BIOENGINEERING OF PROTEIN BASED GENE DELIVERY AGENTS

一种有效的非病毒基因递送剂必须1)结合DNA， 2)促进转染所需的各种步骤。虽然在设计这种药物方面已经付出了很多努力，但有一个基本问题仍然不清楚，即：为了达到基因传递的最大效率，这些化合物与DNA的结合应该有多紧密？一个可能的答案是，化合物的最佳DNA结合稳定性取决于它介导的具体递送步骤。因此，我建议定量和系统地研究dna结合稳定性如何影响基因传递的三个关键步骤，即：细胞特异性摄取，内体易位和核运输。在许多与医学相关的人类细胞系中，所有这些步骤都可以由蛋白质序列介导——这一事实导致了模块化、基于蛋白质的递送系统的发展。我将融合已知催化所有这三个步骤的蛋白质序列到DNA结合域，这些结合域来自两种特征良好的抑制蛋白，LacR和TetR(D)。这两种蛋白质都以高亲和力与各自的操作符结合，更具体地说，具有高度的稳定性。然后，蛋白质-DNA复合物的稳定性将从弱到共价变化，通过简单地修改其特定的DNA结合位点，整体化学变化很小。此外，由于小而无毒的分子诱导Lac和Tet蛋白解离，这些蛋白也将包含一种新的触发释放机制。该项目的长期目标是创造一种安全、转染效率高的合成递送系统，并为设计其他基因递送剂提供一个有用的框架。

An effective non-viral gene delivery agent must 1) bind DNA, and 2) facilitate various steps necessary for transfection. While much effort has gone into the design of such agents, one basic question remains unclear, namely: how tightly should these compounds bind DNA in order to achieve the maximum efficiency of gene delivery? One possible answer is that the optimal DNA- binding stability of a compound depends on which specific step in delivery it mediates. Thus, I propose to quantitatively and systematically examine how DNA-binding stability affects three crucial steps in gene delivery, namely: cell-specific uptake, endosomal translocation, and nuclear transport. In many medically relevant human cell lines, all of these steps can be mediated by protein sequences - a fact that has led to the development of modular, protein-based delivery systems. I will fuse protein sequences known to catalyze all three of these steps to DNA binding domains derived from two well-characterized repressor proteins, LacR and TetR(D). Both of these proteins bind to their respective operators with a high affinity, and more specifically, with a high degree of stability. The stability of the protein-DNA complexes will then be varied from weak to covalent with little change in overall chemistry by simply modifying their specific DNA binding sites. Furthermore, since small, non-toxic molecules induce the dissociation of Lac and Tet proteins, these proteins will also contain a novel triggered release mechanism. The long-term goal of this project is to create a safe synthetic delivery system with high transfection efficiency, and a useful framework with which other gene delivery agents can be designed.