OPTIMIZATION OF A LENTIVIRAL VECTOR FOR TRANSIENT AND STABLE PROTEIN OVEREXPRESSION IN CHO AND HEK 293 CELL LINES

CLAUDIO PRIETO; EDUARDO MUFARREGE; SEBASTIÁN ANTUÑA; RICARDO KRATJE; MARINA ECHEVERRIGARAY

Lille

Congreso; 23rd ESACT Meeting; 2013

The Organising and Scientific Committees of the ESACT Conference and Exhibition

BACKGROUND AND NOVELTY: Recombinant protein overexpression in animal cells constitutes a real challenge in the biomolecule production for therapeutic purposes. Following the intron functionality discovery as gene expression enhancers, various expression vectors that include them in their sequences have been developed. Generally, these are located in the 5? untranslated region (5?UTR), in which case they are called leader introns. In the present work, we have studied various systems leading promoters (CMV or EF1a) and 5? UTRs sequences (HsEF1-a, human Elongation Factor 1-a; CgEF1-a, C. griseus Elongation Factor 1-a; CMV, cytomegalovirus immediate-early 1 gene; CI, Chimeric Intron from the 5?-donor splice site from human s-globin intron 1 plus the 3?-acceptor splice site from the intron of an immunoglobin gene heavy chain variable region) in different combinations, fused to either a gene coding for green fluorescence protein (GFP) or recombinant human Factor VIII (rhFVIII). EXPERIMENTAL APPROACH: Analyzed sequences were amplified by PCR using appropriate primers and templates. Then they were cloned in lentiviral vectors that previously contained either GFP or rhFVIII genes. GFP levels were evaluated by flow cytometry and rhFVIII productivities by sandwich ELISA, after transfection or transduction assays. RESULTS AND DISCUSSION: When evaluating GFP and rhFVIII levels in transient and stable expression conditions in CHO-K1 and HEK293 cell lines, we were able to identify specific promoter/5?UTR combinations with higher expression levels compare to commercial vectors. Expression levels depended on the evaluated expression platform and cell line, reaching increments between 2.5 and 4 times and 1.5 to 4 times in transient and stable conditions, respectively. In summary, in this study we were able to develop new lentiviral vectors for protein overexpression that achieved superior expression levels with respect to widely used vectors and constitute promising candidates as gene expression systems in animal cells in transient and stable conditions.