ssDNA Expression of Triplex-Forming Oligonucleotides

我们建议使用含有报告基因的转基因小鼠模型作为测试系统，以确定从单链DNA表达系统中产生单链DNA的体内效果。1.构建携带三链形成序列的单链DNA表达盒的腺病毒载体。A.将编码AG30三链形成ODN(TFO)序列的双链DNA(DsDNA)序列插入第二代ssDNA表达系统(单载体)，随后将表达盒移除并亚克隆到腺病毒载体中。还将构造一系列控制向量。B.新的腺病毒载体将在细胞培养(转基因小鼠的FL-10细胞和成纤维细胞)中表达TFOS的能力进行测试，以进行小鼠实验。2.小鼠体内靶向性试验。A.我们将使用建立的转基因小鼠检测系统，通过体内生产单链DNA来研究三链介导的基因靶向。B.将比较表达单链DNA的腺病毒载体和全身注射人工合成的ODN。随着这些研究的完成，我们的期望是：(1)建立了单链DNA载体在体内运送和生产单链DNA分子的能力，以便在实验动物的细胞和组织中介导基因靶向。这项工作的成功可能会带来一种新的研究工具，并最终可能提供一种新的方法，利用专门为与三链、反义、适体和DNA酶应用相关的治疗干预而设计的未经修饰的单链单链来进行人类疾病的基因治疗。建议的商业应用：这项工作的成功可能导致一种新的研究工具，并可能最终提供一种新的方法，利用专门为与三链、反义、适体和DNA酶应用相关的治疗干预而设计的未经修饰的单链单链DNA来进行人类疾病的基因治疗。此外，为第二阶段的工作提供了基础，开发了单链DNA生成载体，作为潜在的基因靶向和临床相关基因的基因治疗试剂。

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.