AAV2/8 vector-mediated liver gene therapy for phenylketonuria (PKU)

描述（由申请人提供）： 肝靶向基因治疗是一种很有前途的方法来治疗人类先天性代谢缺陷（IEM）由于特定的肝酶缺乏症。本项目的目标是通过治疗苯丙氨酸羟化酶（PAH）缺陷型Pahenu 2小鼠（一种人苯丙酮尿症（PKU）模型），开发一种安全、有效的肝定向基因转移技术用于IEM。我们最近已经目睹了用新型重组腺相关病毒血清型2载体（用血清型8衣壳假型化）（rAAV 2/8）治疗的Pahenu 2小鼠中血清苯丙氨酸水平的完全校正。我们计划扩展这一观察以评估rAAV 2/8施用后的长期稳定性和不良反应的发生率，并进一步理解支配苯丙氨酸清除的生理因素。我们的具体假设是，完全纠正高苯丙氨酸血症及其伴随的表型特征将需要永久恢复至少10%的Pahenu 2肝细胞中的肝脏PAH活性。在该项目的第一个目标中，我们将研究在小鼠肝脏中控制苯丙氨酸清除的生理阈值。我们将在选择性生长优势下将原代肝细胞移植到PAH缺陷小鼠中，并将准确测定PAH阳性肝细胞的数量和纠正高苯丙氨酸血症所需的PAH酶活性的绝对量。移植野生型肝细胞（因此表达100%正常PAH活性）或Pahenu 2突变杂合型肝细胞（PAH活性< 100%）将使我们能够确定肝脏PAH活性、PAH表达细胞绝对数量和总苯丙氨酸清除率之间的相互关系。在第二个目标中，我们将充分评估rAAV 2/8介导的肝脏定向基因转移作为小鼠PKU治疗的有效性和安全性。我们将评估转导频率、治疗性基因表达的量和持续时间、治疗对小鼠的物理和生化表型的影响、载体整合的频率以及用rAAV 2/8治疗后动物中不良反应的发生率。我们的最终目标是开发一种有效的肝脏定向基因转移方法来治疗小鼠PKU。我们建议从鼠PKU治疗中吸取的经验教训最终将适用于人PKU和其他相关IEM的治疗。

DESCRIPTION (provided by applicant): Liver-directed gene therapy is a promising approach to the treatment of human inborn errors of metabolism (IEM) due to specific liver enzyme deficiencies. The goal of this project is to develop a safe, effective liver- directed gene transfer technique for IEM by treating phenylalanine hydroxylase (PAH)-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). We have recently witnessed complete correction of serum phenylalanine levels in Pahenu2 mice treated with a novel recombinant adeno-associated virus serotype 2 vector pseudotyped with serotype 8 capsid (rAAV2/8). We plan to extend this observation to evaluate long- term stability and incidence of adverse effects following rAAV2/8 administration and to further our understanding of the physiologic factors that govern phenylalanine clearance. Our specific hypothesis is that complete correction of hyperphenylalaninemia and its attendant phenotypic features will require permanent restoration of liver PAH activity in at least 10% of Pahenu2 hepatocytes. In the first aim of the project, we will investigate the physiologic thresholds that govern phenylalanine clearance in murine liver. We will transplant primary hepatocytes under a selective growth advantage into PAH deficient mice and will accurately determine the number of PAH positive hepatocytes and absolute amount of PAH enzyme activity required to correct hyperphenylalaninemia. Transplantation of hepatocytes that are either wild type and therefore express 100% normal PAH activity or are heterozygous for the Pahenu2 mutation and have < 100% PAH activity will allow us to determine the interrelationship between liver PAH activity, the absolute number of PAH-expressing cells and total phenylalanine clearance. In the second aim, we will fully evaluate the efficacy and safety of rAAV2/8-mediated, liver-directed gene transfer as therapy for murine PKU. We will evaluate transduction frequency, the amount and duration of therapeutic gene expression, the effect of therapy upon physical and biochemical phenotypes of the mice, the frequency of vector integration, and the incidence of adverse effects in the animals following treatment with rAAV2/8. Our ultimate goal is to develop an effective liver-directed gene transfer method for the treatment of murine PKU. We propose that lessons learned from the treatment of murine PKU will ultimately be applicable to the treatment of human PKU and other allied IEM.