Improved Efficiency of HIV vectors

提高 HIV 载体的效率

• 批准号: 6850617
• 负责人: Jiing-Kuan YEE
• 金额: $ 28.02万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6850617
• 关键词: AIDS therapy; DNA directed DNA polymerase; HIV infections; Lentivirus; RNA interference; SCID mouse; T lymphocyte; Vesiculovirus; antiAIDS agent; biotechnology; cell differentiation; flow cytometry; gene dosage; gene expression; gene therapy; genetic regulatory element; hematopoietic stem cells; human genetic material tag; human immunodeficiency virus; immunotherapy; methylguanine DNA methyltransferase; polymerase chain reaction; small interfering RNA; therapy design /development; tissue /cell culture; transfection /expression vector

Delivery of genes encoding short interfering RNA (siRNA) to target human immunodeficiency virus (HIV) may represent an effective approach to interfere with virus infection. Human hematopoietic stem cells (HSCs) represent the ideal target for gene delivery since they can readily be isolated with well-established procedures and can be cultured to allow ex vivo gene delivery. For successful treatment of HIV infection with the gene therapy approach, efficient siRNA expression and high percentage transduction are the two most critical factors. In addition, generating stable packaging cell lines that produce clinically acceptable HIV vector stocks would facilitate not only clinical studies but also preclinical experiments in large animals. In this proposal, we will evaluate various vector design and cis elements to optimize siRNA expression in HSCs. We will study the design of HIV vectors for efficient siRNA expression. We will evaluate different pol Ill promoters and shRNA gene dosage on the anti-HIV activity in human T cells and in myelomonocytic cells differentiated from CD34+ cells in culture. We will evaluate different cis-acting DNA elements for their ability to stabilize siRNA expression. To increase the fraction of the transduced cells, we will evaluate the potential of expressing methylguanine methyltransferase (MGMT) or type II inosine-5' monophosphate dehydrogenase (IMPDH2) to enrich and expand the transduced HSCs in vivo. HIV vectors containing the selectable marker will be used to transduce HSCs, followed by cell injection into the SCID-hu mouse model. Upon in vivo drug selection, the engraftment, expansion and resistance to HIV-1 infection of the differentiated T cells will be analyzed. To establish stable packaging cell lines for large-scale vector production, we will test a Cre-loxP-based system for inducible expression for HIV Gag/Pol and the G protein of vesicular stomatitis virus. The cell lines will be tested for their ability to generate high-titer vector stocks under the inducible condition.

将编码短干扰RNA (siRNA) 的基因递送至人类免疫缺陷病毒(HIV) 可能是干扰病毒感染的有效方法。人类造血干细胞 (HSC) 是基因传递的理想靶标，因为它们可以通过完善的程序轻松分离，并且可以培养以允许离体基因传递。为了通过基因治疗方法成功治疗艾滋病毒感染， 高效的 siRNA 表达和高百分比转导是两个最关键的因素。此外，产生可产生临床可接受的HIV载体储备的稳定包装细胞系不仅有利于临床研究，而且有利于大型动物的临床前实验。在本提案中，我们将评估各种载体设计和顺式元件，以优化 HSC 中的 siRNA 表达。我们将研究有效表达 siRNA 的 HIV 载体的设计。我们将评估不同的pol III启动子和shRNA基因剂量对人T细胞和从培养物中的CD34+细胞分化的骨髓单核细胞中的抗HIV活性的影响。我们将评估 不同的顺式作用 DNA 元件具有稳定 siRNA 表达的能力。为了增加转导细胞的比例，我们将评估表达甲基鸟嘌呤甲基转移酶 (MGMT) 或 II 型肌苷-5'单磷酸脱氢酶 (IMPDH2) 在体内富集和扩增转导 HSC 的潜力。含有选择性标记的 HIV 载体将用于转导 HSC，然后将细胞注射到 SCID-hu 小鼠模型中。在体内药物选择后，将分析分化 T 细胞的植入、扩增和对 HIV-1 感染的抵抗力。为了建立用于大规模载体生产的稳定包装细胞系，我们将测试基于 Cre-loxP 的系统，用于 HIV Gag/Pol 和水泡性口炎病毒 G 蛋白的诱导表达。将测试细胞系在诱导条件下产生高滴度载体原液的能力。