Mammalian cells are attractive systems for producing complex proteins for uses ranging from synthetic materials to medical therapeutics, given their abilities to carry out post-translational modifications and mRNA splicing. Commonly, stable gene expression through host cell genomic integration is utilized for high throughput protein production applications. However, it is a difficult and often time-consuming process to transfect and screen cell lines that stably express the protein of interest. Transient protein production is an attractive alternative to constitutive expression, given the lack of cell line screening required. Transient gene expression delivery vehicles (vectors) range from viral (eg. adenovirus, retrovirus) to non-viral (eg. cationic polymers, lipids, metals). Viral vectors are generally much more efficient than non-viral vectors, but they suffer from drawbacks including the potential for toxicity, immunogenicity, and limited nucleic acid cargo load. It is imperative to ultimately improve the efficacy of non-viral gene delivery vehicles, both for therapeutic transient protein production as well as for gene therapy. Kinases are a major class of enzymes ubiquitously expressed in mammalian cells, and they are involved in most cellular processes, including those directly and indirectly involved in transgene expression. We carried out a screening of approximately 200 kinase inhibitors for their ability to enhance transient expression of a reporter gene, when delivered using an in-house cationic polymer. We identified several kinases as transient expression repressors, including those involved in cell cycle and transcription processes, in a prostate cancer cell line employed in the screen. Further inhibitor dosing optimization studies were carried out using an in-house polymer and current gene delivery standard, 25 kDa polyethyleneimine (PEI), for delivery. Combinations of kinase and histone deacetylase inhibitors demonstrated significantly higher levels of transient protein expression compared to cases when only a single inhibitor was employed, with minimal effects on cell viability with the addition of each drug. Given that each inhibitor tested targets a different cellular process, it is clear that simultaneously inhibiting multiple cellular processes involved in transgene expression is an attractive strategy for enhancing transient protein production, a novel finding applicable both in biotechnology and biomedicine.

Kinome-level screening identifies kinase targets for enhancing transient transgene expression

249th National Meeting (2015)

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