Prion disease derives from the accumulation of the misfolded, toxic forms of the proteins called PrnP in the neurons, which might conclude with the death of neurons. Along with the fact that it is a fatal and rare degenerative disorder, there is no treatment yet. It is associated with the misfolded proteins, the chain reaction of which stimulates the improper folding of the others. This might occur spontaneously as well as arising from the genetic mutations. Recently conducted research reveals that decrease in the amount of misfolded proteins not only diminish the progression of the disease but also cease it to occur.
Neumann and his colleagues aimed to silence the PRNP gene causing the prion disease in order to reduce the production of such proteins. Nowadays in order to achieve this aim, DNA arranging technologies based on the CRISPR are utilized in the associated studies; however, these technologies include disadvantages in terms of demanding transfer into cells and toxicity resulting from the alteration in the DNA. The team resorted to epigenetic modulators due to the disadvantages desired to develop novel ones. This was merely because present epigenetic modulators were so large to transfer into the adeno associated viruses (AAVs) and likely to demonstrate toxicity. Therefore, they conducted research on the present epigenetic modulators called CRISPRoff and CRISPRi. In the light of this research, scientists revealed that the PRNP gene, found in both mice and the human, was appropriate for epigenetic silencing ,but this was not the case for the gene of interest. This was attributed to demanding transfer of CRISPRoff into the AAV due to the persistence of the large DNA binding sequence (dSpCas9), the eventuality of showing toxic and antigenic impact ,and diminution of the efficiency of CRISPRi in time. In the consideration of those reasons, a new epigenetic modulator called CHARM (Coupled Histone tail for Autoinhibition Release of Methyltransferase), which contains DNA binding site, histone H3 tail, and DNMT3L site, was developed. This epigenetic modulator activates the enzyme called DNMT3A whereby the gene expression is suppressed via methylation of CpG islands found in the promoter region of the PRNP gene. The active region of DNMT3A was used in the CRISPRoff.
On the other hand, in the CHARM, methylation occurred by resorting to a new cell mechanism in which present enzymes were activated via DNmt3L and the histone H3 tail. Notably, the function of the histone tail constitutes an importance since it binds the ADD site, which is found in an enzyme called DNMT3A and is in charge of suppression of this enzyme, through which the present enzyme is activated. In doing so, via the activation of a cell mechanism, the toxicity based on the excessive activation of a catalytic region as in the CRISPRoff was eliminated. Although CHARM was initially developed in the use of DNA binding side called dSpCas9, eventually alternative sequences such as TALE proteins or zinc finger proteins (ZFPs), which was designed by the Sangamo Therapeutics, were used due to the issue of the transfer into AAV. These binding sequences that occupy less space provide CHARM with optimization and customization. Scientists chose the use of ZFcharms composed of zinc finger proteins despite the facility in the design of the TALE proteins. ZFcharms were added to such sequences so that they are able to silence themselves. In parallel with that, not only the silencing of PRNP genes was maintained to some extent but also it restrained this mechanism from working excessively. In the case of the delivery of ZFcharms into the neurons in the central nerve system, scientists discovered that the expression of the prion protein in the brain reduced up to %80. Moreover, it is quite above the level of expression of proteins that is required to diminish the symptoms of the disease.
In conclusion, ZFcharms are the epigenetic modulators that can silence themselves, fit in the AAV, be customized, and activate the methylation mechanism in the cell without containing any enzyme. In the consideration of the success of the CHARMs in the field of silencing the PRNP gene, it might be deduced that CHARMs are promising therapeutic treatment when it comes to the central nerve system diseases derived from the accumulation of proteins such as not only prion but also Alzheimer or Parkinson.
Translated by Tuğçe Çayır
Editor: Elif Duymaz
Reference: Neumann, E. N., Bertozzi, T. M., Wu, E., Serack, F., Harvey, J. W., Brauer, P. P., Pirtle, C. P., Coffey, A., Howard, M., Kamath, N., Lenz, K., Guzman, K., Raymond, M. H., Khalil, A. S., Deverman, B. E., Minikel, E. V., Vallabh, S. M., & Weissman, J. S. (2024). Brainwide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor. Science, 384(6703). https://doi.org/10.1126/science.ado7082
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