Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors.

To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale.

Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase.

The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty-derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes.

Ecotropic HIV-1 vectors pseudotyped with R-peptide-deleted envelope protein variants reveal improved gene transfer efficiencies.

Viral vectors derived from human immunodeficiency virus type 1 (HIV-1) mediate efficient stable gene transduction. Consequently, these vectors are utilized in gene therapeutic approaches. We here aimed for improving HIV-1 pseudotype vector formation using envelope proteins (Env) of ecotropic murine leukemia virus (MLV) suffering deletions of the R-peptide and further amino acid substitutions in their cytoplasmatic domains. All examined Env variants revealed cell-surface expression and showed elevated fusogenicity as compared to wildtype (eMLV-wt) Env but failed to efficiently pseudotype MLV particles. However, two variants generated ecotropic HIV-1 pseudotype vectors with superior infectivity. Most importantly, pseudotyping with the variant eMLV-GaLVΔR encompassing the R-peptide-deleted cytoplasmic domain of the gibbon ape leukemia virus Env yielded titers three-fold higher than HIV(eMLV-wt) vectors. We anticipate that superior ecotropic HIV(eMLV-GaLVΔR) pseudotype vectors will be of utility in preclinical gene therapy studies aiming at the genetic modification of primary murine cells.

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

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.

Advanced Establishment of Stable Recombinant Human Suspension Cell Lines Using Genotype-Phenotype Coupling Transposon Vectors.

Stable mammalian, namely human, suspension cell lines play a pivotal role in red biotechnology production scenarios for the generation of state-of-the-art biologics. However, selection of genetically modified and highly productive cell populations - prior to the establishment of clonal lines - is often challenging. To overcome this limitation, we first describe an optimized transient transfection protocol using the inexpensive reagent polyethylenimine (PEI) and human 293F cells. Transposon donor vectors derived from Sleeping Beauty encompassing a cassette with the reporter gene encoding for the green fluorescent protein (GFP) coupled with an internal ribosome entry site (IRES) to the expression of puromycin-resistance are employed to readily detect transfected cells. Upon stable transfection in the presence and absence of transposase expression, respectively, and subsequent antibiotic selection, GFP expression using flow cytometry analysis, cell viability, and cell density can be examined over a range of up to 3 weeks. Owing to the integration of high vector copy numbers into the target cell genome, transposase-mediated transposition of transposon donor vectors is instrumental in the faster establishment of recombinant cell population as compared to the classical stable transfection of plasmid DNA.