重组腺相关病毒载体与血友病A基因治疗

基于腺相关病毒(AAV）独特的生物学特性，重组腺相关病毒载体（rAAV)是目前最具希望的因治疗载体之一。在一些目的基因需长期、稳定表达的疾病的基因治疗中，愈来愈受到青睐。本文就AAV的基因结构和生物学特性、rAAV载体、载体改造及在血友病A基因治疗中的应用作一综述。     

重组腺相关病毒载体与血友病A基因治疗

基于腺相关病毒(AAV)独特的生物学特性,重组腺相关病毒载体(rAAV)是目前最具希望的基因治疗载体之一。在一些目的基因需长期、稳定表达的疾病的基因治疗中,愈来愈受到青睐。本文就AAV的基因结构和生物学特性、rAAV载体、载体改造及在血友病A基因治疗中的应用作一综述。     

腺相关病毒在基因治疗中的研究进展

基因治疗为人类一些疑难疾病的治疗带来了新的希望,但如何将外源基因高效导入靶细胞或机体,一直是人们渴望解决的问题。腺相关病毒（Ａｄｅｎｏ－ａｓｓｏｃｉａｔｅｄ Ｖｉｒｕｓ,ＡＡＶ）属细小病毒科,是动物病毒中最小的一种真核病毒,在高效转导、稳定表达方面具有其他病毒载体所没有的优越性。在为人类基因治疗寻找一种理想载体的研究,ＡＡＶ受到广泛重视。近年来其他生物学特性及应用研究均取得了较大进展。     

腺相关病毒在基因治疗中的研究进展

基因治疗为人类一些疑难疾病的治疗带来了新的希望,但如何将外源基因高效导人靶细胞或机体,一直是人们渴望解决的问题。腺相关病毒(Adeno-associated Virus, AAV)属细小病毒科,是动物病毒中最小的一种真核病毒,在高效转导、稳定表达方面具有其他病毒载体所没有的优越性。在为人类基因治疗寻找一种理想载体的研究中,AAV受到广泛重视。近年来其基本生物学特性及应用研究均取得了较大进展。     

用于包装腺相关病毒293细胞的培养

背景:腺相关病毒作为一种主要的基因工程载体,已被广泛应用。但腺相关病毒是缺陷性病毒,需要包装细胞提供E1蛋白。腺相关病毒293细胞作为重要的腺相关病毒特异性包装细胞,能反式产生E1,但腺相关病毒293细胞娇嫩,培养困难,生物学特性易发生改变。因此,有必要建立一种腺相关病毒293细胞的培养方案以满足基因工程的需要。目的:建立一种体外培养腺相关病毒293细胞的方法。设计:开放性实验。单位:华中科技大学同济医学院附属同济医院神经内科。材料:腺相关病毒293细胞株购于Stratagene公司。高糖型DMEM粉(Gibco公司),AAV Helper-Free系统三质粒(Stratagene公司)。方法:实验于2006-10/2007-04在武汉同济医院神经内科实验室完成。在体外将腺相关病毒293细胞复苏,用高糖型DMEM生长培养基培养,当细胞单层融合度达到50%时传代和冻存,并倒置显微镜下观察细胞的生长状态,记录生长曲线。荧光倒置显微镜下观察,根据腺相关病毒293细胞在共转染腺相关病毒系统三质粒后以及被腺相关病毒上清感染后能否激发出绿色荧光,鉴定其包装腺相关病毒的生物学特性。主要观察指标:①腺相关病毒293细胞形态学观察。②生长曲线。③腺相关病毒包装。结果:①倒置显微镜下显示,腺相关病毒293细胞贴壁生长良好,呈现不规则的多角形,胞浆透亮,胞核隐约可见。②生长曲线显示,细胞传代后第1天为生长适应期,2～5d为生长活跃期,6d后细胞进入生长平台期。③荧光倒置显微镜下观察到,腺相关病毒293细胞激发绿色荧光,共转染腺相关病毒系统三质粒成功。腺相关病毒293细胞被腺相关病毒上清感染后激发出绿色荧光,腺相关病毒包装成功。结论:本实验的培养方法简单有用,培养的腺相关病毒293细胞可保持良好的腺相关病毒包装功能。     

腺相关病毒载体和中枢神经系统疾病的基因治疗

中枢神经系统疾病分子改变的深入研究和基因治疗技术的不断发展为临床治疗这些疾病带来了希望.但基因治疗临床应用的最大障碍之一便是转基因技术中载体系统的选择.理想的体内基因转移载体应具有较好的细胞靶向性,目的基因表达的可控性以及转移方法的简单易行.自七十年代末病毒载体的概念提出以来,用于基因治疗研究的缺陷性病毒载体种类日渐增加,应用也愈加广泛.其中,腺相关病毒(AAV)载体是目前病毒类转基因系统中较新的成员.由于该系统最大的优点之一是它的安全性较好,并且最有可能发展成为将外源基因整合入宿主细胞染色体的非病毒载体系统,因此倍受关注,并已开始应用于临床试验[1].     

腺相关病毒载体及其在基因治疗中的应用

1 腺相关病毒的生物学特性 腺相关病毒（adeno—associated virus，AAV）属微小病毒科，病毒颗粒直径约20～26nm，20面体，无包膜，其病毒基因组DNA为单链，线形。AAV属于依赖性病毒，最初被疑为腺病毒培养过程中的污染成分，后来发现它常定居于人类呼吸道和胃肠道，且能够在哺乳动物细胞中长期潜伏存在。     

腺相关病毒载体及其在血液系统基因治疗中的应用及研究进展

腺相关病毒园其独特的生物学特性,在基因治疗的应用中日益受到关注,近年来其基础及应用研究均取得了较大进展.本文就腺相关病毒的生物学特性、重组腺相关病毒载体的构建及其在血液系统基因治疗中的研究作一综述.     

携带肿瘤坏死因子相关细胞凋亡诱导配体重组腺相关病毒的构建及鉴定

目的:构建一种携带肿瘤坏死因子相关细胞凋亡诱导配体(tumor necrosis factor related apoptosis inducing ligand,TRAIL)基因的重组腺相关病毒(recombined adeo-associated virus,rAAV)载体。方法:实验于2005-09/2006-04在中山大学附属第一医院普通外科实验室完成。首先构建携带可溶性肿瘤坏死因子相关细胞凋亡诱导配体基因的穿梭质粒pAAV-sTRAIL,利用磷酸钙共沉淀法将穿梭质粒共转染入HEK293细胞中,采用细胞内质粒DNA同源重组法构建重组腺相关病毒rAAV-sTRAIL。以噬斑分析法筛选单克隆重组腺相关病毒;聚合酶链反应法鉴定阳性重组腺相关病毒;氯化铯密度梯度离心法纯化病毒;紫外分光光度仪测定病毒颗粒数及纯度,噬斑分析法测定病毒感染滴度;Western blot检测rAAV-sTRAIL在293细胞中的表达情况。结果:成功构建了重组腺相关病毒载体rAAV-sTRAIL,制备的病毒纯度好、滴度高,且在293转导细胞中能有效表达目的基因sTRAIL。结论:构建的重组腺相关病毒载体rAAV-sTRAIL,为组织工程相关细胞转基因构建及临床应用提供先进的载体系统。     

重组腺相关病毒载体2型转染骨髓间充质干细胞的体外实验

背景：目前应用于基因治疗的病毒载体中，重组腺相关病毒载体2型载体与腺病毒和反转录病毒载体比较，由于其无致病性，引起学者们的关注。目的：观察体外重组腺相关病毒载体2型转染骨髓间充质干细胞，并用其作为携带基因表达的载体进行急性髓性白血病基因治疗的可能性。设计：开放性实验。单位：南方医科大学南方医院的血液科。材料：实验于2004-02／07在南方医科大学南方医院的血液科实验室完成。本实验所用的骨髓间充质干细胞来自急性髓性白血病6例初发患者和4名健康成年志愿者的第3～5代传代细胞。方法：从初发的急性髓性白血病患者和正常健康志愿者髂后上棘做骨髓穿刺抽取6～10mL肝素抗凝的骨髓，分离培养出间充质干细胞，用包含增强型绿色荧光蛋白的重组腺相关病毒载体2型感染间充质干细胞，将获取的骨髓间充质干细胞加入含一定感染复数(感染复数：1&;#215;10^2．1&;#215;10^3，1&;#215;10^4，1&;#215;10^5，1&;#215;10^6，1&;#215;10^7)的增强型绿色荧光蛋白的重组腺相关病毒载体2型病毒载体的不完全培养液，10～14d后在相差荧光显微镜下或用流式细胞仪观测绿色荧光蛋白的表达。在体外培养条件下观察绿色荧光蛋白在经过重组腺相关病毒载体2型转导的骨髓间充质干细胞中的表达。体外观察经增强型绿色荧光蛋白的重组腺相关病毒载体2型转导并被新霉素筛选后绿色荧光蛋白在被转导的骨髓间充质干细胞中的表达。通过相差荧光显微镜下确证绿色荧光蛋白表达后，在BD流式细胞仪上检测绿色荧光蛋白的表达。主要观察指标：①增强型绿色荧光蛋白的重组腺相关病毒载体2型对骨髓间充质干细胞的转染率分析。②在转染后的不同时间点用相差荧光显微镜和流式细胞仪观察绿色荧光蛋白的表达情况。结果：①转染率分析：增强型绿色荧光蛋白的重组腺相关病毒载体2型对来源于健康志愿者和急性髓性白血病患者的骨髓间充质干细胞的转染率均不高，转染率在0．3％～1．4％。转染后10～14d绿色荧光蛋白开始表达，一般感染条件绿色荧光蛋白阳性骨髓间充质干细胞所占比例为(1．030&;#177;0．034)％，重复3轮感染条件下为(1．140&;#177;0．036)％，脂质体协助感染条件下为(1．380&;#177;0．054)％，改变转染条件(包括重复感染，延长感染时间，增加感染复数，脂质体协助转染)亦不能明显增加转染率(P&;gt;0．05)，而增强型绿色荧光蛋白的重组腺相关病毒载体2型却能高效的转染其包装细胞293细胞。②绿色荧光蛋白在体外长期稳定表达分析：实验观察61d内，绿色荧光蛋白保持低水平长期稳定表达，在转染后的12～33d，绿色荧光蛋白阳性的骨髓间充质干细胞从起始时的1．16％下降到0．5％～0．6％，33～61d一直维持在这一水平；经过新霉素筛选30d后，表达绿色荧光蛋白的骨髓间充质干细胞达到6．6％左右，在体外继续传代培养，在体外观察的100d中，表达绿色荧光蛋白的骨髓间充质干细胞一直维持在6％的水平。结论：重组腺相关病毒载体2型介导的基因转导的优势是安全、无免疫反应，目的基因能够长期稳定表达。重组腺相关病毒载体和骨髓间充质干细胞可用于体外基因治疗，其将来可能成为全身基因治疗的良好载体。     

携带半乳糖苷酶报告基因重组腺相关病毒体内转染骨组织的实验观察

目的:腺相关病毒载体因具有安全性好、免疫源性低、能感染分裂和非分裂细胞且能介导外源基因稳定长期表达等优点而备受瞩目,是最有希望应用于临床的病毒类载体。观察携带半乳糖苷酶报告基因的重组腺相关病毒(rAAV-LacZ)体内转染骨组织的效果。方法:实验于2005-01/2006-01在北京大学医学部第三医院和皖南医学院弋矶山医院骨科实验室完成。实验材料:8周龄雄性SD大鼠8只,体质量220g左右。rAAV-LacZ载体(本元正阳公司,病毒滴度为6×1012v·g/mL);Ⅰ型胶原海绵载体(上海其胜公司)。实验分组:将大鼠随机分为实验组和对照组,每组4只。实验方法:实验组4只大鼠在胫骨前外侧做骨缺损槽后,将滴有6×1011v·g病毒量的rAAV-LacZ和Ⅰ型胶原载体复合物植入骨缺损槽;对照组4只大鼠仅在胫骨骨缺损槽内植入Ⅰ型胶原载体。实验评估:①手术后6周取材行x-gal染色后,分别行标本大体病理观察和组织学观察以了解β-半乳糖苷酶特异表达的情况。②同时提取实验部位以远器官肝和心脏组织RNA,反转录-聚合酶链反应检测这些部位有无β-半乳糖苷酶特异表达。结果:8只SD大鼠均进入结果分析。①大体病理观察结果:实验组动物胫骨骨缺损处取材经x-gal染色后可见骨质蓝染,即有特异性β-半乳糖苷酶表达,而骨缺损临近处肌肉也表达β-半乳糖苷酶,Ⅰ型胶原载体植入部位以远的骨质和肌肉则没有特异性β-半乳糖苷酶的表达。②组织学观察结果:实验组骨缺损处肌肉肌纤维特异性表达β-半乳糖苷酶且没有明显的淋巴细胞局部浸润。蓝染的骨皮质切片显示骨陷窝内的骨细胞特异性表达β-半乳糖苷酶。对照组则没有实验组上述特异性β-半乳糖苷酶的表达。③反转录-聚合酶链反应结果:实验组实验部位有特异性β-半乳糖苷酶的表达,而其以远的心、肝器官内没有特异性β-半乳糖苷酶的表达。而对照组实验部位和实验部位以远的心、肝器官内均没有特异性β-半乳糖苷酶的表达。结论:腺相关病毒载体直接注射到骨缺损和骨折部位可以有效转染骨和临近肌肉组织,并能长期(6周)表达所携带的外源基因,具有一定的安全性。     

Production of recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) is a prototypical gene therapy vector characterized by excellent safety profiles, wide host range, and the ability to transduce differentiated cells. Numerous rAAV-based vectors providing efficient and sustained expression of transgenes in target tissues have been developed for preclinical studies. Interest in rAAV has been driven by advances in production methods originally developed for rAAV serotype 2 vectors and expanded to include alternative serotypes. The transition to clinical trials is dependent on the development of scalable production methods of Good Manufacturing Practice-grade vectors described in this review.     

小鼠脂联素重组腺相关病毒载体的构建

目的:构建脂联素重组腺相关病毒载体,为进一步研究脂联素的功能提供基础。方法:实验于2003年在中山大学附属第二医院林百欣医学研究中心进行。以带有小鼠脂联素基因的质粒pcDNA3.0-Ad为模板,聚合酶链反应克隆脂联素基因,并将聚合酶链反应扩增产物定向克隆于pAAV-MCS载体,重组AAV-MCS-Ad质粒经双酶切和测序鉴定。随后采用磷酸钙转染法将pAAV-MCS-Ad、pAAV-RC,pAAV-Helper质粒共转染AAV-293细胞,包装得到重组腺相关病毒穴rAAV-Ad雪。回收病毒原液,采用PEG8000和氯仿纯化浓缩病毒。SDS-PAGE及透射电镜鉴定纯化的病毒,并采用Southernblot测定重组腺相关病毒的滴度。Westernblotting检测rAAV-Ad转染H4IIE细胞后脂联素蛋白的表达。结果:①重组质粒AAV-MCS-Ad经双酶切和测序鉴定证实将脂联素基因已正确插入。②SDS-PAGE和电镜均证实rAAV-Ad病毒已构建成功。③Southernblotting测定病毒滴度约为约2.5×1014L-1,通过rAAV-LacZ转染细胞后X-gal染色计数,病毒的感染滴度在(0.83～1.2)×1010转导单位/mL。④Westernblotting证实rAAV-Ad转染H4IIE细胞后在30ku处见特异的阳性蛋白条带,证明能有效表达脂联素蛋白。结论:成功构建了携带小鼠脂联素基因的rAAV-Ad载体,经纯化浓缩后具有较好的纯度和较高滴度,能有效转染H4IIE细胞,并表达脂联素蛋白,具有良好的功能,为进一步研究其在真核细胞中的功能奠定了基础。     

Intracellular transport of recombinant adeno-associated virus vectors

Recombinant adeno-associated viral vectors (rAAVs) have been widely used for gene delivery in animal models, and are currently evaluated for human gene therapy after successful clinical trials in the treatment of inherited, degenerative or acquired diseases, such as Leber congenital amaurosis, Parkinson disease or heart failure. However, limitations in vector tropism, such as limited tissue specificity and insufficient transduction efficiencies of particular tissues and cell types, still preclude therapeutic applications in certain tissues. Wild-type adeno-associated viruses (AAVs) are defective viruses that require the presence of a helper virus to complete their life cycle. On the one hand, this unique property makes AAV vectors one of the safest available viral vectors for gene delivery. On the other, it also represents a potential obstacle because rAAV vectors have to overcome several biological barriers in the absence of a helper virus to transduce successfully a cell. Consequently, a better understanding of the cellular roadblocks that limit rAAV gene delivery is crucial and, during the last 15 years, numerous studies resulted in an expanding body of knowledge of the intracellular trafficking pathways of rAAV vectors. This review describes our current understanding of the mechanisms involved in rAAV attachment to target cells, endocytosis, intracellular trafficking, capsid processing, nuclear import and genome release with an emphasis on the most recent discoveries in the field and the emerging strategies used to improve the efficiency of AAV-derived vectors.     

小鼠脂联素重组腺相关病毒载体的构建

目的：构建脂联素重组腺相关病毒载体，为进一步研究脂联素的功能提供基础。方法：实验于2003年在中山大学附属第二医院林百欣医学研究中心进行。以带有小鼠脂联素基因的质粒peDNA3．0-Ad为模板，聚合酶链反应克隆脂联素基因，并将聚合酶链反应扩增产物定向克隆于pAAV-MCS载体，重组AAV-MCS-Ad质粒经双酶切和测序鉴定。随后采用磷酸钙转染法将pAAV-MCS-Ad、pAAV-RC，pAAV-Helper质粒共转染AAV-293细胞，包装得到重组腺相关病毒（rAAV-Ad）。回收病毒原液，采用PEG8000和氯仿纯化浓缩病毒。SDS-PAGE及透射电镜鉴定纯化的病毒，并采用Southern blot测定重组腺相关病毒的滴度。Western blotting检测rAAV-Ad转染H4IIE细胞后脂联素蛋白的表达。 结果：①重组质粒AAV-MCS-Ad经双酶切和测序鉴定证实将脂联素基因已正确插入。②SDS-PAGE和电镜均证实rAAV-Ad病毒已构建成功。③Southern blotting测定病毒滴度约为约2．5&;#215;10^14L^-1，通过rAAV-LacZ转染细胞后X-gal染色计数，病毒的感染滴度在（0．83～1．2）&;#215;10^10转导单位／mL。（少Western blotting证实rAAV-Ad转染H4IIE细胞后在30ku处见特异的阳性蛋白条带，证明能有效表达脂联素蛋白。 结论：成功构建了携带小鼠脂联素基因的rAAV-Ad载体，经纯化浓缩后具有较好的纯度和较高滴度，能有效转染H4IIE细胞，并表达脂联素蛋白，具有良好的功能，为进一步研究其茁真核细胞中的功能奠定了基础。     

Hematopoietic stem cell transduction by recombinant adeno-associated virus vectors: problems and solutions

Recombinant adeno-associated virus 2 (AAV) vectors have taken center stage owing to their potentially safer profile compared with the more commonly used retroviral and adenoviral vectors in human gene therapy clinical trials. Their remarkable versatility and efficacy in a wide variety of preclinical animal models of human diseases have attracted further attention of a number of investigators. Although two particular cell types, muscle and brain, have been shown to be highly transducible by AAV vectors, controversies abound with reference to the efficacy of these vectors in transducing primary hematopoietic cells. Whereas some investigators have claimed that primitive hematopoietic cells are impervious to AAV vectors, others have reported that AAV vectors are capable of transducing these cells, but only at high vector-to-cell ratios. Still other investigators have reported successful transduction of primitive hematopoietic cells at relatively low vector-to-cell ratios. This review attempts to resolve these controversies, and provides a basis for the optimism that safe and high-efficiency transduction of hematopoietic stem and progenitor cells by AAV vectors is well within reach.     

Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus.

Recombinant adeno-associated virus (rAAV) vectors are under consideration for a wide variety of gene therapy applications. One of the limitations of the rAAV vector system has been the difficulty in producing the vector in sufficient quantity for adequate preclinical and clinical evaluation. A common method for vector production involves large-scale transient transfection of multiple plasmids into cultured cells. Because this approach might not be feasible for clinical scale manufacturing, we have sought approaches for rAAV vector production that avoid transient transfection procedures. In previously reported work, we generated an AAV packaging cell line that produces infectious rAAV when the vector genome is transfected into the cell line as plasmid DNA. We have now extended this approach by constructing a hybrid recombinant adenovirus (rAd) that contains a complete rAAV vector genome in the E1 region. This hybrid virus is used to deliver the rAAV genome to the packaging cell line (in the place of plasmid transfection). rAAV is produced when the packaging cell line is infected with the hybrid adenovirus and wild-type adenovirus. This method avoids the need for plasmid transfection and is adaptable to large-scale manufacturing processes.     

Clinical gene therapy using recombinant adeno-associated virus vectors

Recombinant adeno-associated virus (rAAV) vectors possess a number of properties that may make them suitable for clinical gene therapy, including being based upon a virus for which there is no known pathology and a natural propensity to persist in human cells. Wild-type adeno-associated viruses (AAVs) are now known to be very diverse and ubiquitous in humans and nonhuman primates, which adds to the degree of confidence one may place in the natural history of AAV, namely that it has never been associated with any human tumors or other acute pathology, other than sporadic reports of having been isolated from spontaneously aborted fetuses. On the basis of this understanding of AAV biology and a wide range of preclinical studies in mice, rabbits, dogs and nonhuman primates, a growing number of clinical trials have been undertaken with this class of vectors. Altogether, over 40 clinical trials have now been approved. Although all previous trials were undertaken using AAV serotype 2 vectors, at least two current trials utilize AAV2 vector genomes cross-packaged or pseudotyped into AAV1 capsids, which appear to mediate more efficient gene delivery to muscle. The explosion of capsid isolates available for use as vectors to over 120 has now provided the potential to broaden the application of AAV-based gene therapy to other cell types.     

Transfer of activation-dependent gene expression into T cell lines by recombinant adeno-associated virus.

We examined the ability of recombinant adeno-associated virus (rAAV) to transfer regulated gene expression into T cell lines. An AAV-based vector containing the neomycin resistance gene and expressing the firefly luciferase (luc) gene under the regulatory control of the interleukin 2 promoter (pAAV-luc) was generated and adenovirus-free rAAV (rAAV-luc) was produced from this vector. Transfection of pAAV-luc into the human T cell line Jurkat resulted in luciferase expression while infection of Jurkat T cells with rAAV-luc resulted in significant luciferase expression only after selection for neomycin-resistant cells. Long-term growth of transduced Jurkat T cells showed that there was no detectable constitutive expression of luciferase and that luciferase gene expression remained inducible for at least 180 days. Luciferase expression was activated by PMA and ionomycin and by anti-CD3 antibodies and was inhibited by cyclosporin A. Examination of G418-resistant clones showed that rAAV-luc had integrated into the host chromosomes but that some of the clones lost some of the transferred DNA or lost expression from the transferred DNA. These results indicate that rAAV can transfer and integrate regulated gene expression into T cell lines but that the transferred genetic material may be lost or its expression may be silenced over time.     

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus.

It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high titers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ. The lower titer of rAdAAvrep-cap was associated with expression of Rep protein. Non-homologous recombination occurs after high passage and results in deletions within the AAV rep genes. These results indicate that (1) rAdAAVrep-cap can be produced; (2) rAdAAVrep-cap + rAdAAVGFP is a convenient and efficient way to transfect 293 cells to grow high titer rAAV; and (3) frozen stock is required to avoid propagation of rep-deleted pAdAAVrep-cap.