Disrupting the Molecular Pathway in Myotonic Dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults. It consists of two forms: type 1 (DM1) and type 2 (DM2). DM1 is associated with a trinucleotide repeat expansion mutation, which is transcribed but not translated into protein. The mutant RNA remains in the nucleus, which leads to a series of downstream abnormalities. DM1 is widely considered to be an RNA-based disorder. Thus, we consider three areas of the RNA pathway that may offer targeting opportunities to disrupt the production, stability, and degradation of the mutant RNA.

Chronic Exposure to Cadmium and Antioxidants Does Not Affect the Dynamics of Expanded CAG•CTG Trinucleotide Repeats in a Mouse Cell Culture System of Unstable DNA

More than 30 human disorders are caused by the expansion of simple sequence DNA repeats, among which triplet repeats remain the most frequent. Most trinucleotide repeat expansion disorders affect primarily the nervous system, through mechanisms of neurodysfunction and/or neurodegeneration. While trinucleotide repeat tracts are short and stably transmitted in unaffected individuals, disease-associated expansions are highly dynamic in the germline and in somatic cells, with a tendency toward further expansion. Since longer repeats are associated with increasing disease severity and earlier onset of symptoms, intergenerational repeat size gains account for the phenomenon of anticipation. In turn, higher levels of age-dependent somatic expansion have been linked with increased disease severity and earlier age of onset, implicating somatic instability in the onset and progression of disease symptoms. Hence, tackling the root cause of symptoms through the control of repeat dynamics may provide therapeutic modulation of clinical manifestations. DNA repair pathways have been firmly implicated in the molecular mechanism of repeat length mutation. The demonstration that repeat expansion depends on functional DNA mismatch repair (MMR) proteins, points to MMR as a potential therapeutic target. Similarly, a role of DNA base excision repair (BER) in repeat expansion has also been suggested, particularly during the removal of oxidative lesions. Using a well-characterized mouse cell model system of an unstable CAG•CTG trinucleotide repeat, we tested if expanded repeat tracts can be stabilized by small molecules with reported roles in both pathways: cadmium (an inhibitor of MMR activity) and a variety of antioxidants (capable of neutralizing oxidative species). We found that chronic exposure to sublethal doses of cadmium and antioxidants did not result in significant reduction of the rate of trinucleotide repeat expansion. Surprisingly, manganese yielded a significant stabilization of the triplet repeat tract. We conclude that treatment with cadmium and antioxidants, at doses that do not interfere with cell survival and cell culture dynamics, is not sufficient to modify trinucleotide repeat dynamics in cell culture.

Drugging DNA Damage Repair Pathways for Trinucleotide Repeat Expansion Diseases

DNA damage repair (DDR) mechanisms have been implicated in a number of neurodegenerative diseases (both genetically determined and sporadic). Consistent with this, recent genome-wide association studies in Huntington’s disease (HD) and other trinucleotide repeat expansion diseases have highlighted genes involved in DDR mechanisms as modifiers for age of onset, rate of progression and somatic instability. At least some clinical genetic modifiers have been shown to have a role in modulating trinucleotide repeat expansion biology and could therefore provide new disease-modifying therapeutic targets. In this review, we focus on key considerations with respect to drug discovery and development using DDR mechanisms as a target for trinucleotide repeat expansion diseases. Six areas are covered with specific reference to DDR and HD: 1) Target identification and validation; 2) Candidate selection including therapeutic modality and delivery; 3) Target drug exposure with particular focus on blood-brain barrier penetration, engagement and expression of pharmacology; 4) Safety; 5) Preclinical models as predictors of therapeutic efficacy; 6) Clinical outcome measures including biomarkers.

Diagrammatic Theory of RNA Structures and Ensembles with Trinucleotide Repeats

ABSTRACTTrinucleotide repeat expansion disorders (TRED) are associated with the overexpansion of (CNG) repeats on the genome. mRNA transcripts of sequences with greater than 60 to 100 (CNG) tandem units have been implicated in TRED pathogenesis. In this paper, we develop a diagrammatic theory to study the structural diversity of these (CNG)n RNA sequences. Representing structural elements on the chain’s conformation by a set of graphs and employing elementary diagrammatic methods, we have formulated a renormalization procedure to resum these graphs and arrive at a closed-form expression for the ensemble partition function. With a simple approximation for the renormalization and applied to extended (CNG)n sequences, this theory can comprehensively capture an infinite set of conformations with any number and any combination of duplexes, hairpins and 2-way junctions. To quantify the diversity of different (CNG)n ensembles, the analytical equations derived from the diagrammatic theory were solved numerically to derive equilibrium estimates for the secondary structural contents of the chains. The results suggest that the structural ensembles of (CNG)n repeat sequence with n ~ 60 are surprisingly diverse, and they are dominated largely by open segments, with only a small fraction of the nucleotides forming base pairs. At the same time, the variance in the secondary-structural contents on the chains is also quite large, indicating that their structures can undergo strong equilibrium fluctuations and are expected to be rather suspectable to perturbations.STATEMENT OF SIGNIFICANCETrinucleotide repeat expansion disorders (TRED) are associated with the overexpansion of (CNG) repeats on the genome. mRNA transcripts of sequences with critical length greater than 60 to 100 (CNG) tandem units have been implicated in TRED pathogenesis, though their structures remain poorly characterized. Conventional view has tacitly assumed that conformations with maximal C:G base pairing dominate at equilibrium, but here we demonstrate that (CNG) repeat sequences are characterized by diverse ensembles of structurally heterogeneous folds and with a large variance of secondary structural contents. These ensembles of structures also undergo strong equilibrium fluctuations, rendering them rather susceptible to perturbations. These results were based on a novel diagrammatic approach to the ensemble partition function.