Optimization of HD-AD mediated gene transfer in treatment of genetic disorders

HD-AD 介导的基因转移在遗传性疾病治疗中的优化

• 批准号: 6713763
• 负责人: LAWRENCE CHAN
• 金额: $ 5万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6713763
• 关键词: Adenoviridae; Kupffer's cell; Macaca mulatta; apolipoproteins; atherosclerosis; biotechnology; disease /disorder model; dogs; gene delivery system; gene therapy; genetically modified animals; glucose 6 phosphatase; glycogen storage disease type I; helper virus; hypercholesterolemia; laboratory mouse; low density lipoprotein; low density lipoprotein receptor; nonhuman therapy evaluation; reporter genes; technology /technique development; transfection /expression vector; very low density lipoprotein

During the last funding cycle, we demonstrated that HD-Ads are promising vectors for liver-directed gene transfer. However, before we push for clinical trials, it is imperative that we generate the most efficient HD-Ads, develop protocols that will allow the use of low-dose HD-Ads to minimize toxicity, and construct regulatable HD-Ads to enhance its utility. To these ends we propose 4 Specific Aims for this Project. In Specific Aim 1 we will produce three different LDL receptor (LDLR) HD-Ads and test them in LDLR-/- mice. Although HD-Ad has a large cloning capacity of up to 37kb, the native human LDLR is still too large to be inserted into these vectors in its entirety. We will produce different LDLR constructs, including a cDNA construct and two chimeric/cDNA constructs, and test their efficacy and toxicity in LDLR-/- mice. In Specific Aim 2, we take this approach a step further and test the most efficacious of these HD-Ad-LDLR constructs in rhesus monkeys with heterozygous LDLR deficiency. We will further test a protocol developed in a Project that first depletes Kupfer cells in these monkeys before HD-Ad-LDLR treatment, an approach that may enahnce the efficacy and reduce the toxicity of HD-Ads. In Specific Aim 3, we will compare the potential lipid lowering and antiatherogenic effect of HD-Ad-LDLR, apoAI or the two vectors together. We will test whether treatment with a dual LDLR+AI transgene vector produces additive or synergistic effects compared with the individual vectors alone; use of a dual transgene vector may reduce potential toxicity by 50% as the two trasgenes will be delivered in a single vector. In Specific Aim 4, we will use HD-Ads that contain a glucose 6 phosphatase (G6Pase) gene to treat mice and dogs that have with GSD-la (G6Pase deficiency). Three different constructs, a cDNA construct, a genomic construct and a regulatable construct, will be compared. The amelioration of the signs and symptoms of GSD-la and the prevention of long-term complications of the disease may require a substantially lower dose of HD-Ads than that needed for the treatment of dyslipidemia. GSD-la may turn out to be an ideal disease target for the application of HD-Ads in the first clinical trials. Information generated from this project together with that from Projects 2-4 will pave the way toward clinical trials using HD-Ads, a highly efficient gene transfer vector for the treatment of genetic disorders.

在最后一个融资周期中，我们证明了HD-ADS是肝脏定向基因转移的有前途的向量。但是，在我们推动临床试验之前，我们必须生成最有效的HD-ADS，开发协议，以允许使用低剂量HD-ADS最大程度地减少毒性，并构建可调节的HD-ADS来增强其效用。对于这些目的，我们提出了该项目的4个具体目标。在特定目标1中，我们将产生三个不同的LDL受体（LDLR）HD-ADS，并在LDLR - / - 小鼠中测试它们。尽管HD-AD具有高达37KB的较大克隆能力，但本地人类LDLR仍然太大，无法将其全部插入这些向量。我们将生成不同的LDLR构建体，包括cDNA构建体和两个嵌合/cDNA构建体，并测试其在LDLR - / - 小鼠中的功效和毒性。在特定的目标2中，我们将此方法进一步发展，并测试具有杂合LDLR缺乏的恒河猴中这些HD-AD-LDLR构建体中最有效的。我们将进一步测试一个在HD-AD-LDLR处理之前首先在这些猴子中耗尽这些猴子细胞的项目中开发的方案，该方法可能会增强功效并降低HD-ADS的毒性。在特定的目标3中，我们将比较潜在的脂质降低和抗动脉粥样硬化 HD-AD-LDLR，APOAI或两个向量的效果一起。我们将测试是否处理 与单个矢量相比，双LDLR+AI转基因载体会产生累加或协同作用。双重转基因载体的使用可能会将潜在的毒性降低50％，因为两个tasgenes将在单个载体中递送。在特定目标4中，我们将使用含有葡萄糖6磷酸酶（G6Pase）基因的HD-ADS来治疗与GSD-LA（G6Pase缺乏症）的小鼠和狗。将比较三种不同的构造，一种cDNA构建体，一种基因组构建体和可调节构建体。 GSD-LA的体征和症状的改善以及预防疾病的长期并发症可能需要比治疗血脂异常所需的HD-ADS剂量要低得多。 GSD-LA可能是在第一次临床试验中应用HD-ADS的理想疾病靶标。该项目与项目2-4一起产生的信息将铺平使用HD-ADS的临床试验的道路，HD-ADS是一种用于治疗遗传疾病的高效基因转移载体。