Developing Nucleic Acid Therapeutics

描述(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将制造出更好的药物。这个项目的目标是在以下具体目标上解决这些核心问题：目标1-开发一种合理的靶向ASNA的方法-我们已经开发出仅在杂交后才发出信号的自猝灭报告分子(SQRM)，并使用这些探针来“定位”mRNA中的杂交可及部位。我们将通过定位额外的靶向mRNAs，并测量序列指导的ASNA抑制活细胞中基因表达的效率，来确定这一策略在体内的实用性。自然产生的细胞内蛋白质可能促进ASNA/mRNA杂交的假设也将得到检验。候选蛋白质将通过功能鉴定，并通过亲和层析和质谱法进行鉴定。最后，还将探索具有增强的链侵袭特性的DNA骨架的用途，无论是否带有促进杂交的蛋白质；目标2-测试以下假设：合理靶向的ASNA的基因沉默效率可以通过骨架、序列或侧链修饰来增强-ASNA通过在分子中进行工程处理的酶活性或通过激活内源RNAseH来切割mRNA。我们将检验通过各种骨架修饰合成的合理靶向ASNA在体外和体内切割信使核糖核酸靶标的能力。目标3.检测合理设计、活性优化的ASNA在人类白血病动物模型中检测和杀伤肿瘤细胞的能力-将对优化的分子进行药效学研究，并探索它们在人类白血病动物模型中用作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.