A New Protocol for Efficient and Non-Toxic Transfection of siRNA Into Leukemic Cell Lines and Primary Cells – a Direct Comparison Between Nucleofection and Accell Delivery Using Stem Cell Related Antigens As Validation Tools

Abstract

Abstract 4802 BACKGROUND:Transient downregulation of genes in vitro by use of siRNA is a time-honored approach to study gene function. This can readily be applied in haematological malignancies, where many lesions derive from altered RNA activity arising e.g. from balanced translocations. PROBLEM FORMULATION:A crucial requirement to obtain this downregulation is an efficient and non-toxic delivery of the siRNA into the target cells, which has proven difficult to accomplish with some protocols; in particular, cells in suspension, such as leukemic blasts. HYPOTHESIS:Directly comparing the range of new transfection methodologies to unravel protocols suitable for cells in suspension. RESULTS:From pilot data, where a range of other methods were excluded due to either low transfection efficiencies or low viability of transfected cells, we compared the gold standard Nucleofection methodology to the novel non-viral based delivery method Accell in both suspension cell lines as well as primary patient AML and CML cells. As control the housekeeping gene Cyclophilin B was used, while hMICL and CD96, recently suggested to be associated with the putative leukemic stem cells, were chosen as target genes. Comparison of the two transfection methods yielded superior results using Accell methodology, as this technique not only yielded higher transfection rates but also retained superior cell viabilities for both cell lines and primary cells. Three AML and two CML samples were included in this study for the validation of transfection efficiency in primary cell employing the programs T-20 and U-15 for Nucleofection. Here, there was a large span in viabilities pre-experimentation among the five patient samples included in this study, probably reflecting the conditions of the patients. However, following transfection there was a significantly lower viability after Nucleofection (mean 37%, range 23%-65%) compared to Accell (mean 82%, range 57%-95%), in the two groups (p<0.001). Corroborating the cell line data transfection efficiencies were clearly superior after Accell transfection of primary patient cells (mean 85%, range 71%-97%) compared to Nucleofection transfection (mean 38%, range 23%-65%), p<0.01. Transfection efficiencies in primary cells after Accell delivery was 90–97% compared to 25–30% using Nucleofection for siRNA delivery. As a functional correlate to these data we performed CFU-GM assays on selected hMICL+ AML patients exposed to anti-hMICL siRNAs. While the AML blast growth of some patients were unaffected by the siRNA treatment others showed a clear inhibition of blast colony formation, suggesting a differential role of hMICL between AML patients, which needs to be further elucidated. CONCLUSION:Accell delivery is thus the method of choice for non-viral transfection of cells in suspension including primary AML and CML cells. These data should provide a platform for further studies of genes involved in early leukemogenesis. Disclosures:No relevant conflicts of interest to declare.