FP.42 Correction of point mutations in the DMD gene using the prime editing

Abstract

Duchenne muscular dystrophy (DMD) is a X-linked hereditary disease responsible for serious burden, pain and damages to patients and their families. About 20 000 children new cases are diagnosed worldwide each year. Existing experimental treatments with antisense oligonucleotides or aminoglycosides provide limited phenotypic improvement. Our study aimed to experiment the CRISPR-Cas9 PRIME editing technology as therapeutic approach. This technology uses a PRIME editor plasmid (PE2) coding for a Moloney murine leukemia virus reverse transcriptase fused with the Cas9 nickase, and a plasmid coding for a prime editing guide RNA (pegRNA) containing in addition to a sgRNA, a primer binding site (PBS) and a reverse transcriptase template (RTT). This system permits specific nucleotide substitutions, deletions or insertions in the genome. We first designed different pegRNAs targeting several human DMD exons (6, 9, 20, 35, 43, 52, 55, 59 and 61) responsible for DMD in Canadian population to introduce a STOP codon by modifying a single nucleotide. We next designed other pegRNAs for the correction of c.428 G>A and c.8713 C>T point mutations respectively in DMD exon 6 and exon 59 in patients' human myoblast. HEK293T and myoblast cells were harvested three to five days post treatment with PE2 and pegRNA plasmids. Parts of myoblasts were transformed into myotubes to obtain proteins for western blot analysis. Fragment of targeted exons were PCR amplified and sequenced by Sanger and Illumina deep sequencing method. Results were analysed using the EditR and CRISPRESSO programs to estimate the editing percentage. We confirmed that PRIME editing permitted the specific C˃T, A˃G, G˃C, G˃A and G˃T substitutions in the DMD gene with an editing efficiency between 3 to 9% and 6 to 21% respectively using PE2 and PE3 approach in HEK293T cells creating point mutations. An additional mutation in the PAM sequence permitted to achieve up to 40% modification. For the correction of point mutations in patients' myoblasts, the modification of RTT length in combination to synonymous additional mutations around the target mutation showed up to 23% correction for a single electroporation with dystrophin expression detected by western blot. Thus Prime editing permits the correction of point mutations in the DMD gene.