Genetic Reconstitution for Phenylketonuria

描述（由申请人提供）：经典苯丙酮尿症（PKU）是一种隐性单基因氨基酸代谢疾病，由肝脏苯丙氨酸羟化酶（PAH）缺乏引起，易使患者出现严重和永久性的智力迟钝。虽然PKU患者的疾病表型可以通过饮食限制来预防，但治疗需要终身患者管理和患者依从性差。女性PKU患者在怀孕期间停止饮食限制也会导致后代出现各种出生缺陷，无论PAH基因型如何。由于大多数接受治疗的育龄女性PKU患者已经停止了饮食限制，这种综合征被称为母体PKU，已成为一个公共卫生问题。因此，我们已经开始通过体内基因重建来开发PKU和母体PKU的互补治疗模式。为了在肝脏中实现持续和高水平的转基因表达，将使用删除所有病毒基因的重组腺相关病毒（AAV）载体在体内将小鼠多环芳烃烯传递到多环芳烃缺乏的PAH anu2小鼠的肝脏中，PKU是一种非常可靠的PKU动物模型和人类母体PKU。重组AAV载体最近被证明可以有效地在体内转导肝细胞，从而导致转基因产物的持续表达，而没有明显的毒性或引起细胞对病毒转导细胞的免疫反应。然而，它在代谢紊乱治疗中的应用受到限制，因为体内只有一小部分载体转导的肝细胞导致转基因的持续表达，因此需要每个细胞高水平的转基因表达才能有效治疗。此外，大规模生产纯化的rAAV载体以有效地将转基因传递到包括人在内的大型动物体内仍然存在困难。为了克服或规避这些障碍，我们提出了以下分子策略：1)开发和使用具有不同血清型衣壳的杂交rAAV载体，以提高体内肝细胞转导效率；2)将顺式作用遗传元件纳入转基因表达磁带的启动子-增强子区域，导致基因转录水平升高；3)将顺式作用元件插入mrna的3'-非翻译区，增加其在载体转导细胞中的稳定性。我们设想，这些肝脏基因转移和表达的分子策略将协同提高PKU小鼠肝脏中mPAH的活性水平，从而使血液中苯丙氨酸水平恢复正常。这种有益的结果也可以在减少载体剂量的情况下实现，这样就可以最大限度地减少大规模生产纯化载体的需要。这些研究的成功进行将为未来PKU以及其他继发于肝酶缺乏患者的代谢紊乱的基因治疗提供科学基础。

DESCRIPTION (provided by applicant): Classical Phenylketonuria (PKU) is a recessive monogenic disorder in amino acid metabolism that results from a deficiency of hepatic phenylalanine hydroxylase (PAH), and it predisposes affected individuals to severe and permanent mental retardation. While the disease phenotype in PKU patients can be prevented by dietary restriction, the treatment suffers from the need of life-long patient management and poor patient compliance. Discontinuation of dietary restriction during pregnancy in female PKU patients has also caused various birth defects in the offspring regardless of the PAH genotypes. This syndrome, known as maternal PKU, has become a public health concern since most treated female PKU patients of child-bearing age have been off diet restriction. We have therefore begun the development of complementary treatment modalities for PKU and maternal PKU by genetic reconstitution in vivo. To achieve persistent and high level transgene expression in the liver, recombinant adeno-associated virus (AAV) vectors deleted of all viral genes will be used for in vivo delivery of the murine PAH ene to the livers of the PAH-deficient Pah anu2 mice, an extraordinarily faithful animal model of PKU and maternal PKU in humans. Recombinant AAV vectors have recently been shown to effectively transduce hepatocytes in vivo, which resulted in the persistent expression of transgene products without apparent toxicity or eliciting a cellular immune response against the virally transduced cells. Its application in treatment of metabolic disorders however, is limited by the fact that only a small fraction of vector-transduced hepatocytes in vivo resulted in persistent expression of the transgenes, thus requiring high levels of transgene expression per cell for the treatment to be effective. In addition, there still remains the difficulty in large-scale production of purified rAAV vectors for effective transgene delivery into large animals including humans. To overcome or circumvent these obstacles, we propose the following molecular strategies: 1) develop and use of hybrid rAAV vectors with capsids from alternative serotypes to enhance hepatocyte transduction efficiency in vivo; 2) incorporate cis-acting genetic elements into the promoter-enhancer region of transgene expression cassettes that will lead to elevated gene transcription; and 3) insert cis-acting elements into the 3'-untranslated region of mRNAs that will increase their stability in vector-transduced cells. We envision that these molecular strategies for hepatic gene transfer and expression will be synergistic in elevating the level of mPAH activity in the livers of PKU mice, which will restore blood phenylalanine levels to normal. This beneficial outcome might also be accomplished at reduced vector doses so that the need for large-scale production of the purified vectors can be minimized. Successful conduct of these studies will provide the scientific foundation for future applications in the genetic treatment of PKU as well as other metabolic disorders secondary to hepatic enzyme deficiencies in patients.