López Morató, Marta
(2018)
Investigating genome engineering of myotonic dystrophy cells and small molecules as potential therapeutic agents.
PhD thesis, University of Nottingham.
Abstract
Myotonic dystrophy (DM) is a dominantly inherited multisystem disease. There are 2 types of DM: DM1, caused by a CTG repeat expansion in the 3’-UTR of the DMPK gene, and DM2, caused by a CCTG repeat expansion in intron 1 of the CNBP gene. In both types of DM the transcribed RNA, containing the repeat expansions, accumulate in the nucleus and sequester alternative splicing factors, such as MBNL1 proteins, causing the missplicing of many genes and contributing to the disease pathogenesis. These aggregations accumulate in the nucleus in the form of foci and represent a key target for therapeutic development.
In the first part of this thesis I studied the relationship of foci with SC35 speckles and CDK12 protein in the nuclei of DM1 and DM2 cell lines. I also examined a subset of kinase inhibitors, which target mainly CDK12, for their ability to eliminate foci in DM2 cells. Additional effects of the two most potent inhibitors, Dinaciclib and SNS-032, were studied in both DM1 and DM2 cells. Both inhibitors reduce the number of SC35 speckles
in the nucleus, cause a redistribution of MBNL1 and reduce the levels of MBNL1 protein, but do not restore the alternative splicing of the genes analysed. Additionally, attempts were made to knockdown CDK12 in DM1 cells using the CRISPR/Cas9 system. The system achieved a 45% efficiency of cleavage of the target site in hygromycin-resistant clones. Nevertheless, the mutations introduced in the gene did not eliminate the CDK12 protein and they had an unexpected effect on foci production. Transient overexpression of CDK12 increased the number of foci in both DM1 and DM2 cells. Thus far, results suggest that CDK12 is involved in the transcription of the mutant transcripts.
The second part of this thesis focuses on the development of an inducible cell model for DM2 using the CRISPR/Cas9 system. The approach used here aims to overcome the difficulties associated with cloning of very unstable long repeat expansions. The exogenous inducible promoter was integrated into two clones, although in one clone
it was not in the right orientation and in the other, the end of the fragment was missing. Efficiency of the CRISPR system was around 57 to 72%, and many hygromycin-resistant clones showed reduced numbers of foci per cell, suggesting that the mutations in intron 1 of CNBP compromise the transcription of the gene and the production of the mutant transcript.
Thus, the experiments described in this thesis have made a contribution to improve understanding of DM disease pathogenesis and provide platform for further studies.
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