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

AAV 基因治疗在尿素循环障碍小鼠模型中的临床前研究

• 批准号: 7802297
• 负责人: James M Wilson
• 金额: $ 14万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7802297
• 关键词: Acute; Adult; Age; Ammonia; Animal Model; Animals; Arginine; Autopsy; Biochemical; Biology; Birth; Blood specimen; Capsid; Cell membrane; Cells; Chinese Hamster Ovary Cell; Clinical; Clinical Chemistry; Clinical Pathology; Clinical effectiveness; Complementary DNA; Disease; Dominant-Negative Mutation; Engineering; Enzymes; Fluorescent in Situ Hybridization; Follow-Up Studies; Gene Delivery; Gene Expression; Gene Targeting; Gene Transfer; Genome; Glutamine; Goals; Growth; Harvest; Hematology; Histocytochemistry; Histopathology; Human; Hyperammonemia; Immunology; In Situ; In Vitro; Infusion procedures; Life; Liver; Macaca; Measurement; Measures; Mediating; Metabolic; Modeling; Molecular; Mus; Mutation; Neonatal; Newborn Infant; Ornithine Carbamoyltransferase; Ornithine carbamoyltransferase deficiency; Patients; Phase; Phenotype; Plasma; Principal Investigator; Reporter Genes; Role; Safety; Sampling; Series; Serotyping; Speed; Staging; T-Lymphocyte; Testing; Therapeutic; Time; Tissue Harvesting; Transgenes; Viral Genome; Wild Type Mouse; adeno-associated viral vector; argininosuccinate synthase; clinically significant; cohort; enzyme activity; gene therapy; in vivo; juvenile animal; mouse model; mutant; nonhuman primate; novel; orotate; peripheral blood; preclinical study; pregnant; prevent; programs; reconstitution; research study; response; success; transgene expression; urea cycle; urinary; vector

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.

项目II-尿素周期小鼠模型中AAV基因治疗的临床前研究 疾病和非人类灵长类动物 该项目的目的是评估优化临床候选AAV矢量（AAVCC）的潜力 项目I，作为尿素周期疾病的鼠模型中的潜在治疗载体的功效，持续时间和安全性 在新生儿非人类灵长类动物（NHP）中。这个项目建立在我们最近使用新颖AAV的成功基础上 血清型AAV8在保护鸟氨酸经钙化酶（OTC）中缺乏成年SPF和SPFASH小鼠免受 高症血症。特定的目标1将评估AAVCC的成年SPFASH小鼠的治疗。我们将确定如何 在OTC基因转移和代谢持续时间后，迅速保护免受氨挑战的保护 稳定。具体目标2将评估年轻接受者基因治疗的潜力。最初的研究将是 用AAVCC表达记者基因GFP进行 出生，从1天到4周龄。将在不同时间收获动物，以测量 转基因表达。随着时间的推移，将遵循其他队列，以评估AAV介导的基因转移的稳定性 当施用成幼小的动物时。这些实验将定义重要参数，以进一步探索 临床候选者在治疗年轻的SPFSH动物方面的潜力。对临床候选者最严格的测试将 通过靶向基因破坏（OTCKO）在完全缺陷OTC的动物中进行。这条OTCKO系列将是 在项目的早期阶段生成。如果生成此KO模型有延迟，我们将用作 替代物，现有的精氨酸合酶（AS）KO。所有在SPFSH和OTC/AS KO中进行的研究 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 以及收获和死灵的组织的组织病理学。特定于特定的安全性和功效的参数 AIM 2将在新生儿NHP研究中进一步评估特定目标3。新生儿cynomolgus猕猴将是 注射AAVCC表达cernomolgus衍生的OTC cDNA。基因之后将被尸体死亡 转移并评估：1）基因通过荧光原位杂交转移； 2）安全； 3）组织病理学和临床 化学; 4）和针对载体capsid的T细胞。最终的具体目的将评估 可能干扰基因治疗成功的特定人类OTC突变。现有突变可能会干扰 通过正常转基因产物的活性通过主要的负面机制。我们将共同表达 CHO细胞中的突变体和野生型OTC评估具有这些作用的突变。似乎显示的突变体 在体外的主要负面影响将选择使用突变体的AAV基因递送在体内进一步检查 OTC进入野生型小鼠。这些研究将使我们能够确定临床候选者的有效性 用于基因治疗的载体。 外行描述。在第二个项目中，将在项目I中开发的临床候选媒介进行疗效和 动物模型的安全。