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Abstract Details

REMEDY: A Novel CRISPR-based Allele Specific Approach Corrects VCP Mutations
Neuromuscular and Clinical Neurophysiology (EMG)
S21 - Inherited Myopathies and Neuropathies: New Therapeutic Approaches and Observations (2:00 PM-2:12 PM)

Heterozygous mutations in the VCP gene result in a combination of hereditary Inclusion Body Myopathy, Paget’s Disease of Bone, Frontotemporal Dementia and Amyotrophic Lateral Sclerosis named as VCP disease, or MSP. There is no cure for the disease, but one of the highly promising treatment strategies is CRISPR/Cas9 gene-editing technology.  

Valosin-containing protein (VCP) multisystem proteinopathy (MSP) is a neurodegenerative and ultimately fatal disease. Given the genetic background of the disease (dominant negative pathology) we invented a novel method called REMEDY for allele-specific targeting of heterozygous mutations.

We developed a strategy, called REMEDY, to precisely target and correct the mutant allele by recruiting the wild-type homologous chromosome as an endogenous template. We first tested this approach in patients-derived fibroblasts. We designed four allele-specific gRNAs and optimized the electroporation conditions to effectively deliver the gRNA-Cas9 complexes into the fibroblasts.  We then repeated the same experiment in myoblasts obtained from VCP transgenic mice.

In patient-derived fibroblast, three of the four gRNAs designed showed specificity to the mutant allele, and they were tested in VCPR155H/+ mouse myoblasts. All three gRNAs resulted in complete deletion of the mutant allele (100%). Interestingly, we observed a slight correction of the mutant allele as well, and without the need for an exogenous DNA template. Therefore, the frequency of the wild-type allele in the cells targeted by one of the gRNAs increased from 50% (normalized levels) to 58% measured by Sanger sequencing and next generation sequencing (NGS) and analyzed by ICE (interference of CRISPR Edits) and CRISPResso. 

We demonstrated precise targeting of the mutant allele in VCP patient-derived fibroblast and in mouse-derived myoblasts. Our approach has the potential to significantly advance the therapeutics and correct VCP disease in patients-derived cells.  

Eleonora D'Ambrosio (Nationwide Children's Hospital)
Dr. D'Ambrosio has nothing to disclose.
No disclosure on file
Zarife Sahenk (The Research Institute at Nationwide Childrens.org) Dr. Sahenk has nothing to disclose.
Jerry Mendell (The Research Institute at Nationwide Children's Hospital) Dr. Mendell has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Vertex. Dr. Mendell has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Novartis. Dr. Mendell has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Sarepta Therapeutics . The institution of Dr. Mendell has received research support from Sarepta.
No disclosure on file