乙型肝炎表面抗原基因重组2型腺相关病毒的免疫原性研究

目的观察含乙型肝炎表面抗原(HBsAg)基因的2型重组腺相关病毒(rAAV-2-HBsAg)在Balb/c小鼠体内的免疫原性。方法以5×1011的重组病毒颗粒单次注射Balb/c小鼠,RT-PCR扩增肝脏组织中HBsAg基因cD-NA:ELISA检测肝组织中HBsAg的表达;RIA法检测血清中表面抗体(抗一HBs)滴度;51Cr释放分析检测细胞毒性T淋巴细胞(CTL)活性。结果外源性HBsAg基因己整合到肝细胞染色体DNA并有效地转录和翻译,表达水平最高可达33.8pg/(mglivertissue),rAAV-HBsAg免疫小鼠后可以诱导机体产生表面抗体并激发特异性CTL。结论外源性HBsAg基因可以转移到小鼠肝细胞并稳定表达;rAAV-2-HBsAg免疫小鼠可以同时诱导体液和细胞免疫反应的出现。提示基于rAAV-2载体的乙型肝炎(HBV)疫苗,对于防止HBV感染,尤其是作为慢性乙型肝炎的治疗性疫苗可能只有潜在的应用价值,值得做进一步的系统研究。     

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。     

Nurr1基因在大鼠体外培养骨髓基质细胞的表达

目的 :为获得高效表达孤儿核受体 (orphannuclearreceptor,Nurr1)基因的骨髓基质细胞。 方法 :采用分子克隆技术 ,构建携带Nurr1基因的重组腺相关病毒 (adeno associatedvirus,AAV)载体 ;与包装质粒 pAAV RC和辅助质粒phelper一起用磷酸钙法转染包装细胞HEK2 93制备具有感染活性的AAV Nurr1病毒粒子 ;用病毒上清液感染原代培养的大鼠骨髓基质细胞 (Marrowstromalcells,MSCs) ,并用免疫细胞化学方法检测阳性细胞。 结果 :经酶切鉴定和DNA测序 ,得到了序列正确的重组pAAV Nurr1;感染HT10 80细胞进行病毒滴度测定 ,每mL病毒贮存液可达 10 12 个阳性细胞 ;获得了Nurr1阳性的骨髓基质细胞 ,阳性率在 6 0 %以上。结论 :重组AAV携带的Nurr1基因能够在MSCs中表达 ,本文为进一步探讨将该细胞用于帕金森病基因治疗的可能性研究奠定了基础。     

Immunogenicity of an AAV-Based COVID-19 Vaccine in Murine Models of Obesity and Aging

The SARS-CoV-2 pandemic has had a disastrous impact on global health. Although some vaccine candidates have been effective in combating SARS-CoV-2, logistical, economical, and sociological aspects still limit vaccine access globally. Recently, we reported on two room-temperature stable AAV-based COVID-19 vaccines that induced potent and protective immunogenicity following a single injection in murine and primate models. Obesity and old age are associated with increased mortality in COVID-19, as well as reduced immunogenicity and efficacy of vaccines. Here, we investigated the effectiveness of the AAVCOVID vaccine candidates in murine models of obesity and aging. Results demonstrate that obesity did not significantly alter the immunogenicity of either vaccine candidate. In aged mice, vaccine immunogenicity was impaired. These results suggest that AAV-based vaccines may have limitations in older populations and may be equally applicable in obese and non-obese populations.     

重组腺相关病毒载体表达抗表皮生长因子受体抗体对胰腺癌细胞株抑制作用的研究

目的 构建能在体内长期表达抗表皮生长因子受体(EGFR)抗体的重组腺相关病毒(rAAV)载体,观察其对人胰腺癌细胞株的抑制作用.方法 将抗人EGFR单链抗体可变区(SCFV)基因插入腺相关病毒载体的Kpn Ⅰ和BglⅡ位点,构建成含抗EGFR单链抗体基因的重组腺相关病毒载体(rAAV-anfiEGFR).设立对照组(rAAV1-EGFP组)和实验组(rAAV1-anti EGFR组),观察抗EGFR单链抗体蛋白的表达情况.采用Western blot检测抗体蛋白的表达、CCK-8法和流式细胞仪检测6种人胰腺癌细胞株(PCT-3、SW1990、Capan-1、ASPC-1、MiaPaCa-2及PANC-1细胞)的抑制率和凋亡率.两组比较采用t检验.结果 抗EGFR单链抗体蛋白能在6种人胰腺癌细胞株中表达,对照组和实验组胰腺癌ASPC-1细胞抑制率分别为1.1%±2.4%和15.1%±3.5%,两组比较,差异有统计学意义(t=6.598,P＜0.05).对照组和实验组PANC-1细胞凋亡率分别为7.0%±3.0%和11.4%±2.5%,两组比较,差异无统计学意义(t=1.952,P＞0.05);对照组和实验组SW1990、ASPC-1、Capan-1、PCT-3、MiaPaCa-2细胞凋亡率分别为1.1%±0.8%、1.5%±0.7%、1.7%±1.2%、1.1%±0.7%、2.2%±1.1%和17.6%±2.2%、46.9%±3.9%、20.0%±2.8%、12.1%±1.6%、31.1%±2.5%,两组比较,差异均有统计学意义(t=12.208,19.846,10.405,10.909,18.327,P＜0.05).结论 成功构建了携带抗EGFR单链抗体基因的rAAV载体,其表达的抗EGFR单链抗体蛋白对人胰腺癌细胞株有明显的抑制作用.     

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.     

Innovation in Chemistry,Manufacturing, and Controls—A Regulatory Perspective From Industry

This review describes the landscape of novel modalities such as cell and gene therapies, viruses, other novel biologics, oligomers, and emerging technologies, including modern analytics. We summarize the regulatory history and recent landmark developments in some major markets and examine specific chemistry, manufacturing, and controls (CMC) challenges, including suggestions for exploration of potential science-based approaches in support of regulatory strategy development from an industry perspective. In addition, we evaluate the economic factors contributing to patient access to innovation and discuss the impact of regulation. There is a desperate need for a consistent form of regulation where global approaches to regulatory strategies can be harmonized, and specific CMC challenges can be dealt with using the appropriate science and risk-based tools. Although these tools are well described in current guidance documents, the specifics of applicability to complex novel modalities can still result in differing regulatory advice and outcomes. The future goals for efficiently regulating innovative modalities and technologies could be aided by more regulatory harmonization, regulatory education, and industry cooperation through consortia, enabling industry to supply key information to regulators in a transparent yet well-defined manner, and utilizing mutually understood risk-benefit analyses to produce drugs with appropriate safety, efficacy, and quality characteristics.     

靶向敲减KLF4基因的重组腺相关病毒载体的构建及其在肺动脉高压动物模型中的应用

目的 构建靶向敲减KLF4基因的重组腺相关病毒载体,并应用于肺血管疾病,为后期研究肺血管KLF4基因敲减在肺动脉高压大鼠中的作用及相关分子机制奠定基础。方法 根据大鼠KLF4基因序列,设计针对大鼠特异性的siRNA序列,以pHBAAV-U6-MCS-CMV-EGFP作为空白载体,通过酶切技术构建带有GFP荧光标记的KLF4干扰腺病毒载体pHBAAV-r-KLF4 shRNA-GFP,行测序分析,并进行病毒扩增、纯化及滴度测定。本研究对长时间（3个月）接触香烟烟雾的大鼠进行气道注入AAV1-KLF4-shRNA的干预治疗,观察大鼠右心室收缩压、平均右心室压等血流动力学指标改变。结果 经测序检测显示,大鼠AAV1-KLF4-shRNA腺相关病毒载体构建成功。健康对照组、生理盐水模型组、对照病毒模型组、治疗干预组的右心室收缩压和平均右心室压比较,差异均有统计学意义（P <0.05）。结论 对长时间接触香烟烟雾的肺动脉高压模型大鼠应用AAV1-KLF4-shRNA腺相关病毒载体,能有效改善右心室收缩压和平均右心室压,该载体在相关疾病动物模型中应用有效,为后期顺利开展AAV1-KLF4-s...