849 Twin prime editing for restoring type VII collagen expression in primary recessive dystrophic epidermolysis patient cells

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe mucocutaneous disease caused by loss-of-function mutations to the collagen VIIa (COL7A1) gene that encodes type VII peptide (C7). Previously, we used programmable nucleases and DNA base editors (BE) repair COL7A1 through homology-directed (HDR) or deamination, respectively. These efforts represent personalized medicine approach; however, defined reagents with applicability multiple patients desirable. Work has shown exons can be excised create in frame deletions leading restored C7 production. Nucleases generate double-stranded breaks (DSBs) have been accomplish this; DSBs are toxic cell repaired stochastic manner limit efficiency. Prime editing (PE) Cas9-based platform an associated fused reverse transcriptase primes off user provided prime editor guide (peg) RNA accommodate any substitution insertions deletions. Twin (tPE) employs two tandem pegRNAs serve remove large sequences without significant occurrence of DSBs. We tPE delete exon containing inactivating mutation. A single round transfer showed heterozygous targeting restoration primary, patient-derived fibroblasts induced pluripotent stem cells (iPSC). Sequencing loss target vanishingly small rates DSB. Corrected approach for autologous correction iPSC differentiated vitro into keratinocytes mesenchymal stroma cells. The diverse constellation makes individual patient specific impractical our data show offers regional where small, well characterized set could employed across numerous RDEB restore expression DSB-free pathogenic excision.