RECOMBINANT BACULOVIRUS GENE TRANSFER IN OCULAR TISSUES

眼组织中的重组杆状病毒基因转移

• 批准号: 6620366
• 负责人: DAVID A SAPERSTEIN
• 金额: $ 15.16万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620366
• 关键词: Baculoviridae; Sf9 cell line; biotechnology; cellular immunity; confocal scanning microscopy; density gradient ultracentrifugation; enzyme linked immunosorbent assay; flow cytometry; fluorescence microscopy; gene therapy; genetic manipulation; genetically modified animals; green fluorescent proteins; high performance liquid chromatography; humoral immunity; immunocytochemistry; laboratory mouse; molecular cloning; nucleic acid probes; retina; retina degeneration; retina disorder; retinal pigment epithelium; southern blotting; tissue /cell culture; transfection /expression vector

DESCRIPTION: (Applicant's Abstract) This investigation will study modified recombinant Baculovirus (rBV) as a gene vector in ocular tissues in vitro and in vivo. Baculovirus, an insect virus, is able to transfer large amounts of genetic material into target cells. Preliminary data suggests that the rBV is an efficient gene vector in cultured ocular and non-ocular cells as well as in certain ocular tissues in vivo, such as the retinal pigment epithelium (RPE) and corneal endotheflum. Gene transfer to ocular cells in vitro and in vivo is critical to further the understanding of ocular gene structure and function and potentially as a gene therapy vector. The specific aims of this project are 1) To engineer and evaluate rBV as a gene vector for mammalian cells in vitro for both long and short-term transgene expression; 2) To evaluate rBV vectors ability to transfer exogenous genes in the murine retina and other ocular tissues in vivo; and 3) To optimize production, purificat.ion and in vivo delivery of rBV vectors to the murine retina. To achieve these aims, commercially available rBV bacmids will be modifled for mammalian expression and be tested in vitro on RPE cells and HEK cells. Once the rBV synthesis is verified and expression is achieved in vitro, the same vector will be used to transfer marker genes to mouse retinas and other ocular tissue. A vector containing an RPE65 gene will be synthesized and' used to attempt to rescue the retinal degeneration in the RPE65 -/transgenic mouse model. Lastly, increasing the purity and titers of rBV, suppressing the immune system in mice prior to transfection and by adding additional genetic material to promote long-term expression, will optimize the vector system. If successful, the rBV system will prove to be a valuable tool for studying the structure and function of the normal and diseased retina and other ocular tissues in vitro and in vivo. With additional optimization, rBV may be an effective new gene therapy vector to treat retinal and other ocular diseases.

描述：（申请人摘要）本研究将修改 重组杆状病毒（rBV）作为基因载体在体外眼组织中的表达， in vivo.杆状病毒是一种昆虫病毒，能够将大量的 遗传物质进入靶细胞。初步数据表明，rBV是 在培养的眼和非眼细胞中以及在 体内某些眼组织，如视网膜色素上皮（RPE） 和角膜内皮。在体外和体内将基因转移到眼细胞， 这对进一步了解眼部基因结构和功能至关重要， 潜在地作为基因治疗载体。该项目的具体目标是：（1） 目的：设计rBV作为哺乳动物细胞的基因载体， 长期和短期转基因表达; 2）评估rBV载体 在鼠视网膜和其他眼内转移外源基因的能力 3）优化生产、纯化和体内试验 将rBV载体递送至鼠视网膜。为了实现这些目标， 商业上可获得的rBV杆状病毒将被修饰用于哺乳动物表达 并在体外对RPE细胞和HEK细胞进行测试。一旦rBV合成完成， 验证并在体外实现表达后，将使用相同的载体来 将标记基因转移到小鼠视网膜和其他眼部组织。的载体 包含RPE 65基因的基因将被合成并“用于试图拯救 RPE 65-/转基因小鼠模型中的视网膜变性。最后，增加 rBV的纯度和滴度，抑制小鼠的免疫系统， 转染和添加额外的遗传物质，以促进长期 表达，将优化载体系统。如果成功，rBV系统将 证明是一个有价值的工具，研究的结构和功能， 正常和患病视网膜以及其它眼组织的体外和体内研究。与 另外，rBV可能是一种有效的新基因治疗载体， 治疗视网膜和其他眼部疾病。