A next step in adeno-associated virus-mediated gene therapy for neurological diseases: regulation and targeting

Recombinant adeno-associated virus (rAAV) vectors mediating long term transgene expression are excellent gene therapy tools for chronic neurological diseases. While rAAV2 was the first serotype tested in the clinics, more efficient vectors derived from the rh10 serotype are currently being evaluated and other serotypes are likely to be tested in the near future. In addition, aside from the currently used stereotaxy-guided intraparenchymal delivery, new techniques for global brain transduction (by intravenous or intra-cerebrospinal injections) are very promising.Various strategies for therapeutic gene delivery to the central nervous system have been explored in human clinical trials in the past decade. Canavan disease, a genetic disease caused by an enzymatic deficiency, was the first to be approved. Three gene transfer paradigms for Parkinson''s disease have been explored: converting L-dopa into dopamine through AADC gene delivery in the putamen; synthesizing GABA through GAD gene delivery in the overactive subthalamic nucleus and providing neurotrophic support through neurturin gene delivery in the nigro-striatal pathway.These pioneer clinical trials demonstrated the safety and tolerability of rAAV delivery in the human brain at moderate doses. Therapeutic effects however, were modest, emphasizing the need for higher doses of the therapeutic transgene product which could be achieved using more efficient vectors or expression cassettes. This will require re-addressing pharmacological aspects, with attention to which cases require either localized and cell-type specific expression or efficient brain-wide transgene expression, and when it is necessary to modulate or terminate the administration of transgene product. The ongoing development of targeted and regulated rAAV vectors is described.     

9型重组腺相关病毒转染体外培养大鼠心肌细胞的影响

目的:研究含增强型绿色荧光蛋白(EGFP)基因的9型重组腺相关病毒(rAAV9)对大鼠心肌H9C2细胞的转染及对细胞生长的影响。方法:rAAV9-EGFP按转染复数(MOI)1×105、1×106、1×107转染H9C2细胞,转染后在倒置荧光显微镜下观察H9C2细胞中EGFP阳性表达情况,采用流式细胞仪检测rAAV9-EGFP对H9C2细胞的转染效率。观察转染后细胞生长形态,并用AlamarBlue法检测rAAV9-EGFP对H9C2细胞增殖影响。结果:转染后第1天,MOI=1×106、1×107组中EGFP开始表达;第2天,MOI=1×105组开始表达,各组EGFP表达强度随MOI值的增高而增强,同时EGFP的表达强度随着时间延长而逐渐增强;转染后第4天达到高峰,此时流式细胞术检测rAAV9-EGFP对H9C2细胞的转染率分别为MOI=1×105组:(14.1±0.2)%、MOI=1×106组:(35.1±4.8)%、MOI=1×107组:(56.8±0.1)%。rAAV9-EGFP转染H9C2细胞后,细胞生长及形态正常,AlamarBlue法进一步检测表明转染组对H9C2细胞增殖无影响。结论:rAAV9-EGFP能够有效地转染H9C2细胞,且rAAV9-EGFP对H9C2细胞增殖无明显抑制。     

腺相关病毒-1介导增强型绿色荧光蛋白基因在大鼠角膜基质细胞体内外转染的研究

目的:探讨血清1型腺相关病毒(rAAV1)介导增强型绿色荧光蛋白(EGFP)基因体内、外转染大鼠角膜基质细胞后绿色荧光蛋白(GFP)的表达及转染率。方法:采用携带绿色荧光蛋白报告基因的血清1型腺相关病毒(rAAV1-EGFP)感染原代培养的SD大鼠角膜基质细胞,通过荧光显微镜和流式细胞仪观察和检测EGFP的表达及阳性率。建立大鼠异体角膜移植模型,用含rAAV1-EGFP转染液的培养基在37℃孵育SD大鼠角膜植片同时浸泡缝线18h,再用此缝线间断缝合,将植片移植到大鼠角膜植床,术后通过荧光体视镜观察Wistar大鼠角膜出现EGFP荧光的起始时间及分布情况。结果:按rAAV1-EGFP不同转染倍数(MOI)转染角膜基质细胞。转染后3d,在倒置荧光显微镜下观察到角膜基质细胞的细胞质有GFP阳性表达。体视荧光显微镜观察到大鼠角膜中开始有GFP阳性表达;随MOI值的增高而增强,转染后7~9d达到高峰,此时检测rAAV1-EGFP对角膜基质细胞的转染效率,MOI=103时是16.3%,MOI=104时是30.3%,MOI=105时是43.6%,MOI=106时是47.5%。rAAV1-EGFP在大鼠角膜中的表达也明显增强。结论:rAAV1-EGFP可以在体内外稳定、有效转染大鼠角膜基质细胞,是角膜基质细胞理想的报告基因。     

rAAV2载体介导GFP基因转染树突状细胞

目的:在诱导人骨髓CD34 细胞分化成树突状细胞(dendritic cells,DCs)的基础上,探讨2型重组腺相关病毒(Type 2 recombinant adeno-associated virus,rAAV2)载体介导绿色荧光蛋白(green fluorescent protein,GFP)基因转染DCs的条件.方法:采用免疫磁珠法分离纯化人骨髓来源的CD34 细胞.在含有IL-4和GM-CSF的培养体系中体外诱导CD34 细胞生成DCs,于第5天加入TNF-α诱导DCs成熟,在不同时间用rAAV2载体介导GFP基因转染未成熟和成熟的DCs.通过电子显微镜和流式细胞仪鉴定树突状细胞,荧光显微镜及流式细胞仪检测GFP的表达.结果:人骨髓CD34 细胞经诱导分化后,在光镜及透射电镜下均可观察到DCs的形态特征,流式细胞仪检测到DCs表型;荧光显微镜及流式细胞仪检测细胞培养第3天、第5天和加入TNF-α后的rAAV2-GFP转染率分别为0.45%,13.54%和0.25%.结论:本实验中所采用的培养体系可成功将人骨髓CD34 细胞诱导分化成DCs,rAAV2/GFP转染DCs效率随细胞诱导分化时间的延长而增高,且转染未成熟DCs的效率高于转染成熟DCs.     

增强型绿色荧光蛋白基因在永生化大鼠神经前体细胞株的表达

目的:研究重组腺相关病毒载体介导的增强型绿色荧光蛋白基因在永生化大鼠神经前体细胞的转染效率及其表达情况. 方法:携带增强型绿色荧光蛋白基因的重组腺相关病毒转染永生化大鼠神经前体细胞,持续观察转基因神经前体细胞绿色荧光蛋白的表达情况,并计算转染效率. 应用巢蛋白抗体和抗猿肾病毒40大T抗原抗体进行细胞鉴定,50 mL/L胎牛血清诱导细胞分化后,应用神经元和星形胶质细胞特异性标志物抗体检测其分化能力. 结果:荧光显微镜观察显示转染后24 h神经前体细胞即有绿色荧光蛋白表达,转染后72 h,大部分细胞表达绿色荧光蛋白,转染效率可达90%. 经传代培养8 wk后,表达绿色荧光蛋白的阳性细胞仍可高达70%～75%. 免疫细胞化学结果显示转绿色荧光蛋白神经前体细胞巢蛋白和抗猿肾病毒40大T抗原抗体染色均为阳性,50 mL/L胎牛血清可诱导其分化为神经元和星形胶质细胞. 结论:携带增强型绿色荧光蛋白基因的重组腺相关病毒可高效转染永生化大鼠神经前体细胞,并可在细胞中长期稳定地表达,绿色荧光蛋白作为良好的示踪剂可应用于神经前体细胞的体内移植.     

RGD-rAAV2提高卵巢癌细胞体外基因转导效率的研究

目的 进一步证实精氨酸-甘氨酸天冬氨酸-重组腺相关病毒2（RGD-rAAV2）基因表达系统靶向卵巢肿瘤的能力。方法 用流式细胞仪（FACS）检测6种卵巢癌细胞株的硫酸肝素蛋白多糖（HSPG）、整合素（integrin）α，β3和integrin αvβ5的表达；以及野生型腺相关病毒2（wt-AAV2）对上述卵巢癌细胞感染并介导基因转导的能力；通过配体竞争结合实验进一步验证突变体RGD-rAAV2基因转导的特异性。结果 wt-AAV2感染不同卵巢癌细胞的能力各有不同；卵巢癌细胞表达HSPG水平差异很大，而80％卵巢癌细胞表达integrin αvβ3或（和）integrin αvβ5；RGD-rAAV2病毒载体可以有效地感染对wt—rAAV2耐受的卵巢癌细胞SKOV-3ip并介导基因的表达，该效果是非HSPG依赖性的。结论 RGD-rAAV2病毒载体具有较高的卵巢肿瘤靶向性，为卵巢癌基因靶向治疗提供了一种新的策略。     

人CTGF和TIMP1基因重组腺相关病毒双表达质粒的构建及其活性检测

 目的 构建人结缔组织生长因子（connective tissue growth factor, CTGF）和基质金属蛋白酶组织抑制剂1(tissue inhibitor of metalloproteinases 1, TIMP1)基因腺相关病毒（adeno-associated virus, AAV）表达质粒，并观察其在HEK293细胞中的表达，检测目的蛋白的生物学活性。方法 采用PCR技术，分别设计引物扩增所需序列并进行PCR鉴定。再采用分子克隆的方法，以内部核糖体进入位点(internal ribosome entry sites, IRES)序列连接CTGF和TIMP1并克隆到AAV表达质粒pSNAV2.0构建重组质粒pSNAV2-CTGF-IRES-TIMP1。把重组质粒转染HEK293细胞，用双重免疫荧光和Western blot的方法对重组质粒的表达进行研究，MTT法检测细胞培养上清液中CTGF蛋白的生物学活性，ELISA法检测细胞培养上清液中TIMP1蛋白的含量。结果 PCR、酶切、DNA测序等方法均证实成功构建了含有完全正确的CTGF和TIMP1基因序列的AAV表达质粒。双重免疫荧光和Western blot检测到目的蛋白的表达，转染后的细胞上清具有促使成纤维细胞增殖的生物学活性，ELISA法结果证实重组载体能够高表达TIMP1蛋白。结论 成功构建了双表达质粒pSNAV2-CTGF-IRES-TIMP1，转染 HEK293细胞后能够表达具有生物学活性的目的蛋白，为基因治疗椎间盘退变的研究工作奠定了基础。     

Construction of adeno-associated virus coexpression system for human angiopoietin-1 and VEGF gene

Background Ischemic disease is one of the leading causes of death in the world. In order to further study gene therapy for ischemic disease, we constructed a recombinant plasmid for co-expression of human angiopoietin-1 and vascular endothelial growth factor 165 (VEGF165) gene in adeno-associated virus (AAV) gene delivery system.Methods Human angiopoietin 1 and VEGF165 gene were obtained using PCR. The upstream of angiopoietin 1 contained restriction enzyme site Hind Ⅲ,contained restriction enzyme site BamH Ⅰ. The upstreamand the downstream of angiopoietin 1 of VEGF165 contained restriction enzyme site Bgl Ⅱ, and the downstream of VEGF165 contained restriction enzyme site BamH Ⅰ, Using the multiple cloning sites (MCS) in plasmid pZero such as BamH Ⅰ , Bgl Ⅱ, Hind Ⅲ, Not Ⅰ , Xho Ⅰ ,Xba Ⅰ , Sal Ⅰ , BspH Ⅰ , Ksp Ⅰ and the corresponding MCS in plasmid pAAV-MCS, angiopoietin 1 and VEGF165 gene were subcloned into pAAV-MCS.Results DNA sequencing revealed that the PCR- amplified angiopoietin 1 and VEGF165 were consistent with NCBI Gene Bank. The recombinant plasmid was identified using PCR and digestion,which proved to be consistent with our hypothesis. In recombinant plasmid, angiopoietinl and VEGF possessed a CMV promoter and polyA terminator system respectively, thus assuring co-expression of the two genes.Conclusion Successful construction of AAV co-expression system for human angiopoietin 1 and VEGF165 gene will provide the foundation for gene therapy to cure severe ischemic disease.     

腺病毒提高腺相关病毒在耐药与非耐药肿瘤细胞中的基因表达及其机制

目的:研究利用低剂量腺病毒提高重组腺相关病毒2型(recombined adeno-associated virus,rAAV2)在耐药与非耐药肿瘤细胞中基因表达水平的新方法,并初步探讨其可能的作用机制。方法:rAAV2-GFP单独或联合复制缺陷型腺病毒(Ad5-RFP)或条件复制型腺病毒(Ad5-TERT-RFP)感染人非小细胞肺癌细胞系(NCI-H446)、人肺腺癌细胞系(A549)、人胃癌细胞系(SGC7901)、人口腔黏膜上皮癌细胞系(KB)和人口腔黏膜上皮癌耐长春新碱细胞系(KB/VCR)。荧光显微镜观察和流式细胞仪分析肿瘤细胞感染后GFP的表达;Western blotting检测感染后肿瘤细胞GFP蛋白表达及ERK和AKT磷酸化水平;Real-time PCR检测肿瘤细胞内GFP的mRNA表达量、DNA拷贝数,以及细胞表面受体HSPG、α_v integrin和FGFR-1的mRNA表达。结果:流式细胞结果显示,rAAV2-GFP联合Ad5-RFP或Ad-TERT-RFP感染肿瘤细胞24h后,GFP阳性细胞率和GFP平均荧光亮度分别提高了约0.3～3倍和4～8倍。Western blotting证实联合应用Ad5-RFP感染肿瘤细胞后24h,GFP蛋白表达增加约4～6倍,肿瘤细胞内ERK和AKT磷酸化水平升高。联合感染后细胞内GFP的mRNA表达量提高了3.83～7.33倍,DNA拷贝数未见明显改变,HSPG、α_v integrin和FGFR-1的mRNA表达轻微提高。结论:低剂量腺病毒显著提高rAAV2在耐药与非耐药肿瘤细胞中的基因表达水平,可能与激活信号传导通路、增加细胞内转录有关。     

Dysregulation of dopamine signaling in the dorsal striatum inhibits feeding

Dopamine signaling is an important component of many goal-directed behaviors, such as feeding. Acute disruption of dopamine signaling using pharmacological agents tends to inhibit normal feeding behaviors in rodents. Likewise, genetically engineered dopamine-deficient (DD) mice are unable to initiate sufficient feeding and will starve by approximately 3 weeks of age if untreated. Adequate feeding by DD mice can be achieved by daily administration of L-3,4-dihydroxyphenylalanine (L-dopa), a precursor of dopamine, which can be taken up by dopaminergic neurons, converted to dopamine, and released in a regulated manner. In contrast, adequate feeding cannot be restored with apomorphine (APO), a mixed agonist that activates D1 and D2 receptors. Viral restoration of dopamine production in neurons that project to the dorsal striatum also restores feeding in DD mice. Administration of amphetamine (AMPH) or nomifensine (NOM), drugs which increase synaptic dopamine concentration, inhibits food intake in virally rescued DD mice (vrDD) as in control animals. These results indicate that the dysregulation of dopamine signaling in the dorsal striatum is sufficient to induce hypophagia and suggest that regulated release of dopamine in that brain region is essential for normal feeding and, probably, many other goal-directed behaviors.