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.

递送编码简短干扰RNA（siRNA）靶向人免疫缺陷病毒（HIV）的基因可能代表一种干扰病毒感染的有效方法。人造血干细胞（HSC）代表了基因递送的理想目标，因为它们可以轻松地通过完善的程序隔离，并且可以培养以允许离体基因递送。为了成功地通过基因治疗方法治疗HIV感染， 有效的siRNA表达和高百分比转导是两个最关键的因素。此外，产生稳定的包装细胞系以产生临床上可接受的HIV载体种群不仅促进临床研究，而且还促进了大型动物的临床前实验。在此提案中，我们将评估各种向量设计和顺式元素，以优化HSC中的siRNA表达。我们将研究HIV载体的设计，以进行有效的siRNA表达。我们将评估在人T细胞中的抗HIV活性以及培养中与CD34+细胞区分开的抗HIV活性上的不同的促启动子和shRNA基因剂量。我们将评估 不同的顺式作用DNA元素稳定siRNA表达的能力。为了增加转导细胞的分数，我们将评估表达甲基鸟嘌呤甲基转移酶（MGMT）或II型Inosine-5'单磷酸脱氢酶（IMPDH2）的潜力，以富集和扩展体内传染的HSC。含有可选标记的HIV载体将用于传递HSC，然后将细胞注射到SCID-HU小鼠模型中。在体内选择时，将分析分化T细胞的HIV-1感染的植入，扩张和抗性。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，用于诱导表达HIV GAG/POL和囊泡气孔病毒的G蛋白。细胞系将测试其在诱导型条件下产生高点矢量库存的能力。