GENE THERAPY FOR PHENYLKETONURIA

苯丙酮尿症(PKU)是北美和欧洲儿童最常见的遗传性疾病之一，发病率约为每10,000名新生儿中就有1名。苯丙氨酸羟化酶(PAH)的突变是PKU的主要原因。PAH缺乏会导致血清苯丙氨酸(Phe)水平升高，导致大脑发育异常和智力低下。北京大学的频率，加上早期饮食调整的能力，极大地减少了与这种疾病相关的严重神经问题，导致了所有州的新生儿测试计划。不幸的是，有一种普遍的印象是，饮食控制可以治愈这种疾病。在现实中，严格遵守饮食是极其困难的，失误可能会导致严重的长期神经发育后遗症，特别是在PAH缺乏的女性的后代中。鉴于众所周知的单基因缺陷，对PKU进行基因治疗在技术上是可行的，在治疗上也是可取的。最近在基因治疗方面的重要进展来自使用基于AAV的载体来提供有效、稳定和安全的代谢校正。假设AAV载体将在北京大学有用。根据这一假设，将检验AAV载体在以下方面的能力：1)有效挽救Pah/enu2小鼠Phe代谢缺陷。血清Phe水平将与载体基因组和转染细胞的数量以及PKU症状的减轻程度有关。2)在成年和新生Pah/enu2小鼠身上进行载体序列的非生殖线传播，将多环芳烃安全地传递到肝脏将进行测试。潜在的肝脏毒性和对载体的免疫反应也将被检查。3)利用替代调控元件和组织特异性靶向优化PAH的表达和肝细胞递送。这些实验为检验AAV驱动的基因治疗真正治愈PKU的潜力提供了一种合理的方法。

Phenylketonuria (PKU) is one of the most common genetic disorders affecting children in North America and Europe, with an incidence of about 1 in 10,000 births. Mutations in the enzyme phenylalanine hydroxylase (PAH) are the major cause of PKU. PAH deficiency results in elevated serum phenylalanine (Phe) levels, leading to abnormal brain development and mental retardation. PKU's frequency, coupled with the ability of early dietary modification to greatly reduce the severe neurological problems associated with the disorder, has led to neonatal testing programs in all states. Unfortunately, there is a widespread impression that dietary control effects a "cure" for this disease. In reality, strict adherence to the diet is exceedingly difficult and lapses can cause serious long-term neuro-development sequelae, particularly in offspring of PAH-deficient women. Given the well-understood single gene defect, gene therapy for PKU is both technically feasible and therapeutically desirable. Recent important advances in gene therapy have come from the use of AAV-based vectors to provide effective, stable, and safe correction of metabolic. It is hypothesized that AAV vectors will be useful in PKU. To this hypothesis, the ability of AAV vectors to: 1) Provide effective rescue of defective Phe metabolism in the Pah/enu2 mouse will be examined. Serum Phe levels will be related to the number of vector genomes and transfected cells as well as to the degree of reduction of PKU symptoms. 2) Give safe PAH delivery to liver with non germ-line transmission of vector sequences in either adult and neonatal Pah/enu2 mice will be tested. The potential for liver toxicity and immune responses to vector will also be examined. 3) Optimize PAH expression and hepatocyte delivery by using alternative control elements and tissue-specific targeting. These experiments provide a rational approach to examining the potential of AAV-driven gene therapy to truly cure PKU.