Pathophysiology and Gene Replacement Strategies for Arginase Deficiency

描述(申请人提供)：尿素循环是哺乳动物解氨的主要途径。精氨酸酶I缺乏症被认为是尿素循环障碍中最不常见的一种，会导致高精氨酸血症。在人类中，这种酶缺乏的临床特征是进行性智力障碍、痉挛、生长迟缓和周期性的高氨血症。这项建议包括两个方面：1)开发基于基因的病毒载体纠正精氨酸酶缺乏，并密切检查纠正后的动物的行为和生化；2)评估精氨酸和相关代谢物(不仅仅是氨)在精氨酸酶缺乏时大脑发育和智力低下发展中的作用。初步资料：我们的研究小组已经：1)构建并鉴定了精氨酸酶I基因剔除小鼠；2)证明了基因剔除小鼠至少在短期(如果不是更长时间)可以纠正和挽救依赖辅助子的腺病毒载体和腺相关病毒载体；3)证明了基因剔除小鼠神经元中处于S期的细胞比例较高；4)显示来自基因剔除小鼠的分化神经元具有更成熟的形态；5)基因敲除小鼠神经元表现出了抗氧化损伤相关基因的诱导。在目标1中，重组病毒载体将被用于将小鼠从死亡中拯救出来，并更好地了解新生儿基因治疗面临的挑战，这些挑战涉及到细胞的快速增殖和与整合载体相比的潜在损失。在目标2中，将在体外检测高精氨酸血症对神经系统的影响，以及GABA和谷氨酰胺的合成/释放，以及广泛的微阵列分析。在目标3中，将在体内检测高精氨酸血症对神经系统的影响，以及GABA和谷氨酰胺的合成/释放，胍类化合物的测定，以及一氧化氮及其代谢产物在神经系统损伤中的作用。拟议研究的成功完成将提供对精氨酸酶I缺乏和相关疾病对大脑损伤机制的分子理解。此外，预计通过这些研究将开发出一种针对精氨酸酶缺乏症的基因替换策略，并且在治疗具有快速增殖的发育组织的新生儿时获得的信息将适用于其他先天的新陈代谢错误。 这个项目将进一步阐明精氨酸酶在健康中所起的作用，以及疾病和神经性侮辱在其缺陷中的机制。此外，它还将针对这种疾病开发一种基因替换策略。新生儿基因治疗具有与快速细胞增殖相关的特殊挑战，本提案将在这一背景下审查基因治疗，并应用于此类策略如何治愈其他困扰新生儿的类似疾病。

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.