Gene Therapy Clinical Candidate Development for Carbamoyl Phosphate Synthetase Deficiency

项目摘要/摘要尿素循环是哺乳动物体内氨解毒的主要途径。 氨基甲酰磷酸合成酶1(CPS1)缺乏症是一种神经学破坏性的疾病，虽然晚期- 发病确实会发生，这种疾病主要是影响新生儿的一种情况。这种酶的缺陷是 以导致进行性不可逆转的高氨血症周期性发作为临床特征 神经损伤和严重的中枢神经系统损伤，特别是在出生后发育的关键时期； 这种情况与早期新生儿死亡的可能性很高。这项计划的长期目标是 是开发一种临床候选基因治疗载体，将导致氨控制和恢复尿失禁 CPS1缺乏症。基因治疗的最新进展催生了使用腺相关病毒的概念 以AAV为基础的生物技术治疗CPS1缺乏症；目前，这种单基因疾病还没有完全 除肝移植外的有效治疗，尽管通常在婴儿达到一定大小后才会发生 如果移植成功的可能性更大，但已经遭受了多次神经损伤。 除了是临床上最难治疗的尿素循环障碍之一外，一种基因的发展 治疗方法一直受到几个挑战的阻碍。首先，直到最近，还没有动物模型 失调症的症状。其次，必须表达CPS1，这是人类肝脏中含量最丰富的线粒体蛋白 在肝细胞中处于高水平。第三，4.5kb的cps1的大小限制了表达 盒式磁带大小；这可能是临床上可翻译的载体能够在高密度下产生的最大障碍 效价。这项建议寻求利用小肝细胞特异性基因调控来推进基因治疗方法。 产生紧凑的表达CPS1的AAV和混合/双载体AAV的元件作为实现这一点的方法 治疗不当的精神障碍。利普舒茨实验室最近建立了两种CPS1缺乏症和 已经公布了关于AAV方法有效性的初步数据；这项提议是为了完成 开发并推出临床候选载体。初步和公布的数据：研究小组 有：1)建立了条件Cps1缺陷基因敲除小鼠，复制了新生儿发病的CPS1缺陷；2) 单载体AAV肝细胞特异性表达CPS1导致血氨 对照；3)开发了一种双重(或分离)AAV方法在肝脏中表达CPS1；以及4)已经产生了 人源化的小鼠，CPS1缺陷的肝细胞重新填充了小鼠的肝脏；后者对于 测试这些矢量方法。具体目标1：优化肝CPS1表达和表达的AAV构建 找出晋升的最佳人选。特定目标2：检测不同嗜肝型AAV载体- 利用优化的转基因盒在Cps1缺陷人源化小鼠模型中基于CPS1的表达。 这项拟议的研究意义重大，因为预计在完成时，最终的临床候选和血清型 将被选择用于建立针对CPS1缺乏症的肝脏基因替代。

Project Summary/Abstract The urea cycle is the major pathway for detoxification of ammonia in mammals. Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a neurologically-devastating condition that, while late- onset does occur, the disorder primarily is a condition affecting neonates. The deficiency of this enzyme is characterized clinically by periodic episodes of hyperammonemia resulting in progressive irreversible neurological injury and severe CNS impairment, particularly during a period of critical postnatal development; the condition is associated with a high likelihood of early neonatal mortality. The long-term goal of this program is to develop a clinical candidate gene therapy vector that will result in ammonia control and restore ureagenesis in CPS1 deficiency. Recent advances in gene therapy have led to the concept of using adeno-associated virus (AAV)-based biotechnology to treat CPS1 deficiency; at present, this monogenic disorder has no completely effective therapy except liver transplantation, though often not occurring until the infant has reached a size where successful transplantation is more likely but multiple episodes of neurological injury have been sustained. In addition to being one of the most difficult urea cycle disorders to treat clinically, the development of a gene therapy approach has been hampered by several challenges. First, until recently, there were no animal models of the disorder. Second, CPS1, the most abundant mitochondrial protein in the human liver, must be expressed at high level in hepatocytes. And third, the size of the CPS1 cDNA at 4.5kb places limitations on the expression cassette size; this may be the greatest hindrance for a clinically translatable vector that can be produced at high titer. This proposal seeks to advance a gene therapy approach using small hepatocyte-specific gene regulatory elements to produce a compact CPS1-expressing AAV and a hybrid/dual vector AAV as approaches for this poorly treated disorder. The Lipshutz laboratory recently developed two murine models of CPS1 deficiency and has published and preliminary data on the efficacy of an AAV approach; this proposal is to complete the development and bring forth a clinical candidate vector. Preliminary and published data: The research group has: 1) developed a conditional Cps1-deficient knockout mouse, replicating neonatal onset CPS1 deficiency; 2) demonstrated hepatocyte-specific expression of CPS1 from single vector AAV that has led to plasma ammonia control; 3) developed a dual (or split) AAV approach to express CPS1 in the liver; and 4) has produced humanized mice where CPS1-deficient hepatocytes have repopulated the murine liver; the latter is essential for testing these vector approaches. Specific Aim 1: Optimize the AAV constructs for hepatic CPS1 expression and identify the optimal candidate to advance. Specific Aim 2: Test different hepatotropic serotypes of AAV vector- based CPS1 expression utilizing the optimized transgene cassette in a Cps1-deficient humanized mouse model. The proposed research is significant as it is expected that at completion a final clinical candidate and serotype will have been chosen for establishing hepatic gene replacement for CPS1 deficiency.