Patient-derived Keratinocytes Research Articles

Repurposing an epithelial sodium channel inhibitor as a therapy for murine and human skin inflammation.

Inflammatory skin disease is characterized by a pathologic interplay between skin cells and immunocytes and can result in disfiguring cutaneous lesions and systemic inflammation. Immunosuppression is commonly used to target the inflammatory component; however, these drugs are often expensive and associated with side effects. To identify previously unidentified targets, we carried out a nonbiased informatics screen to identify drug compounds with an inverse transcriptional signature to keratinocyte inflammatory signals. Using psoriasis, a prototypic inflammatory skin disease, as a model, we used pharmacologic, transcriptomic, and proteomic characterization to find that benzamil, the benzyl derivative of the US Food and Drug Administration-approved diuretic amiloride, effectively reversed keratinocyte-driven inflammatory signaling. Through three independent mouse models of skin inflammation (Rac1G12V transgenic mice, topical imiquimod, and human skin xenografts from patients with psoriasis), we found that benzamil disrupted pathogenic interactions between the small GTPase Rac1 and its adaptor NCK1. This reduced STAT3 and NF-κB signaling and downstream cytokine production in keratinocytes. Genetic knockdown of sodium channels or pharmacological inhibition by benzamil prevented excess Rac1-NCK1 binding and limited proinflammatory signaling pathway activation in patient-derived keratinocytes without systemic immunosuppression. Both systemic and topical applications of benzamil were efficacious, suggesting that it may be a potential therapeutic avenue for treating skin inflammation.

Identification of novel small molecule-based strategies of COL7A1 upregulation and readthrough activity for the treatment of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic disease caused by loss of function mutations in the gene coding for collagen VII (C7) due to deficient or absent C7 expression. This disrupts structural and functional skin architecture, leading to blistering, chronic wounds, inflammation, important systemic symptoms affecting the mouth, gastrointestinal tract, cornea, and kidney function, and an increased skin cancer risk. RDEB patients have an extremely poor quality of life and often die at an early age. A frequent class of mutations in RDEB is premature termination codons (PTC), which appear in homozygosity or compound heterozygosity with other mutations. RDEB has no cure and current therapies are mostly palliative. Using patient-derived keratinocytes and a library of 8273 small molecules and 20,160 microbial extracts evaluated in a phenotypic screening interrogating C7 levels, we identified three active chemical series. Two of these series had PTC readthrough activity, and one upregulated C7 mRNA, showing synergistic activity when combined with the reference readthrough molecule gentamicin. These compounds represent novel potential small molecule-based systemic strategies that could complement topical-based treatments for RDEB.

Nonselective TRPC channel inhibition and suppression of aminoglycoside-induced premature termination codon readthrough by the small molecule AC1903

Nonsense mutations, which occur in ∼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4–C1, and TRPC5–C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8–18 μM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake.

A Novel Van der Woude Syndrome-Causing IRF6 Variant Is Subject to Incomplete Non-sense-Mediated mRNA Decay Affecting the Phenotype of Keratinocytes.

Van der Woude syndrome (VWS) is a genetic syndrome that leads to typical phenotypic traits, including lower lip pits and cleft lip/palate (CLP). The majority of VWS-affected patients harbor a pathogenic variant in the gene encoding for the transcription factor interferon regulatory factor 6 (IRF6), a crucial regulator of orofacial development, epidermal differentiation and tissue repair. However, most of the underlying mechanisms leading from pathogenic IRF6 gene variants to phenotypes observed in VWS remain poorly understood and elusive. The availability of one VWS individual within our cohort of CLP patients allowed us to identify a novel VWS-causing IRF6 variant and to functionally characterize it. Using VWS patient-derived keratinocytes, we reveal that most of the mutated IRF6_VWS transcripts are subject to a non-sense-mediated mRNA decay mechanism, resulting in IRF6 haploinsufficiency. While moderate levels of IRF6_VWS remain detectable in the VWS keratinocytes, our data illustrate that the IRF6_VWS protein, which lacks part of its protein-binding domain and its whole C-terminus, is noticeably less stable than its wild-type counterpart. Still, it maintains transcription factor function. As we report and characterize a so far undescribed VWS-causing IRF6 variant, our results shed light on the physiological as well as pathological role of IRF6 in keratinocytes. This acquired knowledge is essential for a better understanding of the molecular mechanisms leading to VWS and CLP.

Homozygous NLRP1 gain-of-function mutation in siblings with a syndromic form of recurrent respiratory papillomatosis

Juvenile-onset recurrent respiratory papillomatosis (JRRP) is a rare and debilitating childhood disease that presents with recurrent growth of papillomas in the upper airway. Two common human papillomaviruses (HPVs), HPV-6 and -11, are implicated in most cases, but it is still not understood why only a small proportion of children develop JRRP following exposure to these common viruses. We report 2 siblings with a syndromic form of JRRP associated with mild dermatologic abnormalities. Whole-exome sequencing of the patients revealed a private homozygous mutation in NLRP1, encoding Nucleotide-Binding Domain Leucine-Rich Repeat Family Pyrin Domain-Containing 1. We find the NLRP1 mutant allele to be gain of function (GOF) for inflammasome activation, as demonstrated by the induction of inflammasome complex oligomerization and IL-1β secretion in an overexpression system. Moreover, patient-derived keratinocytes secrete elevated levels of IL-1β at baseline. Finally, both patients displayed elevated levels of inflammasome-induced cytokines in the serum. Six NLRP1 GOF mutations have previously been described to underlie 3 allelic Mendelian diseases with differing phenotypes and modes of inheritance. Our results demonstrate that an autosomal recessive, syndromic form of JRRP can be associated with an NLRP1 GOF mutation.

Deficiency of the human cysteine protease inhibitor cystatin M/E causes hypotrichosis and dry skin

PurposeWe aimed to assess the biological and clinical significance of the human cysteine protease inhibitor cystatin M/E, encoded by the CTS6 gene, in diseases of human hair and skin. MethodsExome and Sanger sequencing was performed to reveal the genetic cause in two related patients with hypotrichosis. Immunohistochemical, biophysical, and biochemical measurements were performed on patient skin and 3D-reconstructed skin from patient-derived keratinocytes. ResultsWe identified a homozygous variant c.361C>T (p.Gln121*), resulting in a premature stop codon in exon 2 of CST6 associated with hypotrichosis, eczema, blepharitis, photophobia and impaired sweating. Enzyme assays using recombinant mutant cystatin M/E protein, generated by site-directed mutagenesis, revealed that this p.Gln121* variant was unable to inhibit any of its three target proteases (legumain and cathepsins L and V). Three-dimensional protein structure prediction confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. ConclusionThe herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis.

379 KB105: An HSV-based gene therapy vector engineered to deliver functional TGM1 to Autosomal Recessive Congenital Ichthyosis (ARCI) keratinocytes

ARCI, a heterogenous group of rare disorders affecting skin cornification, is most commonly caused by mutations in the TGM1 (transglutaminase 1) gene. Current therapeutic options for ARCI are inadequate, providing limited symptomatic relief without restoration of epidermal barrier function or molecular correction of the underlying disease. We have engineered KB105, a novel replication-defective HSV-1 gene therapy vector encoding an optimized TGM1 transgene, amenable to topical treatment, for the molecular correction of ARCI. Here we show that KB105 demonstrates robust efficacy, safety, and functionality in a surrogate in vitro model of ARCI. KB105 efficiently transduced TGM1-deficient ARCI patient-derived keratinocytes, producing physiological levels of functional TGM1 with no impact on cell morphology or viability. The keratinocytes were infected by KB105 at increasing multiplicities of infection (MOIs), and TGM1 expression was evaluated by qPCR, qRT-PCR, western blot, and immunofluorescence (IF). A dose-dependent increase in TGM1 gene/transcript copy number was detected; likewise, a dose-dependent increase in TGM1 protein level was observed by western blotting and IF between an MOI of 0.3 and 1. Interestingly, TGM1 protein concentration was not further increased between MOIs of 1 and 3, potentially due to regulation of TGM1 at the post-transcriptional/translational level. Importantly, the KB105-expressed human TGM1 was shown to be functional in ARCI keratinocytes as assessed by an in situ fluorescent synthetic peptide reporter assay measuring TGM1-mediated glutamine conjugation, indicating successful molecular correction of the underlying TGM1 deficiency in these cells. Taken together, these data, along with in vivo efficacy and safety seen in surrogate disease models, support application of KB105 for treatment of ARCI. A Phase 1 study is planned for the first half of 2019.

Isolation of Biopsy-Derived, Human Cervical Keratinocytes Propagated as Monolayer and Organoid Cultures

The successful isolation and propagation of patient-derived keratinocytes from cervical lesions constitute a more appropriate model of cervical disease than traditional cervical cancer-derived cell lines such as SiHa and CaSki. Our aim was to streamline the growth of patient-obtained, cervical keratinocytes into a reproducible process. We performed an observational case series study with 60 women referred to colposcopy for a diagnostic biopsy. Main outcome measures were how many samples could be passaged at least once (n = 11), and where enough cells could be established, to precisely define their proliferation profile over time (n = 3). Altering cell culture conditions over those reported by other groups markedly improved outcomes. We were also successful in making freeze backs which could be resuscitated to successfully propagate multi-layered, organoids from cervical keratinocytes (n = 3). For best results, biopsy-intrinsic factors such as size and tissue digestion appear to be major variables. This seems to be the first systematic report with a well characterized and defined sample size, detailed protocol, and carefully assessed cell yield and performance. This research is particularly impactful for constituting a sample repository-on-demand for appropriate disease modelling and drug screening under the umbrella of personalized health.

Cord Blood-Derived Stem Cells Suppress Fibrosis and May Prevent Malignant Progression in Recessive Dystrophic Epidermolysis Bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFβ signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFβ signaling was demonstrated in col7a1-/- mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)-9 and MMP-13. Furthermore, human cord blood-derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1-/- mice showed the ability to suppress TGFβ signaling and MMP-9 and MMP-13 expression meanwhile upregulating anti-fibrotic TGFβ3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient-derived fibroblasts, keratinocytes, and cSCC, respectively. The patient-derived cells were constitutively active for STAT, but not TGFβ signaling. Moreover, the levels of MMP-9 and MMP-13 were significantly elevated in the patient derived-keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP-9 and MMP-13, and interferon (IFN)-γ from RDEB patient-derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti-inflammatory and anti-fibrotic functions. Stem Cells 2018;36:1839-12.

198 TALEN-mediated gene editing in epidermolytic ichthyosis patient-derived keratinocytes

198 TALEN-mediated gene editing in epidermolytic ichthyosis patient-derived keratinocytes

COL7A1 Editing via CRISPR/Cas9 in Recessive Dystrophic Epidermolysis Bullosa

Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes. Transplantation of skin equivalents grown from corrected keratinocytes onto immunodeficient mice showed phenotypic reversion with normal localization of type VII collagen at the basement membrane zone, compared with uncorrected keratinocytes, as well as fully stratified and differentiated skin layers without indication of blister development. Next-generation sequencing revealed on-target efficiency of up to 30%, whereas nuclease-mediated off-target site modifications at predicted genomic loci were not detected. These data demonstrate the potential of the CRISPR/Cas9 technology as a possible ex vivo treatment option for genetic skin diseases in the future.

Keratin gene mutations influence the keratinocyte response to DNA damage and cytokine induced apoptosis.

The keratin filament cytoskeleton is vital to the normal function of epithelial cells. It provides structural support and regulates different aspects of cell metabolism. Mutations in keratins 5 and 14 cause a skin fragility disorder, epidermolysis bullosa simplex (EBS). Patients with severe EBS have an increased cumulative risk for basal cell carcinoma. In this study, we tested how keratin 5 and 14 mutant EBS patient-derived keratinocytes behave in the face of two different types of stressors that are able to induce cell death: ionizing radiation and cytokines TNF-α and TRAIL. The data point out to a substantial difference between how normal and keratin mutant keratinocytes deal with such stresses. When case of DNA damage, the ATM/Chk2-pathway is one of the two main tracks that can prevent the progression of mitosis and so allow repair. This was altered in all investigated keratin mutants with a particular down-regulation of the activated form of checkpoint kinase 2 (pChk2). Keratin mutants also appear less sensitive than normal cells to treatment with TNF-α or TRAIL, and this may be linked to the up-regulation of two pro-survival proteins, Bcl-2 and FLIP. Such changes are likely to have a profound effect on mutant keratinocytes ability to survive and withstand stress, and in theory this may be also a contributing factor to cell transformation.

Mutations in SMARCAD1 cause autosomal dominant adermatoglyphia and perturb the expression of epidermal differentiation-associated genes.

Autosomal dominant adermatoglyphia (ADG) is characterized by lack of palmoplantar epidermal ridges. Recently, ADG was found to be caused in one family by a mutation in SMARCAD1, a member of the SNF subfamily of the helicase protein superfamily. To investigate the genetic basis of ADG. We used direct sequencing and global gene expression analysis. We identified three novel heterozygous mutations in SMARCAD1 (c.378 + 2T > C, c.378 + 5G > C and c.378 + 1G > A) in a total of six patients. Surprisingly, all four ADG-causing mutations identified to date disrupt a single conserved donor splice site adjacent to the 3' end of a noncoding exon and are predicted to result in haploinsufficiency for a skin-specific isoform of SMARCAD1. These data indicate a pivotal role for the SMARCAD1-skin specific isoform in dermatoglyph formation. In order to better understand the consequences of ADG-associated mutations, we ascertained the global transcription profiles of primary keratinocytes downregulated for SMARCAD1 and of patient-derived keratinocytes. A total of eight genes were found to be differentially expressed in both patient-derived and knocked down keratinocytes. Of interest, these differentially expressed genes have been implicated in epidermal ontogenesis and differentiation, and in psoriasis, which is characterized by abnormal finger ridge patterns. The present data suggest that ADG is genetically homogeneous and result from perturbed expression of epidermal differentiation-associated genes.

A step closer toward therapies for p63-related disorders.

Small molecules with low molecular weight are of interest for drug development, as they are more likely to be absorbed. In cancer research, p53 is often mutated in many tumors, and many small molecules targeting mutant p53 have been tested. One of such low molecular weight compounds is APR246/PRIMA-1MET that was identified as a compound targeting and reactivating p53 mutants based on a cell-based screening for rescuing the apoptotic activity of p53. Recently, we have reported two different model systems, (1) corneal epithelial cells differentiated from induced pluripotent stem cells (iPSCs) derived from reprogramming of patient fibroblasts and (2) skin organotypic reconstitution of patient-derived keratinocytes. We have shown that APR246/PRIMA-1MET can rescue epithelial differentiation defects caused by mutations in p63 that is a family member of p53 and shares high sequence and structural similarity with the p53 protein.1,2 The rationale of the two cellular models for drug screening and the potential of APR246/PRIMA-1MET to restore visual impairment of patients are discussed (Fig. 1).

Development of Quantitative Molecular Clinical End Points for siRNA Clinical Trials

RNA interference (RNAi) is an evolutionarily conserved mechanism that results in specific gene inhibition at the mRNA level. The discovery that short interfering RNAs (siRNAs) are selective, potent, and can largely avoid immune surveillance has resulted in keen interest to develop these inhibitors as therapeutics. A single nucleotide-specific siRNA (K6a_513a.12, also known as TD101) was recently evaluated in a phase 1b clinical trial for the rare skin disorder, pachyonychia congenita (PC). To develop a clinical trial molecular end point for this type of trial, methods were developed to: (1) isolate total RNA containing amplifiable mRNA from human skin and callus material; (2) quantitatively distinguish the single-nucleotide mutant mRNA from wild-type K6a mRNA in both patient-derived keratinocytes and patient callus; and (3) demonstrate that repeated siRNA treatment results in sustained inhibition of mutant K6a mRNA in patient-derived keratinocyte cultures. These methods allow noninvasive sampling and monitoring of gene expression from patient-collected shavings and may be useful in evaluating the effectiveness of RNAi-based therapeutics, including inhibitors that specifically target single-nucleotide mutations.

Gene Therapy to Fight Infection in Skin Transplants

Wound closure can be problematic in patients with massive burns covering a large area of the body and in diabetic patients suffering from chronic ulcers. Insufficient donor sites in severely burned patients results in delayed wound closure and increases the likelihood of infection, which is a major cause of burn wound mortality [1]. Complications in patients wi th diabetic foot ulcers, most commonly resulting from infection, lead to as many as 50,000 amputations annually in the USA [2]. Tissue-engineered skin substitutes have been developed to begin to meet the needs of these patient populations [3]. The most basic skin substitutes are synthetic, acellular materials that act primarily as barriers to fluid loss and microbial invasion. Among the most innovative products are those containing cultured skin cells. Commercially available cellular skin substitutes, such as Apligraf ® , contain allogeneic dermal fibroblasts and epidermal keratinocytes, derived from donated neonatal foreskin, combined with a bovine collagen matrix. Apligraf is indicated for use in the treatment of venous leg ulcers and diabetic foot ulcers, increasing the percentage of wounds healed and decreasing the time required for wound closure [4–6]. However, as it is prepared using allogeneic cells, Apligraf and similar products act as biological bandages. Cells within the graft secrete growth factors and other proteins that facilitate wound healing and provide a matrix for cell migration, but the grafted cells do not persist indefinitely. Multiple applications are often required for complete wound healing. For burn patients, epidermal sheet grafts such as Epicel ® can be prepared with patient-derived keratinocytes for permanent wound coverage [7]. These grafts contain only epidermal cells; thus, they are prone to blistering and scar formation [7]. Cultured skin substitutes (CSS) are a bilayered autologous skin substitute currently in Phase II clinical trials. Comprised of cultured autologous keratinocytes, fibroblasts and biopolymers, CSS are used as an adjunctive treatment for coverage of burn wounds of greater than 50% of the body’s surface area, and have also been used to treat chronic wounds [8–10]. Despite the clinical successes observed with skin substitutes, they exhibit anatomical deficiencies, such as delayed vascularization, when compared with native skin autograft, which increase the risk for destruction due to microbial contamination. The use of topical antimicrobials in combination with skin substitutes can enhance engraftment and facilitate wound healing, but may contribute to the emergence of resistant strains of microorganisms. The development of a bioengineered skin substitute with increased innate resistance to microbial contamination could reduce or eliminate the requirement for exogenous antibiotics. Hypothetically, this could be achieved using a cutaneous gene therapy approach. Gene-encoded antimicrobial peptides, including members of the α- and β-defensin families and cathelicidins, have demonstrated antimicrobial activity against a wide variety of pathogens. Significantly, antimicrobial peptides have been shown to act against microorganisms commonly found in burns and chronic wounds, including multiply-resistant strains. As their mechanism of action is distinct from pharmaceutical antibiotic agents, defensins and related peptides represent a novel approach for treatment or prevention of infection. Antimicrobial peptides are effectors of the innate immune system and represent the body’s first line of defense against infection. Various antimicrobial peptides are expressed in neutrophils and in tissues that serve as interfaces with the environment, such as gut and lung epithelia and skin [11,12]. Rather than relying on specific antigen recognition, as in adaptive immunity, mediators of innate immunity take advantage of shared structural and functional characteristics of microorganisms. Antimicrobial peptides are cationic, allowing them to interact with the anionic lipid bilayer of microbial cell membranes through electrostatic interactions [13]. Following adsorption, the peptides disrupt the integrity of the outer cell membrane; irreversible injury of the target cell can result from entry of the antimicrobial peptide and other normally excluded molecules and leakage of essential cell components. Defensin-related cell death has also been linked to interference with protein synthesis and