RECOMBINANT BACULOVIRUS GENE TRANSFER IN OCULAR TISSUES

描述：(申请人摘要)本次调查将研究修改 重组杆状病毒(RBV)作为基因载体在眼组织中的应用 在活体内。杆状病毒是一种昆虫病毒，能够传播大量的 遗传物质进入靶细胞。初步数据显示，RBV是 一种高效的眼和非眼细胞培养的基因载体 体内的某些眼组织，如视网膜色素上皮(RPE) 和角膜内花。在体内外将基因转移到眼细胞中 对进一步了解眼睛的基因结构和功能以及 有可能作为基因治疗载体。本项目的具体目标为1) 重组RBV作为哺乳动物细胞基因载体的体外实验研究 长期和短期转基因表达；2)评价RBV载体 在小鼠视网膜和其他眼部转移外源基因的能力 体内组织；3)优化生产、纯化、离子和体内 将RBV载体运送到小鼠视网膜。为了实现这些目标， 商业上可用的RBV杆状病毒将被修饰以在哺乳动物中表达 并在RPE细胞和HEK细胞上进行体外实验。一旦RBV合成 验证并在体外实现了表达，相同的载体将用于 将标记基因转移到小鼠视网膜和其他眼睛组织。一种载体 含有RPE65基因的基因将被合成并用于试图拯救 RPE65-/转基因小鼠模型中的视网膜变性。最后，增加 RBV的纯度和效价，在感染前抑制小鼠的免疫系统 并通过添加额外的遗传物质来促进长期 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来研究的结构和功能 在体外和体内，正常和病变的视网膜和其他眼组织。使用 进一步优化，RBV可能成为一种有效的新的基因治疗载体 治疗视网膜和其他眼部疾病。

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.