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）：该项目的总体目标是开发一种安全、有效、非病毒、肌肉导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错误。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），但需要终生坚持一种令人不快且昂贵的饮食。饮食不合规是常见的。一个有效的，安全的，永久的治疗PKU不依赖于饮食操纵是可取的。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。我们提出，骨骼肌中的多环芳烃活性将作为代谢汇清除循环中的Phe。为此，我们将评估一种新的静脉流体动力学DNA转移方法，在人类PKU模型高苯丙氨酸血症Pahenu2小鼠骨骼肌中表达苯丙氨酸羟化酶（PAH）。多环芳烃需要未偶联的蝶呤，四氢生物蝶呤（BH4）作为辅助因子。骨骼肌含有很少的BH4。初步数据表明，骨骼肌中BH4合成途径的元件表达是维持足够的BH4和支持Phe羟基化所必需的。我们的具体假设是，PAH与BH4合成酶GTP环水解酶（GTPCH）和6-丙酮酰四氢蝶呤合成酶（PTPS）在骨骼肌中的协调表达将导致Pahenu2小鼠血液中足量的BH4生成、持续的PAH活性和生理上显著的Phe水平下降。我们的计划是在Pahenu2骨骼肌中表达PAH、GTPCH和PTPS cdna，方法是使用流体动力学肢体静脉技术，通过静脉注射裸质粒DNA，该方法在小型和大型研究动物中同样有效。我们将评估注射前后小鼠的血液Phe水平、Phe耐受性和身体健康状况。基因表达的数量和持续时间也将被评估。我们的实验还将比较三顺反子质粒载体与三种编码单一基因的质粒载体的基因表达。这些临床前试验将为未来PKU患者临床试验的发展奠定基础。