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- EXPERIMENTAL AND STRUCTURAL STUDIES OF FDA APPROVED EXON SKIPPING TREATMENT DRUGS
- Zhang, Jingwen
DMD is an X-chromosome related genetic disease caused by loss of dystrophin protein expression, and which impacts 1 in 5000 boys born in the...
Show moreDMD is an X-chromosome related genetic disease caused by loss of dystrophin protein expression, and which impacts 1 in 5000 boys born in the world. The usual cause of this at the genetic level is a frame shift due to internal deletions of one or more exons that results in a change of the reading frame. This results in loss of expression of the protein encoded by this gene, dystrophin, which in turn leads to the disease phenotype. Exon skipping is a therapy for DMD which restores dystrophin pre-mRNA reading frame to produce a modified dystrophin. This is done by antisense oligonucleotides, AONs, which disturb the process of exon splicing and exclude targeted exons near the patient’s defect which restore the correct reading frame in the pre-mRNA transcript. In 2016, the first AON was approved for clinical use targeting exon 51, called eteplirsen. This provided the first disease modifying therapy for DMD, but it was only relevant to ~6% of patients who had defects that were correctable by skipping this specific exon. In 2020 two more AONs targeting exon 53 were developed, viltolarsen and golodirsen, providing benefit to an additional 5% of patients, and in 2021 casimersen targeting exon 45 was approved.However, this raises an interesting issue, in that for some patients, with an exon 52 deletion, skipping exon 51 or skipping exon 53 could both restore the reading frame. Which approved exon skipping treatment is better and the differences between them are still unknown. This is the aim of this study: to help patients figure out which AON can have a consequence of less long-term health problems like cardiomyopathy and longer life and get more precise treatment. We selected three exon skipped edits – two that represent exon 53 skipping repair of an underlying Δe52 defect and one targeting exon 51 skipping repair of a Δe52 defect. We then used a panel of biophysical and biochemical including dynamic light scattering, circular dichroism Spectroscopy, thermal denaturation, and protease K challenge to investigate the biophysical characteristics of these different exon skipped edits. From our results we found that Δe51-52 has the more structure (i.e., is less perturbed), compared to e52-53, as assessed by CD or by proteinase K challenge, but it also has lower thermal stability, with a low Tm=48C transition that begins to unfold at the physiological relevant temperature of 37C. On the other hand, e52-53 has less helical structure, but what structure did form had unfolding transitions in the normal range for wild type STRs, Tm> 60C; but this edit also had more non-helical structure. So, the total experimental results of these three edits are very complex, which may be due to the fact that these edits span the normally unfolded H3 region.