Augmented Phenylalanine Clearance by Muscles as Novel Therapy for Phenylketonuria

描述（由申请人提供）：该项目的总体目标是探索肌肉定向基因治疗作为新生儿筛查发现的先天性代谢缺陷（IEM）的替代治疗。 更具体地说，研究人员将把精力集中在苯丙氨酸羟化酶（PAH）缺陷型Pahenu 2小鼠（人类苯丙酮尿症（PKU）模型）上，并将评估肌肉中苯丙氨酸（Phe）代谢系统的表达作为潜在的新型治疗方法。这种疾病。 对于许多IEM，疾病病理是由于循环毒性代谢物的影响，而不是酶缺乏的任何直接组织特异性影响。 例如，PAH主要在肝脏中表达，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。 任何实现血液Phe永久减少的治疗将有效改善PKU表型。 研究人员假设，骨骼肌中强大的Phe代谢系统的表达将增加肌肉中正常的合成代谢Phe摄取，有效代谢循环Phe并纠正高苯丙氨酸血症。 他们将探索两种不同的Phe代谢系统：使PAH沿着在肌肉中的表达与酶GTP环化水解酶1（GTPCH）和6-异戊酰四氢蝶呤合酶（PTPS）（四氢生物蝶呤（BH 4）合成途径的两个元件）协调，以提供支持Phe羟基化或苯丙氨酸解氨酶（PAL）表达所必需的BH 4辅因子，一种不需要任何外部辅因子的苯丙氨酸代谢酶。 在具体目标1中，研究人员将采用标准的种系修饰方法来开发在所有骨骼肌中表达Phe代谢系统的转基因小鼠。 这些小鼠将与Pahenu 2小鼠交配，以产生缺乏肝脏PAH活性但在肌肉中表达Phe代谢系统的后代。 将在这些子代中评估Phe清除率。 在具体目标2中，他们将分别采用重组腺相关病毒血清型1载体（rAAV 2/1）或裸质粒DNA载体，通过流体动力学肢静脉注射递送，以直接在Pahenu 2小鼠后肢肌肉中诱导Phe代谢系统的表达。 有人提出，肌肉中PAH的表达沿着BH 4合成途径的元件或单独PAL的表达将导致Pahenu 2小鼠高苯丙氨酸血症的纠正。 公共卫生相关性：该项目的总体目标是开发新的基因治疗方法，用于治疗通过新生儿筛查在出生时检测到的遗传性代谢紊乱。 研究人员将肌肉基因治疗描述为遗传性代谢紊乱的一种新的治疗方法，并将特别关注人类疾病苯丙酮尿症（PKU）的小鼠模型，这是最常见的代谢疾病之一。

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. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to develop novel gene therapy approach for the treatment of inherited metabolic disorders that are detected at birth through newborn screening. The investigators will delineate muscle gene therapy as a new treatment approach to inherited metabolic disorders, and will focus specifically upon a mouse model of the human disorder phenylketonuria (PKU), one of the most common metabolic diseases.