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，如果解决了与mRNA靶序列选择和分子递送相关的基本问题，ASNA将会制造出更好的药物。本项目的目标是在以下具体目标中解决这些核心问题：目标#1-开发针对ASNA的合理方法-我们已经开发出仅在杂交后发出信号的自猝灭报告分子（SQRM），并使用这些探针“绘制”mRNA中可杂交的位点。我们将通过绘制额外的靶mrna来确定该策略在体内的效用，并测量序列定向ASNA抑制活细胞中基因表达的效率。自然产生的细胞内蛋白可能增强ASNA/mRNA杂交的假设也将被验证。候选蛋白将通过功能、亲和层析和质谱鉴定。最后，还将探讨具有增强链入侵特性的DNA主干的效用，无论有无促进杂交的蛋白质；目标2-验证合理靶向ASNA的基因沉默效率可以通过骨干、序列或垂链修饰来增强的假设- ASNA通过酶活性工程进入分子或激活内源性RNaseH来切割mRNA。我们将在体外和体内研究通过各种骨架修饰合成的合理靶向ASNA切割mRNA靶标的能力。我们还将研究具有光激活垂坠的SQRM在靶mRNA表达的基础上检测和杀死细胞的能力；目标# 3。检验合理设计、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.