title: Lentiviral vector platform for improved erythropoietin expression concomitant with shRNA mediated host cell elastase down regulation.
abstract: 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.
authors: Virology Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.
authors: Hemant
authors: H
authors: Dhamne
authors: Virology Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India.
authors: Ajit G
authors: AG
authors: Chande
authors: Virology Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, India. Electronic address: rmukhopadhyaya@actrec.gov.in.
authors: Robin
authors: R
authors: Mukhopadhyaya
mesh_terms: D003001:Cloning, Molecular; D015536:Down-Regulation; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000302:isolation & purification; D010196:Pancreatic Elastase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D019076:Transgenes
chemical_list: D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068817:Epoetin Alfa; D010196:Pancreatic Elastase
keywords: EPO; Elastase II; Inverse PCR; Lentivirus vector; Metabolic engineering; shRNA delivery