PRE-CLINICAL DEVELOPMENTS IN NEUROMUSCULAR DISORDERS: O.16 Allele-specific gene editing inactivates a dominant-negative, disease-causing, single nucleotide variant in COL6A1 through non-homologous end joining

Abstract

Allele-specific suppression of dominant-negative pathogenic variants via gene editing is a rational approach to treat dominant genetic conditions in which haploinsuffiency is tolerated. Here, we explored a simple and straightforward approach of introducing random frameshifting edits via non-homologous end joining to selectively silence a dominant allele. We focused on a common, single-nucleotide dominant-negative glycine substitution in the COL6A1 gene (c.868G>A; G290R), that causes dysfunction of collagen VI in the extracellular matrix and is associated with a severe form of muscular dystrophy. We nucleofected primary fibroblasts derived from four patients and one control subject with a spCas9-GFP-expressing plasmid that contained one allele-specific guide RNA (gRNA), without providing a repair template. By expanding clonal populations following CRISPR editing, we found that a collagen VI matrix was re-established when a frameshifting edit was stably incorporated on the pathogenic allele, providing proof-of-principle for this strategy. Using a deep-sequencing method, we examined the indel profile generated by the gRNAs and determined that a single frameshifting cytosine deletion accounted for the majority of edits introduced at either allele, across the four patient lines. While the gRNA activity was 2-fold greater at the variant allele, complete allele selectivity was not attained in this experimental framework. Nevertheless, collagen VI matrix deposition was increased in a dish where cells were kept as a mixed population after gene editing, suggesting that the subset of cells that were effectively corrected by this gene editing approach was sufficient to improve the cellular phenotype. To increase allele selectivity, we are currently testing a series of gRNAs that contain a second mismatch. This study strengthens the potential of gene editing to treat dominant disorders, but also underscores the challenge in achieving allele selectivity with gRNAs.