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ADAM10 is A Disintegrin And Metalloproteinase (ADAM) family member that is membrane bound with its catalytic domain present on the cell surface. It is a sheddase that cleaves anchored cell surface proteins to shed them from the cell surface. ADAM10 can cleave at least a hundred different proteins and is expressed in most tissues of the body. ADAM10 is best characterized for its role in Notch signaling. Interestingly, ADAM10 is transported to specific sites on the cell surface by six different tetraspanins. Although the mechanism is not clear, tetraspanins can regulate ADAM10 substrate specificity, which likely contributes to the diversity of ADAM10 substrates. In developing mouse teeth, ADAM10 is expressed in the stem cell niche and subsequently in pre-ameloblasts and then secretory stage ameloblasts. However, once ameloblasts begin transitioning into the maturation stage, ADAM10 expression abruptly ceases. This is exactly when ameloblasts stop their movement that extends enamel crystallites and when the enamel layer reaches its full thickness. ADAM10 may play an important role in enamel development. ADAM10 can cleave cadherins and other cell-cell junctions at specific sites where the tetraspanins have transported it and this may promote cell movement. ADAM10 can also cleave the transmembrane proteins COL17A1 and RELT. When either COL17A1 or RELT are mutated, malformed enamel may occur in humans and mice. So, ADAM10 may also regulate these proteins that are necessary for proper enamel development. This mini review will highlight ADAM10 function, how that function is regulated by tetraspanins, and how ADAM10 may promote enamel formation.

BackgroundEpidermolysis 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).MethodsIn 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.ResultsAs 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.ConclusionHere 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.

Junctional epidermolysis bullosa (JEB) is a hereditary blistering disease caused by reduced dermal-epidermal adhesion due to deficiencies of one of the proteins, laminin-332, type XVII collagen, integrin α6β4 or integrin α3. Significant progress has been achieved in the development of therapies for EB, such as bone-marrow transplantation, local or systemic injections with fibroblasts or mesenchymal stromal cells, readthrough of premature termination codons, or exon skipping. These were tailored in particular for dystrophic EB, which is caused by type VII collagen deficiency and have not yet reached broad clinical practice. Recently, pioneering combined gene and stem cell therapy was successful in treating one boy with junctional EB. Beside these exclusive approaches, no specific therapy to amend the major clinical features, skin and mucosal blistering and non-healing wounds is available to date. Here we extend the mutational spectrum of junctional EB, provide a stratification of COL17A1 mutations and discuss potential molecular therapeutic approaches.