| Summary: | Mammalian expression systems, particularly Chinese hamster ovary (CHO) cells, are used industrially for the production of biopharmaceuticals. Despite improvements over the last few decades with regard to the maximum cell concentrations obtainable and the amount of recombinant protein produced from such expression systems, there is still the potential to engineer and manipulate mammalian cells to generate more robust host cells that grow faster, for longer and produce higher product yields with less heterogeneity. A number of recent studies have reported that mRNA translation is a key limitation in terms of defining the product yield from in vitro cultured mammalian cells. One mechanism by which mRNA translation is controlled is via the expression of microRNAs (miRNA). miRNA are endogenous single stranded non-coding pieces of RNA of approximately 22 nt in length that can either stall mRNA translation or activate cleavage of target mRNAs. This study set out to investigate the role of specific miRNAs on determining CHO cell growth and recombinant protein productivity. Initially, Lonza Biologics undertook an extensive miRNA profiling study using the mercury LNA microarray, and found that -56 miRNA were significantly differentially expressed during the course of a state-of-the-art fed-batch bioreactor culture. In this study we determined the profile of four target miRNA (miR-15b, miR- 16, miR-21 and miR-34c) throughout batch culture in model monoclonal antibody producing CHO cell lines and the effect of transiently over-expressing the corresponding pre-miRNA individually or together. The different miRNA were expressed differentially throughout batch culture, their profile correlating with either growth or productivity characteristics. There was no relationship between pri-microRNA amounts and the mature microRNA amounts observed in the model cell lines investigated. Overexpressing/ knocking-up of the human pre-microRNAs in CHO cells resulted in an increase in the observed mature microRNA amounts. In some cases this over-expression resulted in increased recombinant protein expression and enhanced cell specific productivity when transiently expressed in model cB72.3 monoclonal antibody producing cells. Thus, the transient over-expression of pre-microRNAs resulted in increased product yields and/or changes in growth characteristics. In particular the over-expression of microRNA-34c resulted in an increase in transgene expression. Interestingly, when multiple microRNA were over-expressed transiently together an additive or synergistic effect in growth or product titre was observed in a number of cases. This is one of the first such studies to investigate the effect of manipulating multiple miRNA on recombinant protein synthesis from mammalian cells. When individual pre-microRNAs were stably expressed the amount of mature micro RNA observed in the resulting stable pools was increased. However, the amount of mature microRNA observed could be further increased by transient transfection of the stable pools with a plasmid for the expression of the target pre-microRNA suggesting that the maturation machinery for the generation of mature microRNA was not saturated. Ultimately this work has provided an improved understanding of specific microRNAs in CHO cells and their subsequent effect on industrially relevant phenotypes, providing informed strategies to manipulate microRNAs in order
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