Chimeraplasty for factor IX and VII gene expression

同源重组对缺陷基因的位点特异性校正在基因治疗遗传性代谢性疾病的治疗中仅以极低的频率实现。最近，一种合成的RNA/DNA杂交双工寡核苷酸被设计成与同源基因组序列完美对齐，除了单个碱基错配外，可以促进大鼠肝细胞基因组DNA中的靶向单核苷酸（nt）转换。该过程利用细胞有效的内源性DNA错配修复途径，从而使其成为一种新的基因治疗方法。本研究项目的主要目的是评估这些分子在纠正与血友病相关的单个nt突变方面的效用。这一目标验证了我们的假设，即在受影响的肝细胞中进行基因校正将改善与该疾病相关的表型。第一个具体目标是优化(1)体外非病毒asialal糖蛋白受体肝细胞特异性递送系统，以及（ii）嵌合RNA/DNA寡核苷酸设计，以最大限度地将教堂山犬血友病B因子IX基因在nt 1477处的G转化为A。第二个具体目标是评估这些分子/递送系统促进靶向单nt转化的能力，以纠正犬因子IX体内G到A的转变。将利用特异性靶向1中鉴定的非病毒传递系统和嵌合寡核苷酸。将监测相关代谢参数，以量化原位基因组校正的治疗效果。优化给药方案，建立给药载体和给药途径。第三个具体目标是评估该技术在改变基因组因子VII基因以产生项目2中设计的优化因子VIIa突变方面的潜力。最初的工作将在体外使用培养的肝细胞进行，以优化嵌合寡核苷酸的递送和设计。选择的因子VIIa突变将在体内产生，并在因子VIII缺陷小鼠模型中收集血友病A表型进行评估。将监测相关代谢参数，以量化原位基因组校正的治疗效果。优化给药方案，建立给药载体和给药途径。本研究计划的长期目标是：(i)优化非病毒递送系统和寡核苷酸设计，以促进RNA/DNA寡核苷酸在纠正血友病相关的单个nt突变方面的应用；（ii）阐明在Chapel Hill血友病B犬株中使用该技术对单个nt突变进行体内治疗纠正的最佳参数，以及（iii）评估该技术在体内制造因子VIIa变体的使用及其治疗效果。体外和体内校正结果的比较，将确立利用该技术治疗血友病的体内基因治疗方法的可行性。

Site-specific correction of defective genes by homologous recombination has been achieved at only very low frequencies in the treatment of inherited metabolic diseases by gene therapy. Recently, a synthetic RNA/DNA hybrid duplex, oligonucleotide designed to align in perfect register with the homologous genomic sequence except for a single base mismatch was show to promote targeted single nucleotide (nt) conversion in genomic DNA in rat hepatocytes The process exploits the cell's efficient endogenous DNA mismatch repair pathways, thereby, making it a novel approach to gene therapy. The main objective of t his research project is to evaluate the utility of these molecules in correcting the single nt mutations associated with hemophilia. This objective tests our hypothesis that gene correction in effected hepatocytes will improve the phenotype associated with the disease. The first specific aim is to optimize (1) in vitro our non-viral asialoglycoprotein receptor hepatocyte- specific delivery systems, and (ii) chimeric RNA/DNA oligo oligonucleotide design for maximal conversion of the G to A transition at nt 1477 in the hemophilia B factor IX gene expressed in the Chapel Hill strain of dogs. The second specific aim is to evaluate the capacity of these molecules/delivery systems to promote targeted single nt conversion to correct the G to A transition in the canine factor IX in vivo. The non-viral delivery systems and chimeric oligonucleotides identified in Specific Aim 1 will be utilized. The relevant metabolic parameters will be monitored to quantitate the therapeutic effect of in situ genomic correction. Optimization of the dosing regimen, as well as the delivery vehicle and route of administration will be established. The third specific aim is to evaluate the potential of this technology in altering the genomic factor VII gene to produce the optimized factor VIIa mutations devised in Project 2. The initial work will be performed in vitro using cultured hepatocytes to optimize the delivery and design of the chimeric oligonucleotides. The selected factor VIIa mutation will then be generated in vivo and evaluated in collected hemophilia A phenotype in a factor VIII deficient mouse model. The relevant metabolic parameters will be monitored to quantitate the therapeutic effect of in situ genomic correction. Optimization of the dosing regimen, as well as the delivery vehicle and route of administration will be established. The long term goal of this research proposal is to: (i) to optimize non-viral delivery systems and oligonucleotide design that will promote the utility of RNA/DNA oligonucleotides for correcting single nt mutations associated with hemophilia; (ii) elucidate the optimal parameters for in vivo therapeutic correction of single nt mutations using this technology in the Chapel Hill strain of hemophilia B dogs, and (iii) evaluate the use of this technology for creating factor VIIa variants in vivo and there therapeutic benefit. The comparison of the in vitro and in vivo correction results will establish the feasibility for in vivo gene therapy approach using this technology for treatment of hemophilia.