COL17A1 editing via homology-directed repair in junctional epidermolysis bullosa.

Background: Epidermolysis bullosa (EB), a severe genetic disorder characterized by blister formation in skin, is caused by mutations in genes encoding dermal-epidermal junction proteins that function to hold the skin layers together. CRISPR/Cas9-induced homology-directed repair (HDR) represents a promising tool for editing causal mutations in COL17A1 in the treatment of junctional epidermolysis bullosa (JEB). Methods: In this study, we treated primary type XVII collagen (C17)-deficient JEB keratinocytes with either Cas9 nuclease or nickase (Cas9n) ribonucleoproteins (RNP) and a single-stranded oligonucleotide (ssODN) HDR template in order to correct a causal pathogenic frameshift mutation within the COL17A1 gene. Results: As analyzed by next-generation sequencing of RNP-nucleofected keratinocytes, we observed an HDR efficiency of ∼38% when cells were treated with the high-fidelity Cas9 nuclease, a mutation-specific sgRNA, and an ssODN template. The combined induction of end-joining repair and HDR-mediated pathways resulted in a C17 restoration efficiency of up to 60% as assessed by flow cytometry. Furthermore, corrected JEB keratinocytes showed a significantly increased adhesive strength to laminin-332 and an accurate deposition of C17 along the basement membrane zone (BMZ) upon differentiation into skin equivalents. Conclusion: Here we present a gene editing approach capable of reducing end joining-generated repair products while increasing the level of seamless HDR-mediated gene repair outcomes, thereby providing a promising CRISPR/Cas9-based gene editing approach for JEB.

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6ß4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context.

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

Gene editing via homology-directed repair (HDR) currently comprises the best strategy to obtain perfect corrections for pathogenic mutations of monogenic diseases, such as the severe recessive dystrophic form of the blistering skin disease epidermolysis bullosa (RDEB). Limitations of this strategy, in particular low efficiencies and off-target effects, hinder progress toward clinical applications. However, the severity of RDEB necessitates the development of efficient and safe gene-editing therapies based on perfect repair. To this end, we sought to assess the corrective efficiencies following optimal Cas9 nuclease and nickase-based COL7A1-targeting strategies in combination with single- or double-stranded donor templates for HDR at the COL7A1 mutation site. We achieved HDR-mediated correction efficiencies of up to 21% and 10% in primary RDEB keratinocytes and fibroblasts, respectively, as analyzed by next-generation sequencing, leading to full-length type VII collagen restoration and accurate deposition within engineered three-dimensional (3D) skin equivalents (SEs). Extensive on- and off-target analyses confirmed that the combined treatment of paired nicking and single-stranded oligonucleotides constituted a highly efficient COL7A1-editing strategy, associated with a significantly improved safety profile. Our findings, therefore, represent a further advancement in the field of traceless genome editing for genodermatoses.

Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa.

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.

Context-Dependent Strategies for Enhanced Genome Editing of Genodermatoses.

The skin provides direct protection to the human body from assault by the harsh external environment. The crucial function of this organ is significantly disrupted in genodermatoses patients. Genodermatoses comprise a heterogeneous group of largely monogenetic skin disorders, typically involving mutations in genes encoding structural proteins. Therapeutic options for this debilitating group of diseases, including epidermolysis bullosa, primarily consist of wound management. Genome editing approaches co-opt double-strand break repair pathways to introduce desired sequence alterations at specific loci. Rapid advances in genome editing technologies have the potential to propel novel genetic therapies into the clinic. However, the associated phenotypes of many mutations may be treated via several genome editing strategies. Therefore, for potential clinical applications, implementation of efficient approaches based upon mutation, gene and disease context is necessary. Here, we describe current genome editing approaches for the treatment of genodermatoses, along with a discussion of the optimal strategy for each genetic context, in order to achieve enhanced genome editing approaches.