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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.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αβ-defensins (Csαβ-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins., The ISME Journal, 13, ISSN:1751-7362, ISSN:1751-7370