Exploring the Ethical Puzzle of Epigenome Editing

The CRISPR-Cas9 system rose to prominence as a simple but precise gene-editing tool. Almost 10 years after its discovery, it has been adapted for an ever-increasing number of applications, including RNA editing, base and prime editing, diagnostics, and live imaging. One of the nascent applications of CRISPR technology is epigenome editing – altering the epigenetic marks that regulate the expression of genes.

With every novel application of gene editing technology, however, comes a raft of moral and ethical considerations, and many scientists believe we must address these quandaries sooner rather than later.

Dr. Nikolajs Zeps is a former cancer biologist, having worked as research director of medical institutions including Epworth Healthcare and Saint John of God Healthcare. Currently, Zeps works as an ethicist and partner at Chrysalis Clinical, holding adjunct professorships at Monash University and Curtin University, both in Australia.

Collaborating with colleagues from several universities in Singapore, China, the United Kingdom and the United States, Zeps explores a range of potential issues that could arise from the application of CRISPR for epigenome editing in a recent forum paper published in Stem Cell Reports.

The paper, as Zeps describes it, is really an opinion piece, and the authors didn’t shy away from difficult and speculative topics. Zeps and his colleagues argue that epigenome editing (e-GE) in somatic cells should be viewed through a completely different lens than heritable (germline) CRISPR editing.

The basics of epigenome editing

Epigenetic editing with CRISPR involves conjugating catalytically dead Cas9 (dCas9) with epigenetic enzymes or their catalytic domains (CDs). The DNA-binding ability of dCas9 allows the epigenetic enzymes or CDs to be targeted to specific loci for transcriptional reprogramming; genes can be downregulated (‘switched on’) or upregulated (‘switched off’).

For example, transcriptional repression is modulated by DNA methylation (5mC) at CpG islands in the promoter regions of genes. Fusing dCas9 to a DNA methyltransferase (DNMT) enzyme, such as DNMT3A, can induce methylation on the promoter region of a target gene, which will prevent transcription and silence the gene.

In contrast, demethylation of DNA is controlled via oxidation of 5mC by ten-eleven translocation dioxygenases (TETs). In the case of genes that are repressed by 5mC, dCas9 can be fused to TET1CD to induce targeted demethylation and upregulate expression of the gene.

A discussion of recent progress in CRISPR-based epigenome editing can be found in this 2020 review, published in Current Opinion in Chemical Biology. Currently, no clinical trials using e-GE have been registered, however, e-GE has been achieved both in vitro in human cell lines such as HEK293 cells and in primary animal cells, and in vivo in whole animal models. Because e-GE is only a relatively short time away from clinical trials in human subjects, the ethicist in Zeps saw the recent progress in this field as a perfect opportunity to take on a challenging topic.

»We’re trying to link two different concepts, and give a snapshot summary to people who aren’t necessarily experts in either field – the scientists we’re giving a snapshot into the ethics world and the ethicists we’re giving a snapshot into the science world, and that’s quite a difficult thing to do,« Zeps says.

The authors explore three broad approaches to the clinical translation of somatic cell e-GE in their paper: targeting disease genes directly, augmenting existing therapies, and enhancing desirable phenotypic traits.

A new method for treating sickle cell disease?

The paper also considers the possibility of using e-GE as an alternative method for treating several different genetic disorders by altering the methylation patterns of gene promoters and restoring gene function. Examples of diseases that could be treated in this manner include Fragile X syndrome, Huntington disease, spinocerebellar ataxia, and myotonic dystrophy. Some rare genetic conditions could also be theoretically treated using e-GE by activating silenced alleles – this includes Angelman, Prader-Willi, Pitt-Hopkins, and Rett syndromes.

According to the authors, e-GE could also be employed to derepress genes that are aberrantly silenced and to upregulate genes that can compensate for those which carry pathogenic mutations. In the case of sickle cell disease (SCD), for example, e-GE could be used to ‘switch on’ foetal haemoglobin, which can compensate for a lack of adult haemoglobin in SCD patients.

This method of treating SCD would bypass the need to use CRISPR-Cas9 to make any heritable genetic changes. While this has not yet been attempted in humans, it is certainly feasible. The challenging aspect of e-GE, like any CRISPR application in vivo, is the delivery of editing components to the correct cell type – for such a therapy to work, progenitor cells would need to be targeted.

»No-one will know for sure [if e-GE can cure SCD] until we try it. Erythrocytes have a finite lifespan, so it’s the progenitor cells that we need to transform. The question is, can you actually edit those progenitor cells? If you can’t, then you’ll have to continue giving the treatment regularly over long periods. In that case, the necessary questions are: will it be a transfusion, when do you administer it, what is the mechanism of delivery?« Zeps ponders.

Supplementing existing therapeutic strategies with e-GE

Zeps and his colleagues suggest that e-GE can be used to supplement current treatments for disease, including drug-resistant cancers and certain neurological disorders. In theory, it could be used in any condition in which there is a resistance to other treatments based on a modifiable factor.

»If a person can’t get bioavailability of a drug, because they metabolise it too quickly, or the drug is toxic because they don’t metabolise it adequately, could you use CRISPR to essentially titrate it more effectively? It’s within the realm of possibility. If you can affect the expression of a liver metabolic enzyme, switch it on or off, you could alter the bioavailability of drugs,« Zeps elucidates.

Another exciting avenue for e-GE research is to supplement existing cancer treatments. e-GE can be used to reduce expression of oncogenes, or increase expression of tumour suppressor genes. These treatments could prevent or reduce tumour growth in many types of cancer, including breast and gastric cancers, which are influenced by epidermal growth factor receptor (EGFR) mutations.

Epigenetic ‘doping’: An undetectable performance enhancer?

One of the concerns the authors discuss is the potential application of epigenetic editing to enhance performance in competitive sport. While current performance enhancers are easily detectable by testing, this may not be the case for epigenetic ‘doping’.

As a specific example, recombinant erythropoietin can be taken as a performance enhancer, but it is easily tested for. e-GE could potentially be used to upregulate erythropoietin to increase athletic performance, and this may not be detectable with any currently available testing procedures.

»There’s a whole argument there – if you could do these things, and you could, for example, upregulate your haemoglobin or erythropoietin, and then the question is, would it be detectable in the same way that taking recombinant erythropoietin is? What if you could enhance muscle development, or downregulate your pain receptors?« Zeps speculates.

The big questions: heritability and social justice

Currently, e-GE technology is still in the early stages, but there is certainly progress being made towards testing in human subjects. The uncertainty surrounding the application of this technology is valid, Zeps says, but public reactions to the use of genome-editing tools of any kind in human subjects can be excessive.

»When new technologies emerge, there’s often a reaction to the most extreme version of it, which would be germline editing and selection of human embryos for particular characteristics. Of course should be no circumstances in which you can select the life of a person on the basis of their genetic traits. So then the question becomes, where do you draw the line?«

One of the controversies associated with any type of genome editing is heritability, so Zeps and his colleagues wanted to address the idea that epigenetic editing is heritable. After an exhaustive review of the literature, the team found several lines of evidence that suggest altering the epigenome of somatic cells is likely to have no long-term effects on the germline.

»There were some [scientists] who proposed that if you make changes to the methylome, that’s inheritable, so we wanted to investigate this – what evidence is there that you actually do see germline levels of inheritance [of these changes]? And there’s some highly controversial views around this in the literature. But there’s no firm evidence that if you make somatic changes, it’s any different to giving someone a cytotoxic chemotherapy that could potentially alter their gametes that might be used to develop an embryo,« Zeps elaborates.

Based on these findings, Zeps and his colleagues emphasise that e-GE of somatic cells is akin to other somatic cell therapies in terms of heritability and risk, and suggest that it should therefore be subject to the same ethical guidelines, rather than the much stricter regulations which apply to heritable CRISPR editing.

The application of e-GE in human subjects also raises questions of public availability, economic viability, and social equality – if the treatments are expensive and require regular administration, they may only be available to those patients of higher socio-economic status. Zeps agrees this must be carefully considered, however, this should ideally be the subject of a separate paper.

»We started to consider equity and access, and the justice components of these technologies. For example, should they be made more publicly available? You can argue that about all forms of healthcare, really, it’s not just limited to genetic engineering and technological advances. But that wasn’t something we could really do justice to in this article,« Zeps explains.

Checks and balances

Zeps and his co-authors believe that any form of CRISPR genome editing should be carefully regulated and weighed against associated risks, but they make a convincing argument that e-GE does not carry the same ethical dilemmas of CRISPR germline editing and is therefore a highly desirable alternative to conventional CRISPR gene editing.

»Because this is such a profound issue in terms of how the community thinks about it, you should only [perform germline editing] when you can’t do it any other way. But if you could perform somatic cell alteration using methylation of adult cells, why wouldn’t you just do that? You avoid all the controversy of germline editing. Highlighting the fact that methylation is a pathway, that you can alter the methylome and that it’s reversible - as a scientist, why wouldn’t you want to do that more than editing embryos?« Zeps comments.

Despite being a proponent of e-GE, Zeps is an ethicist at heart, and is primarily concerned with patient safety and informed consent. He cautions that many risks – both in terms of patient safety and social justice – are largely unknown, and insists that discussing the potential ethical issues of e-GE now is paramount if we are to avoid potential problems once it is applied in the first human subjects.

»The overarching message is, we should think about the things that we do carefully before we do them. Those who are engaged in science should equally have a responsibility to think about the kind of outcomes that could be generated. The field’s moving much faster than people can keep up with.« Zeps concludes.

Link to the original article in Stem Cell Reports:

Ethics and regulatory considerations for the clinical translation of somatic cell human epigenetic editing.