LATE BREAKING NEWS E-POSTER PRESENTATION: LBP 6 PRIME editing permits to introduce specific point mutations in the gene coding for dystrophin.

Abstract

Duchenne Muscular Dystrophy is a X-linked hereditary disease with the prevalence of 19.8 per 100 000 males’ birth caused by mutations in the DMD gene coding for dystrophin. Currently available clinical therapies with corticosteroids or with morpholino antisense oligomer injections provides limited phenotypic improvement. Our study aimed to experiment the PRIME editing technology. This technology uses a PRIME editor plasmid (PE2) coding for a Moloney murine leukemia virus reverse transcriptase fused with the Cas9 H840A nickase, and a plasmid coding for a pegRNA containing a primer binding sites (PBS) and a reverse transcriptase template (RTT). It permits specific nucleotide substitutions, deletions or insertions in the genome. We designed different pegRNAs targeting several hDMD exons (9, 20, 35 and 61) to introduce a STOP codon by modifying a single nucleotide. HEK293T cells were harvested from DMEM culture media three days after being simultaneously transfected with the PE2 and pegRNA plasmids. Parts of the targeted exons were PCR amplified and sequenced with the Sanger method. Results were analysed using the EditR program to estimate the editing percentage. We confirmed that PRIME editing permits the specific C to T and G to T substitutions in the DMD gene with an editing efficiency between 6 to 9%. Repeated transfections 6 days after the first one showed up to 15 % edition in exons 9 and 35. Thus PRIME editing permits the specific substitutions in DMD gene and might be used to correct point mutations in the DMD gene to lead to dystrophin expression.