Abstract-Text (inkl. Referenzen)
Collagen VI congenital muscular dystrophy (COL VI-CMD) is a rare spectrum disorder and there is still no treatment. The goal of our project is to establish a therapeutic approach using CRISPRoff targeting dominant-negative acting glycine substitutions in COLVI-CMD. CRISPRoff utilizes a dCas9 that lost its nuclease-function and is coupled to methyltransferase domains. Guided to regulatory elements of the target gene it methylates these regions leading to a potential heritable expression knockdown (Nuñez et al, 2021). Since disease-causing variants are usually very rare, almost private, we want to develop a therapeutic approach that is allele-specific but not mutation-specific using heterozygous common variants to differentiate between the alleles.
Thus, our first step was to phase candidate common variants located in regulatory elements of COL6A2 with pathogenic mutations using nanopore sequencing. After the design of allele-specific gRNAs, we tested different approaches of delivery methods and are now using engineered virus-like particles to deliver CRISPRoff-RNPs into patient-derived primary fibroblasts. The effect of the treatment on RNA and DNA level are assessed by qPCR and modification-sensitive nanopore sequencing.