Gene Therapy Clinical Candidate Development for Carbamoyl Phosphate Synthetase Deficiency

尿素循环是哺乳动物氨解毒的主要途径。 氨甲酰磷酸合成酶1（CPS 1）缺乏是一种神经破坏性疾病，虽然晚期- 一旦发病，该病症主要是影响新生儿的病症。这种酶的缺乏是 临床特征为周期性发作的高氨血症，导致进行性不可逆转 神经损伤和严重的CNS损伤，特别是在出生后发育的关键时期; 该病症与早期新生儿死亡的高可能性相关。该计划的长期目标是 是开发一种临床候选基因治疗载体，其将导致氨控制和恢复尿素生成 CPS 1缺乏症基因治疗的最新进展导致了使用腺相关病毒的概念 （AAV）为基础的生物技术来治疗CPS 1缺乏症;目前，这种单基因疾病还没有完全 有效的治疗，除了肝移植，虽然往往不发生，直到婴儿已经达到一个大小 移植成功的可能性更大，但神经损伤的多次发作持续存在。 除了是临床上最难治疗的尿素循环障碍之一， 治疗方法受到几个挑战的阻碍。首先，直到最近，还没有动物模型 精神紊乱的症状第二，必须表达人类肝脏中最丰富的线粒体蛋白CPS 1 在肝细胞中处于高水平。第三，CPS 1 cDNA的4.5kb大小限制了表达 这可能是临床上可翻译的载体的最大障碍，该载体可以在较高的温度下产生。 滴度该提案旨在推进使用小肝细胞特异性基因调控的基因治疗方法。 元件以产生表达紧凑型CPS 1的AAV和杂合/双重载体AAV作为这种方法。 治疗不当的疾病Lipshutz实验室最近开发了两种CPS 1缺陷的小鼠模型， 已经发表了关于AAV方法有效性的初步数据;该提案将完成 开发并提出临床候选载体。初步和公布的数据：研究小组 已经：1）开发了条件性CPS 1缺陷敲除小鼠，复制新生儿发病CPS 1缺陷; 2） 证明了来自单一载体AAV的CPS 1的肝细胞特异性表达，其导致血浆氨 对照; 3）开发了双重（或分裂）AAV方法以在肝脏中表达CPS 1;以及4）已经产生了 人源化小鼠，其中CPS 1缺陷型肝细胞已重新填充鼠肝脏;后者对于 测试这些向量方法。具体目的1：优化AAV构建体用于肝CPS 1表达和 确定最佳候选人晋升。具体目的2：测试AAV载体的不同嗜肝血清型- 在Cps 1缺陷型人源化小鼠模型中利用优化的转基因盒进行基于CPS 1的表达。 拟议的研究是重要的，因为预计在完成最终的临床候选人和血清型 将被选择用于建立针对CPS 1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.