Gene editing tools such as CRISPR are revolutionizing cell line development and engineering due to the speed and ease of performing edits. Here we look at the intellectual property landscape and epigenetic control of therapeutic proteins.
This extract is part of a whitepaper on the use of CRISPR/CAS9 and other gene editing tools in cell line development and engineering. Download the full whitepaper for free here.
There is some risk that patent filings by academic institutions claiming critical components of the CRISPR–Cas technology might deter or slow down the development and utilization of the technology. However, research organizations (i.e., Universities) through sound management of intellectual property, can establish a workable balance between access and control for essential research tools.
It has been widely publicized that several organizations have been filing patents over fundamental parts of the CRISPR–Cas9 system.
Commercial assignees, Dow AgroSciences and DuPont Nutrition Science, together hold 33 inventions all of them related to the crop and animal agriculture (genome-editing crop and weeds) and food (dairy industry) applications of the technology. Besides, French Cellectis hold rights on a broad patent for gene editing of cells in vitro. Finally, academic institutions, through their licensing, spin-offs (MIT/Broad/Harvard - Editas) and commercial partners (UC Berkeley – Caribou Biosciences; sublicensed to Intellia and Novartis) are primarily in control of medical applications of CRISPR–Cas.
Interestingly, most patent holders appear to be pursuing a strategy of keeping an international option open for their patent portfolios. Fortunately, there are genuine signs that the pace of discovery and development of CRISP is likely to continue, with a high probability of further improvements. MIT/Broad/Harvard are building a portfolio on those principles and diversifying it.
Other gene editing technologies may yet emerge to compete with or possibly even displace CRISPR–Cas. Moreover, it is likely that other follow-on breakthroughs take center stage. How could any of the current patent holders pursue to restrict access to CRISPRCas for research use, since it is already widely used in academic laboratories? What are their options for commercializing follow-on technologies?
Inventors of follow-on applications using a CRISPR-Cas technology will most likely need to seek a commercial sublicense from the respective exclusive commercial licensee that controls that technology—Editas, Caribou, Intellia, CRISPR Therapeutics or Cellectis/Calyxt—rather than from the originating university. How many of the thousands of potential products to come from CRISPR–Cas gene editing technology could this small group of startup companies be expected to successfully manage either internally or in close partnership?
Epigenetic control of therapeutic proteins
Conferring an open chromatin state in targeted chromosome loci (DNA stretches composed of nucleosome-depleted regions) can have a certain benefit for transgene expression. Indeed, cis-acting epigenetic regulatory elements can help to remodel the chromatin environment, maintaining an active transcriptional state around the transgene. One type of epigenetic regulatory element (ERE) is the scaffold/matrix attachment region (S/MAR), thanks to which recombinant proteins could significantly improve the expression levels. Another class of EREs is chromatin opening elements (UCOEs), which confer unmethylated, open chromatin state for transgene expression. UCOEs were also found to be helpful for increasing the productivity of cell lines producers of recombinant proteins. Nonetheless, S/MAR and UCOE proven to decrease the variability of expression between the different clones. Notably, various epigenetic elements not only can help to increase the expression level of biotherapeutics but can also increase the number of clones that have integrated the transgene with a more defined copy number of transgenes per cell, thus accelerating the selection process.
Protein folding and secretion of recombinant cells.
Interestingly, secretory bottlenecks of CHO cells may be relieved by overexpressing soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs). Moreover, new targets for cell engineering approaches can be identified, based on metabolomics profiling. A bottleneck at the malate dehydrogenase II (MDHII) level was characterized for the tricarboxylic acid (TCA) cycle in CHO cells, and pyruvate metabolism was shown to vary between high producing and low producing anti-CD20 CHO clones.
Over the years, many cell engineering strategies were attempted to increase such titers by optimizing selection markers, gene expression, cell growth, and proliferation or protein folding and secretion. Among those engineering tools, CRISPR/Cas9 and RMCE technologies will largely contribute to the advance of glycoprotein production shortly.
We may conclude that with remarkable advances in genome editing technology such as the CRISPR/Cas9 tool as well as the combination of next-generation sequencing with systems biotechnology would exhibit extraordinary potential for discovery and modulation of novel cell engineering targets. These efforts might finally pave the way for the development of rational designer host cells. Efforts are ongoing to engineer post-translational modifications (PTMs) in microbial, insect, and plant cell systems to make these systems more suitable for therapeutic protein production.