Date of Award

5-1-2024

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biological Sciences

Dissertation/Thesis Chair

John Andrew Berglund

Committee Members

Morgan A Sammons, Cara T Pager, Kaalak Reddy, Charles Thornton

Keywords

Disease modifiers, Myotonic Dystrophy, Small molecule therapeutics

Subject Categories

Molecular Biology

Abstract

The expansion of microsatellite repeats within the human genome has been identified as the cause of numerous hereditary diseases, including myotonic dystrophy (DM). DM is an autosomal dominant disease with a worldwide prevalence of ~1:8000 individuals. DM presents multisystemic symptoms such as myotonia, cardiac conduction defects, muscle wasting and weakness, insulin resistance, and cataracts. There are two genetically distinct, yet clinically similar types of DM: DM type 1 (DM1), caused by CTG repeat expansions in the 3’ UTR of the dystrophia myotonica protein kinase (DMPK) gene and DM type 2 (DM2), resulting from CCTG repeat expansions in the first intron of the CCHC-type zinc finger nucleic acid binding protein (CNBP) gene. When transcribed, these repeat expansions produce toxic expansion RNAs that sequester RNA binding proteins (RBPs), like the muscleblind-like (MBNL) family of alternative splicing regulators, leading to the loss of their function. Dysregulation of splicing (mis-splicing) is one of the effects from the expanded toxic RNAs and has been correlated to multiple symptoms observed in DM. While there is currently no cure or effective treatment for DM, different therapeutic approaches are currently being explored for the disease. Our lab has focused on the design and testing of small molecules, such as diamidines and microtubule inhibitors, that have been shown to reduce the toxicity of the expanded repeats. Unfortunately, many of these compounds are toxic, have significant off-target effects or only demonstrate modest splicing rescue. Based on our previous work we have designed and produced a series of novel small molecules referred to as modified polycyclic compounds (MPCs). To assess which MPCs could serve as potential DM1 therapeutics, we screened more than twenty of these new compounds in patient derived fibroblasts for their ability to rescue DM1 mis-spliced exons. We have identified four MPCs which partially rescued mis-splicing in DM1 fibroblasts and based on a splicing score metric selected three of the MPCs for further testing in patient derived myotubes. Our lead MPCs, MPC03 and MPC04, were able to partially rescue mis-splicing at nanomolar concentrations in both fibroblasts and myotubes while showing minimal effects on cell viability. We also assessed global effects of MPCs through RNA sequencing of myotube samples and observed rescue of DM1 associated mis-splicing. We tested our lead MPCs in a DM1 mouse model which contains expanded CTG repeats in a human skeletal actin transgene (HSALR). In a five-day pilot study, MPC03 and MPC04 were able to decrease the transgene levels of expanded CUG repeats in this mouse model and partially rescued mis-splicing. Based on their activity in human cell models with minimal cell viability effects and the reduction of the CUG transgene in the HSALR, MPCs are a promising class of small molecules for DM1. Another aspect of DM1 explored in this dissertation is the existence of disease modifiers as a possible explanation of the heterogeneity observed in DM1 in terms of severity and symptoms. Based on a screen in a CUG repeat expansion HeLa cell line, we observed that knock down of core spliceosome components can modulate expanded CUG RNA levels and reduce the severity of mis-splicing. These findings were also observed in DM1 fibroblasts and myoblasts as knockdown of Small nuclear ribonucleoprotein D2 (SNRPD2) reduced DMPK transcript levels and shifted inclusion levels of exons mis-spliced in DM1 towards unaffected levels. In summary, the research presented in this thesis identified potential new therapeutics for DM as well as showing that components of the spliceosome can act as modifiers in patient-derived cell models. Future studies will determine the potential for MPCs to move into clinical studies and the role of spliceosome components to act as modifiers of the disease.

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