Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process.

Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearized plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single-cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVVs that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Introduction: Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. Methods: High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Results: Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. Discussion: MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.