Pathophysiology and Gene Replacement Strategies for Arginase Deficiency

描述（由申请人提供）：尿素循环是哺乳动物氨解毒的主要途径。精氨酸酶I缺乏被认为是最不常见的尿素循环障碍，并导致高胆固醇血症。在人类中，这种酶缺乏的临床特征是进行性精神损害、痉挛、生长迟缓和高氨血症的周期性发作。这项建议有两个方面：1）开发基于基因的病毒载体对精氨酸酶缺乏症的矫正，并从行为和生化方面仔细检查矫正后的动物; 2）评估精氨酸和相关代谢物（不仅仅是氨）升高对发育中的大脑和精氨酸酶缺乏症中智力迟钝的发展的作用。初步数据：我们的研究小组有：1）构建并表征了转肽酶I敲除小鼠; 2）证实了至少短期的转肽酶I敲除小鼠的表达。（如果不是更长的话）用重组辅助依赖性腺病毒载体和腺相关病毒载体进行校正和拯救; 3）证明敲除小鼠神经元具有更高百分比的细胞处于细胞周期的S期; 4）显示来自敲除小鼠的分化的神经元具有更成熟的形态;和5）显示敲除的神经元显示与保护免受氧化损伤相关的基因的诱导。在目标1中，重组病毒载体将用于拯救小鼠免于致死，并更好地理解新生儿基因治疗所面临的挑战，这些挑战涉及游离型与整合型载体的快速细胞增殖和潜在损失。在目标2中，将在体外沿着GABA和谷氨酰胺合成/释放以及广泛的微阵列分析来检查高胆固醇血症对神经系统的影响。在目标3中，将在体内沿着检查高胆固醇血症对神经系统的影响，以及GABA和谷氨酰胺的合成/释放、胍基化合物的测定，以及一氧化氮及其代谢物可能对神经系统损伤的作用。成功完成拟议的研究将提供一个分子的理解损伤的机制，大脑中的辅酶I缺乏症和相关疾病。此外，预计通过这些研究将开发出一种针对β-淀粉酶缺乏症的基因替代策略，并且在治疗组织正在快速增殖的新生儿中获得的信息将适用于其他先天性代谢缺陷。 公共卫生相关性：与公共卫生的相关性本项目将进一步阐明酶在健康中的作用以及缺乏酶时疾病和神经损伤的机制。此外，它将致力于开发这种疾病的基因替代策略。新生儿基因治疗具有与快速细胞增殖相关的特殊挑战，本提案将在此背景下研究基因治疗，并应用这些策略如何治愈困扰新生儿的其他类似疾病。

DESCRIPTION (provided by applicant): The urea cycle is the major pathway for detoxification of ammonia in mammals. Arginase I deficiency is thought to be the least common of the urea cycle disorders and results in hyperargininemia. In humans, deficiency of this enzyme is characterized clinically by progressive mental impairment, spasticity, growth retardation, and periodic episodes of hyperammonemia. This proposal is two-fold: 1) to develop gene-based correction of arginase deficiency with viral vectors and to closely examine corrected animals behaviorally and biochemically; and 2) to evaluate the role elevated arginine and related metabolites (beyond just ammonia) have on the developing brain and development of mental retardation in arginase deficiency. Preliminary data: Our research group has: 1) constructed and characterized the arginase I knockout mouse; 2) demonstrated at least short-term (if not longer) correction and rescue with recombinant helper-dependent adenoviral vectors and adeno-associated viral vectors; 3) demonstrated that knockout mouse neurons have a higher percentage of cells in the S phase of the cell cycle; 4) shown that differentiated neurons from knockout mice have a more mature morphology; and 5) shown that knockout neurons demonstrate induction of genes related to protection from oxidative damage. In Aim 1, recombinant viral vectors will be used to rescue mice from lethality and to obtain a better understanding of the challenges facing neonatal gene therapy that involve rapid cellular proliferation and potential loss with episomal vs. integrated vectors. In Aim 2, the effect of hyperargininemia on the nervous system will be examined in vitro along with GABA and glutamine synthesis/release, and extensive microarray analysis. In Aim 3, the effect of hyperargininemia on the nervous system will be examined in vivo along with GABA and glutamine synthesis/release, guanidino compound determination, and the role that nitric oxide and its metabolites may have on nervous system injury. Successful completion of the proposed studies will provide a molecular understanding of the mechanism of injury to the brain in arginase I deficiency and related disorders. In addition, it is expected that a gene replacement strategy will have been developed for arginase deficiency through these studies and that the information obtained in treating neonates that have developing tissues undergoing rapid proliferation will be applicable to other inborn errors of metabolism. PUBLIC HEALTH RELEVANCE: Relevance to Public Health This project will further elucidate the role arginase plays in health and the mechanisms of disease and neurological insult in its deficiency. In addition, it will be directed at developing a gene replacement strategy for this disorder. Neonatal gene therapy has particular challenges related to rapid cellular proliferation and this proposal will examine gene therapy in that context with application to how such strategies can cure other similar diseases that afflict newborns.