新型药物PTC124分别在细胞和动物模型上对苯丙酮尿症致病基因PAH无义突变的矫正研究

Phenylketonuria (PKU) is an autosomal recessive inherited birth defect. It is mainly caused by mutations in phenylalanine hydroxylase gene (PAH), an enzyme-coding gene involved in phenylalanine metabolism pathway, and could cause severe damages to neonatal intellectual development. Its prevalence can be up to 1/ 2068 in the North China. Effective treatment is not available at present and diet control is the only clinical approach. In Chinese population, nonsense mutation accounts for more than 15% of all mutation types in PAH. It occurs on limited gene loci and therefore has great clinical values. Recent studies discover that nonsense mutation can be read-through by a new compound, PTC124, which is with high efficiency and has non side-affect on human body. The objects of this study are 1) to establish the cell and animal model of PTC124 read-through of PAH nonsense mutation. The affect of PTC124 on protein activity, cell growth and organ function will be tested and estimated. The optimal treatment of this compound, including treating interval and frequencies, will also be researched; 2) to research the differences of PTC124 read-through on 9 individual PAH nonsense mutations, which account for the most part of nonsense mutations in PAH in Chinese population; 3) to research the role of protein releasing factor and NMD signal pathway in PTC124 read-through by gene silence technology. This study will research the mechanism of PTC124 read-through on PAH nonsense mutation and provide research models and data for PKU treatment. A real effective cure for PKU is expected in this study with social significance and economic benefits.

苯丙酮尿症（phenylketonuria，PKU）是呈常染色体隐性遗传的一类出生缺陷，由酶基因PAH突变产生苯丙氨酸代谢障碍，损害新生儿智力发育，发病率可达1/2068。目前该疾病缺乏治愈手段，仅以饮食控制来缓解病情。无义突变是国内人群PAH基因突变的一种重要类型，占到15%以上，具有临床价值。最新研究发现新药PTC124可以矫正这种基因突变，对人体毒副作用小，适合临床应用。本研究拟通过：1）构建PTC124矫正PAH基因无义突变的细胞及动物模型，分析其对蛋白活性、细胞生长和器官功能的影响，确立药物最适使用方案；2）研究PTC124对国内9个主要PAH无义突变的矫正效率差异；3）对蛋白释放因子和NMD信号通路进行负调控，探索其作为调控工具的可能性。项目将深入研究新药PTC124治疗PAH无义突变机理，为临床治疗PKU提供生物模型和实验数据，提供根本上治愈PKU的方法，具有广泛的社会意义。

苯丙酮尿症（phenylketonuria，PKU）是呈常染色体隐性遗传的一类出生缺陷，由酶基因PAH突变产生苯丙氨酸代谢障碍，损害新生儿智力发育，发病率可达1/2068。目前该疾病缺乏治愈手段，仅以饮食控制来缓解病情。无义突变是国内人群PAH基因突变的一种重要类型，占到15%以上，具有临床价值。最新研究发现新药PTC124可以矫正这种基因突变，对人体毒副作用小，适合临床应用。本研究原计划通过：构建PTC124矫正PAH基因无义突变的细胞及动物模型，分析其对蛋白活性、细胞生长和器官功能的影响，从而摸索药物最适使用方案，并研究PTC124对几个主要PAH无义突变的矫正效率，以及对蛋白释放因子和NMD信号通路的调控。在细胞模型实验中，分别使用了真核表达质粒载体转染和慢病毒感染的方法将PAH野生型及突变型基因转入HEK293细胞，用PTC124、Amlexanox和G418等几种对无义突变有矫正作用的药物处理细胞然后分别检测各药物对细胞生长的影响，对PAH基因mRNA和蛋白的表达水平的影响。细胞实验表明，药物PTC124、Amlexanox和G418可提高野生型及无义突变PAH基因转录水平，且对5个无义突变的增益效果表现出差异性。在蛋白水平上，PTC124和Amlexanox的效果不明显，而G418实现了无义突变蛋白翻译通读，得到了完整的PAH蛋白。除W326X突变外，G418对突变Y356X、R166X、R111X、R176X的通读效果与其浓度呈正相关。由于构建PTC124矫正PAH基因无义突变的细胞模型在探索、稳定实验方法以及蛋白通读实验，在摸索实验条件如质粒蛋白表达元件效能、药物浓度、处理方式等方面投入时间显著超出原计划时间，经过多次失败、反复验证最终证实了PTC124、Amlexanox和G418可提高野生型及无义突变PAH基因转录水平，且对5个无义突变的增益效果表现出差异性，并完成了后续细胞实验工作。原计划建立动物模型工作由于疫情，动物的运输问题而延误。目前我们的动物试验在进行中，我们选用Y356X和R111X两个突 变位点，构建PAH无义突变转基因小鼠，开展了PTC124等氨基糖苷类化合物及其衍生物在转基因动物模型实验。