Gene Therapy Clinical Candidate Development for Carbamoyl Phosphate Synthetase Deficiency

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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.