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

用于体内器官选择性基因编辑的内皮靶向腺病毒

• 批准号: 9810634
• 负责人: David Terry Curiel
• 金额: $ 71.51万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9810634
• 关键词: Address; Adenovirus Vector; Adenovirus hexon capsid protein; Adenoviruses; Animal Model; Animal Testing; Antibodies; Binding Proteins; Biological; Biological Models; CRISPR/Cas technology; Capsid; Cells; Collaborations; Complex; Disease; Endothelium; Engineering; Gene Delivery; Gene Transfer; Genes; Genetic Transduction; Hemophilia A; Hereditary Disease; Human; Immunity; Knock-in; Macaca mulatta; Methods; Modification; Molecular; Mutation; Organ; Pathway interactions; Phase; Proteins; Recombinant Proteins; Reporter Genes; Serum; Site; Site-Directed Mutagenesis; Somatic Cell; Source; System; Technology; Testing; Therapeutic Intervention; Tissues; Tropism; Vascular Endothelium; Viral Vector; adenoviral-mediated; adenovirus mediated delivery; alpha 1-Antitrypsin Deficiency; base; cellular targeting; clinical translation; design; gene therapy; genome editing; in vivo; novel; novel strategies; nuclease; overexpression; particle; practical application; scale up; therapeutic gene; tool; vector

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.

项目摘要 从CRISPR/CAS9特异性核酸酶系统中得出了广泛的生物应用 近年来。值得注意的是，以目标方式完成基因编辑的能力也影响了设计 基因治疗策略，以扩大疾病的曲目。意识到基因编辑功能至关重要 在基因治疗环境中的CRISPR/CAS9系统的要求是完成有效的共同交付的要求 组成部分的体内。已解决此交付问题，同时应用了非病毒和病毒 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已经是 在遗传疾病的模型系统中完成。 尽管有这些优雅的原理研究，但可用矢量技术的限制极大地限制了 CRISPR/CAS9领性基因疗法的应用。在这方面，有效的在CRISPR/CAS9的体内共同交付 许多这些应用都需要靶向体细胞。这种交付应仅限于 在体内关键细胞靶标，以最大程度地减少脱靶效应。此外，必须的共同交付必须是 在潜在的预先形成的抗载体免疫力的情况下完成。最后，限制CAS9的方法 表达式必须努力以限制脱靶编辑的潜力。值得注意的是，这些功能应该 理想地将其配置为单个矢量粒子上下文的上下文，以促进实用的升级和人类 临床翻译。 为此，我们利用了腺病毒（AD）的分子散布来解决 CRISPR/CAS9领域基因疗法。在这方面，我们努力腺病毒的衣壳工程 实现对矢量偏向主义的靶向修改。除了允许重新定向的热潮外，Capsid 工程提供了允许广告规避预成型矢量免疫力的手段。我们还应用了 衣壳工程的策略以完成异源蛋白的短暂表达。在此基础上 UG3阶段（3年）我们将建立基因组编辑机制的原理证明 在体内进入疾病相关的细胞和组织。后续UH3阶段（1年）将解决规模，并 在大型动物模型中测试我们的新方法。这将与SCGE合作完成 大型动物测试中心。