Structure-property study for dendronized polymer vectors for CRISPR delivery

非技术概述：该项目开发了一种新的分子递送系统，以实现基于CRISPR的基因组编辑技术。基于CRISPR的基因组编辑技术可以在体外或体内精确地添加、删除和“纠正”基因；因此，它在治疗应用方面具有巨大的潜力，并正在积极地被用于解决从遗传性疾病、癌症到糖尿病的广泛的人类疾病。然而，实施这项技术的一个主要障碍是缺乏安全和高效的输送系统，可以有效地将所需的分子机械输送到细胞中来执行基因组编辑。为了应对这一挑战，该项目提出了一系列由天然氨基酸和多肽制成的递送分子的设计、合成和研究。这些化合物将被评估它们将两段RNA输送到细胞中进行基于CRISPR的基因组编辑的效率和安全性。结构-功能研究将阐明有效分子载体的重要设计原则。拟议的跨学科研究活动也为不同层次的学生提供了优秀的培训，从K-12、本科生到研究生，特别是女性，少数族裔学生，以及研究机会有限的机构的学生。技术总结：该项目的目标是为基于RNA的CRISPR开发一个新的合成递送平台，并对新的递送系统进行基本结构-性质研究，以获得对设计新的基因递送载体的关键见解。CRISPR/Cas9基因编辑为治疗应用提供了巨大的潜力，并正在积极地被用于治疗广泛的人类疾病。然而，实施CRISPR介导的基因组编辑的一个主要障碍是缺乏安全和高效的传递工具。PI实验室最近开发了一种创新的生物可还原树突化多肽(BDPs)设计，该设计显示出高效率地联合传递Cas9 mRNA和gRNAs。对该系统的进一步研究将产生一种新的平台递送系统，它可以有效和安全地将CRISPR/Cas9 mRNA和gRNA共传递到不同的细胞中。此外，利用BDP良好的分子结构，我们建议对BDP系统进行基础结构-性质研究，以期为设计新的基因递送载体提供重要的见解。如果成功，这项拟议的研究将为CRISPR技术提供一种新的、通用的和生物相容的递送系统，并为指导新的基因递送系统的设计提供重要的见解。研究和广泛影响活动将为不同层次的学生提供全面的培训，从K-12、本科生到研究生，特别是女性，少数族裔学生代表不足。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Nontechnical Summary:This project develops a new molecular delivery system for enabling the CRISPR-based genome editing technology. CRISPR-based genome editing technology can precisely add, delete, and “correct” genes in vitro or in vivo; therefore, it holds tremendous potential for therapeutic applications and is actively being pursued for addressing a wide range of human diseases ranging from hereditary diseases, cancer, to diabetes. However, one major obstacle to implementing this technology is the lack of safe and efficient delivery systems that can effectively transport the required molecular machinery into cells to execute genome editing. To address this challenge, this project proposes the design, synthesis, and investigation of a series of delivery molecules made from natural amino acids and peptides. These compounds will be evaluated for their efficiency and safety for delivering two pieces of RNAs into cells for performing CRISPR-based genome editing. Structure-function studies will shed light on important design principles for effective molecular carriers. The proposed interdisciplinary research activities also provide excellent trainings to students at various levels, ranging from K-12, undergraduate, to graduate students, especially women, underrepresented minority students, and those from institutes with limited research opportunities.Technical Summary:The goal of this project is to develop a new synthetic delivery platform for RNA-based CRISPR and conduct fundamental structure-property studies on the new delivery system to gain critical insights for the design of new gene delivery vectors. CRISPR/Cas9 gene editing offers a tremendous potential for therapeutic applications and is actively being pursued for addressing a wide range of human diseases. However, one major obstacle to implementing CRISPR-mediated genome editing is the lack of safe and efficient delivery vehicles. The PI lab recently developed an innovative design of bioreducible dendronized polypeptides (BDPs) that show high efficiency for co-delivery of Cas9 mRNA and gRNAs. It is proposed that further investigation of this system will generate a new platform delivery system that can effectively and safely co-deliver CRISPR/Cas9 mRNA and gRNA into various cells. Furthermore, capitalizing on BDP’s well-defined molecular structure, we propose to conduct fundamental structure-property studies on the BDP system with the aim to gain critical insights for the design of new gene delivery vectors. If successful, the proposed study will provide a new, general, and biocompatible delivery system for CRISPR technology and offer critical insights for guiding the design of new gene delivery systems. The research and broad impact activities will provide holistic trainings to students at various levels, ranging from K-12, undergraduate, to graduate students, especially women, underrepresented minority students.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.