microRNAs as potential tools for ‘miR’aculous CHO cell phenotypes in Bioprocessing systems

Chinese Hamster Ovary (CHO) cells are the biopharmaceutical industry’s “mini biofactories” for the production of complex, post-translationally modified therapeutic proteins. In order to address the ever-growing market need for these recombinant proteins, various genetic engineering tools have been employed. Here, we describe genetic approaches to improve CHO cell culture longevity, with a view to increasing overall process yield via the manipulation of two miRNAs: Let-7a and miR-7. Previous miRNA profiling studies in our laboratory and in the published literature helped in the identification of these miRNAs, which have shown to be disregulated in various tumor types and are key regulators of the cell cycle. Therefore, this stimulated the interest of our research group to manipulate these miRNAs in CHO cells with a view to positively impact bioprocess-relevant CHO cell phenotypes. In the first approach, we used a Let-7 sponge decoy vector to deplete endogenous Let-7 levels with a view to increasing culture longevity and productivity of CHO-K1 SEAP expressing cells. Despite let-7 having a recognised role in deregulated cell growth no improvement was observed in stable, sponge-transfected clones. Out of a panel of 40 clones, we observed only two with improved cellular viability in 24 well plate format, however, the results were not reproduced in a 5 mL scale-up batch study. In the second approach, we targeted a previously verified miRNA for improved CHO cell growth and productivity i.e. miR-7, using a bacterial genome-editing tool, CRISPR-Cas9. A considerable amount of optimisation work was performed to establish the CRISPR system for use in the lab, initially using eGFP as model target gene in CHO cells. Finally we designed single guide RNAs to target Cas9 to the miR-7a-5p genomic locus to disrupt miR-7 in order to enhance growth of a CHO-K1 cell line producing an IgG-1. We estimated ~ 40% targeting efficiency of miRNAs using this approach. After an extensive screen, one stable clone was identified with what appeared to be a heterozygous deletion of one miR-7a copy. We demonstrate that CRISPR-Cas9 can be successfully used to target miRNA loci in the CHO genome but that functional knockout may be more difficult compared to protein coding genes