Development of Technologies for Efficient In Vivo Prime Editing

项目总结 基因组编辑正在给生物医学和生物技术带来革命性的变化，因为它能够精确地修改 活细胞中的基因组DNA。虽然在过去的十年里已经开发出了各种基因组编辑工具，但 CRISPR-CAS9系统已经成为一种特别通用和高效的DNA编辑技术。 尽管如此，其局限性源于对DNA双链断裂(DSB)的依赖，这可能导致 不可预测的编辑结果，甚至染色体易位，可能会限制它的应用。 基本编辑(BES)和基本编辑(PES)是两类新的基因组编辑工具，能够 在DNA中引入精确的单碱基转换，而不需要DSB。特别是，PES提供了 比BES更灵活，因为它们能够引入任何类型的基本转换，甚至是可编程的 小的插入和删除。与其他技术相比，这种扩展的功能集使pe成为 在生物医学中的应用特别有前景的平台；然而，PE的巨大尺寸阻碍了它们的应用 AAV是一种很有前途的有效的基因传递工具，目前正在进行多项评估 临床试验。 为了克服这些障碍，我们创建了一个与AAV交付和 已经在培养的细胞中展示了这种方法的功能。尽管取得了这些进展，但仍然存在 几个关键挑战，我们在这里建议克服这些挑战，以优化这项技术，使其有效 和特定的活体素材编辑。 为了实现这一目标，我们组建了一个拥有基因组专业知识的多学科团队。 编辑(Perez-Pinera博士)、AAV基因传递(Gaj博士)和计算生物学(Song博士)。我们的协作 努力将产生一个集成和全面的PE工具集，该工具集将混合目标识别和 编辑优化，减少偏离目标的影响和免疫反应的方法，从而启动 未来活体应用的技术。 鉴于PES的灵活性显著扩大了可操作的目标站点的数量，可以 转基因，我们预计我们开发的集成技术将具有大规模、直接和长期的- 不仅使新的基因疗法成为可能，也使基础研究成为可能，从而对生物医学产生持久的影响。特别是， 我们的技术将为研究人员提供独特的能够引入突变的生物工具 在体内的有丝分裂后细胞内，这可以被用来剖析功能元件，甚至确定 以细胞和组织特有的方式发生致病突变。因此，此应用程序创建的技术将 对生物技术和生物医学产生广泛影响。

PROJECT SUMMARY Genome editing is revolutionizing biomedicine and biotechnology by enabling the precise modification of genomic DNA in living cells. While various genome-editing tools have been developed over the past decade, the CRISPR-Cas9 system has emerged as a particularly versatile and efficient technology for editing DNA. Nonetheless, limitations derived from its reliance on DNA double strand breaks (DSB), which can lead to unpredictable editing outcomes and even chromosomal translocations, could limit its applications. Base editors (BEs) and prime editors (PEs) are two novel classes of genome-editing tools capable of introducing precise single-base conversion in DNA without the requirement of a DSB. PEs, in particular, provide greater flexibility than BEs, owing to their ability to introduce any type of base conversion and even programable small insertions and deletions. This expanded set of capabilities compared to other technologies makes PEs a particularly promising platform for applications in biomedicine; however, the large size of PEs precludes their in vivo delivery by AAV, a promising and effective gene delivery vehicle that is currently under evaluation in multiple clinical trials. To overcome these obstacles, we have created a split-PE platform that is compatible with AAV delivery and have demonstrated the functionality of this approach in cultured cells. Despite this progress, there still remain several critical challenges, which we here propose to overcome in order to optimize this technology for effective and specific in vivo prime editing. To accomplish this objective, we have assembled a multidisciplinary team with collective expertise in genome editing (Dr. Perez-Pinera), AAV gene delivery (Dr. Gaj) and computational biology (Dr. Song). Our collaborative efforts will yield an integrated and comprehensive PE toolset that will blend strategies for target identification and editing optimization, with methods for reducing off-target effects and immune responses, thus priming this technology for future in vivo applications. Given that the flexibility of PEs has significantly expanded the number of actionable target sites that can be genetically modified, we anticipate that the integrated technologies we develop will have large, direct and long- lasting impact in biomedicine by enabling not only novel gene therapies, but also basic research. In particular, our technology will provide investigators with biological tools that are uniquely capable of introducing mutations within post-mitotic cells in vivo, which could be used to dissect functional elements or even determine the role of pathogenic mutations in a cell- and tissue-specific manner. The technologies created by this application will thus broadly impact biotechnology and biomedicine.