Genetic Reconstitution for Phenylketonuria

苯丙酮尿症的基因重建

• 批准号: 6680669
• 负责人: Savio L Woo
• 金额: $ 38.5万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680669
• 关键词: SCID mouse; adeno associated virus group; biotechnology; gel mobility shift assay; gene delivery system; gene expression; gene therapy; genetic promoter element; genetic regulatory element; genetic transcription; genetically modified animals; liver; nonhuman therapy evaluation; phenylalanine 4 monooxygenase; phenylketonurias; polymerase chain reaction; transfection /expression vector

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.

描述（由申请人提供）：经典的苯酮尿症（PKU）是氨基酸代谢中的隐性单基因疾病，是由于肝苯丙氨酸羟化酶（PAH）的缺乏而导致的，并且它使个体倾向于对严重和永久性的智力受阻。虽然可以通过饮食限制来预防PKU患者的疾病表型，但该治疗遭受了终身患者治疗和患者依从性不佳的需求。女性PKU患者怀孕期间饮食限制的停用也导致后代的各种出生缺陷，无论PAH基因型如何。这种称为母体PKU的综合征已成为公共健康问题，因为大多数经过治疗的育龄女性PKU患者一直在饮食中限制。因此，我们已经开始通过体内遗传重建来开发PKU和母体PKU的互补治疗方式。 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.最近已显示重组AAV载体在体内有效地转导肝细胞，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针对病毒转导细胞的细胞免疫反应。然而，它在代谢性疾病的治疗中的应用受到以下事实的限制：在体内仅一小部分载体转导的肝细胞会导致转基因的持续表达，因此需要每个细胞的高水平的转基因表达，才能有效。此外，大规模生产纯化的RAAV载体仍将有效转移到包括人类在内的大型动物中，仍然困难。为了克服或避免这些障碍，我们提出了以下分子策略：1）与替代血清型中的衣壳开发和使用杂种raav载体，以提高体内肝细胞转导效率； 2）将顺式作用遗传因素纳入转基因表达盒的启动子增强区域，该区域将导致基因转录升高； 3）将顺式作用元素插入mRNA的3'-非翻译区域，这些元素将增加其在载体转导细胞中的稳定性。我们设想，这些用于肝基因转移和表达的分子策略将在提高PKU小鼠肝脏的MPAH活性水平方面具有协同作用，这将使血液苯丙氨酸水平恢复正常。这种有益的结果也可以在减少的矢量剂量下完成，以便可以最大程度地减少大规模生产纯化的载体。这些研究的成功进行将为未来在PKU的遗传治疗以及患者肝酶缺乏症继发的其他代谢疾病中的科学基础。