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

在上一个资助周期中，我们证明了HD-Ads是一种很有前途的肝脏导向基因转移载体。然而，在我们推动临床试验之前，我们必须产生最有效的HD-ADS，开发允许使用低剂量HD-ADS的方案以将毒性降至最低，并构建可调节的HD-ADS以增强其实用性。为此，我们提出了本项目的4个具体目标。在具体目标1中，我们将生产三种不同的低密度脂蛋白受体(LDLR)HD-ADS，并在LDLR-/-小鼠身上进行测试。虽然HD-Ad具有高达37kb的大克隆容量，但原生人LDLR仍然太大，无法完全插入到这些载体中。我们将生产不同的LDLR构建物，包括一个cdna构建体和两个嵌合/cdna构建体，并在LDLR-/-小鼠身上测试它们的有效性和毒性。在特定的目标2中，我们将这一方法更进一步，并在LDLR杂合性缺陷的恒河猴身上测试了这些HD-Ad-LDLR结构中最有效的。我们将进一步测试在一个项目中开发的方案，该方案在HD-Ad-LDLR治疗之前首先在这些猴子身上耗尽Kupfer细胞，这一方法可能会增强HD-Ads的疗效并降低其毒性。在特定的目标3，我们将比较潜在的降脂和抗动脉粥样硬化。 HD-Ad-LDLR、ApoAI或两种载体共同作用的效果。我们将测试是否用一种 与单独的载体相比，双LDLR+AI转基因载体可以产生相加或协同效应；使用双转基因载体可以将潜在的毒性降低50%，因为两个转基因将在一个载体中传递。在特定的目标4中，我们将使用含有葡萄糖6磷酸酶(G6Pase)基因的HD-ADS来治疗患有GSD-1a(G6Pase缺乏症)的小鼠和狗。将比较三种不同的结构，一种cDNA结构，一种基因组结构和一种可调节的结构。要改善GSD-1a的体征和症状，并预防该病的长期并发症，可能需要比治疗血脂异常所需的剂量低得多的HD-ADS。在第一次临床试验中，GSD-1a可能成为HD-ADS应用的理想疾病靶点。该项目产生的信息与项目2-4产生的信息将为使用HD-ADS进行临床试验铺平道路，HD-ADS是一种用于治疗遗传疾病的高效基因转移载体。

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.