UBC Theses and Dissertations
Development of a novel histone-based reagent to increase retroviral transduction based on an analysis of cell lysate effects Beauchesne, Pascal
Recombinant retroviruses are widely used vectors for engineering cells as they facilitate the delivery and long-term integration of transgenes. Retrovirus-mediated gene transfer is nonetheless challenged by low efficiency due to multiple extracellular rate-limiting steps including mass transport limitations due to the low diffusivity and rapid decay of retroviral vectors as well as limited binding due to electrostatic repulsion between the net negative-charge of both target cells and retroviral vectors. Whole cell lysate was determined to contribute to the variability observed in transduction protocols leading to an increase in the retroviral transduction of TF-1 (human) cells by gibbon ape leukemia virus-pseudotyped vectors and of BaF3 (mouse) cells by ecotropic retroviral vectors. Fractionation of the cell lysate revealed that the bulk of the activity was associated with debris aggregates. These aggregate structures enhanced transduction by increasing mass transport through sedimentation as well as by increasing adsorption of the retroviral vectors to the cells and the culture vessel surfaces. In the presence of this aggregate fraction, transductions of TF-1 and BaF3 cells were enhanced respectively by 59- and 213-fold relative to controls without additives. Further analysis revealed that the aggregate structures were derived from nuclear components sensitive to trypsin digestion, suggesting that nuclear proteins rich in arginines and/or lysines were responsible for the observed enhancement. A subsequent investigation of histone proteins revealed that the arginine-rich fraction, at a concentration of 160 μg/mL, yielded a 22-fold increase in the transduction of TF-1 cells. To mimic the aggregate structures observed in the lysate, histone self-aggregation was stimulated by heat treatment resulting in a 34-fold increase in TF-1 cell transduction while the concentration required was reduced to 10 μg/mL. With BaF3 cells, the transduction exceeded that achieved with lysate under similar conditions. This reagent was also successfully applied to the transduction of primary mouse hematopoietic progenitor cells with long-term reconstitution potential. Overall, a novel histone reagent able to enhance the transduction of primary cells and cell lines with negligible toxicity using both gammaretroviral and lentiviral vectors was designed by isolating the active compounds and analyzing the mechanism of action of lysates on retroviral transduction.
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