Retrovectors for Intron-Enhanced Protein Expression

用于内含子增强蛋白质表达的逆向载体

• 批准号: 6991099
• 负责人: MICHAEL IMBODEN
• 金额: $ 8.97万
• 依托单位: IOGENETICS, LLC
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6991099
• 关键词: CHO cells; Hepadnaviridae; Retroviridae; biological transport; bovine leukemia virus; complementary DNA; gene delivery system; immunoglobulin genes; introns; monoclonal antibody; polymerase chain reaction; posttranslational modifications; technology /technique development; transfection /expression vector; virus RNA

DESCRIPTION (provided by applicant): Retrovectors are widely used for gene transfer in gene therapy, tissue culture and transgenic animals for research or commercial applications.. A long recognized limitation of current retrovectors is that introns are spliced out as retrovector particles are made in packaging cells. Introns may regulate the expression of the genes and convey tissue specificity. The presence of introns boosts expression as a consequence of active splicing. This proposal tests the design of retrovectors which preserve introns by utilizing elements of viral RNA export mechanisms naturally occurring in the complex retroviruses. A packaging cell expressing an RNA export enabling protein (Rex) will be used to produce vectors whose constructs contain the corresponding Rex responsive element (RxRE). In combination this should allow the making of retroviral vectors which contain introns. These vectors subsequently allow the transfer of introncontaining constructs into the final host cell. Relevance: Previously considered "junk DMA", introns are now understood to exert significant effects on the adjacent coding regions of genes. By enabling the transfer of intron-containing genes we anticipate (a) providing a means to make gene therapy vectors with greater predictability of expression and tissue specificity; (b) providing a boost to protein expression in cell culture bioreactor systems and transgenic animals and (c) providing a useful research tool to enhance understanding of RNA biology.

描述（由申请人提供）： 逆转录载体广泛用于基因治疗、组织培养和转基因动物中的基因转移，用于研究或商业应用。目前逆转录载体的一个长期公认的局限性是，当在包装细胞中制备逆转录载体颗粒时，内含子被剪接掉。内含子可以调节基因的表达并传递组织特异性。内含子的存在作为主动剪接的结果促进表达。该提案测试了逆转录病毒载体的设计，该逆转录病毒载体通过利用复杂逆转录病毒中天然存在的病毒RNA输出机制的元件来保留内含子。表达RNA输出使能蛋白（雷克斯）的包装细胞将用于产生载体，所述载体的构建体含有相应的雷克斯应答元件（RxRE）。结合起来，这应该允许制备含有内含子的逆转录病毒载体。这些载体随后允许将含内含子的构建体转移到最终的宿主细胞中。 相关性：以前被认为是“垃圾DMA”，现在人们理解内含子对基因的相邻编码区产生重大影响。通过使含内含子基因的转移成为可能，我们预期（a）提供一种制备具有更高的表达可预测性和组织特异性的基因治疗载体的方法;（B）在细胞培养生物反应器系统和转基因动物中促进蛋白质表达，以及（c）提供一种有用的研究工具来增强对RNA生物学的理解。