GENE THERAPY IN PHENYLKETONURIA

我们首先测试了在苯丙酮尿症突变中使用嵌合DNA/RNA寡核苷酸进行靶向基因纠正的可行性。然而，我们未能观察到美国Kimeragen报道的有效的核苷酸替代，尽管表征影响错配修复机制的各种参数并进一步优化方法似乎很重要，但我们的观察以及其他人的观察可能会对先前报道的可信度产生怀疑。相反，利用重组腺病毒进行的基因转移实验获得了以下丰硕的结果：1)针对外源性苯丙氨酸羟基酶的宿主免疫反应强烈，而不是腺病毒本身；2)药物剂量的苯丙氨酸羟基酶辅助因子四氢生物蝶呤似乎可以增强基因治疗后的酶活性；3)在动物模型中建立了一种有效的基因转移方法；4)建立了定量检测组织中腺病毒颗粒的TaqMan-PCR方法。本研究为基因治疗肝酶缺乏性先天性代谢疾病的临床应用奠定了基础。

We first tested the feasibility of targeted gene correction using chimeric DNA/RNA oligonucleotide in phenylketonuria mutations. However, we failed to observe efficient nucleotide substitution as reported by Kimeragen in U.S.A.Although it seems important to characterize various parameters affecting mismatch repair mechanism and further optimize the method, our observation as well as other's might throw doubt upon the credibility of the previous reports.In contrast, gene transfer experiments using recombinant adenovirus obtained following fruitful results : 1) a strong host immune reaction was demonstrated against extrinsic phenylalanine hydroxylase, rather than adenovirus per se ; 2) a pharmacological dose of tetrahydrobiopterin, a cofactor for phenylalanine hydroxylase, appeared to enhance the enzymatic activity after gene therapy ; 3) an efficient gene transfer method to fetus was established in an animal model ; 4) a TaqMan-PCR method to quantify adenoviral particles in tissues was developed. Our study will facilitate the clinical application of gene therapy in inborn errors of metabolism caused by hepatic enzyme deficiency.