Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation

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• 作者：Hemant Dhamne ; Ajit G. Ch ; e ; Robin Mukhopadhyaya
• 关键词：Lentivirus vector ; EPO ; Elastase II ; Inverse PCR ; Metabolic engineering ; shRNA delivery
• 刊名：Plasmid
• 出版年：January, 2014
• 年：2014
• 卷：71
• 期：Complete
• 页码：1-7
• 全文大小：611 K

文摘

Lentiviral vector (LV) mediated gene transfer holds great promise to develop stable cell lines for sustained transgene expression providing a valuable alternative to the conventional plasmid transfection based recombinant protein production methods. We report here making a third generation HIV-2 derived LV containing erythropoietin (EPO) gene expression cassette to generate a stable HEK293 cell line secreting EPO constitutively. A high producer cell clone was obtained by limiting dilution and was adapted to serum free medium. The suspension adapted cell clone stably produced milligram per liter quantities of EPO. Subsequent host metabolic engineering using lentiviral RNAi targeted to block an endogenous candidate protease elastase, identified through an in silico approach, resulted in appreciable augmentation of EPO expression above the original level. This study of LV based improved glycoprotein expression with host cell metabolic engineering for stable production of protein therapeutics thus exemplifies the versatility of LV and is of significant future biopharmaceutical importance.