(410a) MicroRNA Profiling and Discovery for Process Enhancement in Chinese Hamster Ovary Cells

Mammalian cell culture has become the predominant system used for the production of recombinant protein therapeutics. Chinese hamster ovary (CHO)-derived cell lines are the most common cell type used in industry for this purpose. The productivities of these cell lines today rival that of professional secretor cells in vivo. Cell engineering via over-expression or knock-down of proteins has been an important strategy to further augment the productivity of these cell lines. Transcriptomic and proteomic studies suggest, however, that the complex traits of high productivity may result from the accumulation of changes in gene expression in many pathways. MicroRNAs (miRNAs), an abundant class of short regulatory RNAs, represent an additional level of gene expression control that could be exploited to manipulate global gene expression profiles. MiRNAs carry out simultaneous post-transcriptional regulation of up to hundreds of transcripts, and they have been implicated in the modulation of nearly all cellular processes, including cell cycle control and regulation of apoptosis.

Little work has been published on the global characterization of miRNAs in CHO cells. We show, through ultra high-throughput Illumina Solexa sequencing of seven small RNA libraries prepared from parental and recombinant CHO cell lines of varying productivities, that miRNAs are abundant in CHO cells and are highly conserved with other species. MiRNAs constituted over half of the small RNA sequencing reads, with read counts (a direct measure of transcript abundance) spanning six orders of magnitude. Hundreds of unique miRNAs were identified through sequence orthology across over two dozen different species. Many miRNAs were identified by several reads of slightly different length and sequence. These sequences, termed as isomiRs, are the result of imprecise enzymatic processing or single nucleotide extension during miRNA biogenesis. Importantly, the most abundant of these alternate sequences, often an indication of the most active forms, frequently did not correspond to the most commonly observed sequence in other species.

The sequence data also revealed differential expression of many miRNAs across the seven libraries. Time course miRNA expression dynamics were further investigated using miRNA microarrays in CHO cell lines under productivity-enhancing culture conditions. Potential target mRNAs were identified through the use of publically available target prediction algorithms, and pathway analysis was used to gain biological insight by identifying the major pathways being regulated. These results represent an initial assessment of the extent of miRNA influence in CHO cells, and further studies may facilitate miRNA engineering, representing a new strategy for cell engineering in mammalian cell culture.