AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9

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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...     

α黑素细胞刺激素基因转染对小鼠骨髓来源的树突状细胞成熟的影响

为观察转染α黑素细胞刺激素(-αMSH)基因的树突状细胞(DC)的功能和特性,以腺相关病毒(AAV)为载体将α-MSH基因导入小鼠骨髓来源的未成熟DC(-αMSH-DC),用ELISA检测-αMSH-DC培养上清中-αMSH及IL-12水平,用流式细胞仪分析-αMSH-DC表面分子的表达,以-αMSH-DC提呈OVA抗原刺激致敏T细胞,通过ELISA测定IL-2水平来观察其抗原提呈功能。结果显示,在-αMSH-DC培养上清中检测到-αMSH的分泌,-αMSH-DC表面分子表达下调,分泌IL-12的能力降低,抗原提呈功能被抑制。-αMSH-DC可部分抵抗LPS上调表面分子表达和促进IL-12分泌的作用。表明-αMSH基因可籍AAV载体导入未成熟DC,并有效地表达,α-MSH基因修饰的DC成熟受到抑制。     

腺相关病毒载体优化的研究进展

 腺相关病毒（adeno-associated viruses，AAV）是用于基因传递与表达的常用载体，具有安全性好、免疫原性低、表达稳定等优点，在基因治疗中逐步得到应用。但是AAV载体在基因治疗中仍面临着转导效率低、载体容量小、靶向性弱以及宿主的免疫反应等限制，影响了AAV载体的广泛应用。因此，AAV载体技术的优化研究在持续推进，优化策略能够提高AAV转导效率，降低机体免疫反应，调高靶向性，扩展载体容量，优化调控。本文对相关研究进展进行综述，为AAV载体在临床应用提供一定的参考。     

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.     

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness

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