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（由申请方提供）：经典苯丙酮尿症（PKU）是一种氨基酸代谢的隐性单基因疾病，由肝苯丙氨酸羟化酶（PAH）缺乏引起，易使受影响的个体发生严重和永久性智力迟钝。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患者在妊娠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。这种综合征，被称为孕产妇苯丙酮尿症，已成为一个公共卫生问题，因为大多数接受治疗的育龄女性苯丙酮尿症患者已停止饮食限制。因此，我们已经开始通过体内基因重建开发PKU和母体PKU的补充治疗方式。为了在肝脏中实现持久和高水平的转基因表达，将使用缺失所有病毒基因的重组腺相关病毒（AAV）载体将鼠PAH基因体内递送至PAH缺陷型Pah anu 2小鼠的肝脏，所述小鼠是人类中PKU和母体PKU的非常忠实的动物模型。重组AAV载体最近已显示出在体内有效地使肝细胞增殖，这导致转基因产物的持续表达，而没有明显的毒性或引发针对病毒转导的细胞的细胞免疫应答。然而，其在治疗代谢紊乱中的应用受到以下事实的限制：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以使治疗有效。此外，大规模生产纯化的rAAV载体以有效地将转基因递送到包括人在内的大型动物中仍然存在困难。为了克服这些障碍，我们提出了以下分子策略：1）开发和使用具有来自替代血清型的衣壳的杂合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整合到转基因表达盒的启动子-增强子区域中，这将导致基因转录的提高;和3）将顺式作用元件插入mRNA的3 ′-非翻译区，这将增加它们在载体转导的细胞中的稳定性。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活性的水平，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载体剂量下实现，使得可以最小化对纯化载体的大规模生产的需要。这些研究的成功进行将为未来应用于PKU以及继发于患者肝酶缺乏的其他代谢紊乱的基因治疗提供科学基础。