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