Eduardo Federico Mufarrege1, Sebastián Antuña2, Marina Etcheverrigaray2, Ricardo Kratje2, Claudio Prieto2. 1. Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 242, S3000ZAA Santa Fe, Argentina. Electronic address: mufarrege@fbcb.unl.edu.ar. 2. Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria. Paraje "El Pozo" - c.c 242, S3000ZAA Santa Fe, Argentina.
Abstract
BACKGROUND: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 5'UTR (5' untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. RESULTS: By combining the human cytomegalovirus (CMV) or the elongation factor 1α (EF-1α) promoters along with different 5'UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 5'UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1α promoter in three widely used cell lines. CONCLUSION: In the present work, LVs containing different promoters and 5'UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.
BACKGROUND: Recombinant protein overexpression in mammalian cells constitutes a real challenge in therapeutic protein production. Following the discovery of intron functionality in gene expression, various expression vectors that include them in their sequences have been developed. In this study, the main goal was to develop new lentiviral vectors (LVs) carrying different promoter and intron-containing 5'UTR (5' untranslated region) combinations and the design of LVs for rhFVIII production in Chinese hamster ovary (CHO) cells. RESULTS: By combining the human cytomegalovirus (CMV) or the elongation factor 1α (EF-1α) promoters along with different 5'UTRs that included leader introns, between 2 and 12-fold increases were reached, when transient and stable expression of the enhanced green fluorescent protein (EGFP) and rhFVIII were analyzed. Also, new LVs provided with promoters and 5'UTRs from high expression genes, according to a gene database, were designed. Three of them were shown to be superior to the EF-1α promoter in three widely used cell lines. CONCLUSION: In the present work, LVs containing different promoters and 5'UTRs were designed. In transient and stable assays some of these constructs have shown higher activity compared with commercial promoters and, therefore, constitute promising candidates for therapeutic protein production.
Authors: Chiara De Santi; Frances K Nally; Remsha Afzal; Conor P Duffy; Stephen Fitzsimons; Stephanie L Annett; Tracy Robson; Jennifer K Dowling; Sally-Ann Cryan; Claire E McCoy Journal: Mol Ther Nucleic Acids Date: 2022-08-04 Impact factor: 10.183