December 20, 2022
In partnership with The Hospital for Sick Children (SickKids), Cure Rare Disease is developing a battery of therapeutics to treat rare mutations of Duchenne muscular dystrophy. Read below to learn more about the specific mutations these therapeutics are targeting and our recent successes in correcting those mutations.
Exon 12-20 Duplication
DMD patients with a duplication in exons 12-20 experience a frameshift mutation, meaning that the reading frame of the gene has been disrupted. This therapeutic is currently in stage 2 of development; patient myoblasts have been cultured and genome sequencing has been completed. Using CRISPR/Cas9 technology, researchers at SickKids have developed a guide RNA (gRNA) that allows the Cas9 protein to cut between exons 15 and 16 of the original DNA sequence and the duplicated sequence, thereby restoring the reading frame. The Cas9 and gRNA were packaged into a lentivirus and delivered to myoblast cells. Initial testing has revealed wild-type dystrophin mRNA production and substantial dystrophin protein restoration. The Cas9/gRNA complex is also being packaged into an adeno-associated viral vector called AAV-DJ to be delivered to myotubes (differentiated myoblast cells). Next steps for this project include assessing off-targets and developing a mouse model.
Exon 44 Duplication
CRD’s exon 44 duplication therapeutic is also in stage 2 of development. Similarly to the exon 12-20 duplication, multiple gRNAs have been screened to determine efficacy. Patient myoblast cells were treated with a lentivirus containing the CRISPR/Cas9 system. Wildtype RNA was produced, and a western blot analysis revealed dystrophin protein expression being restored. Future steps include assessing off-targets and testing the Cas9/gRNA complex in myotubes.
Exon 33 Deletion
Our exon 33 deletion therapeutic targets a microdeletion of 4 base pairs within exon 33 of the dystrophin gene. The cell line was successfully corrected using a strategy called reframing. SickKids developed a gRNA molecule that restored the reading frame of the dystrophin gene 32% of the time by adding a single nucleotide. This resulted in significant dystrophin protein restoration as compared to untreated cells.
Overall, we are incredibly excited to share these recent advancements with the community. Having corrected these rare DMD mutations using CRISPR technology in patient cell lines gives us great hope that we can change the course of this disease for even the rarest of the rare. While this is an early accomplishment for these mutations, we march forward toward the clinical application of these technologies.
Our research and development is powered by the generous financial support of families, rare disease communities and organizations unwilling to accept the status quo of drug development practices. As a 501c3 non-profit biotech, 93+% of all donations go directly to the research and development of life-saving therapies.
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