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Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.

Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches., Graphical Abstract, Exploiting the advantages of their highly efficient CRISPR-Cas9 gene editing strategy, in this study, Gálvez et al. abrogate SPINK5, the Netherton syndrome-causing gene, in primary human keratinocytes. SPINK5-knockout cells show disease hallmarks in vitro and in vivo, enabling the development of trustworthy models suitable for the evaluation of advanced therapies for genodermatoses.

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. This work was supported by Spanish grants PI17/01747, PI20/00615, AC17/00054 (MutaEB-E-rare), and CIBERER ER18TRL714 from the Instituto de Salud Carlos III and grant SAF2017-86810-R from the Ministry of Economy and Competitiveness , all co-funded with European Regional Development Funds , and Avancell-CM grant ( S2017/BMD-3692 ). Authors are indebted to Almudena Holguín and Nuria Illera for grafting experiments, and to Jesus Martínez and Edilia De Almeida for animal maintenance and care.

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB., Highly efficient and safe approaches for precise correction of pathogenic mutations are required to realize the full potential of gene-editing protocols for clinical practice. Here we describe a single-step NHEJ-based CRISPR/Cas9 strategy of COL7A1 mutation-bearing exon deletion with unprecedented efficacy for ex vivo correction of recessive dystrophic epidermolysis bullosa.

[EN] Background: A combination of several factors including a change in the paint application system; a lack of proper hygiene; and inadequate safety measures caused a severe health impact in the workers of some textile painting factories. This outbreak, mainly characterized by respiratory disorders, caused the death of six people and it has been classified as Ardystil syndrome. Materials and Methods: Fifty-two workers involved in the outbreak and 48 healthy subjects not known to have exposed to the potentially mutagenic agents participated in the study. The program evaluated possible genotoxic damage through the sister chromatid exchange (SCE) cytogenetic biomarker assay. We determined the frequency of SCE, high-frequency cells (HFCs), and a ratio, which can be considered as a new parameter, allowing for the study of the SCE distribution pattern among the chromosomes. Results: There was no statistically significant difference in the SCE frequency and in the mean number of HFCs between the control and the Ardystil-affected groups. However, smoking increased the incidence of all parameters studied in both the case and control groups. Conclusions: This study shows that workers involved in the Ardystil syndrome did not suffer genotoxic damage as measured by SCE and HFCs when compared with the control group., This research was supported, in part, by Hospital Universitario Politecnico La Fe de Valencia and Conselleria de Sanidad de Valencia. The authors would like to dedicate this article to the memory of Dr. Miguel Almonacid Bujeda, a superb individual, with whom they had the good fortune to work with for a number of years.

Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented geneediting strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patientderived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutationcontaining, functionally expendable COL7A1 exons in patient epidermal stem cells. The study was mainly supported by DEBRA International—funded by DEBRA Austria (grant termed as Larcher 1). Additional funds come from Spanish grants SAF2013-43475-R and SAF2017-86810-R from the Ministry of Economy and Competitiveness and PI14/00931 and PI17/01747 from the Instituto de Salud Carlos III, all of them co-funded with European Regional Development Funds (ERDF).