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Title: Epigenetic modification of the frataxin gene for the treatment of Friedreich's ataxia
Author: Enriquez, Maria Gabriela Vilema
ISNI:       0000 0004 7966 2751
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Friedreich's Ataxia (FRDA) is a recessive neurodegenerative disease caused by the partial silencing of the frataxin gene (FXN). Long GAA triplet repeat expansions in the first intron of FXN are responsible for the reduced levels of frataxin, an essential mitochondrial protein involved in iron-sulphur cluster biosynthesis. The precise mechanisms of FXN silencing remain unclear, but in the last few years, epigenetic silencing of FXN was reported to have a major role in the development of FRDA. The presence of expanded GAA repeats has been shown to induce a repressive heterochromatin environment at the FXN locus, with increased levels of methylated histones H3K9me2/3, H3K27me3 and H4K20me3, as well as reduced acetylation of histones H3 and H4, demonstrating epigenetic repression of FXN. In this thesis, first HDAC3 and G9a were targeted by siRNA-mediated knockdown to increase overall acetylation and reduce methylation and promote transcription of FXN. Using a human FXN-GAA-Luciferase (FXN-GAA-Luc) repeat expansion reporter model of FRDA, HDAC3 knock-down was shown to increase FXN mRNA levels with no effect on protein, while G9a knock-down did not show an effect on either FXN mRNA or protein levels. Next, the Structural Genomics Consortium epigenetic compound library set was screened in the FXN-GAA-Luc reporter model of FRDA to identify compounds that can restore FXN expression. Amongst the hits was a group of compounds targeting methyltransferases which increased frataxin protein expression, assessed by the FXN-GAA-Luciferase assay. Concentration-response curves and direct siRNA-mediated knock-down of the targets showed that the methyltransferases SUV4-20 play an important role in the silencing of FXN. The specific inhibitor of SUV4-20, the probe A-196, was then tested in FRDA patient-derived fibroblasts and was able to increase the expression of mature FXN protein by 1.5-fold in the FRDA line GM04078. When A-196 was tested for its ability to modify a wide range of cellular phenotypes in FRDA patient cells, such as altered mitochondrial membrane potential and mitochondrial ROS generation, it showed only modest effects that did not reach statistical significance. Therefore, in collaboration with Oxford SGC, more stable and potent A-196 analogues were synthesized and tested in the FXN-GAA-Luciferase reporter cell line, and two new variants were found to increase FXN protein expression. In summary, the work has defined SUV4-20 as a novel therapeutic target for FRDA and identified compound A-196 as a potential small molecule candidate for hit-to-lead development as a new drug for this fatal disease.
Supervisor: Wade-Martins, Richard Sponsor: Secretaría de Educación Superior ; Ciencia ; Tecnología e Innovación
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Neurosciences ; Epigenetics ; Friedreich's ataxia