【作者】 易敏；

【导师】 杨林；

【作者基本信息】 四川大学 ， 生物化学与分子生物学， 2022， 硕士

【摘要】 背景:基因治疗是指用遗传手段来治疗人类疾病的生物医学技术。目前来说,基因治疗主要应用于对人类健康威胁严重的疾病,包括遗传病、恶性肿瘤、神经退行性疾病以及免疫缺陷疾病等。早在1990年美国NIH的研究人员就将腺苷酸脱氢酶基因导入病人的淋巴细胞,治疗由于该基因缺损造成的免疫缺陷。腺相关病毒(Adeno-associated virus,AAV)是转基因传递最常用的工具之一,在基因治疗领域展现了良好的安全性和治疗效果,迄今为止,已有三款基于AAV的基因治疗药物获准上市。然而,在AAV的实际运用过程中却存在转导效率不足、衣壳的基因组包装容量较小以及人体预存的AAV中和抗体等问题,让其应用受到了限制。因此在AAV的研究中选择合适的动物模型,并对AAV进行遗传改造优化其生物学特性,可能进一步促进AAV在基因治疗领域的发展。目的:构建AAV2与PPV6(Porcine parvovirus 6)的重组病毒载体(r AAV2/PPV6),以增大AAV的包装容量;构建针对于AAVR(AAV进入组织细胞的主要受体)的AAV饱和突变文库,筛选出较好靶向心脏或者肝脏的AAV突变体;AAV突变体(MG6)病毒的包装与动物模型体内的基因治疗。方法:在构建重组嵌合载体以增大AAV的包装能力课题中,寻找到与AAV是远亲关系细小科病毒——PPV6,构建重组嵌合载体。将ppv6 bj2基因构建于含有AAV2 ITR(反向末端重复序列)的质粒载体上,r AAV2/PPV6质粒与辅助质粒共转染293细胞(三质粒转染法),生产重组病毒并观察病毒的包装能力。利用斑点杂交对病毒滴度进行定量分析,通过透射电镜观察病毒颗粒进一步确定包装性质。设计并改进重组病毒的载体结构,优化重组病毒的包装效率。在构建饱和突变文库增强AAV对组织细胞的趋向性课题中,根据AAV1/AAV2与AAVR结合的氨基酸序列,对AAV8/AAV9与AAVR的结合位点进行分析。将AAV8/AAV9衣壳上对AAVR结合位点的非保守区段的氨基酸序列随机突变,构建饱和突变文库,并对饱和突变文库的突变率以及病毒包装能力进行验证。利用先转染后感染的方式大量包装饱和突变文库病毒并检测病毒滴度,为后续在小鼠模型筛选具有靶向性的AAV变体奠定了基础。在MG6病毒的包装与基因治疗课题中,需要验证通过肽展示库筛选出的对胶质瘤趋向性增强的AAV突变体(MG6),在小鼠模型中基因治疗的应用情况。生产携带单纯疱疹病毒胸苷激酶(hsv-tk)治疗基因的MG6以及野生型AAV2载体,对病毒进行纯化、透析以及浓缩。通过尾静脉将MG6-CMV-TK、AAV2-CMV-TK病毒以及PBS注射进入皮下成瘤小鼠体内,并与更替诺韦(GCV)联合治疗。观察对荷瘤小鼠注射携带治疗基因的MG6病毒后,60天内肿瘤大小与生存情况,进行动物体内神经胶质瘤基因治疗实验。结果:设计构建了AAV2.1-PPV6 BJ2重组载体,通过斑点杂交检测以及透射电镜观察,验证重组病毒能够成功包装。改进并新构建了两种r AAV2/PPV6重组载体(AAV2.1-AAV2-PPV6和AAV2.1-NS/Rep2-PPV6),检测病毒在细胞内的转录、复制以及蛋白表达情况。其中AAV2.1-PPV6 BJ2与AAV2.1-AAV2-PPV6载体均存在基因组的转录与复制,但AAV2.1-AAV2-PPV6较AAV2.1-PPV6 BJ2的转录与复制能力未见增强。通过Western blot检测出AAV2.1-PPV6 BJ2和AAV2.1-AAV2-PPV6载体的衣壳蛋白在细胞内均有表达,且无明显的表达差异。综上所述,衣壳蛋白的表达可能不是AAV2.1-AAV2-PPV6不能进行包装的原因,而PPV6 NS蛋白的存在可能是AAV2.1-PPV6 BJ2载体能够进行包装的基本结构,同时,适当地增加基因组的复制,可能会增加病毒的包装。我们根据以上结果对重组病毒载体质粒结构进行进一步改进,得到另一重组载体AAV2.1-CMV-PPV6也能进行包装,并且包装能力较AAV2.1-PPV6 BJ2略有增强。通过同源建模分析AAV8/AAV9衣壳蛋白与AAVR的结合位点,对AAV8与AAV9在衣壳上识别AAVR的三个可变区的非保守区段,进行饱和突变或缺失,建立饱和突变文库。对合成的文库质粒转化,随机挑取20个单克隆酶切并测序检测病毒质粒载体的正确性,对测序正确的质粒载体进行转染检验病毒的包装能力,根据结果分析文库的突变率与完整性。得出AAV8文库突变率60%,AAV9文库突变率为80%,并且大部分突变病毒能进行包装。将饱和突变病毒文库大量包装,为后续体内筛选做准备工作。通过尾静脉将MG6-CMV-TK、AAV2-CMV-TK病毒以及PBS注射进入荷瘤小鼠体内后与GCV联合治疗,发现应用MG6病毒携带治疗基因较野生型AAV2与PBS对照组而言,肿瘤生长放缓,并且小鼠生存期得到延长。因此,证实了胶质瘤小鼠模型中,利用MG6病毒在体内进行基因治疗相较于野生型AAV的疗效更加显著。结论:成功构建r AAV/PPV6的重组载体,并且对病毒载体的结构进行优化改进,增强了重组病毒的包装能力;对AAV突变体——MG6进行包装,并与GCV联合进行体内的基因治疗实验,验证了MG6病毒在小鼠皮下胶质瘤模型中能发挥有效的治疗作用;构建AAV8/AAV9病毒的饱和突变文库,并对产生的质粒文库进行了鉴定,为后续筛选工作奠定了基础。更多还原

【Abstract】 Background: Gene therapy is a biomedical technique that uses genetic methods to treat human diseases.At present,gene therapy is mainly used for diseases that threaten human health seriously,including: genetic diseases,malignant tumors,neurodegenerative diseases and immunodeficiency disease,etc.As early as 1990,NIH researchers in the United States introduced the adenylate deaminase gene into the lymphocytes of patients to treat the immunodeficiency caused by this gene defect.Adeno-associated virus(AAV)is one of the most commonly used tools for transgenic delivery.It has shown good safety and therapeutic efficacy in the field of gene therapy.So far,three AAV-based gene therapy drugs have been approved for marketing.However,in the practical application of AAV,there are some problems,such as inefficient transduction,small genomic packaging capacity of capsid and pre-existensce of human AAV neutralizing antibodies,which restrict its application.Therefore,genetic engineering of AAV vector,optimization of its biological characteristics,and selection of suitable animal models for study may further promote the development of AAV in the field of gene therapy.Objective: To increase the packing capacity of AAV,a recombinant virus vector of AAV and PPV6 was constructed(r AAV/PPV6);construct an AAV saturation mutation library targeting AAVR(the main receptor for AAV entering tissue cells),and screen out AAV mutants that better target the heart or liver;packaging of AAV mutant viruse(MG6)and gene therapy in animal models.Methods: In the project of constructing a recombinant vector to increase the packaging capacity of AAV,we selected the parvovirus(PPV6)which is a distant relative of AAV,and constructed a recombinant vector.The ppv6 bj2 gene was incorporated into the plasmid vector containing AAV2 ITR(inverted terminal repeat),and the r AAV2/PPV6 plasmid and the helper plasmid were co-transfected into 293cells(three-plasmid transfection method)to produce the recombinant virus and to evaluate the packaging ability of the virus.Virus yield was quantified by dot blotting and virus particles were visualized by transmission electron microscopy.Design and improve the vector structure of recombinant virus and optimize the packaging efficiency of recombinant virus.In order to construct a saturation mutation library to enhance the tropism of AAV to specific tissues,the binding sites of AAV8/AAV9 for AAVR were analyzed according to the amino acid sequences of AAV1/AAV2 and AAVR.The random mutation of the amino acid interaction between the non-conserved segment of the AAVR binding site on the AAV8/AAV9 capsid was used to construct a mutation library,and the mutation rate and virus packaging ability of the saturated mutation library were verified.The saturated mutant library virus was packaged in large quantities by transfection and infection.The virus titer was determined which laid a foundation for the subsequent screening of targeted AAV variants in mouse models.In the project of MG6 package and gene therapy,the purpose was to explore the application of gene therapy in mouse models of AAV mutants with enhanced glioma-tropism screened from the peptide display library.MG6 and wild-type AAV2 vectors carrying the herpes simplex virus thymidine kinase(hsv-tk)gene were produced,purified,dialysed and concentrated for glioma gene therapy in animals.MG6-CMV-TK,AAV2-CMV-TK and PBS were injected into subcutaneous tumor-bearing mice through tail vein,in combination Ganciclovir(GCV),to evaluate the effect of MG6 virus carrying therapeutic gene on tumor size and survival of tumor-bearing mice within 60 days.Results: The recombinant AAV2.1-PPV6 BJ2 vector was designed and constructed,and the successful packaging of the recombinant virus was verified by dot blot and transmission electron microscopy.Two r AAV/PPV6 vectors(AAV2.1-AAV2-PPV6 and AAV2.1-NS/Rep2-PPV6)were improved and constructed to detect the transcription,replication and protein expression of the virus in cells.Both AAV2.1-PPV6 BJ2 and AAV2.1-AAV2-PPV6 vectors had genome transcription and replication,but the transcription and replication ability of AAV2.1-AAV2-PPV6 was not enhanced compared with that of AAV2.1-PPV6 BJ2.Western blot analysis showed that the capsid proteins of AAV2.1-PPV6 BJ2 and AAV2.1-AAV2-PPV6 vectors were expressed in cells without significant difference in expression.In conclusion,the expression of capsid protein may not be the reason why AAV2.1-AAV2-PPV6 cannot be packaged,while the presence of PPV6 NS protein may be the basic structure for AAV2.1-PPV6 BJ2 vector to be packaged.Meanwhile,appropriately increasing the replication of the genome may increase the packaging of the virus.According to the above results,we further improved the structure of recombinant viral vector plasmid,and obtained that another recombinant vector AAV2.1-CMV-PPV6 could also be packaged,and its packaging ability was slightly enhanced with that of AAV2.1-PPV6 BJ2.The binding site between AAV8/AAV9 capsid protein and AAVR was analyzed by homology modeling,and the non-conserved segments of the three variable regions of AAVR recognized by AAV8 and AAV9 on the capsid were subjected to saturation mutation or deletion to establish saturation mutation library.The synthetic library plasmid was transformed into bacteria,and 20 monoclonal enzymes were randomly selected to cut and sequenced to detect the correctness of the virus vector.The correctly sequenced plasmid vector was tested for the packaging ability of the virus,and the mutation rate and integrity of the library were verified.It was concluded that the mutation rate of AAV8 library was 60%,and the mutation rate of AAV9 library was 80%,and most of the mutant viruses could be packaged.A large number of saturated mutant virus libraries were packed to prepare for subsequent screening.MG6-CMV-TK,AAV2-CMV-TK virus and PBS were injected into tumor-bearing mice by tail vein and then treated with GCV.Compared with wild-type AAV2 and PBS control group,MG6 virus carrying therapeutic genes reduced tumor size and significantly prolonged survival time of mice.Therefore,the treatment of MG6 virus proved that MG6 virus in vivo could be better applied in gene therapy in glioma mouse model than wild-type AAV.Conclusion: The recombinant r AAV/PPV6 vector was successfully constructed,and the structure of the virus vector was optimized and improved to enhance the packaging ability of the recombinant virus.The AAV mutant(MG6)was packaged and combined with GCV to conduct in vivo gene therapy experiments,which verified that MG6 virus can play an effective therapeutic role in a mouse subcutaneous glioma model.The saturated mutation library of AAV8/AAV9 virus was constructed and the generated plasmid library was identified,which laid a foundation for subsequent screening.更多还原