Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing

Abstract

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34 + cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.

A non-viral and selection-free COL7A1 HDR approach with improved safety profile for dystrophic epidermolysis bullosa

“…Hence, they achieved an HDR-based gene correction efficiency of %58%. 30 Recently, Bonafont et al 52 demonstrated similar COL7A1 correction efficiencies in primary RDEB keratinocytes following targeting of intron 79 via electroporation of RNPs and adeno-associated virus (AAV)-mediated delivery of single-stranded DNA repair templates. 3D human skin equivalents (HSEs), grafted onto immunedeficient mice, demonstrated accurate deposition of C7 at the BMZ in both studies.…”

“…3D human skin equivalents (HSEs), grafted onto immunedeficient mice, demonstrated accurate deposition of C7 at the BMZ in both studies. 30,52 The inclusion of C7-expressing skin cell types (fibroblasts and keratinocytes) in SEs is likely to be fundamental to create structurally normal C7 anchoring fibrils within the BMZ in order to improve long-term functionality and stability. 30,49 Due to significant concerns that our initial selection-based strategy might lead to random or targeted partial integrations of the donor template sequence, we followed a similar gene-editing strategy to Analyses revealed efficient C7 restoration of intracellular protein forms in treated primary RDEB fibroblasts and secretion of C7 into the supernatant.…”

“…Scale bar, 100 mm. Jacków et al 30 and Bonafont et al, 52 although in our approach, no foreign DNA-reliant selection and no viral transduction were performed. 13 We electroporated Cas9 RNPs together with single-or double-stranded Oligos into patient keratinocytes to correct a prevalent RDEB-associated mutation (c.425A>G) within exon 3 of COL7A1.…”

“…Delivery of CRISPR-Cas9 into target cells via RNP electroporation has been shown to result in high on-target efficiencies accompanied by low off-target activities. 18,20,52 To our knowledge, there is no other alternative in vitro delivery method as efficient as electroporation. Despite this, future investigations into the clinical applicability of this approach must also address the high RNPinduced on-target indel frequencies accompanying the desired perfect repair outcome.…”

Clinical Perspectives of Gene-Targeted Therapies for Epidermolysis Bullosa

“…Gene editing, based on programmable nucleases like the CRISPR/Cas9 system, zinc finger nucleases or transcription activator-like effector nucleases (TALEN), aims to permanently correct the genetic defect at the DNA level and is applicable to both autosomal recessive and dominant diseases [44]. In EB, these technologies are evaluated at the preclinical level, where precise gene correction has been achieved in primary skin cells as well as in induced pluripotent stem cells (iPSCs) [45][46][47][48][49][50]. In essence, these technologies induce targeted single-or double-strand breaks in the DNA, followed by activation of the endogenous repair machinery.…”