ssDNA Expression of Triplex-Forming Oligonucleotides

我们在此建议使用含有报告基因的转基因小鼠模型作为测试系统，该模型具有可适应三联体形成的靶区，以确定ssDNA表达系统在细胞内生成ssDNA的体内功效。1. 腺病毒载体构建以传递带有三联体形成序列的ssDNA表达盒。a.将编码AG30三联体形成ODN （TFO）序列的双链DNA （dsDNA）序列插入第二代ssDNA代ssDNA表达系统（单载体）中，随后将表达盒移除并亚克隆到腺病毒载体中。一系列的控制向量也将被构造。b.在小鼠实验中，将测试新的腺病毒载体结构在细胞培养（FL-10细胞和源自转基因小鼠的成纤维细胞）中表达TFOs的能力。2. 小鼠体内靶向性试验。a.我们将使用一个已建立的转基因小鼠实验系统，通过体内生产ssDNA来研究三体介导的基因靶向。b.将对表达ssDNA的腺病毒载体与系统给药合成ODNs进行比较。在这些研究完成后，我们的期望是：(1)建立ssDNA载体在体内传递和产生ssDNA分子的能力，从而介导实验动物细胞和组织中的基因靶向。这项工作的成功可能会导致一种新的研究工具，并可能最终为人类疾病的基因治疗提供一种新的方法，利用未经修饰的ssDNA链，专门设计用于与三联体、反义、适体和DNA酶应用相关的治疗干预。拟议的商业应用：这项工作的成功可能会导致一种新的研究工具，并可能最终为利用未经修饰的ssDNA链进行人类疾病的基因治疗提供一种新的方法，这些ssDNA链专门用于与三联体、反义体、适体和DNA酶应用相关的治疗干预。同时，为开发ssDNA生成载体作为临床相关基因潜在的基因靶向和基因治疗试剂的二期工作奠定基础。

We propose here to use a transgenic mouse model containing a reporter gene that has a target region amenable to triplex formation as a test system to determine the in vivo efficacy of intracellular ssDNA generation from a ssDNA expression system. 1. Adenovirus vector construct to deliver ssDNA expression cassette with a triplex-forming sequence. a. A double-stranded DNA (dsDNA) sequence encoding for the AG30 triplex-forming ODN (TFO) sequence will be inserted into a second generation ssDNA generation of ssDNA expression system (single- vector), and the expression cassette will be subsequently removed and subcloned into an adenovirus vector. A series of control vectors also will be constructed. b. The new adenovirus vector constructs will be tested for their ability to express TFOs in cell culture (FL-10 cells and fibroblasts derived from transgenic mice) in anticipation of mouse experiments. 2. Test of in vivo targeting in mice. a. We will use an established transgenic mouse-based assay system to investigate triplex-mediated gene targeting via in vivo production of ssDNA. b. Comparison will be made between adenovirus vectors expressing ssDNA versus systemic administration of synthetic ODNs. At the completion of these studies, our expectation os that we will have: (1) established the ability of ssDNA vectors to deliver and produce ssDNA molecules in vivo in order to mediate gene targeting in the cells and tissues of experimental animals. The success of this work may lead to a novel research tool and may eventually provide a new approach to gene therapy for human disease utilizing unmodified strands of ssDNA designed specifically for therapeutic interventions related to triplex, antisense, aptamer, and DNA enzyme applications. PROPOSED COMMERCIAL APPLICATIONS: The success of this work may lead to a novel research tool and may eventually provide a new approach to gene therapy for human disease utilizing unmodified strands of ssDNA designed specifically for therapeutic interventions related to triplex, antisense, aptamer, and DNA enzyme applications. Furthermore, it would provide the basis for Phase II work to develop ssDNA generating vectors as potential gene targeting and gene therapy reagents for clinically relevant genes.