Development of Technologies for Efficient In Vivo Prime Editing

项目摘要 基因组编辑通过精确修改基因组，正在彻底改变生物医学和生物技术。 活细胞中的DNA虽然在过去十年中开发了各种基因组编辑工具， CRISPR-Cas9系统已经成为一种特别通用和有效的DNA编辑技术。 尽管如此，其局限性源于其对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，可能会限制其应用。 碱基编辑器（BE）和主编辑器（PE）是两种新型的基因组编辑工具，能够 在DNA中引入精确的单碱基转换而不需要DSB。特别是PE， 比BE更大的灵活性，因为它们能够引入任何类型的基本转换，甚至可编程 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 特别有前途的平台，在生物医学中的应用;然而，大规模的PE排除了他们的在 通过AAV的体内递送，一种有前途的和有效的基因递送载体，目前正在多个研究中进行评估。 临床试验 为了克服这些障碍，我们创建了一个与AAV递送兼容的拆分PE平台， 已经证明了这种方法在培养细胞中的功能。尽管取得了这些进展，但仍有 几个关键的挑战，我们在这里提出克服，以优化这项技术， 和特异性体内引物编辑。 为了实现这一目标，我们组建了一个多学科团队， 编辑（Perez-Pinera博士）、AAV基因递送（Gaj博士）和计算生物学（Song博士）。我们的协作 这些努力将产生一个综合全面的PE工具集，该工具集将融合目标识别战略， 编辑优化，具有用于减少脱靶效应和免疫应答的方法，从而引发这种 未来在体内应用的技术。 考虑到PE的灵活性显著增加了可操作的目标位点的数量， 转基因，我们预计，我们开发的综合技术将具有巨大的，直接的和长期的， 通过不仅使新的基因疗法，而且使基础研究成为可能，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生物工具， 在体内有丝分裂后的细胞内，这可以用来解剖功能元件，甚至确定 以细胞和组织特异性方式的致病性突变。因此，该应用程序所创建的技术将 广泛影响生物技术和生物医学。

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.