SAFE LENTIVIRAL VECTORS FOR GENE TRANSFER TO NON-DIVIDING CELLS--HIV INFECTION

用于基因转移至非分裂细胞的安全慢病毒载体——HIV感染

• 批准号: 6100267
• 负责人: GARRY P NOLAN
• 金额: $ 22.01万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6100267
• 关键词: 3T3 cells; AIDS therapy; HIV infections; Lentivirus; T lymphocyte; feline immunodeficiency virus; gene expression; gene therapy; genetic manipulation; genetic transduction; hematopoietic stem cells; human immunodeficiency virus 1; molecular cloning; nucleic acid repetitive sequence; plasmids; simian immunodeficiency virus; transfection; transfection /expression vector; virus envelope; virus genetics; virus protein

This proposal is designed to create lentiviral based packaging systems for delivery of gene therapy constructs to HIV-1 infected individuals using Feline Immune Deficiency Virus (FIV) and Simian Immunodeficiency Virus (SIV) based vectors systems. Many current human gene therapy protocols utilize the retrovirus. Moloney murine leukemia virus (MMLV) because of its properties of long-term integration, low copy number, adequate expression and lack of an immune response against the vector. While these systems have been show to work well in mice and other small animals, the transduction efficiency is reduced in primates and humans and long-term expression is variable. The reasons for the decreased transduction efficiencies in humans are not clear, but are thought to be partly due to the inability of these retroviral systems to infect non-dividing or quiescent cells, such as hematopoietic stem cells and hepatocytes. To achieve, others have developed HIV-1 based lentiviral packaging systems (ref) to infect non-dividing cells. Lentiviruses, as a subclass of retroviruses, have the ability to infect non-dividing cells that are metabolically active. Using molecular clones of FIV and SIV, we will dissect their genomes into multiple non-overlapping elements that provide the necessary trans-proteins for synthesis of viral cores and envelope proteins. Other cis elements required for vector delivery will be engineered into safe, high efficiency vector systems for infection of non- dividing human cells of the immune system. Multiple individual vector constructs will be created for testing. We will work closely with Dr. Nabel's group in Project 1 to accommodate vector designs that express RevM10, ribozymes, and single chain variable regions with appropriate marker genes. Cell tested will be model systems, PBL-derived macrophages and hematopoietic stem cells, and T cells. As vector designs continue and progress is made in the first and second specific aims, we will work with Dr. Kohn's group for delivery go genes to be isolated stem cells. We will work closely with Dr. Daniel Littman in Project 4, who has experience with infection of T cells using MMULV, as well as extensive experience in T cell gene expression, to compare directly the abilities of our vector systems to expression at various stages of in vitro and in vivo development after transfer to SCID/Hu models. Several internal promoter elements will be tested, and these will also be tested in the SCID hum model for efficacy and cell subtype expression of inserted genes.

该建议旨在为基于慢病毒的包装系统 使用使用基因治疗构建体向HIV-1感染的个体传递 猫免疫缺陷病毒（FIV）和猿猴免疫缺陷病毒 （SIV）基于矢量系统。许多当前的人类基因治疗方案 利用逆转录病毒。 Moloney鼠白血病病毒（MMLV） 它的长期整合，低拷贝数，足够的特性 对载体的表达和缺乏免疫反应。而这些 系统已显示在小鼠和其他小动物中都可以很好地工作 灵长类动物和人类的转导效率降低了 表达是可变的。转导减少的原因 人类的效率尚不清楚，但被认为部分是由于 这些逆转录病毒系统无法感染非分裂或 静态细胞，例如造血干细胞和肝细胞。到 实现，其他人开发了基于HIV-1的慢病毒包装系统 （参考）感染非分散细胞。慢病毒，作为 逆转录病毒，具有感染非分裂细胞的能力 代谢活跃。使用FIV和SIV的分子克隆，我们将 将其基因组剖析成多个提供的非重叠元素 用于合成病毒核和包膜的必要的转蛋白 蛋白质。向量传递所需的其他顺式元素将是 设计为安全，高效率的矢量系统，以感染非 - 分裂免疫系统的人类细胞。多个单独的向量 将创建用于测试的构造。我们将与博士密切合作。 纳贝尔（Nabel）在项目1中的小组可容纳表达的向量设计 Revm10，核酶和单链变量区域适当 标记基因。经细胞测试的将是模型系统，PBL衍生的巨噬细胞 和造血干细胞和T细胞。随着向量设计的继续， 进度是在第一个和第二个特定目标中取得的进展，我们将与 Kohn博士的分娩GO基因是分离的干细胞。我们将 在项目4中与丹尼尔·利特曼（Daniel Littman）博士紧密合作，他有经验 使用MMULV感染T细胞，以及T的丰富经验 细胞基因表达，直接比较我们的向量的能力 在体外和体内各个阶段表达的系统 转移到SCID/HU模型后的开发。几个内部启动子 元素将进行测试，这些元素也将在SCID嗡嗡声中进行测试 插入基因的功效和细胞亚型表达的模型。