Augmented Phenylalanine Clearance by Muscles as Novel Therapy for Phenylketonuria

描述(申请人提供)：这个项目的总体目标是探索肌肉定向基因疗法作为新生儿筛查发现的先天性代谢错误(IEM)的替代治疗方法。更具体地说，研究人员将把他们的努力集中在苯丙氨酸羟基酶(PAH)缺陷的Pahenu2小鼠上，这是人类苯丙酮尿症(PKU)的模型，并将评估苯丙氨酸(Phe)代谢系统在肌肉中的表达，作为治疗这种疾病的潜在新方法。 对于许多IEM来说，疾病的病理是由于循环中的有毒代谢物的影响，而不是酶缺乏所产生的任何直接的组织特异性影响。例如，PAH主要在肝脏表达，但与PKU相关的病理完全是由于循环Phe的影响；肝脏本身不受PAH缺乏的影响。任何永久性降低血Phe的治疗都将有效地改善PKU表型。研究人员假设，在骨骼肌中表达强大的苯丙氨酸代谢系统将增加肌肉对正常合成代谢苯丙氨酸的摄取，有效代谢循环中的苯丙氨酸，纠正高苯丙氨酸血症。他们将探索两个不同的苯丙氨酸代谢系统：在肌肉中协调PAH的表达以及四氢生物蝶呤(BH4)合成途径的两个元素-GTP环水解酶1(GTPCH)和6-丙酮酰四氢蝶呤合成酶(PTPS)，以提供支持苯丙氨酸羟基化或苯丙氨酸解氨酶(PAL)表达所需的BH4辅因子，苯丙氨酸解氨酶(PAL)是苯丙氨酸代谢酶，不需要任何外部辅助因子。在具体目标1中，研究人员将使用标准的生殖系修饰方法来开发在所有骨骼肌中表达苯丙氨酸代谢系统的转基因小鼠。这些小鼠将被培育成Pahenu2小鼠，产生的后代缺乏肝脏PAH活性，但在肌肉中表达Phe代谢系统。Phe清除将在这些后代中进行评估。在特定的目标2中，他们将分别使用重组腺相关病毒血清1型载体(rAAV2/1)或通过流体动力肢体静脉注射传递的裸质粒DNA载体直接在Pahenu2小鼠的后肢肌肉中诱导Phe代谢系统的表达。提示PAH的肌肉表达与BH4合成途径的元件或单独的PAL将导致Pahenu2小鼠高苯丙氨酸血症的纠正。

DESCRIPTION (provided by applicant): The overall goal of this project is to explore muscle-directed gene therapy as an alternative treatment for inborn errors of metabolism (IEM) detected by newborn screening. More specifically, the investigators will focus their efforts upon phenylalanine hydroxylase (PAH)-deficient Pahenu2 mice, a model of human phenylketonuria (PKU), and will evaluate expression of a phenylalanine (Phe) metabolizing system in muscle as a potential novel therapeutic approach to this disorder. For many IEM, disease pathology is due to effects of a circulating toxic metabolite rather than any direct tissue-specific effect from the enzyme deficiency. For example, PAH is primarily expressed in liver, but the pathology associated with PKU is entirely due to effects of circulating Phe; the liver itself is unaffected by PAH deficiency. Any treatment that achieves permanent reduction of blood Phe will effectively ameliorate the PKU phenotype. The investigators' hypothesized that expression of a robust Phe-metabolizing system in skeletal muscle will augment normal anabolic Phe uptake in muscle, effectively metabolize circulating Phe and correct hyperphenylalaninemia. They will explore two different Phe-metabolizing systems: coordinate expression in muscle of PAH along with the enzymes GTP cyclohydrolase 1 (GTPCH) and 6-pyruvoyltetrahydropterin synthase (PTPS), both elements of the tetrahydrobiopterin (BH4) synthetic pathway, to provide the BH4 cofactor necessary to support Phe hydroxylation or expression of phenylalanine ammonia lyase (PAL), a Phe-metabolizing enzyme that does not require any external cofactors. In Specific Aim 1, the investigators will employ standard germline modification methods to develop transgenic mice that express a Phe metabolizing system throughout all skeletal muscle. These mice will be bred to Pahenu2 mice to yield progeny that lack liver PAH activity but express the Phe metabolizing system in muscle. Phe clearance will be assessed in these progeny. In Specific Aim 2, they will separately employ either recombinant adenoassociated virus serotype 1 vectors (rAAV2/1) or naked plasmid DNA vectors delivered by hydrodynamic limb vein injection to induce expression of a Phe metabolizing system directly in hindlimb muscles of Pahenu2 mice. It is proposed that muscle expression of PAH along with elements of the BH4 synthetic pathway or of PAL alone will lead to correction of hyperphenylalaninemia in Pahenu2 mice.