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EXPERIMENTAL AND STRUCTURAL STUDIES OF FDA APPROVED EXON SKIPPING TREATMENT DRUGS
Title
EXPERIMENTAL AND STRUCTURAL STUDIES OF FDA APPROVED EXON SKIPPING TREATMENT DRUGS
Creator
Zhang, Jingwen
Date
2021
Description
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.
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COMPREHENSIVE ANALYSIS OF EXON SKIPPING EDITS WITHIN DYSTROPHIN D20:24 REGIONS
Title
COMPREHENSIVE ANALYSIS OF EXON SKIPPING EDITS WITHIN DYSTROPHIN D20:24 REGIONS
Creator
Niu, Xin
Date
2020
Description
Exon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to...
Show moreExon skipping is a disease modifying therapy that operates at the RNA level. In this strategy, oligonucleotide analog drugs are used to specifically mask specific exons and prevent them from being included in the mature mRNA. Exon skipping can also be used to restore protein expression in cases where a genetic frameshift mutation has occurred, and this how it is applied to Duchenne muscular dystrophy, DMD. DMD most commonly arises as a result of large exonic deletions that juxtapose flanking exons of incompatible reading frame, which abolishes dystrophin protein expression. This loss leads to the pathology of the disease, which is severe, causing death generally in the second or third decade of life. Here, the primary aim of exon skipping is to restore the reading frame by skipping an exon adjacent to the patient’s original. While restoring some protein expression is good, how removing some region from the middle of protein affects its structure and function is unclear. Complicating this in this case is that the dystrophin gene is very large, containing 79 exons. Many different underlying deletions are knowns, and exon skipping can be applied in many ways. It has previously been shown that many exon-skip edits result in structural perturbations of varying degrees. Very few studies are focused on the protein biophysical study and it is still basically unclear whether and how such editing can be done to minimize such perturbations. In order to provide the solid evidences which prove the significant variation among those cases (especially for the clinically relevant cases) and better understanding the general principles of “what makes a good edit”, we examine a systematic and comprehensive panel of possible exon edits in a region of the dystrophin protein. The domain D20:24 of dystrophin rod region are selected for its entirety which is separated by hinge region (mostly random coiled structure) and addition of other STRs will not disrupt the structure stability. Also D20:24 regions lie in the Hot Spot region II (HS2) which holds the most number of DMD patients. During the comprehensive scan, we identify for the first time, exon edits that appear to maintain structural stability similar to wild-type protein and those clinically relevant edits. Then we figure out the factors that appear to be correlated with the degree of structural perturbation, such as the number of cooperative protein domains, as well as how the edited exon structure interacts with the protein domain structure. Our study is the first systematic and comprehensive scan for an entire multiple STRs domain. This would help us understand the protein nature of various exon skipping edits and provide useful target for clinical treatment. Also the knowledge we learned may be applied to produce more sophisticated CRISPR edits in the future work.
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