Preclinical Studies of AAV Gene Therapy in MOuse Models of Urea Cycle Disorders

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PROJECT II - PRECLINICAL STUDIES OF AAV GENE THERAPY IN MOUSE MODELS OF UREA CYCLE DISORDERS AND IN NONHUMAN PRIMATES The goal of this project is to evaluate the potential of an optimized clinical candidate AAV vector (AAVcc) developed in Project I, for efficacy, duration and safety as a potential therapeutic vector in murine models of urea cycle disorders and in neonatal nonhuman primates (NHP). This project builds upon our recent success with the use of a novel AAV serotype, AAV8, in protecting ornithine transcarbamylase (OTC) deficient adult spf and spfash mice from hyperammonemia. Specific Aim 1 will evaluate the AAVcc in the treatment of adult spfash mice. We will determine how rapidly protection from an ammonia challenge is conferred after gene transfer of OTC and the duration of metabolic stability. Specific Aim 2 will evaluate the potential of gene therapy in younger recipients. Initial studies will be performed with AAVcc expressing the reporter gene GFP, administered to wild-type mice at various stages following birth, from 1 day to 4 weeks of age. Animals will be harvested at various times to measure the rate of onset of transgene expression. Additional cohorts will be followed over time to assess stability of AAV-mediated gene transfer when administered into young animals. These experiments will define important parameters to further explore the potential of the clinical candidate in treating young spfsh animals. The most stringent test for the clinical candidate will be performed in animals completely deficient in OTC through targeted gene disruption (OtcKO). This OtcKO line will be generated during the early phase of the project. If there is a delay in generating this KO model, we will use as a surrogate, the existing argininosuccinate synthase (AS) KO. All studies performed in the spfsh and OTC/AS KO animals will also involve measures of safety, including a series of clinical chemistry and hematology measurements as well as histopathology of tissues harvested and necropsied. The parameters of safety and efficacy defined in Specific Aim 2 will be further evaluated in neonatal NHP studies in Specific Aim 3. Newborn cynomolgus macaques will be injected with AAVcc expressing cynomolgus-derived OTC cDNA. Animals will be necropsied subsequent to gene transfer and evaluated for: 1) gene transfer by fluorescent in situ hybridization; 2) safety; 3) histopathology and clinical chemistry; 4) and T cells directed against the vector capsid. The final specific aim will evaluate the potential role of specific human OTC mutations that could interfere with the success of gene therapy. Existing mutations may interfere with the activity of the product of the normal transgene through a dominant negative mechanism. We will coexpress mutant and wild-type OTC in CHO cells to evaluate mutations that have these effects. Mutants that appear to show dominant negative effects in vitro will be selected for further examination in vivo using AAV gene delivery of the mutant OTC into wild-type mice. Together, these studies will allow us to determine the effectiveness of the clinical candidate vector for use in gene therapy. Lay description. In Project II, the clinical candidate vector, developed in project I, will be assessed for efficacy and safety in animal models.