Development of Technologies for Efficient In Vivo Prime Editing

高效体内 Prime 编辑技术的开发

• 批准号: 10184207
• 负责人: Pablo Perez-Pinera
• 金额: $ 53.59万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10184207
• 关键词: Amyotrophic Lateral Sclerosis; Basic Science; Biological; Biotechnology; CRISPR/Cas technology; Carrying Capacities; Cells; Chromosomal translocation; Clinical Research; Clinical Trials; Computational Biology; Cultured Cells; DNA; DNA Double Strand Break; DNA Sequence; Data; Development; Elements; Engineering; Evaluation; Experimental Designs; Future; GTP-Binding Protein alpha Subunits, Gs; Gene Delivery; Genes; Genome; Genomic DNA; Genomic approach; Immune response; Knowledge; Lead; Machine Learning; Mediating; Methodology; Methods; Mitotic; Modification; Mutation; Nonsense Mutation; Open Reading Frames; Outcome; Pathogenicity; Peptide Signal Sequences; Poly A; Positioning Attribute; Regulatory Element; Research; Research Personnel; Role; Site; Site-Directed Mutagenesis; Specificity; System; Technology; Testing; Therapeutic; Time; Tissues; Trans-Splicing; Variant; adeno-associated viral vector; base; biophysical techniques; cell type; computational suite; design; experience; experimental study; flexibility; gene therapy; genome editing; human disease; immunogenic; improved; in vivo; innovation; insertion/deletion mutation; intein; miniaturize; multidisciplinary; novel; nuclease; particle; predictive modeling; prevent; programs; promoter; reconstitution; response; success; technology development; technology research and development; tool

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.

项目概要 基因组编辑通过精确修改基因组来彻底改变生物医学和生物技术 活细胞中的基因组 DNA。尽管在过去十年中已经开发了各种基因组编辑工具， CRISPR-Cas9 系统已成为一种特别通用且高效的 DNA 编辑技术。 尽管如此，其对 DNA 双链断裂 (DSB) 的依赖仍存在局限性，这可能会导致 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) 是两类新颖的基因组编辑工具，能够 在 DNA 中引入精确的单碱基转换，无需 DSB。 PE 尤其提供 比 BE 具有更大的灵活性，因为它们能够引入任何类型的基数转换，甚至是可编程的 小的插入和删除。与其他技术相比，这种扩展的功能使 PE 成为 特别有前景的生物医学应用平台；然而，PE 的大尺寸阻碍了它们的应用 AAV 体内递送是一种有前途且有效的基因递送载体，目前正在多个方面进行评估 临床试验。 为了克服这些障碍，我们创建了一个与 AAV 交付兼容的 split-PE 平台 已经在培养细胞中证明了这种方法的功能。尽管取得了这些进展，但仍然存在 我们在此建议克服几个关键挑战，以优化该技术以实现有效的 和特定的体内prime编辑。 为了实现这一目标，我们组建了一支具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）。我们的合作 努力将产生一个综合的、全面的 PE 工具集，它将融合目标识别和 编辑优化，采用减少脱靶效应和免疫反应的方法，从而启动这一过程 未来体内应用的技术。 鉴于 PE 的灵活性显着增加了可操作的目标站点的数量 转基因，我们预计我们开发的综合技术将具有大规模、直接和长期的作用。 不仅可以实现新型基因疗法，还可以进行基础研究，从而对生物医学产生持久影响。尤其， 我们的技术将为研究人员提供具有独特能力引入突变的生物工具 在体内有丝分裂后细胞内，可用于剖析功能元件，甚至确定 以细胞和组织特异性方式发生致病性突变。因此，该应用程序创建的技术将 广泛影响生物技术和生物医学。