ASSESSMENT, AND STREAMLINED PREPARATION OF LOW CYTOTOXICITY LENTIVIRAL VECTORS FOR MOBILIZED HUMAN HEMATOPOIETIC STEM CELL TRANSDUCTION.

ABSTRACT As important vectors for ectopic protein expression, gene silencing and progenitor cell barcoding, lentiviruses continue to emerge as versatile research and clinical tools. For studies employing cell types that are relatively resistant to transduction, high titer lentivirus preparations with low cytotoxicity are required. During lentivirus production, carryover plasmid DNA endotoxins, transfection reagents, damaged packaging cells, and virus concentration procedures are potential sources of cytotoxicity. As an often unevaluated property of lentivirus preparations, cytotoxicity can unwittingly skew estimates of functional titers, and complicate interpretations of transduced cell phenotypes. By employing hematopoietic UT7epo cells cultured in EPO below maximal dosing, we first define a sensitive flow cytometric bioassay for critically assessing cytotoxicities (and titers) of lentivirus preparations. When custom preparations of research- grade lentiviruses from six commercial sources were bioassayed, substantial cytotoxicity unexpectedly was revealed (with certain preparations additionally registering titers several logs below designated values). To overcome such limiting properties, we further report on unique, efficient workflows for reproducibly preparing and processing high titer low cytotoxicity (HTLC) lentiviruses at research scale. These HTLC lentiviruses reliably transduce PB-HSPCs at ≥40% frequencies, with low cytotoxicity. In addition, by employing cyclosporin-H (to inhibit IFITM3), PB-HSPC can be transduced at heightened efficiency with nominal cytotoxicity. Overall, this work provides straightforward approaches to: 1) critically assess the cytotoxicity of lentivirus preparations; 2) reproducibly generate (and concentrate) high quality lentiviruses via a streamlined workflow; and 3) transduce PB-HSPC at benchmark levels with nominal cytotoxicity.