DM2 pathomechansims and therapeutic development

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are dominantly inherited muscular dystrophies caused by
short tandem repeat, or microsatellite, expansions in the noncoding regions of two different genes. While DM1
results from CTG trinucleotide expansions in the DMPK 3' untranslated region, DM2 is caused by structurally
similar CCTG tetranucleotide expansions in intron 1 of the CNBP gene. DM1 and DM2 share underlying
pathomechanisms since transcription of mutant DMPK and CNBP genes produces expanded CUG and CCUG
RNAs, respectively, that are pathogenic because they alter the activities of developmentally regulated RNA
processing factors while also serving as templates for repeat-associated non-AUG (RAN) translation.
Importantly, DM2 represents an emerging and prevalent class of STR expansion diseases caused by intronic
mutations. Therapeutic development for DM1 has been prioritized since it is the most common form of adult-
onset muscular dystrophy, and several mouse models such as HSALR have been used extensively to evaluate
potential therapies in vivo. In contrast, DM2 mouse models that replicate the DM2 clinical presentation have
not been reported. To address this deficiency, we have recently generated both an authentic DM2 mouse model,
or human CNBP-DM2 BAC transgenic mice, and an intensive DM2 model, HSA-DM2 mice which express 1,100
CTG repeats inserted into the highly expressed HSA gene. Preliminary studies show that CNBP-DM2 mice
reproduce DM2 clinical manifestations, such as repeat instability, nuclear RNA foci, centralized myonuclei,
muscle weakness, RAN proteins, while HSA-DM2 mice reproduce DM2 mis-splicing, myotonia and myopathy.
This project will use these new transgenic models to improve our understanding of DM2 pathomechanisms and
as a tool to accelerate therapeutic testing in vivo. Aim 1 will test the hypothesis that CNBP-DM2 transgenic lines
varying in CCTG repeat expansion length and insertion sites provide an authentic DM2 mouse model. CNBP-
DM2 mice with >4,200 repeats will be tested to determine if they reproduce key pathophysiological hallmarks
of DM2 disease and if RAN translation occurs in skeletal and cardiac muscle. Aim 2 will focus on HSA-DM2
transgenic mice to define DM2 biochemical and pathophysiological mechanisms and compare these outcomes
to the HSALR model for DM1. A key question is to understand why an apparently larger burden of CCUG RNA
repeats causes a milder phenotype than in HSALR mice. Aim 3 will use CNBP-DM2 and HSA-DM2 mice to test
two potential therapies for DM2, including cell penetrating phosphorodiamidate morpholinos (cpPMOs) and
MyoAAV-dCas9, that selectively target mutant CNBP RNAs and CNBP transcription, respectively.