Duchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a... Show moreDuchenne muscular dystrophy (DMD) is a degenerative genetic disease caused by a genetic defect that results in the absence of dystrophin, a protein with an important
stabilizing role in muscle cells. DMD causes progressive muscle degeneration leading to
the loss of ambulation, and typically results in death before the third decade of life.
Treatments for DMD aim to restore dystrophin expression and typically do so by producing
edited or modified dystrophins. The only FDA approved therapy, exon skipping, produces
dystrophin edits at exon boundaries but emerging therapeutic approaches like gene
replacement therapy and CRISPR-Cas9-based gene editing techniques like CinDel allow
for greater flexibility and are not constrained to exon boundary edits. However,
understanding of what makes a “good”, functional edit is limited so it is not clear how to
make use of this increased flexibility to produce optimal edits which are believed to be
necessary for robust treatment. In an effort to improve understanding of the biophysics of
these non-exon edits, we have embarked on a mixed experimental and computational study
of a set of CinDel edits in the D19-D21 region of the dystrophin central rod domain. First,
we have conducted an Alphafold structure prediction-based screen of a subset of possible
edits in this region and selected one edit for follow-up characterization. We then compared
this computationally-selected edit to three other heuristically designed edits experimentally
and computationally by molecular dynamics simulations.
We found that the computationally selected edit is significantly more
thermodynamically stable than the other edits in the cohort. This edit also generally
exhibited more favorable properties in MD simulations across multiple measures such as
helicity, STR-junction unwinding and conformational variability. Show less
