Intravenous non-viral gene therapy for phenylketonuria (PKU)

苯丙酮尿症 (PKU) 的静脉内非病毒基因治疗

• 批准号: 7108140
• 负责人: Cary O. Harding
• 金额: $ 18.64万
• 依托单位: MIRUS BIO CORPORATION
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-20 至 2008-09-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7108140
• 关键词: aminoacid metabolism; aminohydrolases; biotechnology; cofactor; disease /disorder model; enzyme activity; gene delivery system; gene expression; gene therapy; genetically modified animals; guanosine triphosphate; intravenous administration; laboratory mouse; muscle metabolism; nonhuman therapy evaluation; phenylalanine; phenylalanine 4 monooxygenase; phenylketonurias; plasmids; striated muscles; tetrahydrobiopterin; therapy design /development; transfection; transfection /expression vector

DESCRIPTION (provided by applicant): The overall goal of this project is to develop a safe, effective, non-viral, muscle-directed gene therapy approach to the treatment of phenylketonuria (PKU), a relatively common inborn error of metabolism. Contemporary dietary therapy for PKU, although preventing the major manifestations of the disease (seizures, microcephaly, mental retardation), requires life-long commitment to an unpalatable and expensive diet. Dietary noncompliance is common. An effective, safe, permanent treatment for PKU that does not rely upon dietary manipulation is desirable. The pathology associated with PKU is caused by the accumulation of phenylalanine (Phe); any method that successfully reduces Phe levels will ameliorate the PKU phenotype. We propose that PAH activity in skeletal muscle will act as a metabolic sink to clear Phe from the circulation. To this end, we will evaluate a novel intravenous hydrodynamic DNA transfer method to express the enzyme phenylalanine hydroxylase (PAH) in skeletal muscle of hyperphenylalaninemic Pahenu2 mice, a model of human PKU. PAH requires the unconjugated pterin, tetrahydrobiopterin (BH4), as a cofactor. Skeletal muscle contains little BH4. Preliminary data demonstrate that expression of elements of the BH4 synthetic pathway in skeletal muscle is required to maintain sufficient BH4 and support Phe hydroxylation. Our specific hypothesis is that coordinated expression of PAH along with the BH4-synthetic enzymes, GTP cyclohydrolase (GTPCH) and 6-pyruvoyltetrahydropterin synthase (PTPS) in skeletal muscle will lead to sufficient BH4 production, sustained PAH activity, and a physiologically significant decrease in blood Phe levels in Pahenu2 mice. Our plan is to express the PAH, GTPCH, and PTPS cDNAs in Pahenu2 skeletal muscle by delivering naked plasmid DNA intravenously using a hydrodynamic limb vein technique, a procedure that is equally effective in small and large research animals. We will evaluate blood Phe levels, Phe tolerance and physical well-being of the mice before and after injection. The amount and duration of gene expression will also be evaluated. Our experiments will also compare gene expression from a triple-cistronic plasmid vector vs. co-administration of 3 separate plasmid vectors encoding single genes. These preclinical trials will form the basis for the development of future clinical trials in humans with PKU.

描述（由申请人提供）：本项目的总体目标是开发一种安全、有效、非病毒、肌肉定向的基因治疗方法来治疗苯丙酮尿症（PKU），这是一种相对常见的先天性代谢缺陷。当代PKU的饮食疗法，虽然可以预防疾病的主要表现（癫痫发作，小头畸形，智力迟钝），但需要终身致力于难吃和昂贵的饮食。不遵守饮食规定是很常见的。一个有效的，安全的，持久的治疗PKU，不依赖于饮食控制是可取的。与PKU相关的病理是由苯丙氨酸（Phe）的积累引起的;任何成功降低Phe水平的方法都将改善PKU表型。我们建议，多环芳烃活动在骨骼肌将作为一个代谢汇清除苯丙氨酸从循环。为此，我们将评估一种新的静脉内流体动力学DNA转移方法，以表达酶苯丙氨酸羟化酶（PAH）在高苯丙氨酸血症Pahenu 2小鼠，人类PKU模型的骨骼肌。PAH需要非共轭蝶呤，四氢生物蝶呤（BH 4），作为辅因子。骨骼肌含有少量的BH 4。初步数据表明，需要在骨骼肌中表达BH 4合成途径的元件以维持足够的BH 4并支持Phe羟基化。我们的具体假设是，PAH沿着与BH 4合成酶、GTP环化水解酶（GTPCH）和6-乙酰四氢蝶呤合酶（PTPS）在骨骼肌中的协调表达将导致Pahenu 2小鼠中充足的BH 4产生、持续的PAH活性和血液Phe水平的生理学显著降低。我们的计划是表达PAH，GTPCH，和PTPS的cDNA在Pahenu 2骨骼肌通过提供裸质粒DNA静脉注射使用流体动力学肢静脉技术，一个程序，是同样有效的小型和大型研究动物。我们将评估注射前后小鼠的血液Phe水平、Phe耐受性和身体健康。还将评价基因表达的量和持续时间。我们的实验还将比较来自三顺反子质粒载体的基因表达与编码单个基因的3个单独质粒载体的共施用。这些临床前试验将成为未来PKU人体临床试验的基础。