Optimized Delivery of Small Regulatory RNAs into Industrial CHO Cell Lines Enables High-Throughput microRNA Screening - Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control mRNA abundance and thus critically regulate cellular phenotypes. Inter alia, efforts were made to engineer Chinese hamster ovary (CHO) cells which represent the most frequently applied host cell type for commercial production of therapeutic proteins. In the present study, we established a high-throughput screening approach which enabled us to assess the power of miRNAs in CHO cell line engineering approaches. To this end, a robust cultivation method was developed mimicking appropriate cultivation conditions for CHO manufacturing cell lines in micro-scale format. Different chemical transfection reagents were tested for their capability to efficiently introduce small RNAs such as siRNAs and miRNA mimics into CHO cells. We found that the novel cationic lipid Screen Fect®A plus exhibits excellent transfection efficiency and functionality in various CHO cell lines cultivated in complex production media. Recent approaches using miRNAs in CHO cells were limited to model cell lines that were far away from industry standard or produced standard IgG molecules. The experiments described here were performed using industrial production CHO cell lines stably expressing different monoclonal antibodies as well as novel antibody-derived molecules at various expression levels. This study demonstrates that the identification of a suitable transfection reagent and the careful optimization of the transfection conditions in combination with a micro-scale cultivation process led to the development of a high-throughput miRNA screening platform. A feasibility study using known pro-productive miRNAs rendered this approach suitable for future attempts aiming at the identification of novel miRNA candidates for innovative CHO cell engineering strategies.