Quinine polymer beacons for probing encapsulation and cellular delivery of CRISPR-Cas9

非技术摘要：在材料研究部生物材料项目的支持下，Reineke和Frontiera教授正在开发一系列新的聚合物材料，这些材料可以将核酸和基因编辑系统结合、压缩和运输到活细胞中。开发新的运载工具对于降低基因编辑疗法的成本和毒性至关重要。基因编辑的目标是通过递送治疗性核酸和/或核糖核蛋白复合物来调节基因产物的表达以治疗疾病。传统上，基因编辑系统是由经过修饰的病毒提供的，这些病毒非常昂贵，并且可能导致有害的免疫反应。聚合物载体是有前途的候选系统，因为它们将核酸和蛋白质结合到紧凑的纳米级载体中，同时减少传统使用的病毒载体的副作用。在这里，PI建议使用一系列含有奎宁的可调聚合物，奎宁是一种长期用于治疗疟疾的天然产物，以促进活细胞中基因治疗成分的递送和解包。PI将合成和表征一系列为高效基因编辑构建体递送而合理设计的聚合物，并全面表征其结构和包装效率。这些聚合物将被筛选用于基因组编辑效率和缺乏毒性。最有希望的候选制剂将用先进的光谱法进行检查，以确定细胞递送的机制。此外，PI将通过吸引学生参加McNair Scholars计划并举办暑期研究和指导体验，建立互动的指导和学习环境。技术摘要：开发新材料，以高效，无毒，不干扰正常细胞功能的方式将核酸和基因编辑系统结合，压缩和运输到活细胞中，对于医学研究的许多方面的成功至关重要。聚合物核酸载体在规避与病毒载体相关的问题（免疫原性、难以扩展和高成本）方面显示出巨大的前景，因为它们的结构可以被合成修饰以赋予它们无毒和高效运输的性质。该项目的目标是开发一种新型的七种运载工具“信标”，其含有奎宁，与核酸和Cas9蛋白形成复合物，毒性低，并且可以容易地跟踪有效载荷包装和细胞内运输。该项目将需要合成一系列可调的含奎宁聚合物，并通过全面的光学和振动光谱进行定量表征。将评估这套聚合物的细胞活力和基因组编辑效率，并对最有前途的配方进行统计鉴定。这些系统将被详细检查，发现细胞内传递的机制，解包装的动力学，和编辑有效载荷的位置。探索具有独特特性的新递送系统，以促进多组分复合物的形成，有效载荷包装的光谱探测，细胞递送和成像，可以为非病毒基因编辑系统开发领域提供变革潜力。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Non-Technical Abstract:With support from the Biomaterials Program in the Division of Materials Research, Professors Reineke and Frontiera are developing a series of new polymeric materials that bind, compact, and transport nucleic acids and gene editing systems into living cells. The development of new delivery vehicles is crucial to reducing the cost and toxicity of gene editing therapies. The goal of gene editing is to modulate the expression of gene products to treat diseases by delivering therapeutic nucleic acids and/or ribonucleoprotein complexes. Traditionally, gene editing systems, have been delivered by modified viruses, which are extremely expensive and can lead to detrimental immune responses. Polymeric vehicles are promising candidate systems for delivery as they bind nucleic acids and proteins into compact nanoscale vehicles, while simultaneously reducing side effects from traditionally used viral vehicles. Here, the PIs propose using a tunable series of polymers containing quinine, a natural product long used to treat malaria, to promote delivery and unpackaging of gene therapy components in living cells. The PIs will synthesize and characterize a family of polymers rationally designed for efficient gene editing construct delivery, and comprehensively characterize their structure and packaging efficiency. These polymers will be screened for genome editing efficiency and lack of toxicity. The most promising candidate formulations will be examined with advanced spectroscopies in order to determine the mechanism of cellular delivery. Additionally, the PIs will establish an interactive mentoring and learning environment by attracting students to the McNair Scholars program and hosting them for summer research and mentoring experiences.Technical Abstract:Development of new materials that bind, compact, and transport nucleic acids and gene editing systems into living cells in a manner that is efficient, nontoxic, and does not interfere with normal cell function is essential for the success of many facets of medical research. Polymeric nucleic acid vehicles show great promise in circumventing the problems associated with viral vectors (immunogenicity, difficulty in scalability, and high cost) because their structures can be modified synthetically to endow them with properties for nontoxic and highly efficient transport. The objective of this project is to develop a novel family of seven delivery vehicle "beacons" containing quinine that form complexes with nucleic acids and Cas9 protein, are low in toxicity, and can be readily tracked for payload packaging and intracellular trafficking. The project will entail synthesis of a family of tunable quinine-containing polymers, with quantitative characterization by comprehensive optical and vibrational spectroscopies. This suite of polymers will be assessed for cell viability and genome editing efficiency, with statistical identification of the most promising formulations. These systems will be examined in detail, with discovery of the mechanisms of intracellular delivery, kinetics of unpackaging, and editing payload location. The exploration of new delivery systems with unique properties to facilitate multicomponent complex formation, spectroscopic probing of payload packaging, cellular delivery, and imaging could offer transformative potential to the field of non-viral based gene editing system development.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.