Abstract
A successful immunotherapeutic strategy for the treatment of relapsed Acute Lymphoblastic Leukemia (ALL) post stem cell transplantation requires the optimisation of multiple parameters. The aim of this project was to optimise RNA loading conditions into dendritic cells (DC) for eventual induction of leukemia specific cytotoxic T lymphocyte (CTL) responses. We hypothesized that electroporation would be an efficient technique with which to introduce IVT-RNA into cord blood CD34+derived DC. The aims of this project were to: 1) to optimise variables affecting transfection efficiency (using IVT-enhanced green fluorescent protein (eGFP) mRNA as a model antigen), 2) optimise variables affecting cell viability, 3) determine the optimal day of RNA loading and 4) show that the electroporated CD34+ derived DC were functional. We designed experiments to investigate the effects of cuvette size, cell concentration, voltage, capacitance and electroporation medium on the viability and transfection efficiency of CD34+derived DC. Additionally, we investigated factors pre and post electroporation including: incubation times and exposure of cells to different temperature environments that can influence viability and GFP uptake by CD34+derived DC. Using our optimal conditions we achieved 91% GFP transfection efficiency with 90% cell viability, and 94% of Lin−CD11c+HLA-DR+ DC have taken up RNA. A crucial finding was the effect of temperature on cell membranes, significantly affecting transfection efficiency and cell survival. Cells will take up more RNA and survive better in a warm (37°C) environment compared to room temperature or ice. Transfection efficiency of CD34+derived DC electroporated on day 9 of culture was consistently higher that that observed on day 7 of culture. Interestingly, electroporation did not further upregulate activation markers on CD34+derived DC. CD34+derived DC electroporated with Flu Matrix protein RNA under the optimised conditions are able to present antigen and be killed by flu CTL clones. In conclusion, we have developed an efficient and safe electroporation procedure that results in functional RNA loaded CD34+derived DC that can now be used to stimulate CTL for adoptive immunotherapy.
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