CN116790610A - 一种治疗骨科疾病的基因及以aav为载体的基因治疗药物 - Google Patents
一种治疗骨科疾病的基因及以aav为载体的基因治疗药物 Download PDFInfo
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Abstract
本发明提供一种治疗骨科疾病的基因,其为SEMA3Aopt,并提供了一种复制缺陷型腺相关病毒AAV6‑SEMA3A,其包载了SEMA3Aopt基因,该复制缺陷型腺相关病毒能够在病灶过表达SEMA3Aopt,该重组腺相关病毒改变患病部位炎症微环境,并调节病灶部位淋巴细胞分群,恢复免疫平衡以改善CIA模型小鼠脚掌炎症和骨侵蚀以及OVX模型小鼠骨质疏松的情况。另外由于该重组腺相关病毒能够有效抑制关节部位炎症水平,因此还具有治疗骨关节炎的能力。综上,本发明的AAV6‑SEMA3A复制缺陷型腺相关病毒是一种针对骨科疾病的基因治疗药物,所述骨科疾病包括类风湿性关节炎、骨质疏松和/或骨关节炎。
Description
技术领域
本发明涉及一种骨科疾病治疗基因SEMA3A及复制缺陷型重组腺相关病毒,其过表达SEMA3A,可作为基因治疗药物用于治疗类风湿性关节炎、骨关节炎和/或骨质疏松。本发明属于生物工程技术领域。
背景技术
类风湿性关节炎(Rheumatoid Arthritis,RA)是一种自身免疫疾病,其特征表现为持续性滑膜炎、关节损伤以及全身性免疫反应失调。在工业化国家,有0.5%-1.0%的成年人罹患这种疾病,而且女性和老年人是这种疾病的高发人群。若对该疾病不进行控制会导致患者关节损伤、残疾、生活质量下降以及心血管及其他合并症。
目前治疗RA的药物可以分为四大类:(1)抗炎药物,即糖皮质激素类和非甾体抗炎药物;(2)传统抗风湿药物(DMARDs),主要是甲氨蝶呤;(3)生物DMARDs,主要是TNF-α抑制剂(例如阿达木单抗)、IL-6抑制剂(例如托珠单抗)、T细胞共刺激阻断剂(例如阿巴西普)和B细胞耗竭疗法(例如利妥昔单抗);(4)靶向合成DMARDs,目前主要是JAK抑制剂,例如托法替尼。当关节炎不受控制时或传统DMARDs联合类固醇药物出现不良反应时,可以考虑使用生物DMARDs或靶向合成DMARDs。但是这类方案都有一定的局限性,例如生物DMARDs的使用会抑制免疫系统的运行,往往提高患者罹患感染和肿瘤的风险。
骨关节炎是一种退行性病变,通常是由衰老、肥胖、劳损、创伤、先天性关节异常和畸形等因素引起的关节软骨退化损伤,常表现为关节功能障碍及炎症症状。目前广泛认为促炎型巨噬细胞是该种疾病的驱动因素,由于促炎巨噬细胞(即M1型巨噬细胞)能够分泌大量促炎细胞因子和基质金属蛋白酶,前者加剧关节部位炎症微环境,后者则直接针对软骨,导致软骨破坏。
由于骨关节炎是由多种因素引起,因此目前尚无有效的手段来阻止骨关节炎的进展,主要的治疗目的是缓解疼痛、改善关节功能,从而提高患者生活质量。目前主流的药物是糖皮质激素,虽然糖皮质激素能够显著改善炎症反应,但是往往会导致关节软骨的破坏加剧,这可能是由于糖皮质激素会促进软骨细胞凋亡并抑制软骨细胞分化而导致的。
骨质疏松是一种由于成骨细胞分化受到抑制,破骨细胞分化增强而导致骨稳态被打破,从而造成骨吸收大于骨形成的骨质流失性疾病。骨质疏松会导致骨骼强度明显降低,使骨折的风险提升。骨质疏松会影响约一半的女性和三分之一的男性,该种疾病患者的晚期生活水平会显著降低。
目前用于治疗骨质疏松的药物主要可以分为雌激素类制剂、双膦酸盐类制剂和甲状旁腺激素制剂。但是雌激素制剂的长期给药可能会增加乳腺癌的发病几率;双膦酸盐则大多通过口服给药,容易出现胃肠道刺激;而甲状旁腺激素制剂一般指特立帕肽,虽然治疗效果很好,但是患者终身只能接受一次为期24个月的治疗,这是由于特立帕肽会在体内起到PTH样作用,当体内PTH量过大时则会过度激活破骨细胞活性,导致骨质流失严重,从而加重骨质疏松的病症。
Semaphorin3A是信号素家族(Semaphorins)的一员,简称SEMA3A。SEMA3A是一种80KDa的分泌型蛋白,是一种新兴治疗靶点。类风湿性关节炎患者体内SEMA3A含量较正常人群显著减少,同时人们了解到SEMA3A主要由内皮细胞、T细胞和DC细胞分泌,其作用有三个方面:(1)能够促进成骨细胞生成并抑制破骨细胞的分化;(2)SEMA3A可以直接作用于T细胞,阻断细胞肌动蛋白骨架重组,从而影响TCR极化,从而抑制T细胞的活化、诱导Treg细胞的产生(3)SEMA3A可以诱导巨噬细胞复极化,使促炎型的M1巨噬细胞复极化为M2型,从而改善炎症环境。因此SEMA3A能够直接影响成骨和破骨细胞分化,同时通过调控免疫微环境间接影响成骨和破骨细胞分化从而恢复骨稳态,并抑制炎症,从而达到治疗类风湿性关节炎、骨关节炎和骨质疏松的目的。
相较于传统直接递送目标蛋白的策略,基因疗法由于其能够有效转移目的基因而逐渐走入人们的视野。生物DMARDs如阿达木单抗等,多采用全身给药,这导致需要大量且重复多次给药,才能满足给药剂量,达到治疗作用,这导致一方面成本增加,另一方面也增加了副作用产生的可能性同时降低患者顺应性。
目前已上市基因递送载体主要可分为病毒类载体和非病毒类载体。病毒类载体主要包括逆转录病毒、腺病毒和腺相关病毒;非病毒类载体则主要是阳离子脂质体和脂质纳米颗粒(Lipid Nanoparticle,LNP)。其中逆转录病毒存在基因组整合风险,腺病毒则免疫原性较高且大部分人群具有很高的预存中和抗体,有研究证明过高的预存中和抗体会使腺病毒作用效果大大减弱;而阳离子脂质体则由于毒性大,给药途径受限,LNP则制备难度高,且目前应用范围仅限于疫苗领域。腺病毒载体与非病毒类载体都存在表达时间短,针对慢性疾病的治疗需要面临反复多次给药的问题。
腺相关病毒(Adeno-Associated Virus,AAV)作为一种基因递送病毒类载体,其属于细小病毒科内的依赖细小病毒属,在多款基因治疗上市药物中被使用,其具有低免疫原性,高安全性,长期稳定表达目的基因等优点,这对于治疗基因递送有着显著的帮助。因此构建一种重组腺相关病毒,其可过表达SEMA3A,从而作为治疗骨科疾病的药物,具有重要意义。
发明内容
本发明的目的在于针对现有技术的不足,提供一种治疗骨科疾病的基因。
本发明的目的还在于提供含有所述的治疗骨科疾病的基因的蛋白表达框及载体系统。
本发明的目的还在于提供表达SEMA3A蛋白的重组腺相关病毒。
本发明的目的还在于提供所述的治疗骨科疾病的基因、蛋白表达框、载体及重组腺相关病毒在制备治疗类风湿性关节炎、骨质疏松症和/或骨关节炎的药物中的应用。
本发明的目的可通过以下技术方案实现:
本发明提供了一种治疗骨科疾病的多核苷酸,所述多核苷酸的序列如SEQ ID NO:1所示。
本发明提供了一种包含所述多核苷酸序列(SEQ ID NO:1)的载体,其特征在于,所述载体的蛋白表达框由增强子-启动子-目的基因序列-polyA信号或由启动子-目的基因序列-polyA信号组成,其中,启动子选自CAG启动子、CMV启动子、SV40启动子、CBh启动子、SFFV启动子、MSCV启动子、EF1α启动子、软骨细胞特异的COL2A1启动子、软骨细胞特异的COL1A1启动子、肌肉特异的MHCK7启动子、肌肉特异的ACTA1启动子的一种或多种,启动子优选为CAG启动子,载体优选为pAAVCAG-MCS
本发明提供了一种复制缺陷型重组腺相关病毒,其特征在于,所述重组腺相关病毒包含如SEQ ID NO:1所示多核苷酸及蛋白表达框。
其次基于背景所提到的安全长效的目的,本发明选择使用以天然和人工设计的复制缺陷型腺相关病毒作为基因递送载体,其特征在于所述的复制缺陷型腺相关病毒的血清型,包括但不限于天然的AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV-DJ、AAV-DJ8、AAV-DJ9、AAVrh8、AAVrh8R和AAVrh10,以及人工设计的腺相关病毒血清型包括但不限于7m8,优选为天然的AAV6。
本发明还包括将上述所述的多核苷酸、所述的蛋白表达框、所述的载体、所述的重组腺相关病毒用于在制备治疗类风湿性关节炎、骨质疏松症和或骨关节炎的药物中的应用。
进一步,所述药物为液体制剂、固体制剂。
进一步,所述液体制剂的给药途径为关节腔内注射、肌肉注射、静脉注射、经皮给药的一种或多种,优选的经皮给药为皮下注射、微针的任一一种或多种,所述类风湿性关节炎、骨关节炎治疗优选为关节腔内注射,所述骨质疏松治疗优选为肌肉注射。
本发明还提供一种制备所述重组腺相关病毒的制备方法,所述方法包括以下步骤:
(1)构建含多核苷酸的顺式质粒载体;
(2)构建含有血清型6的AAV衣壳质粒;
(3)将步骤(1)和(2)所述质粒与辅助质粒一起在PEI的作用转染至宿主细胞;
(4)37℃5%CO2条件下培养宿主细胞;
(5)将步骤(4)所述宿主细胞收集裂解,收货含有复制缺陷型重组腺相关病毒的病毒液;
(6)对步骤(5)所述病毒液进行纯化即得。
优选的,步骤(1)所述顺式质粒载体为pAAVCAG-MCS。
优选的,步骤(2)所述衣壳质粒载体为pAAV2/6。
优选的,步骤(3)所述辅助质粒为pHelper。
优选的,步骤(4)所述宿主细胞为AAV293细胞。
优选的,步骤(6)所述纯化方法为POROSTM CaptureSelectTM AAVX亲和层析法。
本发明提供如上所述的制备方法制备的可表达SEMA3A蛋白的复制缺陷型重组腺相关病毒在作为/制备治疗类风湿性关节炎、骨质疏松症和/或骨关节炎的药物中的应用。
有益效果:
本发明提供一种多核苷酸序列,其如SEQ ID NO:1所示,经密码子优化后可明显提高SEMA3A蛋白的表达量,同时本发明提供了一种治疗类风湿性关节炎、骨质疏松症和/或骨关节炎的基因及以复制缺陷型腺相关病毒为载体的基因治疗药物(AAV6-SEMA3A),其在CIA小鼠模型(胶原诱导的关节炎模型)上表现出良好的抑制炎症和骨保护的效果,相较于上市药物Adalimumab,AAV6-SEMA3A能够更加有效地减轻小鼠脚掌肿胀和炎症程度,同时在改善关节部位的炎症环境和保护关节及脚掌部位骨骼方面有更好的效果。CIA小鼠模型是由胶原蛋白诱导产生的关节部位炎症,能够一定程度代表人类骨关节炎的大部分症状,由于AAV6-SEMA3A能够显著抑制关节部位的炎症同时保护软骨,因此AAV6-SEMA3A也能治疗骨关节炎;同时在OVX小鼠模型(绝经后骨质疏松模型)上,AAV6-SEMA3A相较于雌二醇,虽在恢复骨量方面相当,但是在调节骨免疫稳态方面效果更佳,表明其有治疗骨质疏松症的作用。相较于上市药物Adalimumab和雌二醇,AAV6-SEMA3A无需频繁给药,大大提高患者顺应性,同时降低多次给药带来的不良反应,降低了治疗成本,为患者降减轻了经济负担。
附图说明
图1.顺式质粒pAAVCAG-SEMA3Aopt图谱。
图2.SEMA3A蛋白由AAV293细胞表达并分泌的Western blot鉴定图。
图3.密码子优化后SEMA3A蛋白表达载体体外表达。
图4.重组腺相关病毒(AAV6-SEMA3A)的银染和透射电镜图。
图5.AAV5-Luc,AAV6-Luc,AAV8-Luc在DBA/1小鼠踝关节腔注射后荧光素酶表达活体图。
图6.AAV5-Luc,AAV6-Luc,AAV8-Luc在DBA/1小鼠踝关节腔注射后荧光素酶表达水平随时间变化比较图。
图7.AAV6-SEMA3A在CIA模型上的脚掌厚度及临床评分随时间变化比较图。
图8.CIA小鼠治疗终点第52天各组踝关节及脚掌部位Micro-CT重构图。
图9.CIA小鼠治疗终点第52天各组关节部位促炎细胞因子水平图。
图10.CIA小鼠治疗终点第52天各组关节部位T细胞活化、巨噬细胞极化及滑膜巨噬细胞流式分析图。
图11.CIA小鼠治疗终点第52天各组关节H&E染色、SF染色、TRAP染色分析图。
图12.OVX小鼠治疗终点(造模后12周)各组股骨骨小梁Micro-CT重构图。
图13.OVX小鼠治疗终点(造模后12周)各组股骨骨小梁指标。
图14.OVX小鼠治疗终点(造模后12周)各组骨髓巨噬细胞极化及Treg细胞流式分析图。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的权利要求所限定的保护范围构成任何限制。
实施例1.构建编码SEMA3A的6型复制缺陷型重组腺相关病毒
一、载体构建和SEMA3A体外表达鉴定
1、载体构建
全基因合成SEMA3A野生型序列(SEMA3A wt,NCBI数据库NM_001243072.1(382bp-2700bp))和经过密码子优化后的SEMA3A基因序列(SEMA3A opt,见SEQ ID NO:1)通过酶切,将其连接至pAAVCAG-MCS顺式质粒(addgene),转化stable3感受态,涂布Ampr LB平板,挑单克隆进行菌落PCR鉴定,并对PCR鉴定为阳性的克隆进行测序验证。构建得到pAAVCAG-SEMA3Awt(图1A,SEQ ID NO:2)和pAAVCAG-SEMA3Aopt(图1B,SEQ ID NO:3)表达载体。
2、SEMA3A的体外表达鉴定
使用PEI 40000(Polyscience)将pAAVCAG-SEMA3Aopt质粒转染至AAV293细胞,48小时后收集细胞与培养上清液进行Western Blot(WB)检测。实验方法如下:
转染:将AAV293细胞以6×105细胞/孔接种于6孔板中,于37℃、5%CO2条件下培养过夜。转染前2h,将培养基换为新鲜的含10%FBS的DMEM培养基。转染时,每个转染孔取对应质粒2ug,加入到200ul无FBS的DMEM培养基中,混匀,加入PEI40000(2.6ul,1mg/ml),涡旋15s混匀后,室温静置15min。然后,将质粒转染混合液按上述质粒计量加入6孔板中,混匀。细胞于37℃、5%CO2细胞培养箱中培养孵育8h,将培养基换成新鲜的含5%FBS的DMEM培养基,48h后收集培养上清和细胞,制备样品,进行WB检测。
样品制备:培养48h后,分别收集培养上清和细胞,上清液记为细胞培养上清样品。使用预冷的PBS润洗细胞2次,每孔加入200ul RIPA裂解液(含1%PMSF),冰浴条件下裂解细胞,15min后将细胞吹下,4℃ 12000rpm离心15分钟,取上清液,记为细胞沉淀样品。各取一份细胞上清样品与细胞沉淀样品,分别加入适量含有β-巯基乙醇的5×SDS-PAGE上样缓冲液,95℃加热10min,冻存备用,即可用于WB检测。
WB检测:使用10%SDS-PAGE胶,每孔上样量为20ul。电泳条件为80V,30min;120V,1.5h。跑胶结束后,使用湿法转膜,将SDS-PAGE胶上的蛋白质条带转移至PVDF膜上,电转条件为300mA,1.5h。转膜结束后,将PVDF膜使用5%脱脂奶粉室温封闭1h,将PVDF膜用TBST洗涤3次,每次于摇床上80rpm摇动10min,然后按照1:1000的稀释度加入抗SEMA3A蛋白鼠单克隆抗体(Abcam,货号199475),4℃摇床60rpm孵育过夜。将PVDF膜洗涤3此后,加入用5%脱脂奶粉以1:5000稀释的HRP标记的山羊抗兔IgG抗体(thermo scientific,31430),室温60rpm摇动下孵育1h。使用TBST洗涤PVDF膜3次后,使用ImmobilonTM Western ChemiluminescentHRP Subsrate(MILLIPORE,Cat#WBKLS0500)进行化学发光反应,使用凝胶成像仪采集不同曝光时间的图像。
结果如图2所示,其中SEMA3A supernatant为转染pAAVCAG-SEMA3Aopt质粒细胞的细胞沉淀样品;SEMA3A lysate为转染pAAVCAG-SEMA3Aopt质粒细胞的细胞培养上清样品;Blank supernatant为未做转染的空白细胞的细胞沉淀样品;Blank lysate为未做转染的空白细胞的细胞培养上清样品。结果表明该质粒表达框能够成功表达SEMA3A蛋白,并将其分泌至胞外。
3、密码子优化SEMA3A表达验证
使用PEI 40000(Polyscience)将pAAVCAG-SEMA3Awt质粒和pAAVCAG-SEMA3Aopt质粒分别转染至AAV293细胞,48小时后收集细胞,提取细胞蛋白后通过Western Blot对SEMA3A蛋白水平进行比较。结果显示经过密码子优化后可以显著提高蛋白表达水平(图3A),通过灰度分析比较,密码子优化后SEMA3A蛋白表达水平提高了3.23倍(图3B)。
二、重组腺相关病毒包装、纯化和鉴定
1、重组腺相关病毒包装
将以上构建好的顺式质粒pAAVCAG-SEMA3Aopt、辅助质粒pHelper分别与pAAV2/6腺相关病毒衣壳质粒共转染AAV293细胞,进行重组腺相关病毒的包装。过程如下:
(1)将AAV293细胞接种于40个150mm培养皿中,培养基为含10%FBS的DMEM培养基,置于37℃ 5%CO2细胞培养箱中培养过夜。
(2)待细胞生长至融合度为80%时,转染前2h将培养基更换为新鲜的含10%FBS的DMEM培养基。取上述质粒按PEI 40000(Polyscience,USA)所附说明书进行转染。具体步骤为:
(a)取顺式质粒pAAVCAG-SEMA3Aopt 260μg,野生型衣壳质粒pAAV2/6 260μg,辅助质粒pAdDeltaF6 520μg混和均匀;质粒使用40mL不含血清的DMEM培养基进行稀释。
(b)取1.25mL PEI 40000(1mg/mL)加入到稀释于培养基中的质粒,涡旋30秒使其充分混匀,室温下静置15min备用。
(c)将PEI与质粒混合液均匀加至40皿细胞中。
(3)转染16h后,将培养基更换为含4%FBS的DMEM培养基继续培养56小时。
(4)将细胞收集于离心管中,4000rpm离心15min,弃上清,加入10ml RB缓冲液(1mMMgCl2,50mM Tris·Cl,pH 7.4)重悬细胞沉淀,室温下静置10min后,14000g离心15min,收集上清液,加入2ml NB缓冲液(2MHEPES,pH 8.0)混合均匀,加入适量5M NaCl溶液调整NaCl浓度至200mM,得到含有病毒的上清液,使用0.45umPES膜过滤后,得到病毒液备用。
2、重组腺相关病毒纯化
(1)平衡纯化柱柱:使用平衡缓冲液(含200mM NaCl的PBS缓冲液,pH 7.4)以2ml/min的流速冲洗纯化柱,直至洗脱液在UV280nm吸收峰不再变化。
(2)上样:使用POROSTM CaptureSelectTM AAX亲和介质进行后续重组腺相关病毒的分离纯化。将上述病毒液以1ml/min流速上柱,结束后使用平衡缓冲液重复步骤(1)操作。
(3)洗脱:使用洗脱缓冲液(50mM柠檬酸,50mM柠檬酸钠,pH3.0)以2ml/min流速洗脱,待洗脱液出现明显UV280nm吸收峰时,收集对应洗脱液,并立即加入中和缓冲液(1MTris,pH 8.0)中和病毒纯化液。将表达SEMA3A的6型复制缺陷型重组腺相关病毒记为:AAV6-SEMA3A。
此外,为方便实验更好地开展与结果展示,本文引入报告基因Luciferase携载顺式质粒pAAVCAG-Luc(Addgene,货号83281)进行不同血清型报告基因携载重组病毒包装纯化,其包装纯化方法同上述一致,将表达Luciferase的各血清型重组腺相关病毒记为:AAV5-Luc,AAV6-Luc,AAV8-Luc。
3、重组腺相关病毒滴度与纯度鉴定
(1)滴度测定
设计目的基因序列的qPCR特异性引物,序列5’-3‘如下:
SEMA3A-F:AGACAGCCACTTCGAGAACG;
SEMA3A-R:GTCCCTTCCCATGAAGTCGG;
Luc-F:CCCATCTTCGGCAACCAGAT;
Luc-R:CCCATCTTCGGCAACCAGAT。
病毒预处理:
分别取10μL病毒液+5μL DNA酶反应缓冲液+1μL DNase I+34μLDEPC水,于37℃孵育30min后,立即95℃孵育5min以终止反应,结束后立即置于冰上,并按下表制备成不同稀释倍数样品:
标准曲线样品处理:
将携带目的基因的顺式质粒pAAVCAG-SEMA3Aopt或pAAVCAG-Luc,使用BamHI酶进行线性化,经酚氯仿-乙醇沉淀纯化回收后,按如下计算公式,配制成2×109molecules/μL储备液,作为制备标准曲线的样品:
Cmol代表质粒分子数浓度单位molecules/μL;Cm代表质粒质量浓度单位μg/μL;l代表质粒大小单位bp;NA代表阿伏伽德罗常数数值6.02×1023。
随后按下表配制标准曲线各浓度梯度:
反应体系:
总体积20μL:模板5μL,TaqMan Fast Advanced Master Mix 10μL(Thermo,4444557),上下游引物、探针终浓度均为0.2μM,剩余体积无酶水补齐。
反应条件(Roche,Lightcycler96):
50℃ UNG酶孵育2分钟,95℃预变性20秒;95℃变性3秒,60℃退火并延伸30秒(获取信号),共39个循环;熔解程序(默认)。病毒滴度(Genome Copies/mL)根据标曲与样品对应稀释倍数求得。AAV6-SEMA3A,AAV5-Luc,AAV6-Luc,AAV8-Luc的滴度分别为:1.03E+12vg/ml,3.28E+12vg/ml,1.37E+12vg/ml,3.8E+11vg/ml。
(2)病毒纯度鉴定
取10μL待测样品(AAV6-SEMA3A)加入2.5μL 5×SDS-PAGE上样缓冲液,于95℃变性10min后,使用10%SDS-PAGE胶进行SDS-PAGE电泳。结束后参考银染试剂盒说明书(赛维尔,G2080-25T)进行银染确定病毒纯度,结果如图4-A所示,泳道内只有代表衣壳蛋白的三条带,分别是60kDa左右的VP3,75kDa代表病毒左右的VP2,以及90kDa左右的VP1,显示病毒样品的纯度高。取15μL待测样品(AAV6-SEMA3A)进行磷钨酸负染,透射电镜拍摄病毒形态,结果如图4-B所示,显示病毒纯度高同时空壳率低。
实施例2.不同血清型复制缺陷型重组腺相关病毒对小鼠进行踝关节腔内注射后感染效率
按照实施例1中方法得到的AAV5-Luc,AAV6-Luc,AAV8-Luc进行下述实验:
选取6周龄雄性DBA/1小鼠(购自江苏集萃药康),使用Hamilton注射器通过双侧踝关节关节腔内注射AAV5-Luc,AAV6-Luc,AAV8-Luc(病毒用量均为3.84E+9vg/ankle),分笼饲养保证小鼠正常进食进水,注射后3天、7天、14天、21天分别对小鼠腹腔注射底物D-荧光素钾盐(200ul/只),使用小动物活体成像仪拍摄检测给药部位发光信号强度。
荧光素酶表达强度通过小动物活体成像系统(Lumina LT Series III,PerkinElmer,德国)分析,结果见图5,并将荧光强度水平随时间变化曲线以图6表示。结果表明,AAV6-Luc对小鼠踝关节的感染效率最高,且能维持一定时间,因此AAV6适合作为小鼠踝关节腔基因递送载体。
实施例3.AAV6-SEMA3A在胶原诱导的关节炎小鼠模型的药效学考察一、胶原诱导的关节炎小鼠模型(CIA)的构建:
选取29只6周龄雄性DBA/1小鼠,按照下述方法进行CIA模型的构建:
(1)第0天初次造模:选用6周龄的DBA/1雄性小鼠,先将0.6ml牛2型胶原蛋白溶液(2mg/ml)与0.6ml(4mg/ml)完全弗氏佐剂装入挤膜器中,制备得到1ml左右乳白色乳剂。小鼠每只尾根部皮下注射100μl乳剂,观察尾根部肿胀且乳剂未漏出,小鼠未死亡,即说明给药基本成功。
(2)第21天二次造模:将0.6ml牛2型胶原蛋白溶液(2mg/ml)与0.6ml(4mg/ml)不完全弗氏佐剂装入挤膜器中,制备得到1ml左右乳白色乳剂,小鼠每只尾根部皮下注射100μl乳剂。
二、给药方案及分组:
将造模后的29只DBA/1小鼠随机分为5组,5只健康DBA/1小鼠作为组,根据表1所示分组情况于第22天给药。/>为未造模健康小鼠;Control为造模后未给药组;Adalimumab为阿达木单抗(AbbVie,America)给药组;AAV6-SEMA3A i.a为AAV6-SEMA3A双侧踝关节腔注射组;AAV6-SEMA3A i.m为AAV6-SEMA3A双侧小腿腓肠肌注射组。阿达木单抗作为上市药品中治疗类风湿性关节炎的经典药物,因此选用作为对照。
表1.DBA/1小鼠分组和给药情况
药效学评价及机制探究
1、脚掌厚度监测及临床评分
给药后开始对各组小鼠双侧后脚掌进行厚度监测及临床评分。隔天测量并评分一次,采用游标卡尺测量脚掌厚度,临床评分标准见表2,临床评分采取双侧后脚掌评分之和。
表2.临床评分标准
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各时间点脚掌厚度及临床评分见图7。结果表明,AAV6-SEMA3A踝关节腔内注射临床评分与脚掌厚度效果最好,肌肉注射组也有药效但效果略差于关节腔给药组,并且两组药效都优于阿达木单抗给药组。可见,本发明提供的表达SEMA3A基因的腺相关病毒效果显著优于现有经典药物阿达木单抗,且给药仅需一次,有助于提高患者顺应性。(ns,P≥0.05;*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001)
2、踝关节及脚掌micro-CT
微计算机断层扫描(Micro Computed Tomography,Micro-CT),是一种非破坏性的3D成像技术,可以在不破坏样本的情况下清除观察到样本内部的显微结构,分辨率高,可以达到微米级别。采用Micro-CT对各组小鼠踝关节及脚掌进行3D拍照,可以直观了解骨表面的侵蚀程度,结果见图8。
结果表明,Control组由于发病,踝关节及脚掌部位骨表面侵蚀严重,关节结构不清晰;Adalimumab组脚趾关节部位有明显骨侵蚀现象,关节结构不清晰;而经AAV6-SEMA3A治疗后踝关节及脚掌骨表面平整,关节结构清晰,说明AAV6-SEMA3A能够有效保护类风湿性关节炎所造成的骨损伤,可见,本发明的AAV6-SEMA3A具有骨保护作用,且效果优于Adalimumab。
3、关节提取液细胞因子水平测定
(1)关节提取液的制备
于治疗终点(第52天)将各组小鼠断颈处死后,将踝关节及跗骨关节部位剪下,去除皮肤,称量后低温冷冻研磨,加入1ml预冷PBS(含1%PMSF)再度研磨后,10000g离心15min,取上清液,即为关节提取液。
(2)ELISA法测定关节提取液细胞因子
酶联免疫吸附法(Enzyme-linked immunosorbent assay,ELISA)是依据抗原抗体特异性结合而定量测定样品中细胞因子或抗体的一种免疫学方法,特异性好,灵敏度高,常用于免疫学研究。为了探索AAV6-SEMA3A在CIA模型上的药效机制,采用ELISA法分析了各组小鼠关节提取液中的多种细胞因子水平,包括TNF-α、IL-6、IL-1β,结果见图9。
结果表明AAV6-SEMA3A治疗组无论是关节腔注射还是肌肉注射,都能有效降低小鼠踝关节促炎细胞因子水平,阿达木单抗组虽然也有类似效果,但是AAV6-SEMA3A关节腔给药组效果最好,说明AAV6-SEMA3A能有效降低炎症细胞因子水平。(ns,P≥0.05;*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001)
3、踝关节淋巴细胞表型
类风湿性关节炎患者的关节部位常出现淋巴细胞浸润的现象,主要是活化的T细胞以及促炎巨噬细胞,同时滑膜细胞主要可以分为滑膜巨噬细胞和滑膜成纤维细胞,其中滑膜巨噬细胞为类风湿性关节炎的主要致病细胞。巨噬细胞可按照表面分子不同,可分为经典活化的M1型和替代性活化的M2型。M1型巨噬细胞主要分泌促炎细胞因子,如TNF-α等,M2型巨噬细胞主要分泌抑炎细胞因子,如IL-10等。正常生理条件下,M1和M2型巨噬细胞维持相对平衡,然而在类风湿性关节炎存在的情况下,平衡被打破,分泌促炎细胞因子的M1型巨噬细胞占据主导,M2型巨噬细胞减少。因此我们通过检测各组踝关节部位T细胞活化、巨噬细胞极化情况以及滑膜巨噬细胞量,考察AAV6-SEMA3A能否降低T细胞活化、诱导巨噬细胞复极化以及减少滑膜巨噬细胞从而治疗类风湿性关节炎。方法如下:
于治疗终点(第52天)将各组小鼠处死后,将踝关节及跗骨关节部位剪下,置于5mlEP管中,加入2ml四型胶原酶(1mg/ml)37℃摇床振摇消化25min,消化后用剪刀剪碎,置于70um筛网研磨至PBS中,3000rpm离心4min,弃掉上清,加入1mlPBS洗涤一遍,3000rpm离心4min,弃掉上清,即可进行染色,流式检测。其中,采用F4/80+、CD86+、CD206-标记M1型巨噬细胞,采用F4/80+、CD86+、CD206+标记M2型巨噬细胞;采用CD3+、CD4+、CD69+标记活化的T细胞;采用CD68+标记滑膜巨噬细胞,结果见图10。
结果表明Adalimumab并不能有效降低关节部位的M1型巨噬细胞,而AAV6-SEMA3A无论是关节腔给药还是肌肉注射给药,均能明显降低关节部位T细胞活化程度,同时诱导M1型巨噬细胞复极化,恢复M1/M2平衡,并能显著减少滑膜巨噬细胞,从而达到治疗类风湿性关节炎的效果,可见AAV6-SEMA3A相较于Adalimumab能更好地改善关节部位的炎症微环境。(ns,P≥0.05;*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001)
4、关节部位H&E、SF及TRAP染色情况
为了更直观表现各组关节部位炎症及软骨破坏情况,将各组小鼠踝关节部位剪下固定、脱钙、切片、染色。其中H&E染色是组织病理学研究常用的方法,可以反映炎症关节的病理特征;SF染色能观察关节部位软骨的含量与结构从而间接反映炎症对软骨的侵蚀情况;TRAP染色,能够将关节部位破骨细胞含量清晰表示,破骨细胞具有骨吸收的作用而能降低骨密度,侵蚀骨组织,可以间接反映关节部位骨损伤情况,染色结果见图11。
结果表明AAV6-SEMA3A关节腔给药组,H&E染色关节组织结构清晰,软骨表面光滑,软骨细胞数量丰富,且未见明显炎症细胞浸润;SF染色软骨表面光滑,数量丰富,排列规则;而TRAP染色,相较于Control组,破骨细胞较少,几乎没有。说明AAV6-SEMA3A关节腔给药能明显改善踝关节部位炎症情况,并减少炎症细胞浸润,同时保护软骨,减少破骨细胞生成。虽然肌肉注射组相对于关节腔给药效果略差,但依旧远好于Adalimumab组和Control组。
实施例4.AAV6-SEMA3A在卵巢摘除的小鼠骨质疏松模型上的药效学考察.
一、卵巢摘除的小鼠骨质疏松模型(OVX)的构建:
选取53只8周龄雌性C57BL/6J小鼠,腹腔注射2%三溴乙醇麻醉后,手术摘取双侧卵巢。同时建立假手术组(Sham),即制造同样的创口但是不摘取卵巢。
二、给药方案及分组
将造模后的小鼠随机分为6组,10只健康小鼠作为组,根据表3所示分组情况于造模后第7周开始给药。/>为未造模健康小鼠;Sham为假手术组;Control为造模后不给药组;E2为雌二醇肌肉注射;AAV6-SEMA3A i.a为AAV6-SEMA3A双侧膝关节腔注射组;AAV6-SEMA3A i.m为AAV6-SEMA3A双侧小腿腓肠肌注射。雌二醇常在临床用于骨质疏松的治疗,因此作为阳性对照。
表3.C57BL/6J小鼠分组和给药情况
三、药效学评价
1、小鼠股骨micro-CT
于治疗终点(造模后12周)将各组小鼠断颈处死,分离股骨并去除肌肉后,使用4%多聚甲醛固定过夜后,用PBS洗涤漂洗三次,保存于PBS中,使用micro-CT(SCANCO MedicalAG vivaCT80)自骨垢线起往下纵向扫描100层,对其分析重构股骨3D图像并测量小鼠股骨骨小梁指标,包括骨小梁数量(Tb.N)、骨小梁分离度(Tb.sp)、小梁骨体积/组织体积(BV/TV)、骨小梁结构模型指数(SMI),结果见图12和图13。结果表明AAV6-SEMA3A通过肌肉注射和膝关节腔内注射均可改善小鼠股骨骨小梁缺损情况,但是肌肉注射相较于膝关节腔内注射能更好地恢复股骨的骨体积比,表明效果更优。AAV6-SEMA3A在恢复骨量上与雌二醇相当,但是雌二醇给药次数更加频繁,而AAV6-SEMA3A仅需一次给药,这有助于提高患者的顺应性同时减少频繁给药带来的针刺伤害。(ns,P≥0.05;*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001)
2、小鼠骨髓巨噬细胞极化及Treg
骨质疏松患者常伴有骨部位的炎症反应,而M1型巨噬细胞是主要的促炎细胞因子分泌细胞,同时调节性T细胞(Treg)能够分泌抑炎细胞因子并维持免疫稳态,因此考察骨髓部位M1型巨噬细胞和Treg细胞能够反映治疗药物的抑炎能力。
(1)骨髓细胞悬液制备
处死小鼠后,使用75%乙醇溶液消毒,剪开小鼠后肢双腿,分离股骨和胫骨,并将毛发、皮肤和肌肉尽量剔除,放置于75%乙醇溶液中浸泡除菌,之后转移至超净台内,使用无菌PBS溶液冲洗3遍,转移至1640培养基中,将骨头两端剪去后,使用1ml无菌注射器吸取1640培养基,从骨头一端插入冲洗骨髓腔内的骨髓,反复冲洗3次用70um无菌细胞筛网过滤骨头碎片及组织团块后,收集骨髓悬液于离心管中,1000rpm离心10min,得到细胞沉淀,使用500ul 1640培养基重悬后,稀释50倍计数,调整细胞浓度为2E+7cell/ml,得到骨髓细胞悬液,进行后续操作。
(2)骨髓巨噬细胞极化及Treg细胞
将上述骨髓细胞种于12孔板中,每孔加入400ul骨髓细胞悬液+600ul1640培养基,37℃孵箱孵育3天,离心收集细胞培养上清,用于细胞因子检测,细胞沉淀加入PBS重悬后,进行流式染色,其中,采用CD80+/F4/80+标记为M1型巨噬细胞;采用CD25+FoxP3+/CD4+标记为Treg细胞,结果见图14。
结果表明,AAV6-SEMA3A能够有效减少骨髓内M1型巨噬细胞的极化,这有助于减少促炎细胞因子的生成,从而降低破骨细胞前体细胞的分化能力,而雌二醇并不能降低骨髓内的M1型巨噬细胞;同时AAV6-SEMA3A能够使骨髓内Treg细胞的增加,有助于抑炎细胞因子的产生,从而抑制骨质疏松造成的炎症破坏并恢复免疫稳态。可见AAV6-SEMA3A相较于雌二醇能更全面地改善骨质疏松引起的炎症环境,恢复正常的骨免疫稳态。(ns,P≥0.05;*,P<0.05;**,P<0.01;***,P<0.001;****,P<0.0001)
综上,研究结果提示AAV6-SEMA3A能够有效减少关节部位炎症,并提供优良的骨保护作用,同时由于其仅需一次给药的特性,AAV6-SEMA3A无需像Adalimumab和雌二醇需要频繁多次给药,明显提高患者顺应性。而Adalimumab作为单抗药物,在多次给药后易在体内产生特异性中和抗体,会导致疗效明显减弱;雌二醇作为激素类药物,长期给药易提高罹患子宫内膜癌和乳腺癌的概率。同时本发明还针对不同类型的骨科疾病筛选了不同的给药方式,如类风湿性关节炎及骨关节炎主要针对关节部位炎症,关节腔内注射效果更佳;如骨质疏松则肌肉注射效果更佳。因此AAV6-SEMA3A具备成为治疗骨科疾病的基因治疗药物的潜力。
以上所述仅是本发明的优选实施例而已,并非对本发明做任何形式上的限制,虽然本发明已以优选实施例揭露如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案的范围内,当可利用上述揭示的技术内容做出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案的内容,依据本发明的技术实质对以上实施例所做的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (10)
1.一种治疗骨科疾病的多核苷酸,其特征在于,所述多核苷酸的序列如SEQ ID NO:1所示。
2.一种包含权利要求1所述多核苷酸的载体,其特征在于,所述多核苷酸的序列如SEQID NO:1所示。
3.根据权利要求2所述的载体,其特征在于,所述载体的表达框由增强子-启动子-目的基因序列-polyA信号或由启动子-目的基因序列-polyA信号组成,其中,启动子选自CAG启动子、CMV启动子、SV40启动子、CBh启动子、SFFV启动子、MSCV启动子、EF1α启动子、软骨细胞特异的COL2A1启动子、软骨细胞特异的COL1A1启动子、肌肉特异的MHCK7启动子或肌肉特异的ACTA1启动子,启动子优选为CAG启动子,载体优选为pAAVCAG-MCS。
4.一种复制缺陷型重组腺相关病毒,其特征在于,所述重组腺相关病毒包含如SEQ IDNO:1所示的多核苷酸。
5.根据权利要求4所述的复制缺陷型重组腺相关病毒,其特征在于,所述重组腺相关病毒血清型包括所有天然和人工设计的腺相关病毒血清型,其特征在于所述的天然腺相关病毒血清型包括但不限于天然的AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV-DJ、AAV-DJ8、AAV-DJ9、AAVrh8、AAVrh8R和AAVrh10,以及人工设计的腺相关病毒血清型包括但不限于7m8,优选为天然的AAV6。
6.如权利要求1所述的多核苷酸、权利要求2-3任一所述的载体或权利要求4-5任一所述的复制缺陷型重组腺相关病毒在制备治疗类风湿性关节炎、骨质疏松症和/或骨关节炎的药物中的应用。
7.根据权利要求6所述的应用,其特征在于,所述药物为液体制剂、固体制剂。
8.根据权利要求7所述的应用,其特征在于,所述液体制剂的给药途径为关节腔内注射、肌肉注射、静脉注射、经皮给药的一种或多种,优选的经皮给药为皮下注射、微针的任一一种或多种,所述类风湿性关节炎、骨关节炎治疗优选为关节腔内注射,所述骨质疏松治疗优选为肌肉注射。
9.一种权利要求4-5任一所述的复制缺陷型重组腺相关病毒的制备方法,所述方法包括以下步骤:
(1)构建含多核苷酸的顺式质粒载体;
(2)构建含有血清型6的AAV衣壳质粒;
(3)将步骤(1)和(2)所述质粒与辅助质粒一起在PEI的作用转染至宿主细胞;
(4)37℃5%CO2条件下培养宿主细胞;
(5)将步骤(4)所述宿主细胞收集裂解,收货含有复制缺陷型重组腺相关病毒的病毒液;
(6)对步骤(5)所述病毒液进行纯化即得。
10.如权利要求9所述的复制缺陷型重组腺相关病毒的制备方法制备的复制缺陷型重组腺相关病毒在制备治疗类风湿性关节炎、骨质疏松症和/或骨关节炎的药物中的应用。
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