Epigenetic Editing is an emerging field that has sparked interest across a number of research groups. By using a fusion of TET we can now target specific genomic regions for demethylation in order to learn about their function and regulation. We got in touch with one of the field’s experts, Dr. Marianne Rots, commissioning editor of Clinical Epigenetics, Professor of Molecular Epigenetics at the University of Groningen in the Netherlands, and head of their Epigenetic Editing laboratory since 2007, to get her thoughts on the this booming field.
Epigenie: Can you comment on the growth of interest in 5hmC and active demethylation since the discovery of the TET enzymes in 2009?
Dr. Rots: Until very recently (2009), DNA methylation had been considered a very stable epigenetic mark and the initial quest for active DNA demethylation faced serious disbelief. Although active DNA demethylation was known to occur in plants, no mammalian homologues to such enzymes (ROS1, DEMETER) were identified. The TET enzymes were identified as homologues to modification enzymes in Trypanosoma and their DNA demethylation activity has been unequivocally demonstrated through the TET3 -demethylation process of the genome in the paternal pronucleus.
This realization of the concept of active DNA demethylation set the stage for long-awaited approaches to induce demethylation that do not require active cell division. Obviously, targeting of TET enzymes to loci of interest (e.g. tumor suppressor genes) as we and others recently showed, allows novel therapeutic options: although some epigenetic therapies have been approved in the clinic, such approaches have genome-wide and non-chromatin effects. Active DNA demethylation is of special significance for Epigenetic Editing purposes, since sustained re-expression of epigenetically silenced genes requires removal of DNA methylation marks to prevent heterochromatinization to re-occur eventually.
EpiGenie: Do you have any plans with industry to commercialize this Epigenetic Editing process?
Dr. Rots: I am involved in two different approaches to commercialize Epigenetic Editing: one is for therapeutic applications and would involve clinical trials for which solid proof of principle in in vivo contexts is required first. Clinicians in different areas have expressed their interest, but general issues such as delivery, specificity and heritability need to be thoroughly addressed. Different aspects are being tackled in different labs worldwide. With my background in gene therapy, both viral and non-viral, I am closely associated with progress in this field and I expect some breakthroughs in the nearby future to allow also Epigenetic Editing to be clinically realistic. In this respect, I closely collaborate with Synvolux Therapeutics, which recently demonstrated targeted non-viral delivery of siRNA specifically to endothelial cells in an in vivo inflammation mouse model. We are in the process of further exploiting the proven efficacy for active protein delivery by Synvolux-formulations for ZFP-fusion proteins. Moreover, clinical trials are ongoing in the field of genome editing, enforcing progress from which the field of Epigenetic Editing will directly benefit.
The second promising field of application would be the upcoming field of Functional Foods. Again, issues here need to be addressed first including bio-availability and bio-stability of the approach. In this respect it is interesting to note that the pharmaceutical formulation of so called Epigenetic Editors is not limited to gene or protein based DNA targeting, but can readily be envisioned as chemically-based DNA binding domains. Renewed interest would have to be raised to increase research efforts into these fields but several publications demonstrate the opportunities of such synthetic approaches (e.g. polyamides or Triplex Forming Oligonucleotides).
EpiGenie: What do you see as the most practical application of this technology?
Dr. Rots: Epigenetics is an exploding field of science, not only with respect to basic chromatin biology research but also within medical sciences: researchers are dedicated to find epigenetic mutations underlying the pathophysiology of their diseases of interest. This yields massive amounts of data, but the majority of claims on the involvement of epigenetics in disease are through “guilt by association”approaches. Validation of observed epimutations has been a laborious, indirect process. Nowadays, mimicking of epimutations in the relevant contexts can easily be achieved by Epigenetic Editing (especially when achieved through CRISPR-based approaches, provided that specificity is carefully addressed), resulting in validation of new therapeutic targets.
Next to expected benefits for the field of Clinical Epigenetics, many basic questions still exist in the field of Functional Epigenetics: cause-versus-consequence of epi-marks versus gene expression and order-of-events are still largely unresolved. By targeting epigenetic enzymes or catalytically (in)active domains unique insights will be obtained which will surely advance the field. I am convinced that Epigenetic Editing will result in dogma-challenging new insights in chromatin biology, while providing unique tools to fully exploit clinically-relevant observations.
EpiGenie: What do you think the biggest contribution of this technology will be to the basic sciences?
Dr. Rots: Basic science is still in need of a robust way to investigate the function of a protein-coding gene or a non-protein-coding RNA in its normal context. As Epigenetic Editing is opening up chromatin allowing transcription instead of ectopic overexpression, the advantage over cDNA approaches are numerous and likely include the expression of all intended isoforms, in their natural ratios, without enforced (over)expression and the expression takes place only when associated cell signaling pathways are activated. Moreover, Epigenetic Editing can induce permanent silencing in expressing cells, which has as a clear advantage over siRNA which has to be continuously expressed for permanent effects. Epigenetic Editing provides a hit-and-run approach for gene expression modulation and since it is one similar delivery approach for both up- AND downregulation it allows for the ideal controls within one experimental setting.
EpiGenie: Can you comment on your current research?
Dr. Rots: To optimally exploit the power of Epigenetic Editing, my lab sets out to unravel underlying (chromatin) rules to permanently overwrite epigenetic signatures in order to induce/repress gene expression and maintaining such intended effects on gene expression.
EpiGenie: What’s your dream application of Epigenetic Editing?
Dr. Rots: I am convinced that Epigenetic Editing will allow full realization of the drug-able genome concept. However, for this to become clinically relevant, we need to demonstrate mitotic stability in vivo. I decided to establish a research line together with (pre)clinical collaborators to focus on prevention of organ failure after transplantation, which is due to fibrosis. Organ transplantation is one of the few clinically relevant examples where organ-specific delivery of the therapeutic agent is easily achieved via ex vivo organ perfusion. Such early-stage interventions, however, would need to be able to survive the cell proliferations which occur upon grafting to effectively prevent later-stage fibrosis: Epigenetic editing is uniquely suited to provide such a hit-and-run approach, allowing early intervention for long term effectivity. From a translational perspective, fibrosis is an unmet medical need affecting huge numbers of people worldwide via various diseases without effective treatment options. The identification of the key fibrosis players is not yet fully crystallized and gene expression modulation allows to identify and validate the key players. My intended studies will not only yield unique chromatin insights with respect to sustainability of epigenetic marks, but will have clear clinical relevance by validating novel anti/pro-fibrotic targets.
EpiGenie: Where do you see Epigenetic Editing heading in the next few years?
Dr. Rots: As a start, Epigenetic Editing will enter many research laboratoria as it is a convenient addition to siRNA and cDNA approaches likely to result in sustained modulation of gene expression without harmful integration of foreign DNA. As a matter of fact, the explosion on the use of CRISPR-Cas for genome targeting is clearly illustrating the need to interfere with gene function. As this CRISPR system can easily be adapted for Epigenetic Editing, the field will experience a burst in activities. Currently, several clinical trials are ongoing to explore the promise of genome editing by Zinc Finger-based nucleases, and I expect that the stage is soon set for Zinc Finger-based Epigenetic Editing therapeutics. Epigenetic Editing will provide ways to realize the full potential of the druggable genome concept, thereby adding to the quest of finding a cure for the uncurable.