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。虽然这类药物的设计已经付出了很多努力，但有一个基本问题仍然不清楚，那就是：为了实现基因传递的最大效率，这些化合物应该与DNA结合得多紧密？一种可能的答案是，一种化合物的最佳DNA结合稳定性取决于它在传递过程中所介导的具体步骤。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、内体转位和核运输。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的、基于蛋白质的递送系统的发展。我将把已知的催化所有这三个步骤的蛋白质序列融合到DNA结合域，这些DNA结合域来自两个特性良好的阻遏蛋白LACR和TetR(D)。这两种蛋白质都以高亲和力结合到各自的操纵子上，更具体地说，具有高度的稳定性。然后，通过简单地修改其特定的DNA结合部位，蛋白质-DNA复合体的稳定性将从弱到共价变化，而总体化学变化很小。此外，由于无毒的小分子诱导Lac和Tet蛋白的解离，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建一个安全的、高转染率的合成递送系统，以及一个有用的框架，可以用来设计其他基因递送剂。

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.