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

项目II -AAV基因治疗在小鼠尿素循环模型中的临床前研究 疾病和非人类灵长类动物 本项目的目标是评估在美国开发的优化的临床候选AAV载体（AAVcc）的潜力。 项目I，作为尿素循环障碍小鼠模型中的潜在治疗载体的有效性、持续时间和安全性， 新生非人灵长类动物（NHP）。该项目建立在我们最近成功使用一种新的AAV 血清型AAV 8在保护鸟氨酸转氨甲酰酶（OTC）缺陷型成年spf和spfash小鼠免受 高氨血症具体目标1将评估AAVcc在成年spfash小鼠治疗中的作用。我们将决定如何 在OTC的基因转移和代谢的持续时间后， 稳定具体目标2将评估基因治疗在年轻受者中的潜力。初步研究将 用表达报告基因GFP的AAVcc进行，在施用后的各个阶段施用给野生型小鼠， 出生，从1天到4周大。将在不同时间收获动物，以测量 转基因表达将随时间推移随访其他队列，以评估AAV介导的基因转移的稳定性。 当给药到年轻的动物。这些实验将确定重要的参数，以进一步探索 临床候选物在治疗幼年SPFSH动物中的潜力。对临床候选人最严格的测试将 通过靶向基因破坏（OtcKO）在完全缺乏OTC的动物中进行。这条OtcKO线将是 在项目的早期阶段。如果在生成此KO模型时有延迟，我们将使用 替代物，现有的氨基琥珀酸合成酶（AS）KO。在spfsh和OTC/AS KO中进行的所有研究 动物还将涉及安全性的测量，包括一系列临床化学和血液学测量， 以及采集和尸检的组织的组织病理学。安全性和有效性参数定义见特定 将在具体目标3的新生儿NHP研究中进一步评价目标2。新生的食蟹猴 注射表达食蟹猴衍生的OTC cDNA的AAVcc。在基因治疗后对动物进行尸检。 转移并评价：1）通过荧光原位杂交进行基因转移; 2）安全性; 3）组织病理学和临床 化学; 4）和针对载体衣壳的T细胞。最后的具体目标将评估以下方面的潜在作用： 可能干扰基因治疗成功的特定人类OTC突变。现有的突变可能会干扰 通过显性负性机制与正常转基因产物的活性相关。我们将共同表达 突变体和野生型OTC，以评价具有这些效应的突变。突变体显示 将选择体外显性负效应用于使用突变体的AAV基因递送的体内进一步检查 OTC至野生型小鼠。这些研究将使我们能够确定临床候选药物的有效性 用于基因治疗的载体。 Lay描述。在项目II中，将评估项目I中开发的临床候选载体的有效性， 动物模型的安全性。

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.