Chimeraplasty for factor IX and VII gene expression

通过同源重组对缺陷基因进行位点特异性校正在通过基因疗法治疗遗传性代谢疾病中仅以非常低的频率实现。最近，一种合成的RNA/DNA杂交双链体，设计成与同源基因组序列完全对齐的寡核苷酸，除了一个碱基错配之外，被证明可以促进大鼠肝细胞基因组DNA中的靶向单核苷酸（nt）转换。该过程利用了细胞有效的内源性DNA错配修复途径，从而使其成为基因治疗的新方法。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nt突变中的效用。这一目标验证了我们的假设，即受影响的肝细胞中的基因校正将改善与疾病相关的表型。第一个具体目的是优化（1）体外我们的非病毒脱唾液酸糖蛋白受体肝细胞特异性递送系统，和（ii）嵌合RNA/DNA寡核苷酸设计，以使在查佩尔山品系狗中表达的血友病B因子IX基因中在nt 1477处的G至A转变最大化.第二个具体目的是评估这些分子/递送系统促进靶向单nt转化以在体内校正犬因子IX中的G至A转变的能力。将使用特定目标1中确定的非病毒递送系统和嵌合寡核苷酸。将监测相关代谢参数，以定量原位基因组校正的治疗效果。将确定给药方案以及给药溶媒和给药途径的优化。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突变中的潜力。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。然后在体内产生选定的因子VIIa突变，并在因子VIII缺陷小鼠模型中收集的血友病A表型中进行评价。将监测相关代谢参数，以定量原位基因组校正的治疗效果。将确定给药方案以及给药溶媒和给药途径的优化。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寡核苷酸用于纠正与血友病相关的单核苷酸突变的效用;（ii）阐明在血友病B狗的查佩尔山品系中使用该技术进行单nt突变的体内治疗校正的最佳参数，和（iii）评价该技术在体内产生因子VIIa变体的用途及其治疗益处。体外和体内校正结果的比较将确立使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.