体家族13成员5(由SLC13A5编码)、T-细胞可诱导共刺激分子(由ICOS编码)、甲酰肽受体2(由FPR2)编码、基质金属肽酶12(由MMP12编码)、肌腱蛋白N(由TNN编码)、天冬蛋白(由ASPN编码)、粒酶A(由GZMA编码)、骨诱导因子(由OGN编码)、Wnt家族成员2(由WNT2编码)、神经肽Y(由NPY编码)、骨形态发生蛋白2(由BMP-2编码)、具有序列相似性家族169成员A(由FAM169编码)、视黄醇结合蛋白1(由RBP1编码)、ISG15泛素样修饰因子(由ISG15编码)、三碱基假激酶3(由TRIB3编码)、泛素特异性肽酶18(由USP18编码)、紧密连接蛋白(由CLDN1编码)、肌纤蛋白(由MYOC编码)、C-X-C基序趋化因子配体9(由CXCL9编码)、具有四肽重复序列3的干扰素诱导蛋白(由IFIT3编码)、RRAD、RAS相关糖酵解抑制剂和钙通道调节剂(由RRAD编码)干扰素α诱导蛋白27(由IFI27编码)。
根据一个方面,本发明涉及上述组合物,其用于诱导或促进人或动物受试者中组织的修复、再生或产生的方法中,所述方法包括将所述组合物施用至待修复、再生或产生的组织的部位。
根据一些实施方式,组织是骨组织。
在一些实施方式中,这些方法与脊柱融合和在骨组织中的植入植入物有关,例如假体关节和牙齿植入物。
在一些实施方式中,采用这样的方法以促进或改善由于创伤和/或疾病,如骨折和创伤性骨裂损伤而受伤的组织的愈合。
在一个方面,本发明涉及用于诱导或促进人或动物受试者中组织的修复、再生或产生的方法,所述方法包括将根据本发明的组合物施用至待修复、再生或产生的组织的部位。
在一个实施方式中,所述组织是骨组织。
在一些实施方式中,这些方法与脊柱融合和在骨组织中的植入植入物有关,例如假体关节和牙齿植入物。
在一些实施方式中,采用这样的方法以促进或改善由于创伤和/或疾病,如骨折和创伤性骨裂损伤而受伤的组织的愈合。
用本发明的组合物和方法治疗的受试者可以是人或动物受试者。动物受试者包括哺乳动物物种,例如马、猫、狗、牛、猪、绵羊、骆驼和啮齿动物,包括小鼠和大鼠。当制备用于非人物种的本发明组合物时,编码目的蛋白质的mRNA序列可以是编码来自相关物种的目的蛋白质的序列。
从以下给出的详细描述中,本公开会清楚。详细描述和具体示例仅通过说明的方式公开了本公开的优选实施例。本领域技术人员从详细描述的指导中理解,可以在本公开的范围内进行改变和修改。
因此,应当理解,本文公开的公开内容不限于所描述的装置的特定组成部分或所描述的方法的步骤,因为这样的装置和方法可以变化。还应理解,本文所用的术语仅是为了描述特定实施方式,而不是为了限制。应当注意,如在说明书和所附权利要求中所使用的,冠词“一”、“一个”、“该”和“所述”旨在表示存在一个或多个元件,除非上下文明确地另外规定。因此,例如,对“一个单元”或“该单元”的引用可以包括若干设备等。此外,词语“包括”、“包含”、“含有”和类似的措辞不排除其他元件或步骤。此外,本文引用的所有公开文献均全文以引用方式并入。
附图说明
图1是展示在DMEM(暴露的)或盐水(对照)中预培养的MnO分析期间通过tof-sims检测的Ca3PO5 +离子的相对强度(检测的离子数,标准化为总离子检测)的图。
图2:人胚胎干细胞(SA16.7MFG-hesc)在0.5mg/ml的MnO下生长24小时,MnO已在DMEM中预培养。VonKossa染色揭示细胞内和细胞外间隙中HA的沉淀。在集落内粘附细胞的表面覆盖率>80%。
图3:在细胞培养基(DMEM)或盐水对照中,在MnO或MgO与RNA培养后,通过ToF-SIMS对氮碱基特异的二次离子的检测。
图4:在含有三种不同化学改性淀粉凝胶的细胞培养基(DMEM)中,将MnO或MgO与RNA一起培养后,通过ToF-SIMS对氮碱基特异的二次离子的检测。
具体实施方式
在生理条件下,在腐蚀的Mg2+(Nygren等,2017)和Mn2+(参见实施例1)表面上形成磷酸钙,包括羟基磷灰石(HA)。
已知,核酸分子如RNA,在磷酸盐缓冲溶液中与羟基磷灰石颗粒结合(Fadrosh等,2011)。因此,在金属Mg或Mn或其二价离子的存在下,mRNA可用作磷酸盐源时,在生理条件下,mRNA分子将被结合到Mg或Mn颗粒上的羟基磷灰石中(参见实施例2)。
在小干扰RNA(siRNA)的情况下已经表明,作为羟基磷灰石颗粒的组成部分存在的RNA,对血清酶的降解具有抗性,并且容易被靶细胞吸收(Zantine等,2021)。也已经表明,HA-siRNA颗粒溶解在细胞的细胞质中并释放siRNA(Liu等,2020)。当应用于mRNA时,释放的mRNA分子将被靶细胞翻译成目的蛋白质(参见实施例3)。
本公开的第一方面提供药学上可接受的含水凝胶组合物,其包含胶凝剂、镁和/或锰或其二价离子,和编码目的蛋白质的mRNA分子。
在一个实施方式中,镁和/或锰或其二价离子,以在生理条件下足以诱导羟基磷灰石形成的量存在,例如在细胞培养基中培养时或当对受试者原位给药时。
凝胶的功能是将mRNA和金属离子保持在注射部位或注射部位附近,并使向周围组织的渗漏最小化。凝胶应该是在药物递送中药学上可接受的,并且是生物可降解的,以允许mRNA在组织中的应用部位从含有mRNA的羟基磷灰石颗粒中释放。可以使用的凝胶的类型例如但不限于,基于琼脂/琼脂糖的凝胶或琼脂/琼脂糖的衍生物、基于透明质酸或透明质酸衍生物的凝胶、基于壳聚糖、明胶、葡聚糖或淀粉或其衍生物的凝胶。凝胶的浓度应在0.5-10%w/w的范围内,制备药学上可接受的凝胶制剂的指导可参见例如“Pharmaceutics:TheScience of Dosage Forms”(Aulton,2002);“Encyclopedia ofPharmaceuticalTechnology”(Swarbrick,2006);“Modern Pharmaceutics”(Banker等,2002);“The Theoryand Practice of Industrial Pharmacy”(Lachman等,1986),所有这些文献在此引入作为参考。
在优选的实施方式中,胶凝剂是淀粉。淀粉在药物组合物中的应用是公知的,并且可从多个来源和商业供应商获得。作为药物赋形剂的淀粉通常由玉米、马铃薯、木薯或大米生产,并且进一步描述于例如European Pharmacopoeia中。包含可降解淀粉微球(DSM)的组合物已用于药物递送和医学中的其它应用,例如体液渗漏的检测(例如WO2019/122120)和经动脉化疗栓塞术(Ludwig等,2021),并且可用于本发明。在美国专利4,124,705中公开了生产DSM的方法。淀粉可以是交联的,如本领域已描述的(Atyabi等人,2006),(Fang等人,2008)。示例性的交联剂是戊二醛、甲醛、环氧氯丙烷(epichlorohydrine)和三偏磷酸钠。
在一些实施方式中,根据本发明的含水凝胶组合物是酸性的,即pH<7,例如低于6.5、6.0、5.5、5.0、4.5或4.0的pH。在一些实施方式中,根据本发明的含水凝胶组合物是碱性的,即pH>7,例如高于7.5、8.0或8.5的pH。通常认为药学上可接受的溶液具有约4.5至约8.0的pH。
在一些实施方式中,目的蛋白质是由人基因编码的人源蛋白质。
按照Uhlen等人(Uhlén等人,2015)所述分析愈合大鼠胫骨中mRNA的表达。在愈合骨中表达但在未处理对照中不表达的基因包括编码选自以下的蛋白质的基因:磷酸调节性中性内肽酶,X-连接的(由PHEX编码),热休克蛋白90-α(由HSP90AA1编码),腱蛋白样2(由CHRDL2编码),短瞬时受体电位通道4(由TRPC4编码),泛连接蛋白3(由PANX3编码),胶原XXIV型α1(由COL24A1编码),ATP283的基因产物,含有普列克底物蛋白同源结构域的B1(由PLEKHB1编码),白细胞免疫球蛋白样受体亚家族B成员4(由LILRB4编码),茴香胺5(由ANO5编码),ChaC谷胱甘肽特异性-谷氨基环转移酶1(由CHAC1编码),轴丝中链动力蛋白(由DNAI2编码),聚糖(由ACAN编码),整合素α10(由ITGA10编码),纤调蛋白(由FMOD编码),分泌型磷蛋白1(又称骨桥蛋白,由SPP1编码),载脂蛋白C1(由APOC1编码),负调控蛋白2(由PTCH2编码)、载脂蛋白E(由APOE编码)、髓系细胞上触发受体2(由TREM2编码)、平足蛋白(由PDPN编码)、溶质载体家族13成员5(由SLC13A5编码)、T-细胞可诱导共刺激分子(由ICOS编码)、甲酰肽受体2(由FPR2)编码、基质金属肽酶12(由MMP12编码)、肌腱蛋白N(由TNN编码)、天冬蛋白(由ASPN编码)、粒酶A(由GZMA编码)、骨诱导因子(由OGN编码)、Wnt家族成员2(由WNT2编码)、神经肽Y(由NPY编码)、骨形态发生蛋白2(由BMP-2编码)、具有序列相似性家族169成员A(由FAM169编码)、视黄醇结合蛋白1(由RBP1编码)、ISG15泛素样修饰因子(由ISG15编码)、三碱基假激酶3(由TRIB3编码)、泛素特异性肽酶18(由USP18编码)、紧密连接蛋白(由CLDN1编码)、肌纤蛋白(由MYOC编码)、C-X-C基序趋化因子配体9(由CXCL9编码)、具有四肽重复序列3的干扰素诱导蛋白(由IFIT3编码)、RRAD、RAS相关糖酵解抑制剂和钙通道调节剂(由RRAD编码)干扰素α诱导蛋白27(由IFI27编码)。因此,在一些实施方式中,目的蛋白质是选自该组的蛋白质。目的蛋白质也可以是上述蛋白质的哺乳动物直系同源物。
关于编码本发明目的蛋白质的人基因的详细信息可见例如数据库(www.genecards.org)。
在一个实施方式中,目的蛋白质是参与组织修复或再生的蛋白质。
在一些实施方式中,所述组合物用于治疗受试者中受损组织的方法中,所述方法包括将所述组合物施用至受损组织的部位。
在一些实施方式中,受损组织是骨组织。
本领域技术人员认识到,本公开不限于上述优选实施例,而是在所附权利要求的范围内可以进行修改和变化。另外,本领域技术人员在实践所要求保护的公开内容时,通过研究附图、公开内容和所附权利要求,可以理解和实现对所公开的实施例的变型。
本文引用的所有参考文献都明确地通过引用并入。
包括以下实施例以进一步说明本发明,并且不应认为是对本发明的限制。
实施例
包括以下实施例,以根据本发明的一些实施方式说明本发明,并且不应解释为限制本发明的范围,本发明的范围是所附权利要求的范围。
实施例1
MnO样品制备。
将粉末形式(粒度1-2μm)的商业纯MnO(Sigma-Aldrich,瑞典,99.9%)在细胞培养基(DMEM)中培养24-72小时,在盐水和蒸馏水中冲洗并在160℃下干燥灭菌2小时。
如(Nygren等,2017)所述,在DMEM中培养之前和之后,通过ToF-SIMS分析样品的化学组成。
人间充质干细胞培养。
本研究中使用的人胚胎干细胞(hESC)系分别是在我们以前的研究中衍生和表征的第12代和第44代的SA167MFG-hESC和AS034.1MFG-hESC(Bigdeli等,2007)。注意,干细胞粘附于塑料培养皿上,并可在培养皿中培养。
hESC的扩增
在该研究中,在没有任何支持性涂层的情况下,hESC直接在组织培养塑料上扩增并向成骨谱系分化。简言之,如前所述(Bigdeli等人,2007),在含有80%KnockOutTM DMEM(Gibco-BRL/英杰公司(Invitrogen),盖瑟斯堡,马里兰州,美国)、20%KnockOutTM血清替代物(SR;Gibco-BRL/英杰公司)、2mM L-谷氨酰胺(Gibco-BRL/英杰公司)、0.1mM-巯基乙醇(Gibco-BRL/英杰公司)和1%NEAA(非必需氨基酸;Gibco-BRL/英杰公司)条件的hES培养基中,在培养皿(Falcon,表面修饰的聚苯乙烯非致热的;贝克顿·迪金森公司(Becton Dickinson),富兰克林湖,美国)上扩增细胞,并在37℃和5%CO2(Herus BBD6220)的潮湿气氛中培养。SA167MFG-hESC和AS034.1MFG-hESC每4到6天传代,并且培养基每两天更换一次。
干细胞暴露于金属氧化物/CHA
未分化的hESC在常规组织培养塑料上培养,没有诸如胚状体(EB)形成的分化前阶段。
通过将不同浓度的CHA涂覆的金属氧化物加入培养基中,进行24小时细胞暴露。
Von Kossa染色
使用von Kossa染色研究钙沉淀物(主要是羟基磷灰石),通过在PBS中洗涤细胞,然后在戊二醛溶液(25%H2O Sigma-Aldrich,以1:10稀释)中固定2小时进行所述染色。加入AgNO3溶液(2%w/v:Sigma-Aldrich),将板在黑暗中保持10分钟。然后用蒸馏水将板漂洗三次,然后暴露于亮光15分钟。用蒸馏水洗涤后,在显微镜检查前加入100%乙醇使样品快速脱水。
细胞活力。
将hMSC以10000个细胞/孔的密度接种到24孔板上。在存在或不存在金属氧化物的生长培养基中培养细胞24小时以允许附着。附着的细胞被认为是活的,而漂浮的细胞是非活的。
ToF-SIMS。
ToF-SIMS分析用TOF.SIMS 5仪器(ION-TOF GmbH,Münster,德国)进行。使用Bi3+簇离子枪作为一次离子源。用焦点约为2μM,质量分辨率为M/ΔM=5×103fwhm的脉冲一次离子束(Bi3+,25keV下的0.24pA,剂量密度1.12×1011)在m/z为500的情况下,分析60μM×60μM至105μM××105-66105μM的多个(n=5)区域。所有的光谱都是用表面实验室软件(6.3版,ION-TOF GmbH,Münster,德国)获得和处理的,并且用于计算的离子强度被标准化为每次测量的总离子剂量。ToF-SIMS分析是表面敏感的,且检测表面处的第一纳米中的原子和分子。不认为ToF-SIMS是定量分析。
统计分析。
使用t检验进行统计分析。统计学显著性的极限被设置为p>0.05。
结果
如图1所示,在DMEM中培养MnO导致羟基磷灰石的形成,而在盐水对照中培养MnO基本上不形成羟磷灰石。图1中的图表显示了在仪器中轰击期间从羟磷灰石释放的离子(Ca3PO5 +)的Tof-SIMS分析结果。该离子是在分析过程中形成的,并且揭示了在细胞培养基中而不是在盐水中培养的MnO样品中羟基磷灰石的存在。
图2显示了hESC暴露于MnO的结果,MnO已经在MEM预培养了24-72小时。粘附于皮氏培养皿并暴露于0.5mg/ml预培养后的MnO下的hESC,显示菌落中粘附在培养皿上的细胞覆盖率>90%。用von Kossa染色对固定的培养物进行染色,在暴露于在DMEM预培养后的该浓度的MnO后,细胞含有HA并被HA包围。细胞反应可以是细胞的特异性激活,或是在暴露于MnO时的应激反应。在暴露于在DMEM预培养后的MnO后,生长培养基的pH是稳定的。
实施例2
通过用细胞培养基(DMEM)培养制备结合金属氧化物的HA-RNA
金属氧化物MnO和MgO(粉末)通过在160℃的烘箱中加热2小时来灭菌。然后将金属氧化物在离心管(Falcon tubes)中与6ml下列物质一起培养:
a.含有10%无菌胎牛血清和10μg RNA的无菌DMEM,或
b.含有10μg RNA的无菌盐水作为对照
6小时后,将氧化物以1300rpm旋转3分钟。用无菌水冲洗沉淀3次。将沉淀在60℃下干燥过夜,然后用ToF-SIMS分析。
用正负极性的ToF-SIMS分析样品。使用ToF.SIMS 5仪器(ION-TOF GmbH,Münster,德国)对粉末样品进行ToF-SIMS分析。使用25keVBi簇离子枪作为一次离子源。使用焦点为约2μm的脉冲一次离子束(Bi3 ++,在50keV下0.34pA),使用高电流聚束模式(Sodhi,2004)分析样品,以获得高质量分辨率光谱。该装置的质量分辨率在m/z 500下至少为M/ΔM=5000fwhm。所有的光谱都是用表面实验室软件(6.4版,ION-TOF GmbH,明斯特,德国)获得和处理的。对于正离子模式,光谱内部校准为[C]+、[CH2]+、[CH3]+、[C5H15PNO4]+和[C27H45]+的信号,对于负离子模式,光谱内部校准为[C]-、[CH]-、[C2]-、[C3]-的信号。
分析了胞嘧啶、鸟嘌呤和腺嘌呤的特异性峰。收集来自每个峰的平均离子信号并将其标准化为每个样品的总离子数。结果示于图3中。
在金属氧化物上检测到腺嘌呤、胞嘧啶和鸟嘌呤。在用含Ca2+离子的细胞培养基培养的样品中,吸附的碱的水平较高。这表明RNA和Ca2+在金属氧化物颗粒的表面上形成羟基磷灰石。
实施例3
含水凝胶的类型对RNA与金属氧化物颗粒结合的影响
1.在实验中使用了三种DSM,下面称为A、B和C。
“d(0.5)”表示50%的微球具有小于指定值的直径
在实验前,所有三个DSM都作为干粉进行高压灭菌(所有3种干粉在密封小瓶中121℃下稳定20分钟)。
2.在160℃下在烘箱中加热2h对MnO和MgO进行消毒。
3.DMEM培养基、胎牛血清、注射用水、离心管(Falcontubes)是无菌的。
4.在实验当天制备1mg/ml的RNA溶液(在无菌盐水中)并直接使用。
5.在实验当天,制备含有以下组分的反应:
a.0.3g DSM(粉末)+6ml DMEM含血清培养基+200μl MnO+10μg RNA(10μl)
b.0.3g DSM(粉末)+6ml DMEM含血清培养基+200μl MgO+10μg RNA(10μl)
总共制备6个反应(每个DSM2个管)。
上述混合物中DSM的最终浓度为5%。所得凝胶用“A”、“B”和“C”表示,对应于制备凝胶时所用的DSM。
将每个DSM在DMEM培养基中溶胀过夜,然后加入其它组分,将整个混合物轻轻旋转(室温)培养6小时。
6小时后,将氧化物+凝胶离心(约1300rpm,3分钟)。用水(5-6ml)冲洗沉淀。将旋转/洗涤步骤重复3次。
凝胶+氧化物沉淀在50-60℃下干燥过夜。干燥的样品送去ToF-SIMS分析。
结果(图4)显示,凝胶组合物对RNA与MgO和MnO的结合有主要影响。凝胶DSM 480中RNA的结合比凝胶DSM 52中高10倍,这些凝胶之间化学组成的差异是酸性。凝胶B通过磷酸盐交联并具有酸性,而凝胶A通过磷酸盐交联并具有碱性。凝胶C为环氧氯丙烷交联,具有酸性。结果表明,为了支持RNA与金属氧化物的结合,凝胶基质应该是酸性的,最可能是通过在Ca2+离子存在下,在金属氧化物表面上形成羟基磷灰石层
实施例4
本实施例说明了,使用根据本发明的一个实施方式的组合物评估有效mRNA递送的方案。
交联淀粉(无菌0.05g)在1ml无菌盐水中溶胀形成凝胶,所述无菌盐水还含有0.5mg MnO和200μg编码目的蛋白质的mRNA。如前所述(Nygren等,2017),将一部分凝胶(100μl)注射到具有钻孔裂缝的大鼠胫骨中,并使骨愈合24-96小时。将大鼠安乐死,解剖出骨。制备组织切片的制品,并通过免疫组织化学检测由注射的mRNA编码的目的蛋白(Uhlén等,2015)。
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Claims (10)
1.一种药学上可接受的含水凝胶组合物,其中,其包含胶凝剂、镁和/或锰或其二价离子,和编码目的蛋白质的mRNA分子。
2.根据权利要求1所述的组合物,其中,其包含锰或其二价离子。
3.根据权利要求1或2所述的组合物,其中,镁和/或锰或其二价离子以在生理条件下足以诱导羟基磷灰石形成的量存在。
4.根据权利要求1-3中任意一项所述的组合物,其中,所述胶凝剂选自淀粉或其衍生物;琼脂/琼脂糖或琼脂/琼脂糖的衍生物;透明质酸或透明质酸的衍生物;壳聚糖;明胶;和葡聚糖。
5.根据权利要求1-4中任意一项所述的组合物,其中,所述目的蛋白质是参与组织修复、组织再生或组织生成的蛋白质。
6.根据权利要求1-5中任意一项所述的组合物,其中,所述蛋白质选自磷酸调节性中性内肽酶,X-连接的(由PHEX编码),热休克蛋白90-α(由HSP90AA1编码),腱蛋白样2(由CHRDL2编码),短瞬时受体电位通道4(由TRPC4编码),泛连接蛋白3(由PANX3编码),胶原XXIV型α1(由COL24A1编码),ATP283的基因产物,含有普列克底物蛋白同源结构域的B1(由PLEKHB1编码),白细胞免疫球蛋白样受体亚家族B成员4(由LILRB4编码),茴香胺5(由ANO5编码),ChaC谷胱甘肽特异性-谷氨基环转移酶1(由CHAC1编码),轴丝中链动力蛋白(由DNAI2编码),聚糖(由ACAN编码),整合素α10(由ITGA10编码),纤调蛋白(由FMOD编码),分泌型磷蛋白1(又称骨桥蛋白,由SPP1编码),载脂蛋白C1(由APOC1编码),负调控蛋白2(由PTCH2编码)、载脂蛋白E(由APOE编码)、髓系细胞上触发受体2(由TREM2编码)、平足蛋白(由PDPN编码)、溶质载体家族13成员5(由SLC13A5编码)、T-细胞可诱导共刺激分子(由ICOS编码)、甲酰肽受体2(由FPR2)编码、基质金属肽酶12(由MMP12编码)、肌腱蛋白N(由TNN编码)、天冬蛋白(由ASPN编码)、粒酶A(由GZMA编码)、骨诱导因子(由OGN编码)、Wnt家族成员2(由WNT2编码)、神经肽Y(由NPY编码)、骨形态发生蛋白2(由BMP-2编码)、具有序列相似性家族169成员A(由FAM169编码)、视黄醇结合蛋白1(由RBP1编码)、ISG15泛素样修饰因子(由ISG15编码)、三碱基假激酶3(由TRIB3编码)、泛素特异性肽酶18(由USP18编码)、紧密连接蛋白(由CLDN1编码)、肌纤蛋白(由MYOC编码)、C-X-C基序趋化因子配体9(由CXCL9编码)、具有四肽重复序列3的干扰素诱导蛋白(由IFIT3编码)、RRAD、RAS相关糖酵解抑制剂和钙通道调节剂(由RRAD编码)干扰素α诱导蛋白27(由IFI27编码)。
7.根据权利要求1-6中任意一项所述的组合物,其中,其用于诱导或促进人或动物受试者中组织的修复、再生或产生的方法中,所述方法包括将所述组合物施用至待修复、再生或产生的组织的部位。
8.根据权利要求7所述的用于应用的组合物,其中,所述用于诱导或促进组织的修复、再生或产生的方法是用于促进或改善由于创伤和/或疾病而损伤的组织的愈合。
9.根据权利要求7或8所述的用于应用的组合物,其中,所述组织是骨组织。
10.根据权利要求9所述的用于应用的组合物,其中,用于诱导或促进组织修复、再生或产生的方法与脊柱融合或在骨组织中植入植入物有关,所述植入物例如假体关节和牙齿植入物。
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