Abstrakt

A new approach to introduction specific point mutations in the gene coding for the dystropin : PRIME editing technology

Cédric Happi Mbakam

Mutations in the dystrophin gene lead to neuromuscular dis¬orders such as Duchenne Muscular Dystrophy which is a lethal X-linked hereditary disease with the prevalence of 19.8 per 100 000 males’ birth. Currently available clinical therapies with corti¬costeroids or with morpholino antisense oligomer injections pro¬vide limited phenotypic improvement. Our study aimed to mea¬sure the PRIME editing technology efficiency. This technology uses a PRIME editor plasmid (PE2 or PE3) 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 target-ing several hDMD exons (9, 20, 35,43, 51, 55, and 61) to intro¬duce 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. Ex¬ons were PCR amplified and sequenced using the Sanger method. Results were analysed using the EditR program to estimate the ed¬iting 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 11 % (PE2) and 21% (PE3). Repeated transfections 6 days after the first one showed up to 15 % (PE2) edition in exons 9 and 35. An additional mutation in PAM sequence (exon 35) improved a PE2 result to 38% for a single transfection. Thus, PRIME editing permits the specific sub¬stitutions in the DMD gene and might be used to correct point mutations in the DMD gene to lead to dystrophin expression. 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 11 % (PE2) and 21% (PE3). Repeated transfections 6 days after the first one showed up to 15 % (PE2) edition in exons 9 and 35. An additional mutation in PAM sequence (exon 35) improved a PE2 result to 38% for a single transfection. Thus, PRIME editing permits the specific substitutions in the DMD gene and might be used to correct point mutations in the DMD gene to lead to dystrophin expression. This technology uses a PRIME editor plasmid (PE2 or PE3) cod¬ing 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 sub¬stitutions, deletions or insertions in the genome.