Developing Nucleic Acid Therapeutics

描述（由申请人提供）：使用反义核酸（ASNA）治疗人类恶性肿瘤的前景仍然诱人，但尚未实现。我们假设，如果与mRNA靶序列选择和分子递送相关的基本问题得到解决，ASNA将成为更好的药物。本项目的目标是解决这些核心问题，具体目标如下：目标#1-开发一种合理的方法，为靶向ASNA-我们已经开发了自淬灭报告分子（SQRM），信号仅在杂交后，并已使用这些探针“地图”杂交可访问的网站在mRNA中。我们将通过绘制额外的靶mRNA，并测量序列指导的ASNA抑制活细胞中基因表达的效率来确定这种策略的体内效用。也将测试天然存在的细胞内蛋白质可能增强ASNA/mRNA杂交的假设。候选蛋白将通过功能，并通过使用亲和色谱法和质谱法进行鉴定。最后，还将探索具有增强的链侵入性质的DNA主链（具有或不具有促进杂交的蛋白质）的效用;目的#2-测试合理靶向的ASNA的基因沉默效率可以通过主链、序列或悬垂修饰增强的假设- ASNA通过工程化到分子中的酶活性或通过激活内源性RNaseH切割mRNA。我们将研究合理靶向的ASNA的能力，与各种骨架修饰合成，在体外和体内切割mRNA靶点。还将检查具有可被光活化的悬垂物的SQRM基于靶mRNA表达检测和杀死细胞的能力;目的#3。检查合理设计的、活性优化的ASNA在人类白血病动物模型中检测和杀死肿瘤细胞的能力-将进行优化分子的药效学研究，并将探索其在人类白血病动物模型中作为诊断和治疗分子的能力。

DESCRIPTION (provided by applicant): The prospect of employing antisense nucleic acids (ASNA) for treating human malignancies remains tantalizing, but unrealized. We hypothesize that ASNA would make better drugs if fundamental problems related to mRNA target sequence selection, and molecule delivery, were solved. The goal of this project is to address these core issues in the following specific aims: Aim #1- Develop A Rational Method For Targeting ASNA- We have developed self-quenching reporter molecules (SQRM) that signal only after hybridization, and have used these probes to "map" hybridization accessible sites in mRNA. We will determine the in vivo utility of this strategy by mapping additional target mRNAs, and measuring the efficiency with which sequence directed ASNA inhibit gene expression in living cells. The hypothesis that naturally occurring intracellular proteins might enhance ASNA/mRNA hybridization will also be tested. Candidate proteins will be identified by function, and by using affinity chromatography, and mass spectrometry. Finally, the utility of DNA backbones with enhanced strand invasion properties, with or without proteins that facilitate hybridization, will also be explored; Aim #2- Test the Hypothesis that Gene Silencing Efficiency of Rationally Targeted ASNA Can be Enhanced by Backbone, Sequence, or Pendant Modifications- ASNA cleave mRNA by enzymatic activity engineered into the molecule, or by activating endogenous RNaseH. We will examine the ability of rationally targeted ASNA, synthesized with various backbone modifications, to cleave mRNA targets in vitro and in vivo. The ability of SQRM with pendants that can be photoactivated to detect, and kill, cells on the basis of target mRNA expression will also be examined; Aim #3. Examine the ability of rationally designed, activity optimized ASNA to detect, and kill, tumor cells in animal models of human leukemia- Pharmacodynamic studies of optimized molecules will be undertaken, and their ability to serve as diagnostic, and therapeutic molecules in animal models of human leukemia will be explored.