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.

项目摘要 基因组编辑正在通过实现精确修改生物医学和生物技术的革新 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，但 CRISPR-CAS9系统已成为一种用于编辑DNA的广泛和高效的技术。 尽管如此，限制源于其对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。 基础编辑（BES）和Prime Editor（PES）是两种新颖的基因组编辑工具，能够 在不需要DSB的情况下引入DNA中的精确单基转换。特别是PES提供 由于其能够引入任何类型的基础转换甚至可以编程的能力，因此比BES更高的灵活性 小插入和删除。与其他技术相比，这套扩展的功能集使PES成为 特别有希望的生物医学应用平台；但是，大尺寸的PE排除了它们 AAV的体内传递，这是一种有前途有效的基因输送工具，目前正在评估多个 临床试验。 为了克服这些障碍，我们创建了一个与AAV交付兼容的拆分PE平台 已经证明了这种方法在培养的细胞中的功能。尽管取得了这种进展，仍然存在 我们在这里提出的几个关键挑战要克服，以优化这项技术以有效 和特定的体内素数编辑。 为了实现这一目标，我们组建了一个具有基因组集体专业知识的多学科团队 编辑（Perez-Pinera博士），AAV基因分娩（GAJ博士）和计算生物学（Song）。我们的合作 努力将产生综合而全面的PE工具集，将目标识别策略融合在一起 编辑优化，并采用减少脱靶效应和免疫反应的方法，从而对此进行启动 用于未来体内应用的技术。 鉴于PES的灵活性显着扩大了可行的目标位点的数量 基因修饰，我们预计我们开发的集成技术将具有大型，直接和长期的 生物医学对生物医学的持久影响不仅可以实现新颖的基因疗法，还可以实现基础研究。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学工具 在体内有丝分裂细胞内，可用于剖析功能元素，甚至确定 细胞和组织特异性方式的致病突变。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。