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

项目II-AAV基因治疗尿素循环小鼠模型的临床前研究 非人灵长类中的疾病和 该项目的目标是评估一种优化的临床候选AAV载体(AAVcc)的潜力 项目I，作为尿素循环障碍小鼠模型的潜在治疗载体的有效性、持续时间和安全性 在新生非人灵长类(NHP)中。这个项目建立在我们最近成功的基础上，使用了一种新型的AAV AAV8血清型在保护鸟氨酸转氨酰胺酶(OTC)缺陷的成年SPF和Spfash小鼠中的作用 高氨血症。特异性目标1将评价AAVcc对成年Spfash小鼠的治疗作用。我们将决定如何 在OTC基因转移和代谢持续时间后，对氨攻击的快速保护被授予 稳定性。具体目标2将评估年轻受者进行基因治疗的潜力。初步研究将是 用表达报告基因GFP的AAVcc免疫野生型小鼠 出生，从1天到4周大。将在不同的时间采集动物以测量发病速度 转基因表达。随着时间的推移，将对其他队列进行跟踪，以评估AAV介导的基因转移的稳定性 当给幼年动物注射时。这些实验将定义重要的参数，以进一步探索 临床候选治疗幼年spfsh动物的潜力。对临床候选人最严格的测试将是 通过靶向基因破坏(OtcKO)在非处方药完全缺乏的动物身上进行。这条OtcKo系列将是 在项目的早期阶段生成。如果在生成此KO模型时出现延迟，我们将使用 代用，现有精氨酸琥珀酸合成酶(AS)KO。在SPFSH和OTC/AS KO进行的所有研究 动物还将涉及安全措施，包括一系列临床化学和血液学测量，如 以及收获和尸检的组织的组织病理学。具体中定义的安全性和有效性参数 目标2将在特定目标3的新生儿NHP研究中进一步评估。新生食蟹猴将被 注射表达食蟹猴OTC基因的AAVcc。动物将在基因之后被解剖 基因转移和评价：1)荧光原位杂交；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。最终的具体目标将评估 可能会干扰基因治疗成功的特定人类非处方药突变。现有突变可能会干扰 与正常转基因产物的活性通过显性负向机制。我们将联合快递 CHO细胞中的突变型和野生型OTC，以评估具有这些影响的突变。突变体似乎显示出 体外显性负效应将被选择用于进一步的体内检测，使用AAV基因转导突变体 OTC变成野生型小鼠。总之，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。 版面描述。在项目二中，将评估项目一中开发的临床候选载体的疗效和 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.