Epigenetic therapies and addiction
potential therapeutic were to alter the regulation of just this one gene, however, a far more directed approach than simply inhibiting an array of different epigenetic enzymes would be required.
Fortunately, in recent years it has been discovered that the Cas9 enzyme (most commonly used in genetic engineering) can be repurposed by replacing its ability to cleave DNA with a different catalytic effect, depending on the researchers’ needs, resulting in a so- called ‘dead Cas (dCas9)’ (Ganesan et al., 2019). With this technology, modulation of gene expression could be achieved with far more precision and predictability, making reprogramming of the epigenome far more feasible
Figure 5 – Reprogramming of Cas9 to carry out epigenetic alterations instead of cleaving DNA. dCas9 is still able to be recruited to a gene with the same specificity as Cas9.
than before (Figure 5) (Geel et al., 2018). Indeed, recent studies which implemented the catalytic domain of the TET1 enzyme, capable of demethylating DNA (and thus increasing expression of a specific gene), into dCas9 systems found that target genes could be successfully demethylated by as much as 90% (Morita et al., 2016; Choudhury et al., 2016). Meanwhile, another study in the same year found that dCas9 could alternatively be conjugated with the DNMT3a catalytic domain to introduce entirely new methylation onto desired sequences, thus limiting their expression (Liu et al., 2016). Together, these two different uses of dCas9 provide a powerful tool with which further research, and possibly the development of future epigenetic treatments, may be achieved. However, one concern with using CRISPR-Cas9 systems for epigenetic modifications is its propensity for causing ‘ significant off-target mutagenesis’ (Fu et al., 2013) to regions of a patient’s DNA with sequences that are similar, but not identical, to the intended target gene (Anderson et al., 2018). Whilst it has so far been observed that ‘off - target epigenetic modifications are usually transient’ (Brezgin et al., 2019), and thus are unlikely to tangibly alter the expression of incorrect genes, any apprehension surrounding the possibility of inadvertent outcomes in patients could hinder development or even outright preclude such therapies from regulatory approval until extensive research has been carried out into safety precautions. One possible solution to CRISPR- Cas9’s lack of true specificity would be to limit the use of modifications to ‘truly unique genomic sequences’ (Davies, 2019), thereby preventing any risk of off-target genes being affected. However, in order to realistically achieve this, each individual patient would require personalized genome sequencing and tailored design of the therapeutic in order to account for the natural variation in genetic sequences that exist from patient to patient (Davies, 2019). Similarly, the use of ‘variant - aware’ guide RNAs, which a re precisely engineered to direct dCas9 activity away from unintended regions of the genome, may appear to be a viable solution, but they still do not eliminate the dependence on personalized genome sequencing, making the future potential for clinical application of dCas9 technology somewhat ambiguous for now (Lessard et al., 2017). In conclusion, research into the epigenetic mechanisms responsible for morphine addiction has thus far returned highly diverse results, with some studies providing promise for future therapeutic applications, and others posing more questions about the safety of certain approaches to treatment. Regardless, in various cases, it still remains unknown as to exactly how long certain drug-induced alterations in neuronal gene expression persist, making it somewhat harder to justify targeting a given
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