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

项目总结 CRISPR/Cas9位点特异性核酸酶系统在许多生物学领域有着广泛的应用 最近几年。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 为不断扩大的疾病保留剧目提供基因治疗策略。实现基因编辑功能的关键 CRISPR/Cas9系统在基因治疗方面的应用是实现有效联合传递的必要条件 在体内的组成成分。这一递送问题已经通过非病毒和病毒两种方式解决了 向量系统。在选定的实例中，成功的基因编辑促进的基因疗法 在遗传性遗传病的模型系统中完成。 尽管有这些优雅的原则证明研究，但可用的载体技术的限制极大地限制了 CRISPR/Cas9促进的基因治疗的应用在这方面，有效的体内联合交付CRISPR/CAS9 其中许多应用都需要以体细胞为靶点。此类交付应仅限于 体内的关键细胞靶点，以最大限度地减少偏离靶点的影响。此外，强制联合交付必须是 在可能存在预先形成的抗病媒免疫的情况下完成。最后，限制Cas9的方法 必须努力表达，以限制非目标编辑的可能性。值得注意的是，这些功能应该 理想地配置到单个矢量粒子上下文的上下文中，以便于实际的升级和人类 临床翻译。 为此，我们使用腺病毒(Ad)的分子杂乱来解决以下要求 CRISPR/Cas9促进的基因治疗。在这方面，我们努力对腺病毒进行衣壳工程，以 实现对媒介取向的有针对性的修改。除了允许重定向定向外，衣壳 工程学提供了允许Ad绕过预先形成的媒介免疫的方法。我们还应用了一个 外源蛋白瞬时表达的衣壳工程策略。在此基础上，在 UG3阶段(3年)我们将建立关于提供基因组编辑机器的原则证明 进入体内与疾病相关的细胞和组织。后续UH3阶段(1年)将解决规模扩大和 在大型动物模型上测试我们的新方法。这将通过与常设咨询小组合作完成。 大型动物检测中心。

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.