Actinomycin D Specifically Reduces Expanded CUG Repeat RNA in Myotonic Dystrophy Models.

Myotonic dystrophy type 1 (DM1) is an inherited disease characterized by the inability to relax contracted muscles. Affected individuals carry large CTG expansions that are toxic when transcribed. One possible treatment approach is to reduce or eliminate transcription of CTG repeats. Actinomycin D (ActD) is a potent transcription inhibitor and FDA-approved chemotherapeutic that binds GC-rich DNA with high affinity. Here, we report that ActD decreased CUG transcript levels in a dose-dependent manner in DM1 cell and mouse models at significantly lower concentrations (nanomolar) compared to its use as a general transcription inhibitor or chemotherapeutic. ActD also significantly reversed DM1-associated splicing defects in a DM1 mouse model, and did so within the currently approved human treatment range. RNA-seq analyses showed that low concentrations of ActD did not globally inhibit transcription in a DM1 mouse model. These results indicate that transcription inhibition of CTG expansions is a promising treatment approach for DM1.

Biological Efficacy and Toxicity of Diamidines in Myotonic Dystrophy Type 1 Models.

Myotonic dystrophy type 1 (DM1) is a disease characterized by errors in alternative splicing, or "mis-splicing". The causative agent of mis-splicing in DM1 is an inherited CTG repeat expansion located in the 3' untranslated region of the DM protein kinase gene. When transcribed, CUG repeat expansion RNA sequesters muscleblind-like (MBNL) proteins, which constitute an important family of alternative splicing regulators. Sequestration of MBNL proteins results in the mis-splicing of its regulated transcripts. Previous work has demonstrated that pentamidine, a diamidine which is currently FDA-approved as an antiparasitic agent, was able to partially reverse mis-splicing in multiple DM1 models, albeit at toxic concentrations. In this study, we characterized a series of pentamidine analogues to determine their ability to reverse mis-splicing and their toxicity in vivo. Experiments in cell and mouse models demonstrated that compound 13, also known as furamidine, effectively reversed mis-splicing with equal efficacy and reduced toxicity compared to pentamidine.

Reducing levels of toxic RNA with small molecules.

Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded noncoding CTG/CCTG repeats, and these CUG/CCUG repeats sequester the Muscleblind-like (MBNL) family of RNA binding proteins. The MBNL proteins are regulators of alternative splicing, and their sequestration has been linked with mis-splicing events in DM. A previously reported screen for small molecules found that pentamidine was able to improve splicing defects associated with DM. Biochemical experiments and cell and mouse model studies of the disease indicate that pentamidine and related compounds may work through binding the CTG*CAG repeat DNA to inhibit transcription. Analysis of a series of methylene linker analogues of pentamidine revealed that heptamidine reverses splicing defects and rescues myotonia in a DM1 mouse model.

Neonatal DHT but not E2 speeds induction of sexual receptivity in the musk shrew.

Neural aromatization of testosterone (T) to estrogen during development is thought to be important for sexual differentiation of many altricial mammals. We evaluated the effects of neonatal injections of the non-aromatizable androgen dihydrotestosterone propionate (DHTP) and estradiol (E2) on the copulatory behavior of the female musk shrew, an altricial insectivore. Following adult ovariectomy and replacement T, animals were paired with a stimulus female for two 60-minute copulatory behavior tests. The latency to induce sexual receptivity (in the form of tail-wagging by the female), mount latency and total number of mounts were recorded in experimental females and in a group of untreated control males. While neither hormone treatment significantly affected mounting behavior, DHTP-treated animals induced receptivity faster and with latencies not significantly different from intact males, suggesting that early non-aromatizable androgens can have masculinizing actions by either increasing sexual motivation or making the treated animal more attractive to the stimulus female. Reliance on androgenic rather than estrogenic metabolites for the differentiation of courtship behaviors conforms to the pattern seen more typically in primates than rodents.