Rational Design of Degradable Polymers for Gene Delivery

智力优势：该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传递到细胞核的系统。没有病毒感染可能产生的不良影响的替代给药系统引起了相当大的兴趣。另一种策略是用带正电的生物相容性聚合物络合和凝聚带负电的DNA。由此产生的“复合体”已被证明能够进入细胞，穿透细胞核，并释放部分复杂的DNA，从而使其可用于转染。这个过程的效率仍然很低，可能是因为离子稳定的复合体不容易释放其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，但对构建在聚合物主链中的-硫酯、-氨基酯、-氨基和硫酰胺键的水解敏感性不同。多聚体的解离，一旦传递到细胞，将通过裂解敏感键的聚合物降解促进。降解速率原则上可以通过调节可水解键的比例和这种键附近的取代基模式来调节。降解动力学和复杂，凝聚和保护DNA的能力将在体外研究一个多阳离子库。挑战将是调整多聚体的水解敏感性，使多聚体的稳定性保留在细胞质中，而不是在细胞核中。最后，将在选定的细胞系中研究复合物的解离、细胞内运输和体外转染效率。聚合物将用适当的荧光标签进行标记，以方便使用荧光共振能量转移光谱研究细胞内的贩运。更广泛的影响：这项工作在基因治疗领域有潜在的重大影响。该计划包括教育推广计划，提供本科生研究机会和课程开发。PI将与怀俄明大学多元文化事务办公室合作，从整个落基山脉农村地区（加上内布拉斯加州和达科他州）招募高中生参加为期七周的有偿暑期学徒计划。将特别强调从该地区大量的美洲土著人口中招募人员。该项目将为两名本科生提供在他的实验室进行项目研究的机会。他还在怀俄明校区开设了高分子合成、工程和应用的第一门课程。这将包括一个实验室部分，包括各种合成方法的实验，聚合物分子量和分子量分布的测定，反应性聚合物的制备，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。

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.