miR-acle Grow: Improving CHO cell Growth by miRNA Dysregulation

The “CHO group” here in NICB is a sizable collection of young researchers, incorporating people with a wide variety of cultural and academic backgrounds. Collectively, we effort to tackle many real challenges facing the biopharmaceutical industry today. Some members such as myself, use targeted genetic engineering approaches to improve Chinese Hamster Ovary (CHO) cell characteristics, while others focus on bioprocess design related problems, contribution to publicly available databases and understanding the fundamental biology of CHO cells.  The group also has strong relationships with other academic labs in National Institute for Bioprocessing Research & Training (NIBRT) and Trinity College Dublin (TCD) among others, along with industry collaborations with Pfizer, Biogen and Eli Lilly. To meet a few group members, please take the time to view our most recent video:

[embedyt] http://www.youtube.com/watch?v=cyPV-GFYh18[/embedyt]

When people ask me what I do and I reply, I’m a PhD candidate, they don’t always get it. The best example I can think of is my Nan, she thinks I’m a bit dim because I’m still in “school” at the age of 24. It can be a struggle to relate what you do, not only to the non-scientific community, but also scientists in different fields. When friends ask, I tell them a PhD is a 300- to 400-page book that only a handful of people will ever read and my day to day involves adding small volumes of liquid to other small volumes of liquid, with documentation of the results. Why, you may ask?

The biopharmaceutical industry is one of Irelands largest, with capital investment exceeding €8 billion in the last 10 years. Currently 9 of the world’s top 10 pharmaceutical companies have a base in Ireland, with annual exports of €39 billion, as recently published in The Irish Times. This industry not only exists, but thrives due to the inevitability that people get sick. A limitation of the biopharma sector, by no fault of their own, is the inability to provide affordable treatment options for many debilitating and life threatening diseases. In some instances, this is not because a bio-therapeutic does not exist, but simply the drug cannot be manufactured in reliable, robust and cost-effective manner. That’s where we come in… kind of!


CHO cells have become a favourite of the biopharma industry due to their uncanny ability to synthesise complex human-like proteins and their adaptiveness regarding culture format. Numerous factors contribute to the generation of a stable recombinant CHO (rCHO) cell line. CHO cells are enabled  to produce therapeutic drugs by the stable integration of DNA, encoding for the therapeutic, into their genome. Traditional cell line development is essentially a numbers game; rCHO cells are screened by the thousand for what is termed high producers, single cells from an original pool are isolated and their drug production capabilities monitored, with the best candidates progressing to process optimisation. The most pressing drawbacks to current clonal screening strategies include the vast number of clones that must be vetted, and the naivety of this practice, we don’t know why one cell outperforms another.


Figure 1: Schematic representation of recombinant CHO cell line development. Genetically engineered CHO cell lines are used to make stable transgenic mixed pools expressing the drug of choice. Clonal isolation and screening is used to find the top candidates. Subsequent process development is required to maximize product output and CHO cell production efficiency.

The biopharmaceutical industry requires robust, reliable and reproducible bioprocesses, yielding a high level of good quality product to ensure, above all else, patient safety. Reproducibility is achieved and enhanced by understanding and controlling bioprocess variables. Evolution of process analytical technology (PAT), has aided this greatly at experimental, pilot and industrial scales, involving both on-line, in situ, and off-line monitoring of culture variables. Real-time assessment of the bioprocess enables the manufacturer to identify issues or irregularities and act on them, saving any potential loss in yield.


This is all great but we still don’t know what is going on inside the cell and why some cells make good quality homogeneous product while so many others fail in this regard. This is where genetic engineering of CHO cells comes in.  In the simplest of terms, my research, which is funded by Science Foundation Ireland, aims to improve the productive capability of a CHO cell culture. This is achieved by altering natural cellular pathways to improve the growth of these cells. The idea being, if we can increase the number of cells present in the culture with no negative effects on how they make a product, it is back to a numbers game – more cells equates to more product. To do this I am altering specific gene regulatory elements, microRNAs (miRNAs), at defined points in the culture in the hope to maximize the process yield.

[embedyt] http://www.youtube.com/watch?v=StWDQgSg3UE[/embedyt]

The rational for CHO cell engineering by miRNA over conventional gene expression or repression strategies is simple: miRNAs with their short functional nucleotide sequence have the ability to target many genes, and in some instances play a key role in the regulation of fundamental biological pathways. miRNAs are highly conserved between species, however their function has been seen to be not only cell type but also cell stage specific. The cell type and cell stage specificity of these molecules makes them appealing for CHO cell engineering, profiling the relative abundance of miRNA in a cell line with attractive characteristics provides us with our miRNA targets for engineering.


Figure 2: Stable miRNA manipulation strategies. (i) Expression vector is transfected and stably integrated into the host genome. (ii) Transcription of transgene gives rise to one of two options, miRNA over-expression or depletion via sponge decoy. (iii) miRNA over-expression is achieved by expression of the pri-miRNA hairpin. The pri-miRNA undergoes the same miRNA maturation process as native miRNA, and functions in the same manner. (iii) Stable depletion of native miRNA is achieved by expression of a miRNA sponge decoy. The miRNA responsive elements (MRE)s, deplete free native miRNA and inhibit its natural functions.

Once we have our miRNA candidates they must be screened to elucidate their potential for improving CHO cell characteristics. We have two options, over-expression or depletion, see Figure 2. By over-expressing a miRNA we can essentially enhance the potency this molecule has on the cell, this is achieved by the generation of a genetic construct encoding for the desired miRNA and subsequent integration of this DNA into our host CHO cell of choice. When it comes to miRNA depletion, we need to design a synthetic binding site, or decoy, for our miRNA of choice. By expressing a gene with this decoy sequence we can inhibit the miRNA’s natural function, we call this sequence a miRNA sponge, as it “soaks up” all the targeted miRNA. Think of the miRNA as a stain, and you remove it with a sponge.

[embedyt] http://www.youtube.com/watch?v=tb_wZJSN4yY[/embedyt]

These stable miRNA manipulation studies have been conducted in the past by the CHO group here in NICB for a number of different miRNAs, see DECOY-7 video, along with other labs in BOKU University of Natural Resources and Life Sciences, Vienna and Hochschule Biberach University of Applied Sciences (HBC), Germany among others. Where my strategy differs, is the addition of control. The miRNA over-expression or sponge mediated depletion is controlled by a drug inducible promoter. Simply speaking, the addition of a particular antibiotic to the culture media tells the cell to make our miRNA or sponge of interest. So why does this matter? If we return to the idea of process control and couple this with the fact that miRNA expression/ role/ abundance change over the stages of mammalian cell growth, now we can begin to join the dots…we have the ability to control the free abundance of specific regulatory molecules at defined stages of culture. What we need is a list of miRNA with a potent phenotypic impact. In our lab, we routinely screen miRNA for impact of CHO cell growth, cell viability and product titer. Unfortunately, it’s difficult to identify miRNA that significantly improve all three of these characteristics consistently across different CHO cell lines. However, the miRNA manipulation technology I have at my disposal is flexible, there is the ability to utilize multiple miRNA, either in combination or controlled independently at defined points of the culture. The idea of multi-miR engineering of CHO cells is exciting, coupling this with the control of these technologies only increases the potential of miRNA engineering technology in the future.