腺相关病毒载体基因治疗的研究进展

腺相关病毒 (ＡＡＶ)属于病毒中最小、也是最简单的一种———细小病毒属 ,它是一种有缺陷的非致病性的人类细小病毒 ,重组的腺相关病毒 (ｒＡＡＶ)作为基因治疗的载体 ,对长期基因矫正和基因治疗具有优越性。新的生产工艺提高了ｒＡＡＶ的滴度 ,去除了腺病毒的污染 ,并构建了适用于不同靶细胞的可诱导载体。这些改进有望加速进一步的动物实验研究 ,使临床治疗人类疾病早日成为可能。1 .ｒＡＡＶ的制备方法在 1 989年 ,首次产生了没有野生型ＡＡＶ帮助复制的重组的ＡＡＶ ,这以后 ,为了增加病毒颗粒数 ,人们制造了许多ＡＡＶ辅助质粒 ,其中…     

腺相关病毒载体与中枢神经系统的基因治疗

本文对ＡＡＶ的分子生物学结构,重组和其对细胞的整合及ｒＡＡＶ载体在基因治疗中的作用,尤其是ｒＡＡ载体在中枢神经系统疾病基因方面的最新应用作了较全面阐述。     

重组腺相关病毒：很有潜力的基因治疗载体

腺相关病毒(AAV)是细小病毒家族的一员,为无包膜的线性单链DNA病毒.由于AAV具有长期潜伏于人体而不具有任何明显致病性等优点,人们对AAV作为一种理想的基因治疗载体给予了很大期望.但是,近来发现,这类载体在应用上有许多明显的缺陷,包括某些细胞膜上病毒受体数量极少,重组AAV载体位点特异性整合不足,AAV衣壳成分和转基因产物引起宿主的免疫反应等等.这些缺陷促使人们加大对AAV生物学特性和转染过程的研究,从而更好地对AAV载体进行改进,使新一代重组AAV载体具备基因治疗所必需的安全性、高效性和靶向性,以期更广泛地应用于临床.     

重组腺相关病毒介导的RNAi靶向治疗作用研究

目的:采用前期成功构建的靶向沉寂CDK2基因的重组腺相关病毒r AAV-sh RNA-CDK2转染人肝癌Hep G2细胞,研究其对人肝癌细胞增殖的抑制作用。方法:取人肝癌Hep G2细胞于裸鼠前肢腋下接种,构建裸鼠皮下移植瘤模型,将成瘤裸鼠随机分为三组:肿瘤组、NC对照组、r AAV-sh RNA-CDK2给药组。各试验组均通过尾静脉注射给药,每隔五天用游标卡尺测量肿瘤的长径(a)、短径(b),计算肿瘤体积。根据每组裸鼠移植瘤体积的平均值,绘制移植瘤生长曲线。于给药24 h后处死,称取瘤重,计算抑瘤率,应用实时荧光定量PCR和Western blot方法检测各组肝癌组织中CDK2基因m RNA和蛋白的表达量,观察r AAV-sh RNA-CDK2对肝癌组织CDK2表达的影响;结果:r AAV-sh RNA-CDK2能够显著抑制肝癌Hep G2细胞的增殖,其抑瘤率为72.18%;并能够下调肝癌组织中CDK2基因m RNA与蛋白表达量;结论:r AAV-sh RNA-CDK2实现了体内靶向治疗肝癌的目的,并确定静脉定量给药方式。     

腺相关病毒在脂代谢研究和降脂基因治疗中的应用

动脉粥样硬化性心脑血管疾病是当前全球死亡率最高的疾病,而脂代谢紊乱是动脉粥样硬化性心脑血管疾病的主要病因,其既可以导致心肌梗死、脑卒中、急性胰腺炎等急性病,也可以导致慢性肾脏疾病。近年来基因治疗技术的快速发展,为脂代谢机制研究提供了有效的手段,特别是使先天性脂代谢异常患者的治愈成为可能。腺相关病毒（adeno associated virus,AAV）宿主范围广、安全性高、免疫原性低、表达长期稳定,是当前单基因遗传病基因治疗首选的递送工具。以AAV为载体的多种降脂基因治疗药物目前已经得到临床应用或正在进行临床试验。本文综述了AAV载体在脂质代谢机制研究的新进展及其在降脂基因治疗中的应用和前景。     

重组腺相关病毒载体的研究进展

重组腺相关病毒(rAAV)载体作为基因治疗的工具,具有无致病性、长期表达、低免疫原性和感染多种细胞等特点.因此,rAAV载体越来越受到重视.就rAAV载体在生物学特性、构建、安全性和应用方面作一介绍.     

重组腺相关病毒载体在肝疾病基因治疗中的应用及局限性

基因治疗(gene therapy)是近十年来随着现代分子生物学技术的发展而诞生的新的生物医学治疗技术.重组腺病毒伴随病毒是一种具有开发潜质的病毒载体,因此近年来对它的研究很是受人关注.肝炎、肝硬化、肝癌在我国乃至全世界是严重损害人们的健康,据此,国内外的研究人员对rAAV在肝疾病基因治疗方面做了大量的工作,本文对rAAV在肝疾病基因治疗中的应用和局限性研究进行综述.     

重组腺相关病毒载体的整合性研究进展

重组腺相关病毒(rAAV)载体是一种具有高靶向性和良好安全性的病毒载体,在基因治疗中得到了较为广泛的应用。目前全球范围内已有70余项以rAAV为基因药物的临床研究已经完成或正在进行中。与野生型AAV(wtAAV)定点整合不同,不表达Rep蛋白的rAAV载体与宿主染色体发生的是随机整合,而这给临床应用带来了可能的潜在的安全隐患。该文在综述wtAAV和rAAV整合机理的基础上对rAAV的因随机整合而可能导致的致癌性及其他后果进行探讨,并总结了相应应对策略,特别是目前利用Rep蛋白所开展的定点整合研究。     

重组腺相关病毒载体相关性杂质

可以稳定表达治疗基因而无明显不良反应的重组腺相关病毒 (rAAV) 载体被认为是最有发展前景的基因治疗载体。但如何建立可以有效去除rAAV载体内具有潜在致病危害的杂质、产品质量符合临床使用要求的纯化工艺是研究人员面临的巨大挑战。其中针对载体相关性杂质的纯化工艺尤为关键,因为该类杂质的性质与真正的rAAV载体极其相似,一旦存在便难以去除,且会引起严重不良反应。以下总结了该类杂质形成的过程及有别于rAAV载体的特点,并对可以防止其生成或将其与rAAV载体有效分离的技术手段进行了评价。     

基因治疗的新载体——腺联病毒

基因治疗将一种全新的概念引入医学界,给现有条件尚难治愈的疾病患者带来一线希望。基因治疗主要是利用病毒介导的基因转移,腺联病毒ＡＡＶ载体,由于其得天独厚的特点,正广泛地用于基因治疗研究。文中简单地介绍了ＡＡＶ的生物学特点、载体结构及其在基因治疗研究中的优越性。     

新型重组AAV5/5载体及包装系统

本研究组建了一种可用于规模化生产的以重组单纯疱疹病毒为辅助病毒的AAV5/5载体包装系统。首先,将5型腺相关病毒 (AAV5) 的rep和cap基因插入I型单纯疱疹病毒 (HSV-1) 基因组非必需基因UL2中,获得重组病毒rHSV1-rep5cap5。其次,构建一种携带AAV5 ITR的通用型载体质粒pAAV5neo,将报告基因EGFP插入pAAV5neo中,得到pAAV5neo-EGFP质粒。将pAAV5neo-EGFP质粒导入BHK-21细胞,用G418选择培养,挑选出表达EGFP并在重组病毒rHSV1-rep5cap5感染下能高效产生rAAV5/5-EGFP的单克隆载体细胞株C020。用rHSV1-rep5cap5感染C020细胞制备rAAV5/5-EGFP,用“氯仿处理-聚乙二醇/氯化钠-氯仿抽提”方法粗纯化rAAV5/5-EGFP。用100 kDa分子量截流超滤方法进一步纯化和浓缩,获得高纯度的rAAV5-EGFP。SDS-PAGE电泳分析可见3条特征性外壳蛋白带。电镜分析显示病毒颗粒以实心颗粒为主。用rAAV5/5-EGFP病毒按1×105 vg/cell感染体外培养的HEK293细胞,可见30％细胞呈现绿色荧光。本研究提出了一种高效AAV5/5载体生产系统和纯化方法,为重组AAV5载体的进一步应用提供了基础。     

Comparative transductions of breast cancer cells by three DNA viruses

Defining the ideal vectors to transduce breast cancer using viruses is currently under intense pre-clinical evaluation. Our study constitutes the first direct comparison of the infection efficiencies of a human serotype 5 (Ad5), a canine serotype 2 (CAV-2) adenovirus, and a human serotype 2 adeno-associated virus (AAV-2) in breast cancer cells. We observed an excellent infection efficiency for Ad5 vector, whereas both CAV-2 and AAV-2 vectors lead to low infection of these cells. Real-time PCR, flow cytometry, and antibody blocking studies suggest that Ad5 and CAV-2 infection ability is not strictly dependent on coxsackie adenovirus receptor (CAR) or alpha(v) integrin levels. In conclusion, our data suggest that human adenoviruses are excellent transducers of breast cancer cells, though it may be difficult to predict the extent of infection solely on CAR or alpha(v) integrin levels.     

Viral vector-mediated [corrected] expression of human collagen Q in cultured cells

Congenital myasthenic syndromes are caused by mutations in molecules expressed at the neuromuscular junction. Collagen Q (ColQ) makes a triple helical structure and anchors the catalytic subunit of acetylcholinesterase (AChE) to the synaptic basal lamina in the form of asymmetric AChE. Mutations in the collagen Q gene (COLQ) cause endplate AChE deficiency. As an initial step to develop a novel therapeutic strategy for endplate acetylcholinesterase deficiency, we expressed AChE species in cultured cells using retrovirus and adeno-associated virus (AAV). The retroviral vectors carried human ACHE and COLQ either in a single construct (EF1alpha-ACHE-IRES-COLQ) or in two separate constructs (EF1alpha-ACHE and EF1alpha-COLQ). We produced high-titer retroviruses using the PLAT-E retrovirus packaging cells. We also confirmed expression of asymmetric AChE in the PLAT-E cells. We infected NIH3T3 and confirmed expression of the transgenes by RT-PCR. The AAV vector carried human COLQ-IRES-EGFP downstream of the CMV promoter (pAAV-CMV-COLQ-IRES-EGFP). We produced recombinant AAV using HEK293 cells carrying pDF6 encoding the AAV6 capsid gene. We infected AAVHT1080 cells and confirmed expression of COLQ by RT-PCR and EGFP by flow cytometry. We are currently trying to achieve further higher expression levels of transgenes in cultured cells to make the current strategy applicable to an animal model.     

AAV mediated expression of anti-sense neuropeptide Y cRNA in the arcuate nucleus of rats results in decreased weight gain and food intake

Neuropeptide Y (NPY) is the most potent stimulant of feeding when administered by intracerebroventricular injection. Despite this, there is conflicting evidence as to its importance in the regulation of daily food intake and energy balance. It has been suggested that whilst it is important in the response to starvation it has little role in the regulation of daily food intake. To investigate the role of NPY in the regulation of food intake, anti-sense cRNA to NPY was expressed in the arcuate nucleus of adult male rats. The anti-sense NPY (AS-NPY) construct was initially tested in vitro and there was a decrease of approximately 50% in NPY release from anti-sense treated cells compared to controls (16.3 +/- 2.0 fmol/L [AS-NPY] vs 37.3 +/- 7.7 fmol/L [control], mean +/- SEM p < 0.05). NPY release from hypothalamic explants from anti-sense injected animals was decreased by over 50% compared to those from controls at both 15 and 20 days after AAV injection (15 days 42% +/- 6.5% [AS-NPY] vs 100% +/- 36% [control], 20 days 41% +/- 6% [AS-NPY] vs 100% +/- 27% [control] mean+/-SEM, p < 0.05). In a study lasting for 50 days, weight gain was significantly lower in anti-sense injected animals from day 16 (day 16: 6.25 +/- 1.10 g [AS-NPY] vs 9.42 +/- 0.65 g [control] mean +/- SEM, p < 0.05) and remained so until the end of the study when they had gained approximately 40% less weight than controls (day 50: 52.0 +/- 9.6 g [AS-NPY] vs 82.0 +/- 6.3 g [control] mean +/- SEM, p < 0.01). Cumulative food intake was significantly lower in the anti-sense injected animals from day 23 (day 23: 225.8 +/- 1.9 g [AS-NPY] vs 250.6 +/- 8.7 g [control], mean +/- SEM, p < 0.05) and remained so until the end of the study (day 50: 834.5 +/- 14.8 g [AS-NPY] vs 926.0 +/- 31.7 g [control], mean +/- SEM, p < 0.05). Similarly mean daily food intake was also reduced in the anti-sense injected animals (days 7-14: 24.9 +/- 0.4 g/day [AS-NPY] vs 27.2 +/- 0.4 g/day [control], mean +/- SEM, p < 0.01). These data are supportive of a role for NPY in the regulation of daily food intake as well as in response to starvation.     

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.     

2型腺相关病毒转导人胎肝造血细胞及其介导的β珠蛋白基因的表达

β地中海贫血是一种因β珠蛋白基因缺陷导致的遗传性贫血性疾病,基因治疗是唯一有望治愈该病的方法.2型腺相关病毒(adeno-associated virustype2,AAV2)是一种非致病性病毒,作为一种基因治疗载体,其应用潜力日益受到关注.目前还未见AAV2转导人早期胎肝造血细胞及其介导β珠蛋白基因在动物体内表达的实验报道.有研究表明,AAV2转导人造血干细胞的效率,因各实验室包装和纯化rAAV2的方法不同而存在差异,其中辅助病毒的污染被认为是一重要原因.制备了无辅助病毒污染的rAAV2,经体外检测其滴度,纯度及功能后,再转导人早期胎肝造血细胞,将被转导的胎肝造血细胞移植入受亚致死量剂量照射的8只BALB/C裸鼠体内,检测rAAV2介导的β珠蛋白基因在裸鼠体内的表达.结果显示:制备的无辅助病毒污染的rAAV2具有较高的滴度、纯度,并能够在体外介导β基因的表达;在8只受试BALB/C裸鼠中,RT-PCR在2只BALB/C裸鼠骨髓中检测到β珠蛋白基因的表达.提示,rAAV2能够转导人早期胎肝细胞并介导β珠蛋白基因的表达,但同时也存在表达量较低的缺点,应用于β地中海贫血的基因治疗还需要对AAV2生物学特性做深入的研究.     

Valproic acid,a mood stabilizer and anticonvulsant,protects rat cerebral cortical neurons from spontaneous cell death: a role of histone deacetylase inhibition

Overexpression of a dominant-negative truncated Kv1.1 (Kv1DN) polypeptide in the mouse heart resulted in marked attenuation of a 4-aminopyridine (4-AP)-sensitive current, I_K,slow1. We used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of Kv1.5 into the mouse myocardium in order to normalize the action potential duration (APD) 6 months after injection. The injection of rAAV-Kv1.5 reconstituted the 4-AP-sensitive outward potassium currents, shortened the APD, and eliminated spontaneous early afterdepolarizations. Immunoblots detected the FL-Kv1.5 polypeptides only in rAAV-Kv1.5-infected hearts. These data demonstrate long-term expression of 4-AP-sensitive potassium currents in ventricular myocytes by gene transfer using rAAV vector encodes Kv1.5.