Current Strategies for the Gene Therapy of Autosomal Recessive Congenital Ichthyosis and Other Types of Inherited Ichthyosis

Mutations in genes such as transglutaminase-1 (TGM1), which are responsible for the formation and normal functioning of a lipid barrier, lead to the development of autosomal recessive congenital ichthyosis (ARCI). ARCIs are characterized by varying degrees of hyperkeratosis and the presence of scales on the body surface since birth. The quality of life of patients is often significantly affected, and in order to alleviate the manifestations of the disease, symptomatic therapy with moisturizers, keratolytics, retinoids and other cosmetic substances is often used to improve the condition of the patients’ skin. Graft transplantation is commonly used to correct defects of the eye. However, these approaches offer symptomatic treatment that does not restore the lost protein function or provide a long-term skin barrier. Gene and cell therapies are evolving as promising therapy for ARCIs that can correct the functional activity of altered proteins. However, these approaches are still at an early stage of development. This review discusses current studies of gene and cell therapy approaches for various types of ichthyosis and their further prospects for patient treatment.     

An Evaluation of Human Induced Pluripotent Stem Cells to Test for Cardiac Developmental Toxicity

To prevent congenital defects arising from maternal exposure, safety regulations require pre-market developmental toxicity screens for industrial chemicals and pharmaceuticals. Traditional embryotoxicity approaches depend heavily on the use of low-throughput animal models which may not adequately predict human risk. The validated embryonic stem cell test (EST) developed in murine embryonic stem cells addressed the former problem over 15 years ago. Here, we present a proof-of-concept study to address the latter challenge by updating all three endpoints of the classic mouse EST with endpoints derived from human induced pluripotent stem cells (hiPSCs) and human fibroblasts. Exposure of hiPSCs to selected test chemicals inhibited differentiation at lower concentrations than observed in the mouse EST. The hiPSC-EST also discerned adverse developmental outcomes driven by novel environmental toxicants. Evaluation of the early cardiac gene TBX5 yielded similar toxicity patterns as the full-length hiPSC-EST. Together, these findings support the further development of hiPSCs and early molecular endpoints as a biologically relevant embryotoxicity screening approach for individual chemicals and mixtures.     

Genetic Correction of Stem Cells in the Treatment of Inherited Diseases and Focus on Xeroderma Pigmentosum

Somatic stem cells ensure tissue renewal along life and healing of injuries. Their safe isolation, genetic manipulation ex vivo and reinfusion in patients suffering from life threatening immune deficiencies (for example, severe combined immunodeficiency (SCID)) have demonstrated the efficacy of ex vivo gene therapy. Similarly, adult epidermal stem cells have the capacity to renew epidermis, the fully differentiated, protective envelope of our body. Stable skin replacement of severely burned patients have proven life saving. Xeroderma pigmentosum (XP) is a devastating disease due to severe defects in the repair of mutagenic DNA lesions introduced upon exposure to solar radiations. Most patients die from the consequences of budding hundreds of skin cancers in the absence of photoprotection. We have developed a safe procedure of genetic correction of epidermal stem cells isolated from XP patients. Preclinical and safety assessments indicate successful correction of XP epidermal stem cells in the long term and their capacity to regenerate a normal skin with full capacities of DNA repair.     

Revisiting the Roles of Filaggrin in Atopic Dermatitis

The discovery in 2006 that loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and can predispose to atopic dermatitis (AD) galvanized the dermatology research community and shed new light on a skin protein that was first identified in 1981. However, although outstanding work has uncovered several key functions of filaggrin in epidermal homeostasis, a comprehensive understanding of how filaggrin deficiency contributes to AD is still incomplete, including details of the upstream factors that lead to the reduced amounts of filaggrin, regardless of genotype. In this review, we re-evaluate data focusing on the roles of filaggrin in the epidermis, as well as in AD. Filaggrin is important for alignment of keratin intermediate filaments, control of keratinocyte shape, and maintenance of epidermal texture via production of water-retaining molecules. Moreover, filaggrin deficiency leads to cellular abnormalities in keratinocytes and induces subtle epidermal barrier impairment that is sufficient enough to facilitate the ingress of certain exogenous molecules into the epidermis. However, although FLG null mutations regulate skin moisture in non-lesional AD skin, filaggrin deficiency per se does not lead to the neutralization of skin surface pH or to excessive transepidermal water loss in atopic skin. Separating facts from chaff regarding the functions of filaggrin in the epidermis is necessary for the design efficacious therapies to treat dry and atopic skin.     

Multitargeted Approach for the Optimization of Morphogenesis and Barrier Formation in Human Skin Equivalents

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen–chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.     

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.     

Homozygous ALOXE3 Nonsense Variant Identified in a Patient with Non-Bullous Congenital Ichthyosiform Erythroderma Complicated by Superimposed Bullous Majocchi’s Granuloma: The Consequences of Skin Barrier Dysfunction

Non-bullous congenital ichthyosiform erythroderma (NBCIE) is a hereditary disorder of keratinization caused by pathogenic variants in genes encoding enzymes important to lipid processing and terminal keratinocyte differentiation. Impaired function of these enzymes can cause pathologic epidermal scaling, significantly reduced skin barrier function. In this study, we have performed a focused, genetic analysis of a probrand affected by NBCIE and extended this to his consanguineous parents. Targeted capture and next-generation sequencing was performed on NBCIE associated genes in the proband and his unaffected consanguineous parents. We identified a homozygous nonsense variant c.814C>T (p.Arg272*) in ALOXE3 ({"type":"entrez-nucleotide","attrs":{"text":"NM_001165960.1","term_id":"260166611","term_text":"NM_001165960.1"}}NM_001165960.1) in the proband and discovered that his parents are both heterozygous carriers of the variant. The clinical manifestations of the proband’s skin were consistent with NBCIE, and detailed histopathological assessment revealed epidermal bulla formation and Majocchi’s granuloma. Infection with Trichophyton rubrum was confirmed by culture. The patient responded to oral terbinafine antifungal treatment. Decreased skin barrier function, such as that caused by hereditary disorders of keratinization, can increase the risk of severe cutaneous fungal infections and the formation of Majocchi’s granuloma and associated alopecia. Patients with NBCIE should be alerted to the possible predisposition for developing dermatophytoses and warrant close clinical follow-up.     

An Interleukin-4 and Interleukin-13 Induced Atopic Dermatitis Human Skin Equivalent Model by a Skin-On-A-Chip

Currently, the mechanism of progression of atopic dermatitis (AD) is not well understood because there is no physiologically appropriate disease model in terms of disease complexity and multifactoriality. Type 2 inflammation, mediated by interleukin (IL)-4 and IL-13, plays an important role in AD. In this study, full-thickness human skin equivalents consisting of human-derived cells were fabricated from pumpless microfluidic chips and stimulated with IL-4 and IL-13. The morphological properties, gene expression, cytokine secretion and protein expression of the stimulated human skin equivalent (HSE) epidermis were investigated. The results showed epidermal and spongy formations similar to those observed in lesions in AD, and decreased expression of barrier-related filaggrin, loricrin and involucrin genes and proteins induced by IL-4Rα signaling. In addition, we induced the expression of carbonic anhydrase II (CAII), a gene specifically expressed in the epidermis of patients with AD. Thus, AD human skin equivalents can be used to mimic the key pathological features of atopic dermatitis, overcoming the limitations of existing studies that rely solely on mouse models and have been unable to translate their effects to humans. Our results will be useful for future research on the development of therapeutic agents for atopic dermatitis.     

Effect of Neferine on DNCB-Induced Atopic Dermatitis in HaCaT Cells and BALB/c Mice

Atopic dermatitis (AD) is a chronic and persistent inflammatory skin disease characterized by eczematous lesions and itching, and it has become a serious health problem. However, the common clinical treatments provide limited relief and are accompanied by adverse effects. Therefore, there is a need to develop novel and effective therapies to treat AD. Neferine is a small molecule compound isolated from the green embryo of the mature seeds of lotus (Nelumbo nucifera). It has a bisbenzylisoquinoline alkaloid structure. Relevant studies have shown that neferine has many pharmacological and biological activities, including anti-inflammatory, anti-thrombotic, and anti-diabetic activities. However, there are very few studies on neferine in the skin, especially the related effects on inflammatory skin diseases. In this study, we proved that it has the potential to be used in the treatment of atopic dermatitis. Through in vitro studies, we found that neferine inhibited the expression of cytokines and chemokines in TNF-α/IFN-γ-stimulated human keratinocyte (HaCaT) cells, and it reduced the phosphorylation of MAPK and the NF-κB signaling pathway. Through in vivo experiments, we used 2,4-dinitrochlorobenzene (DNCB) to induce atopic dermatitis-like skin inflammation in a mouse model. Our results show that neferine significantly decreased the skin barrier damage, scratching responses, and epidermal hyperplasia induced by DNCB. It significantly decreased transepidermal water loss (TEWL), erythema, blood flow, and ear thickness and increased surface skin hydration. Moreover, it also inhibited the expression of cytokines and the activation of signaling pathways. These results indicate that neferine has good potential as an alternative medicine for the treatment of atopic dermatitis or other skin-related inflammatory diseases.     

The Fate of Epidermal Tight Junctions in the stratum corneum: Their Involvement in the Regulation of Desquamation and Phenotypic Expression of Certain Skin Conditions

We evaluated the presence of tight junction (TJ) remnants in the stratum corneum (SC) of in vitro reconstructed human epidermis and human skin explants subjected or not to an aggressive topical treatment with beta-lipohydroxy salicylic acid (LSA) for 24 h. LSA-treated samples showed an increased presence of TJ remnants in the two lowermost layers of the SC, as quantified with standard electron microscopy. The topical aggression-induced overexpression of TJ-like cell–cell envelope fusions may influence SC functions: (1) directly, through an enhanced cohesion, and (2) indirectly, by impeding accessibility of peripheral corneodesmosomes to extracellular hydrolytic enzymes and, thus, slowing down desquamation. Observations of ichthyotic epidermis in peeling skin disease (PSD; corneodesmosin deficiency; two cases) and ichthyosis hypotrichosis sclerosing cholangitis syndrome (IHSC/NISCH; absence of claudin-1; two cases) also demonstrated increased persistence of TJ-like intercellular fusions in pathological SC and contributed to the interpretation of the diseases’ pathological mechanisms.     

IgG and IgA with Potential Microbial-Binding Activity Are Expressed by Normal Human Skin Epidermal Cells

The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative V_HDJ_H rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity.     

Docosahexaenoic Acid Modulates Paracellular Absorption of Testosterone and Claudin-1 Expression in a Tissue-Engineered Skin Model

Healthy skin moLEdels produced by tissue-engineering often present a suboptimal skin barrier function as compared with normal human skin. Moreover, skin substitutes reconstructed according to the self-assembly method were found to be deficient in polyunsaturated fatty acids (PUFAs). Therefore, in this study, we investigated the effects of a supplementation of the culture media with docosahexaenoic acid (DHA) on the barrier function of skin substitutes. To this end, 10 μM DHA-supplemented skin substitutes were produced (n = 3), analyzed, and compared with controls (substitutes without supplementation). A Franz cell diffusion system, followed by ultra-performance liquid chromatography, was used to perform a skin permeability to testosterone assay. We then used gas chromatography to quantify the PUFAs found in the epidermal phospholipid fraction of the skin substitutes, which showed successful DHA incorporation. The permeability to testosterone was decreased following DHA supplementation and the lipid profile was improved. Differences in the expression of the tight junction (TJ) proteins claudin-1, claudin-4, occludin, and TJ protein-1 were observed, principally a significant increase in claudin-1 expression, which was furthermore confirmed by Western blot analyses. In conclusion, these results confirm that the DHA supplementation of cell culture media modulates different aspects of skin barrier function in vitro and reflects the importance of n-3 PUFAs regarding the lipid metabolism in keratinocytes.     

Effect of Scutellaria baicalensis extract on skin barrier function via peroxisome proliferator-activated receptor-α

Scutellaria baicalensis extract (SBE) was investigated to determine its effects on the transactivation activity of peroxisome proliferator-activated receptors (PPAR)-responsive element (PPRE) and anti-oxidant activity for improvement of skin barrier function. The treatment with SBE resulted in a significant increase in the transactivation activity of PPRE such as PPAR-α and effect in anti-oxidant and anti-microbial activity. In addition, SBE promotes the expression of protein related to cornified envelope (CE) formation such as involucrin. Therefore, these results indicate that SBE can promote keratinocyte differentiation and restore skin barrier homeostasis, and is suggested to be an appropriate skin therapeutic agent for improving epidermal permeability barrier function.     

miR-155 Contributes to Normal Keratinocyte Differentiation and Is Upregulated in the Epidermis of Psoriatic Skin Lesions

The role of microRNAs (miRNAs) during keratinocyte (KC) differentiation and in skin diseases with epidermal phenotypes has attracted strong interest over the past few years. However, combined mRNA and miRNA expression analyses to elucidate the intricate mRNA–miRNA networks of KCs at different stages of differentiation have not been performed yet. In the present study, we investigated the dynamics of miRNA and mRNA expression during KC differentiation in vitro and in normal and psoriatic epidermis. While we identified comparable numbers of up- and downregulated mRNAs (49% and 51%, respectively), miRNAs were predominantly upregulated (76% vs 24%) during KC differentiation. Further bioinformatics analyses suggested an important inhibitory role for miR-155 in KC differentiation, as it was repressed during KC differentiation in normal skin but strongly upregulated in the epidermis of psoriatic skin lesions. Mimicking the inflammatory milieu of psoriatic skin in vitro, we could show that the pro-inflammatory cytokines IL17, IL1β and INFγ synergistically upregulated miR-155 expression in KCs. Forced over-expression of miR-155 in human in vitro skin models specifically reduced the expression of loricrin (LOR) in KCs, indicating that miR-155 interferes with the establishment of a normal epidermal barrier. Together, our data indicate that downregulation of miR-155 during KC differentiation is a crucial step for epidermal barrier formation. Furthermore, its strong upregulation in psoriatic lesions suggests a contributing role of miR-155 in the altered keratinocyte differentiation observed in psoriasis. Therefore, miR-155 represents as a potential target for treating psoriatic skin lesions.     

Lamin-Related Congenital Muscular Dystrophy Alters Mechanical Signaling and Skeletal Muscle Growth

Laminopathies are a clinically heterogeneous group of disorders caused by mutations in the LMNA gene, which encodes the nuclear envelope proteins lamins A and C. The most frequent diseases associated with LMNA mutations are characterized by skeletal and cardiac involvement, and include autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B, and LMNA-related congenital muscular dystrophy (LMNA-CMD). Although the exact pathophysiological mechanisms responsible for LMNA-CMD are not yet understood, severe contracture and muscle atrophy suggest that mutations may impair skeletal muscle growth. Using human muscle stem cells (MuSCs) carrying LMNA-CMD mutations, we observe impaired myogenic fusion with disorganized cadherin/β catenin adhesion complexes. We show that skeletal muscle from Lmna-CMD mice is unable to hypertrophy in response to functional overload, due to defective fusion of activated MuSCs, defective protein synthesis and defective remodeling of the neuromuscular junction. Moreover, stretched myotubes and overloaded muscle fibers with LMNA-CMD mutations display aberrant mechanical regulation of the yes-associated protein (YAP). We also observe defects in MuSC activation and YAP signaling in muscle biopsies from LMNA-CMD patients. These phenotypes are not recapitulated in closely related but less severe EDMD models. In conclusion, combining studies in vitro, in vivo, and patient samples, we find that LMNA-CMD mutations interfere with mechanosignaling pathways in skeletal muscle, implicating A-type lamins in the regulation of skeletal muscle growth.     

Carboxamide Derivatives Are Potential Therapeutic AHR Ligands for Restoring IL-4 Mediated Repression of Epidermal Differentiation Proteins

Atopic dermatitis (AD) is a common T-helper 2 (Th2) lymphocyte-mediated chronic inflammatory skin disease characterized by disturbed epidermal differentiation (e.g., filaggrin (FLG) expression) and diminished skin barrier function. Therapeutics targeting the aryl hydrocarbon receptor (AHR), such as coal tar and tapinarof, are effective in AD, yet new receptor ligands with improved potency or bioavailability are in demand to expand the AHR-targeting therapeutic arsenal. We found that carboxamide derivatives from laquinimod, tasquinimod, and roquinimex can activate AHR signaling at low nanomolar concentrations. Tasquinimod derivative (IMA-06504) and its prodrug (IMA-07101) provided full agonist activity and were most effective to induce FLG and other epidermal differentiation proteins, and counteracted IL-4 mediated repression of terminal differentiation. Partial agonist activity by other derivatives was less efficacious. The previously reported beneficial safety profile of these novel small molecules, and the herein reported therapeutic potential of specific carboxamide derivatives, provides a solid rationale for further preclinical assertation.     

Exploring the efficacy of AHA–BHA infused nanofiber skin masks as a topical treatment for acne vulgaris

This study investigates the efficacy of AHA–BHA infused nanofiber skin masks as a topical treatment for acne vulgaris. The skin is a vital organ that functions as a barrier to protect the body from external substances. In recent years, there has been a growing emphasis on skin treatment research. Hyaluronic acids (HAs) are commonly used in cosmetic and drug formulations to regulate excessive skin cornification, making them promising candidates for acne treatment. The introduction of HAs in dermatology has revolutionized the field of skin care, and they are used to treat various skin disorders, such as acne, ichthyosis, keratoses, warts, psoriasis, and photoaged skin. The antiaging benefits of HA have gained considerable attention in cosmetic dermatology, resulting in a surge in cosmetic products and skin care systems that contain HA. The study found that AHA–BHA-infused nanofiber skin masks are effective in treating acne vulgaris. The nanofiber masks were found to reduce inflammation, sebum production, and acne lesions. The study suggests that AHA–BHA-infused nanofiber skin masks could be a promising topical treatment for acne vulgaris.     

SMA Human iPSC-Derived Motor Neurons Show Perturbed Differentiation and Reduced miR-335-5p Expression

Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by mutations in the Survival Motor Neuron 1 gene, resulting in very low levels of functional Survival of Motor Neuron (SMN) protein. SMA human induced Pluripotent Stem Cells (hiPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. MicroRNAs (miRNAs) are often reported as playing a key role in regulating neuronal differentiation and fate specification. In this study SMA hiPSCs have been differentiated towards early motor neurons and their molecular and immunocytochemical profile were compared to those of wild type cells. Cell cycle proliferation was also evaluated by fluorescence-activated cell sorting (FACS). SMA hiPSCs showed an increased proliferation rate and also higher levels of stem cell markers. Moreover; when differentiated towards early motor neurons they expressed lower levels of NCAM and MN specific markers. The expression of miR-335-5p; already identified to control self-renewal or differentiation of mouse embryonic stem cells (mESCs); resulted to be reduced during the early steps of differentiation of SMA hiPSCs compared to wild type cells. These results suggest that we should speculate a role of this miRNA both in stemness characteristic and in differentiation efficiency of these cells.