Rational Design of Degradable Polymers for Gene Delivery

用于基因传递的可降解聚合物的合理设计

• 批准号: 0705298
• 负责人: Youqing Shen
• 金额: $ 37.5万
• 依托单位: University of Wyoming
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0705298&HistoricalAwards=false
• 关键词: Rational; Design; Degradable; Polymers; Gene

INTELLECTUAL MERIT: This project explores a strategy for the delivery of genetic material to target cells for gene therapy. Viral vectors provide one system capable of delivering DNA to the nucleus of a cell. Alternative delivery systems, free of the possible adverse effects of viral infection, are of considerable interest. An alternative strategy involves complexing and condensing the negatively charged DNA with a positively charged biocompatible polymer. The resulting "polyplex" has been shown to be capable of entering a cell, penetrating the nucleus, and releasing some portion of the complexed DNA, thus making it available for transfection. The efficiency of this process remains low, presumably because an ionically stabilized polyplex does not readily release its DNA component. In this work the PI plans to synthesize a series of cationic polymers, similar in backbone structure but differing in their susceptibility to hydrolysis of beta-thioester, beta-aminoester, beta-amino, and thioamide linkages built into the polymer backbone. Dissociation of the polyplex, once delivered to the cell, will be facilitated by degradation of the polymer through cleavage of the susceptible linkages. Rates of degradation can in principle be tuned by adjusting the proportions of hydrolysable linkages and the substitutent patterns near such linkages. The degradation kinetics and ability to complex, condense, and protect DNA will be investigated in vitro for a library of polycations. The challenge will be to tune the hydrolytic susceptibility of the polycations so that the stability of the polyplex is retained in the cytoplasm but not in the nucleus. Finally the dissociation, intracellular trafficking, and in vitro transfection efficiency of the polyplexes will be investigated in selected cell lines. The polymers will be labeled with appropriate fluorescence tags to facilitate study of trafficking within the cells using fluorescence resonance energy transfer spectroscopy.BROADER IMPACTS: The work has the potential for substantial impact in the gene therapy domain. The program includes a program of educational outreach, provision of undergraduate research opportunities, and curriculum development. The PI will work with the University of Wyoming Office of Multicultural Affairs to recruit high school students from throughout the rural Rocky Mountain area (plus Nebraska and the Dakotas) to participate in a paid , seven-week Summer Apprentice Program. Special emphasis will be placed on recruiting from the substantial population of Native Americans in the region. The PI will provide opportunities for two undergraduates to work on the project in his laboratory. He is also establishing the first course in Polymer Synthesis, Engineering, and Applications on the Wyoming campus. This will include a laboratory component that incorporates experiments in various synthetic approaches, determination of polymer molecular weights and molecular weight distributions, preparation of responsive polymers, preparation and characterization of amphiphilic block copolymers, and cellular uptake of nanoparticles prepared by the students.

智力优势：这个项目探索了一种将遗传物质输送到靶细胞进行基因治疗的策略。病毒载体提供了一种能够将DNA运送到细胞核的系统。没有病毒感染可能的不利影响的替代递送系统引起了人们的极大兴趣。另一种策略包括将带负电的DNA与带正电的生物相容聚合物络合和缩合。由此产生的“多聚体”已被证明能够进入细胞，穿透细胞核，并释放部分复杂的DNA，从而使其可用于转基因。这一过程的效率仍然很低，可能是因为离子稳定的多聚体不容易释放其DNA成分。在这项工作中，PI计划合成一系列阳离子聚合物，它们在主链结构上相似，但对聚合物主链中的β-硫代酯、β-氨基酸酯、β-氨基和硫代酰胺键的水解性不同。一旦多聚体被输送到细胞，聚合物的降解将通过切割敏感的连接来促进。原则上可以通过调整可水解键的比例和这种键附近的取代模式来调整降解速度。聚阳离子文库的降解动力学和络合、浓缩和保护DNA的能力将在体外进行研究。挑战将是调整聚合阳离子的水解性，使多聚体的稳定性保留在细胞质中，而不是细胞核中。最后，将在选定的细胞系中研究这些复合体的解离、细胞内转运和体外转染率。聚合物将被标记上适当的荧光标签，以便于使用荧光共振能量转移光谱仪研究细胞内的运输。溴影响：这项工作可能在基因治疗领域产生重大影响。该计划包括一个教育推广计划，提供本科生研究机会，以及课程开发。PI将与怀俄明大学多元文化事务办公室合作，从整个落基山区农村地区(以及内布拉斯加州和达科他州)招募高中生参加一个为期七周的带薪暑期学徒计划。将特别重视从该地区庞大的美洲原住民人口中招募人才。PI将为两名本科生提供在他的实验室从事该项目的机会。他还在怀俄明州校园开设了第一门聚合物合成、工程和应用课程。这将包括一个实验室部分，包括各种合成方法的实验，聚合物相对分子质量和分子量分布的测定，响应性聚合物的制备，两亲性嵌段共聚物的制备和表征，以及学生制备的纳米颗粒的细胞摄取。