Transposon vector-mediated stable gene transfer for the accelerated establishment of recombinant mammalian cell pools allowing for high-yield production of biologics

Abstract

Stable recombinant mammalian cells are of growing importance in pharmaceutical biotechnology production scenarios for biologics such as monoclonal antibodies, growth and blood factors, cytokines and subunit vaccines. However, the establishment of recombinant producer cells using classical stable transfection of plasmid DNA is hampered by low stable gene transfer efficiencies. Consequently, subsequent selection of transgenic cells and the screening of clonal cell populations are time-and thus cost-intensive. To overcome these limitations, expression cassettes were embedded into transposon-derived donor vectors. Upon the co-transfection with transposase-encoding constructs, elevated vector copy numbers stably integrated into the genomes of the host cells are readily achieved facilitating under stringent selection pressure the establishment of cell pools characterized by sustained and high-yield recombinant protein production. Here, we discuss some aspects of transposon vector technologies, which render these vectors promising candidates for their further utilization in the production of biologics.

piggyBac system to co-express NKG2D CAR and IL-15 to augment the in vivo persistence and anti-AML activity of human peripheral blood NK cells

“…PB systems offer several advantages, including high cargo capacity of up to 100 Kb DNA fragment for the delivery of multiple transgenes, significantly lower manufacturing costs, and potentially fewer regulatory restrictions in clinical translations. 49 , 50 Considering these appealing features, PB transposons have been recommended as an alternative gene transfer system to viral vectors for CAR-T cell production. 31 , 51 , 52 Compared with PB-mediated CAR gene transfer in T cells, we noticed a low transfection efficiency for CAR gene transfer into NK cells.…”

Progress of Transposon Vector System for Production of Recombinant Therapeutic Proteins in Mammalian Cells

“…A series of individual DNA transposons from different donor organisms have been identified in detail, including the hAT gene family element-Tol2 from the medaka fish ( Kawakami et al, 2000 ), the engineered Tc1/mariner transposon, “ Sleep Beauty ” (SB) ( Mikkelsen et al, 2003 ), and the insect-derived natural element PiggyBac (PB) ( Tschorn et al, 2020 ; Rajendran et al, 2021 ). Although PB, Tol2, and SB do not show a preference against specific host cell chromosomes, they differ in their phylogenetic origin, biochemical properties, size of integrated target genes, and DNA sequence preference for translocation ( Ding et al, 2005 ; Huang et al, 2010 ; Li et al, 2011 ; Meir et al, 2011 ; Wang et al, 2014 ).…”

“…Recombinant proteins are produced in heterologous cells using genetic engineering techniques by obtaining the gene of interest (GOI), constructing the expression vector, and expressing the protein of interest in the host cell. The trend of using mammalian cell lines in RTPs production has accelerated dramatically in recent years, 84% of approved RTPs were produced using mammalian cells in 2018 ( Walsh 2018 ; Tschorn et al, 2020 ). The protein produced from Chinese hamster ovary (CHO) cells have similar post-translational modification (PTM) system to those of mammalian cell, therefore approximately 70% of the approved recombinant therapeutic protein (antibody) are produced in CHO cells.…”

Current view on novel vaccine technologies to combat human infectious diseases

“…A mammalian expression system would be an ideal choice because it will post-translationally modify expressed proteins in a human-like fashion. Still, the process involves the laborious and time-consuming generation of stable cell lines and the high cultivation costs, while the quantities of the expressed proteins are lowest compared to other expression systems (Gutiérrez-Granados et al 2018;Tschorn et al 2020).…”