With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.

Epidermolysis bullosa simplex (EBS) is a rare genetic, blistering skin disease for which there is no cure. Treatments that address the pathophysiology of EBS are needed. Compare the impact of 1% diacerein cream with placebo in reducing the number of blisters in EBS. In a randomized, placebo-controlled, phase 2/3 trial we used a 1% diacerein topical formulation to treat defined skin areas in 17 patients. In a 2-period crossover trial, patients were randomized to either placebo or diacerein for a 4-week treatment and a 3-month follow-up in period 1. After a washout, patients were crossed over during period 2. The prespecified primary end point was the proportion of patients with a reduction of number of blisters by more than 40% from baseline in selected areas over the treatment episode. Of the patients receiving diacerein, 86% in episode 1 and 37.5% in episode 2 met the primary end point (vs 14% and 17% with placebo, respectively). This effect was still significant after the follow-up. Changes in absolute blister numbers were significant for the diacerein group only. No adverse effects were observed. Low patient numbers and no invasive data acquisition because of clinical burden in children. This trial provides evidence of the impact of 1% diacerein cream in the treatment of EBS.

The efficient treatment of hereditary disorders, especially of those caused by dominant-negative mutations still remains an obstacle to be overcome. Allele specificity is a critical aspect that must be addressed by silencing therapies such as small interfering RNA, which has the potential risk of also reducing expression of the normal allele. To overcome this hurdle, we used spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA exon segments in an in vitro disease model. In this model, a heterozygous insertion of a leucine codon into exon 9 of the plectin gene (PLEC1) leads to aggregation of plectin peptide chains and subsequent protein degradation recapitulating, together with a nonsense mutation on the other allele, the blistering skin disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Transient transfection of EBS-MD fibroblasts with a 5′ pre-trans-splicing molecule encoding wild-type exons 2–9 led to specific replacement of the mutated 5′ portion of the endogenous PLEC1 transcript through trans-splicing. This treatment reduced the levels of mutant mRNA and restored a wild-type pattern of plectin expression as revealed by immunofluorescence microscopy. When EBS-MD fibroblasts were transfected with retroviral constructs, the level of full-length plectin protein in the corrected fibroblasts increased by 58.7%. Thus, SMaRT may be a promising new tool for treatment of autosomal-dominant genetic diseases.

The major challenge to a successful gene therapy of autosomal dominant genetic diseases is a highly efficient and specific knock-down or repair of the disease-causing allele. In epidermolysis bullosa simplex-type Dowling–Meara (EBS-DM), a single amino acid exchange in exon 1 of the keratin 14 gene (K14) triggers a severe skin phenotype, characterized by blistering of the skin and mucous membranes after minor trauma. We chose spliceosome-mediated RNA trans-splicing to specifically replace exons 1–7 of the K14 gene. In this approach, the mutated coding region is replaced by an RNA-trans-splicing molecule (RTM) that incorporates a binding domain (BD) and the wild-type sequence of K14. Since the BD is crucial for the trans-splicing functionality, we developed a fluorescence-based RTM screen consisting of an RTM library containing random BDs. Co-transfection of the library with a target molecule enabled us to identify highly functional RTMs. The best RTMs were adapted for endogenous trans-splicing in an EBS-DM patient cell line. In this cell line, we were able to detect functional, efficient and correct trans-splicing on RNA and protein levels. Scratch assays confirmed phenotypic reversion in vitro. Owing to concomitant knock-down and repair of the mutated allele, we assume that trans-splicing is a promising tool for the treatment of autosomal dominant genetic disease.

Blistering in epidermolysis bullosa simplex type Dowling-Meara (EBS-DM) is associated with an inflammatory phenotype, which can be disrupted by diacerein in vitro. In this pilot study we hypothesized, that a topical formulation of diacerein 1% reduces blistering. Five patients initially applied diacerein underneath both armpits. Then, each participant received 1% diacerein-cream for one armpit, and placebo for the other (randomized withdrawal). The number of blisters was reduced significantly (left: -78%; right: -66% of baseline) within two weeks and remained significantly below the initial level even during withdrawal in four patients. These findings point to a relevant effect of diacerein and provide important information for a confirmative study.