OR05

Efficient in vitro correction of a highly recurrent COL7A1 pathogenic variant using Cytosine Base editing to treat recessive dystrophic epidermolysis bullosa

M Hautbois(1) A Peynet(2) M Nouvel(1) C Masson(2) C Bole(2) M Titeux(1) A Hovnanian(1) A Izmiryan(1)

1:Laboratory of Genetic skin diseases, INSERM UMR 1163, Imagine Institute, Paris, 75014, France; 2:Bioinformatics Platform. Imagine Institute, Paris, France 3: Genomics Platform. Imagine Institute, Paris, 75014, France

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease resulting in blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of variants in COL7A1 encoding type VII collagen (C7), the major component of anchoring fibrils that form key attachment structures for dermal-epidermal adhesion.

Here, we achieved highly efficient COL7A1 correction in primary RDEB cells using Cytosine Base editors (CBEs). We designed four guide RNAs (gRNAs) targeting c.425A>G (p.K142R), a highly recurrent variant changing the last nucleotide of exon 3.

Four in vitro transcribed CBEs together with four chemically modified gRNAs were delivered as mRNA by nucleofection to RDEB keratinocytes and fibroblasts (K and F) in culture. Two gRNAs showed up to 73 and 91% editing in RDEB cells at the gDNA level, as evaluated by Sanger and high-throughput sequencing. RT-PCR and sequence analysis showed the presence of a correct transcript in gene-edited RDEB-F. Assessment of C7 protein expression and secretion after editing revealed levels of restored C7 similar to C7 in normal human cells. Rescued C7 protein expression was also confirmed by immunofluorescence staining.

We concluded that COL7A1 editing and desired phenotypic correction (up to 91%) could be achieved in primary RDEB-K and F by base editing using combined gRNA and CBE mRNA delivery.

Grafting of genetically corrected 3D skin equivalents onto nude mice is ongoing to demonstrate functional correction for future ex vivo clinical applications.