Chimeraplasty for factor IX and VII gene expression

在遗传性代谢性疾病的基因治疗中，通过同源重组对缺陷基因进行定点纠正的频率很低。最近，一种合成的RNA/DNA杂合双链寡核苷酸被证明可以促进大鼠肝细胞基因组DNA的靶向性单核苷酸(NT)转化，该过程利用了细胞有效的内源性DNA错配修复途径，从而使其成为一种新的基因治疗方法。该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突变方面的有效性。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疾病相关的表型。第一个具体目标是优化(1)体外我们的非病毒去唾液酸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，以最大限度地转化在犬血友病B因子IX基因中表达的血友病B因子IX基因中1477位核苷酸的G到A转变。第二个具体目的是评估这些分子/递送系统在体内促进靶向单个NT转换以纠正犬凝血因子IX的G到A转变的能力。将利用在特定目标1中确定的非病毒递送系统和嵌合寡核苷酸。相关的代谢参数将被监测，以量化原位基因组矫正的治疗效果。将优化给药方案，以及递送车辆和给药路线。第三个具体目标是评估这项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。初步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传递和设计。然后，选定的因子VIIa突变将在体内产生，并在收集的血友病A表型中对因子缺乏的小鼠模型进行评估。相关的代谢参数将被监测，以量化原位基因组矫正的治疗效果。将优化给药方案，以及递送车辆和给药路线。这项研究建议的长期目标是：(I)优化非病毒递送系统和寡核苷酸设计，以促进RNA/DNA寡核苷酸用于纠正与血友病相关的单个NT突变；(Ii)阐明在血友病B犬的Chapel Hill品系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)评估该技术在体内创造因子VIIa变体的使用及其治疗益处。体外和体内校正结果的比较将为利用该技术治疗血友病的体内基因治疗方法建立可行性。

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.