LENTIVIRAL VECTOR BASED GENE THERAPY FOR LIVER DISEASES

简介(摘自申请者摘要)：本研究的总体目标是 为了验证我们的假设，即慢病毒载体可以作为一种有效的 以及向肝组织输送治疗性基因的安全平台。这个 HIV-1和其他慢病毒转导未分裂细胞的能力提示 基于HIV-L的基因递送系统的开发。新型慢病毒 载体被证明在体内有效地转导各种组织(脑、肝、 肌肉、视网膜和造血干细胞)，没有任何可检测到的病理。 最近，我们发现一次腹腔注射血友病小鼠 慢病毒载体导致治疗水平的长期表达 犬凝血因子IX。经处理的小鼠的aPTT值相当于 从杂合的产仔仔中获得的。此外，我们的初步调查 结果表明，慢病毒中的顺式调控序列下调 转基因表达。这些研究是最令人鼓舞的，但我们认为 在载体生产、转基因表达和 监管，以及更好地描述导致 我们需要开发抑制性抗体才能考虑 慢病毒系统作为一种安全有效的肝脏病毒载体的应用 基因疗法。 为了促进安全的媒介生产，我们建议生成一个新的第三个 代包装细胞株，其中将不含TAT和全部艾滋病毒--L 辅助性蛋白质。作为安全的额外衡量标准，我们将分开 新的包装系统被包装成四个稳定整合的质粒(载体， 信封、包装和版本)。 为了提高慢病毒载体盒的转基因表达，我们将 尝试从慢病毒载体中识别和删除抑制序列 基因组。 为了改善对转基因表达的调控，我们将产生一种改进的新的 具有最小基本可诱导启动子的可诱导慢病毒载体 活动。在血友病小鼠和狗身上测试拟议的改进 动物模型将使我们能够表征潜在的免疫反应 新合成的第IX因子。我们相信，有能力保持 这些动物模型中凝血因子IX的治疗水平将决定 慢病毒载体基因治疗血友病B的可行性研究 以及其他肝脏代谢性疾病。

DESCRIPTION (Copied from Applicant Abstract): The overall goal of this study is to validate our hypothesis that lentivirus vectors can serve as an efficient and safe platform for therapeutic gene delivery to the liver tissue. The ability of HIV-1 and other lentiviruses to transduce non-dividing cells prompt the development of an HIV-l based gene delivery system. The novel lentivirus vectors proved efficient at transducing various tissues in vivo (brain, liver, muscle, retina, and hematopoietic stem cells) without any detectable pathology. Recently, we showed that a single intraperitoneal injection of hemophilic mice with lentivirus vectors resulted in long term expression of therapeutic levels of canine factor IX. The treated mice demonstrated aPTT values equivalent to those obtained from heterozygous littermates. In addition our preliminary results indicated that cis-regulatory sequences in the lentivirus down-regulate transgene expression. These studies are most encouraging, however we believe that further improvements in: vector production, trsansgene expression, and regulation, and better characterization of the mechanism responsible for the development of inhibitory antibodies are required before we can consider the use of the lentiviral system as a safe and efficient viral vector for liver gene therapy. To facilitate safe vector production we propose to generate a novel third generation packaging cell line, which will be devoid of the Tat and all HIV-l accessory proteins. As an additional measurement of safety, we will separate the new packaging system into four stably integrated plasmids (vector, envelope, packaging, and rev). To improve transgene expression from the lentivirus vector cassette we will attempt to identify and delete inhibitory sequences from the lentivirus vector genome. To improve regulation of transgene expression we will generate an improved new inducible lentivirus vector which will exhibit minimal basal inducible promoter activity. Testing the proposed improvements in hemophilic mouse and canine animal models will allow us to characterize potential immune response against the newly synthesized factor IX. We believe that the ability to maintain therapeutic levels of factor IX in these animal models will determine the feasibility of using lentivirus vector based gene therapy to cure hemophilia B and other hepatic metabolic diseases.