Clinically Relevant Correction of Recessive Dystrophic Epidermolysis Bullosa by Dual sgRNA CRISPR/Cas9-Mediated Gene Editing.

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.

The regenerative potential of fibroblasts in a new diabetes-induced delayed humanised wound healing model.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human-skin-engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell-based wound therapy consisting of the application of plasma-derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re-epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.

Epidermolysis bullosa simplex with mottled pigmentation: a family report and review.

Epidermolysis bullosa simplex with mottled hyperpigmentation (EBS-MP) is an uncommon subtype of EBS. Its clinical features depend on the age of diagnosis, and clinical variations have been described even within family members. We present six cases from two unrelated Spanish families each with several affected members with EBS-MP and review the clinical and genetic findings in all reported patients. We highlight the changing clinical features of the disease throughout life.

Revertant mosaicism due to a second-site mutation in COL7A1 in a patient with recessive dystrophic epidermolysis bullosa.

Despite the high incidence of revertant mosaicism (35%) in patients with the genetic skin disease epidermolysis bullosa (EB) due to correcting mutations in the genes COL17A1 and LAMB3, revertant mosaicism has not been described for COL7A1 until recently. Mutations in COL7A1 are responsible for the most devastating form of EB in adults, which is characterized by cocooned "mitten" deformities of the hands. This report shows in vivo reversion of an inherited COL7A1 mutation in a patient with recessive dystrophic EB who was homozygous for the frameshift mutation COL7A1:c.6527insC,p.2176FsX337. The patient exhibited a patch of clinically healthy revertant skin on her left forearm. The second-site mutation c.6528delT, which is present in revertant keratinocytes, resulted in correction of the reading frame. As the new CCC codon codes for the same amino acid proline as the wild-type codon CCT, the revertant cells expressed wild-type type VII collagen polypeptide, leading to restoration of skin function. We hypothesize that, on careful examination, revertant mosaicism might be found to be more common in patients with type VII collagen-deficient EB. Furthermore, the revertant keratinocytes might offer the possibility to explore cell-based therapeutic strategies, by culturing in vitro and subsequently grafting as part of bioengineered dermo-epidermal substitutes on affected skin.

Assessment of optimal virus-mediated growth factor gene delivery for human cutaneous wound healing enhancement.

Using a recently described skin-humanized model based on the engraftment of human bioengineered skin equivalents onto immunodeficient mice, we compared the efficacy of different in vivo gene transfer strategies aimed at delivering growth factors to promote skin wound healing. The approaches involving transient delivery of keratinocyte growth factor (KGF) to wounds performed in the engrafted human skin included (1) KGF gene transfer by intradermal adenoviral injection; (2) KGF gene transfer by adenoviral vector immobilized in a fibrin carrier; and (3) KGF-adenoviral gene-transferred human fibroblasts embedded in a fibrin matrix. All delivery systems achieved KGF protein overproduction at the wound site, with a concomitant re-epithelialization enhancement. However, although direct gene delivery strategies exhibited variability in terms of the number of successfully transduced humanized mice, the use of genetically modified fibroblast-containing matrix as an in situ protein bioreactor was highly reproducible, leading to a significant improvement of the overall healing process. This latter approach appeared to be the most reliable means to deliver growth factors to wounds and also avoided the potential danger of scoring cases of faulty administration as therapeutic failures and direct exposure to viral vectors. The combined use of cell and gene therapy appears a robust tool to aid healing in a clinical context.

In vitro and in vivo wound healing-promoting activities of human cathelicidin LL-37.

The human antimicrobial peptide LL-37 plays an important role in host defense against infection. In addition to its antimicrobial action, other activities have been described in eukaryotic cells that may contribute to the healing response. In this study, we demonstrated that in vitro human cathelicidin activates migration of the human keratinocyte cell line HaCaT, involving phenotypic changes related to actin dynamics and associated to augmented tyrosine phosphorylation of proteins involved in focal adhesion complexes, such as focal adhesion kinase and paxillin. Other events involved in the LL-37 response were the induction of the Snail and Slug transcription factors, activation of matrix metalloproteinases and activation of the mitogen-activated protein kinase , and phosphoinositide 3-kinase/Akt signaling pathways. These signaling events could be mediated not only through the transactivation of EGFR but also through the induction of G-protein-coupled receptor FPRL-1 expression in these cells. Finally, by in vivo adenoviral transfer of the antimicrobial peptide to excisional wounds in ob/ob mice, we demonstrated that LL-37 significantly improved re-epithelialization and granulation tissue formation. The protective and regenerative activities of LL-37 support its therapeutic potential to promote wound healing.

Inhibition of xenografted human melanoma growth and prevention of metastasis development by dual antiangiogenic/antitumor activities of pigment epithelium-derived factor.

Human melanoma mortality is associated with the growth of metastasis in selected organs including the lungs, liver, and brain. In this study, we examined the consequences of overexpression of pigment epithelium-derived factor (PEDF), a neurotrophic factor and potent angiogenesis inhibitor, on both melanoma primary tumor growth and metastasis development. PEDF overexpression by melanoma cells greatly inhibited subcutaneous tumor formation and completely prevented lung and liver metastasis in immunocompromised mice after tail vein injection of metastatic human melanoma cell lines. Whereas the effects of PEDF on primary tumor xenografts appear mostly associated with inhibition of the angiogenic tumor response, abrogation of melanoma metastasis appears to depend on direct PEDF effects on both migration and survival of melanoma cells. PEDF-mediated inhibition of melanoma metastases could thus have a major impact on existing therapies for melanoma.