重组腺伴随病毒载体在肿瘤基因治疗中的作用

腺伴随病毒(adeno-associated virus,AAV)是一种辅助依赖型的微小病毒。在基因治疗研究中,重组逆转录病毒和重组腺伴随病毒在基因转移载体中一直占主导地位,特别是用AAV作为基因转移载体已成为基因治疗研究的热点。本文仅就重组腺伴随病毒载体在肿瘤基因治疗中的作用的相关研究     

表达胰高血糖素样肽1重组腺伴随病毒载体的构建

背景：胰高血糖素样肽1(glucagon-like peptide-1, GLP-1)在人体内半衰期过短限制了其应用。 目的：构建可表达GLP-1的重组腺伴随病毒。 方法：将NT4-GLP-1融合基因插入腺伴随病毒包装质粒pSSHG-CMV中,构建pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒。采用磷酸钙共沉淀法将辅助质粒pAAV/Ad、腺病毒质粒pFG140及pSSHG/NT4-GLP-1转染至293细胞系,用其感染Hela细胞。 结果与结论：重组质粒pSSHG/NT4-GLP-1经限制性内切酶EcoRⅠ酶切鉴定可见342 bp的目的片段,说明NT4-GLP-1融合基因已经成功重组于腺伴随病毒包装质粒pSSHG-CMV内。免疫细胞化学结果显示,转染pSSHG/NT4-GLP-1重组腺伴随病毒包装质粒的Hela细胞内有大量棕黄色颗粒,阳性率达到70%以上,说明NT4-GLP-1重组腺伴随病毒在细胞中可以表达GLP-1。     

肿瘤血管抑制肽alphastatin基因克隆及序列分析

目的克隆获得编码具有生物学活性的肿瘤血管抑制肽alphastatin基因。方法采用非对称引物／模板法，依据基因库提供的alphastatin基因序列，设计合成alphastatin基因的引物／模板链，利用聚合酶链反应（PCR）法，从DNA中扩增出人肿瘤血管抑制肽alphastatin基因片段；将获得的基因片段插入质粒载体pGEM．TEasy中，转化到大肠杆菌DH5ct后挑选阳性克隆，利用限制性内切酶酶切鉴定重组质粒。结果经质粒DNA酶切分析及序列测定，获得了人肿瘤血管抑制肽alphastatin基因片段序列。结论首次体外克隆获得了人肿瘤血管抑制肽alphastatin基因，为肿瘤血管抑制肽alphastatin的基因治疗奠定了基础。     

NT4-GFP-Ant重组腺相关病毒载体的构建及病毒包装、滴度测定

目的为使基因治疗时,较大分子的功能蛋白能通过血脑屏障,构建NT4-GFP-Ant重组腺相关病毒载体,包装重组病毒,并测定滴度。为进一步动物实验研究该重组病毒液感染鼻粘膜细胞,在鼻粘膜内表达的大分子融合蛋白沿“鼻-脑”通路人脑的可行性奠定基础。方法将已构建成功的NT4-GFP-Ant融合基因插入病毒载体质粒PSSHG中,构建重组腺相关病毒载体质粒,酶切电泳鉴定。用腺病毒辅助质粒PFG140、包装质粒pAAV/Ad及已构建的重组腺伴随病毒载体质粒,三质粒磷酸钙共沉淀法转染80％融合的293细胞系,包装重组腺病毒（rAAV）。回收病毒、斑点杂交方法测定重组病毒滴度。结果成功构建了重组腺相关病毒质粒PSSHG/NT4-GFP- Ant。包装、回收病毒后斑点杂交方法测定重组病毒滴度为3．36×10^9 PFU/ml。结论成功构建了PSSHG/NT4- GFP-Ant重组腺相关病毒载体,并成功包装了较高浓度的重组病毒。     

携带cdc2-siRNA重组腺相关病毒的包装

背景：细胞分裂周期2(cell division cycle 2, cdc2)在神经变性疾病如阿尔茨海默病的神经元变性过程中扮演了重要角色。以腺相关病毒为载体对cdc2基因进行沉默,可能对神经变性疾病的神经元起保护作用。 目的：包装携带cdc2-siRNA的重组腺相关病毒(recombinant adeno-associated virus, rAAV)。 设计、时间及地点：空白对照实验,于2007-10/2008-08在武汉同济医院神经科实验室完成。 材料：Helper Free 腺相关病毒系统(pAAV-MCS-EGFP、p-RC、p-Helper)及AAV-293细胞为Stratagene公司产品。 方法：应用分子生物学方法构建生成pAAV-MCS-U6-cdc2-siRNA-EGFP表达质粒;用磷酸钙法将该质粒和p-RC、p-Helper质粒共转染AAV-293细胞,包装生成携带cdc2-siRNA的重组腺相关病毒 (rAAV-U6-cdc2-siRNA-EGFP)。病毒感染AAV-293细胞后行Western Blot 检测cdc2-siRNA对细胞内cdc2基因的沉默效果,并用斑点杂交方法测定该病毒的滴度。 主要观察指标：pAAV-MCS-U6-cdc2-siRNA-EGFP质粒中插入片段U6-cdc2-siRNA的测序;3质粒pAAV- MCS-U6-cdc2-siRNA-EGFP、p-RC、p-Helper共转染AAV-293细胞; rAAV-U6-cdc2-siRNA-EGFP感染AAV-293细胞后cdc2的表达。 结果：①DNA测序证明U6-cdc2-siRNA已成功构建到表达质粒pAAV-MCS-EGFP中。②AAV-293细胞表达绿色荧光蛋白,共转染成功。③包装得到的重组腺相关病毒 (rAAV-U6-cdc2-siRNA-EGFP)感染AAV-293细胞后能显著下调 AAV-293细胞cdc2基因的表达量。④rAAV-U6-cdc2-siRNA-EGFP的滴度为1x1012 v.g/mL。 结论：携带cdc2-siRNA重组腺相关病毒的包装获得成功,它具有沉默cdc2基因表达的功能。     

重组腺相关病毒载体在中枢神经系统转基因研究中的应用

重组腺相关病毒（recombinant adeno-associated virus,rAAV）作为转基因载体具有无神经毒性和低免疫原性等优点,因此在基因治疗的基础研究和临床应用领域倍受重视.在中枢神经系统（central nervous system,CNS）,以rAAV为载体已经成功转导了脑和脊髓的多个部位.随着国内外对rAAV载体研究的日趋深入,其优势已为学术界所公认,现已应用于数项CNS疾病临床前期的基因治疗,其有可能成为转基因治疗CNS疾病的最佳载体.     

重组腺相关病毒对中枢神经系统特异性转染的基础研究

中枢神经系统(CNS)疾病的基因治疗研究中，基因转染载体的选择非常重要。在现有的病毒载体中，重组腺相关病毒(recombined adeno—associated virus，rAAV)安全性好，无致突变性和免疫原性，可以重复多次使用；同腺病毒相比，rAAV的弥散性更强；同单纯疱疹病毒相比，rAAV无神经毒性；rAAV     

低氧特异性重组腺相关病毒VEGF165的构建与表达

目的构建VEGF165低氧启动rAAV载体,用于缺血性脑血管病的治疗.方法通过分子生物学的方法,构建VEGF165低氧启动rAAV载体,采用磷酸钙和氯仿-PEG8000/NaCl-氯仿法构建低氧启动rAAV病毒.通过体内外低氧诱导,验证VEGF165低氧启动rAAV对低氧的特异性.结果体内外实验证实,VEGF165低氧启动rAAV仅在低氧诱导后表达具有生物活性的VEGF蛋白,对低氧具有特异性.结论VEGF165低氧启动rAAV构建成功,可望用于缺血性疾病的基因治疗.     

低氧启动重组腺相关病毒的构建

目的构建低氧启动rAAV,介导目的基因仅在低氧时特异性高效表达.方法通过分子生物学的方法,分别构建pGL3-6 HRE-CMV-mp和pGL3-6 HRE-CMV-mp-WPRE载体,比较2种低氧启动子的特异性和在低氧时的启动能力.选择最佳的低氧启动子构建低氧启动rAAV载体.采用磷酸钙和氯仿-PEG8000/NaCl-氯仿法构建低氧启动rAAV病毒.rAAV病毒蛋白电泳和EGFP报告基因验证低氧启动rAAV病毒的构建和介导目的基因在低氧时的表达. 结果HRE-CMV-mp-WPRE是最佳的低氧启动子,在低氧时高效、特异的表达目的基因.低氧启动rAAV仅在低氧时介导报告基因EGFP的表达.采用6 HRE-CMV-mp-WPRE启动子的rAAV具有良好的低氧特异性和高效启动能力.结论采用最佳的低氧启动子,本研究实现了低氧启动rAAV的构建.     

表达hVEGF165重组腺相关病毒对脊髓损伤的作用

血管内皮细胞因子曾经被认为是具有促血管生成作用的血管生长因子,如今,更多的研究将其应用于神经细胞缺血损伤的保护中。然而,一些研究却认为VEGF的应用使得脊髓损伤后脊髓的二次打击更加严重。为了探讨VEGF是否能够有效保护损伤的脊髓,我们在大鼠脊髓损伤模型中应用了表达hVEGF165的重组腺相关病毒。我们将大鼠随机分为假手术组,单纯脊髓损伤对照组,AAV-GFP组及AAV-VEGF 组。在相应时间点评估完神经功能后,我们将脊髓组织迅速取出。我们通过透射电子显微镜观察及TUNEl凋亡组织学染色观察在形态学角度评估凋亡的发生。应用免疫组化及Western blot评估凋亡蛋白Bax 和Bcl-2的表达。通过水通道蛋白-4（AQP-4）的免疫组化染色观察VEGF表达与脊髓损伤的相关性。实验结果显示,AAV-VEGF 组神经功能较对照组及AAV-GFP组有显著差异。(P < 0.05). VEGF能够明显抑制神经细胞的凋亡,减少运动神经元的损伤和丢失,减少细胞凋亡率。VEGF治疗组BAX蛋白表达减弱,Bcl-2蛋白表达加强,AQP-4表达减弱。所有结果表明VEGF具有对脊髓损伤神经的保护效应。     

利用Helper-Free系统构建cdk5-siRNA腺相关病毒载体并鉴定

目的构建能表达CDK5特异性小干扰RNA(siRNA)(cdk5-siRNA)的重组腺相关病毒(AAV)载体,体外观察其对CDK5基因的沉默作用。方法采用基因克隆技术将合成的特异性cdk5 RNA干扰寡核苷酸序列克隆至AAV Helper-Free System中的表达质粒pAAV-MCS,构建出质粒pAAV-MCS-cdk5-siRNA,通过酶切测序鉴定重组质粒;将该质粒与系统中的控制质粒pAAV-RC、辅助质粒pHelper用磷酸钙法共转染HEK293细胞,包装得到表达cdk5-siRNA的重组腺相关病毒载体(rAAV-cdk5-siRNA);用斑点杂交法测定重组病毒的滴度,重组病毒感染体外培养的PC12细胞,W estern b lot检测其抑制cdk5表达的效果。结果成功构建并包装出重组腺相关病毒载体rAAV-cdk5-siRNA,病毒滴度达4×1013/m l,重组病毒感染后的PC12细胞cdk5表达明显下调。结论构建的重组腺相关病毒载体rAAV-cdk5-siRNA能明显干扰cdk5的表达,为将其进一步应用于神经变性疾病的治疗研究奠定了基础。     

不同血清型的重组腺相关病毒载体对成熟肌纤维转导的研究

目的探讨不同血清型对成熟肌纤维的转导效率.方法我们用不同的血清型AAV（rAAV-1,rAAV-2,rAAV-3和rAAV-5）表达LacZ和GDNF基因研究在小鼠骨骼肌转导效率,转基因表达用组化方法或ELISA来评定. 结果在3周龄的小鼠中,LacZ组化染色显示rAAV5在慢和快的肌纤维中都有效的转导,而rAAV2和rAAV3优先在慢纤维中转导.在8周龄的小鼠（成年鼠）中,rAAV3和rAAV5在慢纤维和快纤维中都有效的转导.用rAAV3-LacZ或rAAV5-LacZ优先转导的慢纤维与rAAV2相比没有显著性差异.用8周龄的小鼠肌肉注射不同血清型的rAAV-GDNF后,结果显示rAAVl-GDNF表达最高;rAAV2、rAAV3和rAAV5在骨骼肌的表达也有效.结论在肌肉基因转导中,rAAV3和rAAV5介导的载体都是有效的,能克服rAAV2所受到的限制.     

Phenotype correction of hemophilia A mice with adeno-associated virus vectors carrying the B domain-deleted canine factor VIII gene

Adeno-associated virus (AAV) vectors carrying the B domain-deleted canine FVIII (BDD cFVIII) gene utilizing the beta-actin minimum promoter (167b) pseudotyped with serotype 1 (AAV1-beta-actin-cFVIII) and serotype 8 (AAV8-beta-actin-cFVIII) were developed to express cFVIII in hemophilia A mice. FVIII clotting activities measured by the APTT method increased in hemophilia A mice with intramuscular injection of AAV1-beta-actin-cFVIII in a dose-dependent manner. Therapeutic FVIII levels (2.9+/-1.0%) in hemophilia A mice with the AAV1-beta-actin-cFVIII dose of 1 x 10(12) gc/body were achieved, suggesting partial correction of the phenotype with AAV1-beta-actin-cFVIII vectors. FVIII clotting activity levels in hemophilia A mice with intravenous injection of AAV8-beta-actin-cFVIII also were increased dose-dependently, achieving therapeutic FVIII levels (5-90%) in hemophilia A mice with the AAV8-beta-actin-cFVIII doses of 1-3 x 10(11) gc/body and supernormal FVIII levels (180-670%) in hemophilia A mice with the AAV8-beta-actin-cFVIII dose of 1 x 10(12) gc/body. Transduction of the liver with AAV8-beta-actin-cFVIII is superior to transduction of skeletal muscles with AAV1cFVIII regarding the FVIII production and antibody formation. These data suggested that both AAV1 and AAV8 vectors carrying the FVIII gene utilizing a minimum promoter have a potential for hemophilia A gene therapy.     

Establishment of a recombinant adeno-associated virus expressing hVEGF165*☆

BACKGROUND： Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE： To construct a non-pathogenic, recombinant adeno-associated virus （AAV） simultaneously expressing human vascular endothelial growth factor 165 （hVEGF165） and green fluorescent protein （GFP）. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS： AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS： The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC （the rep/cap-gene containing plasmid）, and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES： Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter （FACS） upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS： The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system prov     

Establishment of a recombinant adeno-associated virus expressing hVEGF_(165)

BACKGROUND: Because certain gene vectors could have deleterious effects in the central nervous system, the choice of a safe and effective vector system has become more important for gene therapy of nerve regeneration. OBJECTIVE: To construct a non-pathogenic, recombinant adeno-associated virus (AAV) simultaneously expressing human vascular endothelial growth factor 165 (hVEGF165) and green fluorescent protein (GFP). DESIGN, TIME AND SETTING: A randomized controlled experiment was performed at the Virology Laboratory of Shaanxi Provincial Center for Disease Control and Prevention between March and September 2007. MATERIALS: AAV helper-free system, AAV-293 packaging cell line, and AAV HT-1080 cells were purchased from Stratagene, USA. E. Coli DH5α was a stocked strain from Centers for Disease Control and Prevention of Shaanxi, China. Plasmid pUC18-hHVEGF165 was a gift from Zhibin Shi. METHODS: The hVEGF165 gene was amplified by PCR from pUC18-hHVEGF165 and inserted into plasmid pAAV-IRES-hrGFP to construct recombinant plasmid pAAV-hVEGF165-IRES-hrGFP. Subsequently pAAV-hVEGF165-IRES-hrGFP, pAAV-RC (the rep/cap-gene containing plasmid), and pHelper were co-transfected into AAV-293 cells to complete rAAV-hVEGF165-IRES-hrGFP packaging through homologous recombination. The efficiency of AAV packaging was monitored under a fluorescent microscope, and the recombinant viral particles were harvested from infected AAV-293 cells, and further concentrated and purified. AAV HT-1080 cells were infected with the recombinant virus AAV-hVEGF165-IRES-hrGFP. MAIN OUTCOME MEASURES: Recombinant virus titer was measured by fluorescent cell counting, and infection efficiency was detected by Fluorescence Activated Cell Sorter (FACS) upon infecting AAV-HT1080 cells. The recombination with the exogenous gene was verified by PCR. RESULTS: The PCR amplified products were verified as hVEGF165 gene by DNA sequencing, and the recombinant pAAV-hVEGF165-IRES-GFP was confirmed by double digestion. The system provided a high packaging ratio of 95%, and the purified recombinant virus had a high titer of 5.5×1011 virus particles/mL. The AAV-HT1080 cells were infected at a ratio of 90.4%. The recombinant virus was confirmed by PCR to contain the exogenous hVEGF165 gene. CONCLUSION: The non-pathogenic rAAV-hVEGF165-GFP vector, carrying hVEGF165 and GFP reporter gene, was successfully constructed with a high titer and infection efficiency.