Endothelial-targeted adenovirus for organ-selective gene editing in vivo

项目摘要 CRISPR/Cas9位点特异性核酸酶系统已经衍生出广泛的生物学应用， 近年值得注意的是，以有针对性的方式完成基因编辑的能力也影响了设计 基因治疗策略的一个扩展库的疾病。实现基因编辑功能的关键 CRISPR/Cas9系统在基因治疗背景下的应用是实现有效共递送的要求 在组成成分的体内。这个交付问题已经处理应用非病毒和病毒 向量系统在选定的情况下，成功的基因编辑促进的基因疗法已经被证明是有效的。 在遗传性疾病的模型系统中完成。 尽管有这些优雅的原理验证研究，但可用载体技术的限制极大地限制了 CRISPR/Cas9促进的基因疗法的应用。在这方面，CRISPR/Cas9的有效体内共递送是有效的。 靶向体细胞是许多这些应用所需要的。这种交付应仅限于 体内关键细胞靶点，以最大限度地减少脱靶效应。此外，规定的共同交付必须 在预先形成的抗载体免疫的潜在存在下完成。最后，限制Cas9 表达式必须是可终止的，以限制脱靶编辑的可能性。值得注意的是，这些功能应该 理想地被配置到单个矢量粒子上下文的上下文中，以便于实际的放大和人类 临床翻译 为此，我们已经利用腺病毒（Ad）的分子混杂性来解决以下要求： CRISPR/Cas9促进的基因治疗。在这方面，我们已经将腺病毒衣壳工程化， 实现载体向性的靶向修饰。除了允许重新定向的向性，衣壳蛋白 工程化提供了允许Ad规避预先形成的载体免疫的手段。我们还应用了 衣壳工程化策略以实现异源蛋白的瞬时表达。在此基础上， UG 3阶段（3年），我们将建立关于基因组编辑机器交付的原理证明 转化为体内疾病相关的细胞和组织。后续UH 3阶段（1年）将解决规模扩大问题， 在大型动物模型中测试我们的新方法。这项工作将与专家咨询小组合作完成 大型动物试验中心

PROJECT SUMMARY A wide range of biological applications have derived from the CRISPR/Cas9 site-specific nuclease system in recent years. Of note, the capacity to accomplish gene editing in a targeted manner has also impacted the design of gene therapy strategies for an expanding repertoire of disorders. Critical to realizing the gene editing functions of the CRISPR/Cas9 system in a gene therapeutic context is the requirement to accomplish effective co-delivery in vivo of the constituent components. This delivery issue has been approached applying both non-viral and viral vector systems. In selected instances, successful gene-editing facilitated gene therapies have been accomplished in model systems of inherited genetic disease. Despite these elegant proof-of-principle studies, limits in available vector technology have greatly restricted the application of CRISPR/Cas9-facilitated gene therapy. In this regard, effective in vivo co-delivery of CRISPR/Cas9 to target somatic cells is required for many of these applications. Such delivery should be restricted exclusively to the key cellular targets in vivo to minimize off-target effects. In addition, the mandated co-delivery must be accomplished in the potential presence of pre-formed anti-vector immunity. Finally, methods to limit Cas9 expression must be endeavored to limit the potential of off-target editing. Of note, these functionalities should ideally be configured into the context of a single vector particle context to facilitate practical upscaling and human clinical translation. To this end, we have exploited the molecular promiscuities of adenovirus (Ad) to address the requirements of CRISPR/Cas9-facilitated gene therapy. In this regard, we have endeavored capsid engineering of adenovirus to achieve targeted modifications of vector tropism. In addition to allowing for re-directed tropism, capsid engineering provides the means to allow Ad to circumvent pre-formed vector immunity. We have also applied a strategy of capsid engineering to accomplish transient expression of heterologous proteins. On this basis, during the UG3 Phase (3 years) we will establish proof-of-principle with respect to delivery of genome editing machinery into disease relevant cells and tissues in vivo. The follow-on UH3 Phase (1 year) will address scale up and testing of our novel approach in a large animal model. This will be accomplished in collaboration with the SCGE Large Animal Testing Centers.