Allele-specific CRISPR-Cas9 editing of dominant epidermolysis bullosa simplex in human epidermal stem cells.

Epidermolysis bullosa simplex (EBS) is a rare skin disease inherited mostly in an autosomal dominant manner. Patients display a skin fragility that leads to blisters and erosions caused by minor mechanical trauma. EBS phenotypic and genotypic variants are caused by genetic defects in intracellular proteins whose function is to provide the attachment of basal keratinocytes to the basement membrane zone and most EBS cases display mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes. Besides palliative treatments, there is still no long-lasting effective cure to correct the mutant gene and abolish the dominant negative effect of the pathogenic protein over its wild-type counterpart. Here, we propose a molecular strategy for EBS01 patient's keratinocytes carrying a monoallelic c.475/495del21 mutation in KRT14 exon 1. Through the CRISPR-Cas9 system, we perform a specific cleavage only on the mutant allele and restore a normal cellular phenotype and a correct intermediate filament network, without affecting the epidermal stem cell, referred to as holoclones, which play a crucial role in epidermal regeneration.

Posttranslational modifications of keratins and their associated proteins as therapeutic targets in keratin diseases.

The keratin cytoskeleton protects epithelia against mechanical, nonmechanical, and physical stresses, and participates in multiple signaling pathways that regulate cell integrity and resilience. Keratin gene mutations cause multiple rare monoallelic epithelial diseases termed keratinopathies, including the skin diseases Epidermolysis Bullosa Simplex (EBS) and Pachyonychia Congenita (PC), with limited available therapies. The disease-related keratin mutations trigger posttranslational modifications (PTMs) in keratins and their associated proteins that can aggravate the disease. Recent findings of drug high-throughput screening have led to the identification of compounds that may be repurposed, since they are used for other human diseases, to treat keratinopathies. These drugs target unique PTM pathways and sites, including phosphorylation and acetylation of keratins and their associated proteins, and have shed insights into keratin regulation and interactions. They also offer the prospect of testing the use of drug mixtures, with the long view of possible beneficial human use coupled with increased efficacy and lower side effects.

Dystonin modifiers of junctional epidermolysis bullosa and models of epidermolysis bullosa simplex without dystonia musculorum.

The Lamc2jeb junctional epidermolysis bullosa (EB) mouse model has been used to demonstrate that significant genetic modification of EB symptoms is possible, identifying as modifiers Col17a1 and six other quantitative trait loci, several with strong candidate genes including dystonin (Dst/Bpag1). Here, CRISPR/Cas9 was used to alter exon 23 in mouse skin specific isoform Dst-e (Ensembl GRCm38 transcript name Dst-213, transcript ID ENSMUST00000183302.5, protein size 2639AA) and validate a proposed arginine/glutamine difference at amino acid p1226 in B6 versus 129 mice as a modifier of EB. Frame shift deletions (FSD) in mouse Dst-e exon 23 (Dst-eFSD/FSD) were also identified that cause mice carrying wild-type Lamc2 to develop a phenotype similar to human EB simplex without dystonia musculorum. When combined, Dst-eFSD/FSD modifies Lamc2jeb/jeb (FSD+jeb) induced disease in unexpected ways implicating an altered balance between DST-e (BPAG1e) and a rarely reported rodless DST-eS (BPAG1eS) in epithelium as a possible mechanism. Further, FSD+jeb mice with pinnae removed are found to provide a test bed for studying internal epithelium EB disease and treatment without severe skin disease as a limiting factor while also revealing and accelerating significant nasopharynx symptoms present but not previously noted in Lamc2jeb/jeb mice.

Allele-Specific Inactivation of an Autosomal Dominant Epidermolysis Bullosa Simplex Mutation Using CRISPR-Cas9.

Epidermolysis bullosa simplex (EBS) is a rare mechanobullous disease caused by dominant-negative mutations in either keratin 5 (KRT5) or keratin 14 (KRT14) genes. Until now, there is no cure for EBS and the care is primarily palliative. The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system raised hope for the treatment of EBS and many other autosomal dominant diseases by mutant allele-specific gene disruption. In this study, we aim to disrupt the mutant allele for the heterozygous EBS pathogenic variation c.449T>C (p.Leu150Pro) within KRT5. This mutation generates, naturally, a novel protospacer-adjacent motif for the endonuclease Streptococcus pyogenes Cas9. Thus, we designed a single-guide RNA that guides the Cas9 to introduce a DNA cleavage of the mutant allele in patient's keratinocytes. Then, transfected cells were single-cell cloned and analyzed by deep sequencing. The expression of KRT5 and KRT14 was quantified, and the keratin intermediate filament stability was assessed. Results showed successful stringent mutant allele-specific knockout. An absence of synthesis of mutant transcript was further confirmed indicating permanent mutant allele-specific inactivation. Edited EBS patient keratinocytes produced a lower amount of K5 and K14 proteins compared with nonedited EBS cells, and no disturbance of cellular properties was observed.

Kinase Inhibition by PKC412 Prevents Epithelial Sheet Damage in Autosomal Dominant Epidermolysis Bullosa Simplex through Keratin and Cell Contact Stabilization.

Epidermolysis bullosa simplex (EBS) is a severe and potentially life-threatening disorder for which no adequate therapy exists. Most cases are caused by dominant sequence variations in keratin genes K5 or K14, leading to the formation of cytoplasmic keratin aggregates, profound keratinocyte fragility, and cytolysis. We hypothesized that pharmacological reduction of keratin aggregates, which compromise keratinocyte integrity, represents a viable strategy for the treatment of EBS. In this study, we show that the multikinase inhibitor PKC412, which is currently in clinical use for acute myeloid leukemia and advanced systemic mastocytosis, reduced keratin aggregation by 40% in patient-derived K14.R125C EBS-associated keratinocytes. Using a combination of epithelial shear stress assay and real-time impedance spectroscopy, we show that PKC412 restored intercellular adhesion. Molecularly, global phosphoproteomic analysis together with immunoblots using phosphoepitope-specific antibodies revealed that PKC412 treatment altered phosphorylated sites on keratins and desmoplakin. Thus, our data provide a proof of concept to repurpose existing drugs for the targeted treatment of EBS and showcase how one broad-range kinase inhibitor reduced keratin filament aggregation in patient-derived EBS keratinocytes and the fragility of EBS cell monolayers. Our study paves the way for a clinical trial using PKC412 for systemic or local application in patients with EBS.

Human-Induced Pluripotent Stem Cell‒Derived Keratinocytes, a Useful Model to Identify and Explore the Pathological Phenotype of Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS), an autosomal dominant skin disorder, is characterized by skin fragility. Genetically, the majority of cases are related to missense sequence variations in two keratin genes K5 or K14, leading to cytolysis of basal keratinocytes (KCs) and intraepidermal blistering. Progress toward the identification of treatments has been hampered by an incomplete understanding of the mechanisms underlying this disease and availability of relevant and reliable in vitro models recapitulating the physiopathological mechanisms. Recent advances in stem cell field have fueled the prospect that these limitations could be overcome, thanks to the availability of disease-specific human induced pluripotent stem cells (hiPSCs). In this study, we generated hiPSC-derived KCs from patients carrying keratin gene K5-dominant sequence variations and compared them with nonaffected hiPSC-derived KCs as well as their primary counterparts. Our results showed that EBS hiPSC-derived KCs displayed proliferative defects, increased capacity to migrate, alteration of extracellular signal‒regulated kinase signaling pathway, and cytoplasmic keratin filament aggregates as observed in primary EBS KCs. Of interest, EBS hiPSC-derived KCs exhibited downregulation of hemidesmosomal proteins, revealing the different effects of keratin gene K5 sequence variations on keratin cytoskeletal organization. With a combination of culture miniaturization and treatment with the chaperone molecule 4-phenylbutyric acid, our results showed that hiPSC-derived KCs represent a suitable model for identifying novel therapies for EBS.

Pathogenic DST sequence variants result in either epidermolysis bullosa simplex (EBS) or hereditary sensory and autonomic neuropathy type 6 (HSAN-VI).

DST encodes bullous pemphigoid antigen-1 (BPAG1), a protein with eight tissue-specific isoforms expressed in the skin, muscle, brain and nerves. Accordingly, mutations in this gene are associated with epidermolysis bullosa simplex (EBS) and hereditary sensory and autonomic neuropathy type 6 (HSAN-VI). The genotypic spectrum is attested to by 19 distinct mutations but genotype-phenotype correlation for both disorders is not well established. In this study, we performed next-generation sequencing (NGS) on two families with different phenotypic presentations, one foetus (P1) with musculoskeletal and neurological malformations established by prenatal ultrasound and family history, and a 15-year-old female patient (P2) with skin blistering. P1 had a novel homozygous nonsense mutation, DST: NM_001144769, c.3805C>T, p.R1269* within a region of genetic homozygosity (ROH). This mutation resides within the plakin domain of BPAG1 and ablates all isoforms of this protein, leading to novel extracutaneous phenotypes consistent with HSAN-VI in P1. P2 had a recurrent homozygous mutation DST: NM_001723.7, c.3370C>T, p.Gln1124* that presented with giant, trauma-induced skin blisters without extracutaneous involvement. This mutation is located within the coiled-coil domain present in the skin isoform of DST, BPGA1-e, associated with EBS. In summary, we report two families with pathogenic DST variants and expand the spectrum of DST genotype and phenotypes.

Proteasome-mediated degradation of keratins 7, 8, 17 and 18 by mutant KLHL24 in a foetal keratinocyte model: Novel insight in congenital skin defects and fragility of epidermolysis bullosa simplex with cardiomyopathy.

Epidermolysis bullosa simplex (EBS) with cardiomyopathy (EBS-KLHL24) is an EBS subtype caused by dominantly inherited, gain-of-function mutations in the gene encoding for the ubiquitin-ligase KLHL24, which addresses specific proteins to proteasomal degradation. EBS-KLHL24 patients are born with extensive denuded skin areas and skin fragility. Whilst skin fragility rapidly ameliorates, atrophy and scarring develop over time, accompanied by life-threatening cardiomyopathy. To date, pathogenetic mechanisms underlying such a unique disease phenotype are not fully characterized. The basal keratin 14 (K14) has been indicated as a KLHL24 substrate in keratinocytes. However, EBS-KLHL24 pathobiology cannot be determined by the mutation-enhanced disruption of K14 alone, as K14 is similarly expressed in foetal and postnatal epidermis and its protein levels are preserved both in vivo and in vitro disease models. In this study, we focused on foetal keratins as additional KLHL24 substrates. We showed that K7, K8, K17 and K18 protein levels are markedly reduced via proteasome degradation in normal foetal keratinocytes transduced with the mutant KLHL24 protein (ΔN28-KLHL24) as compared to control cells expressing the wild-type form. In addition, heat stress led to keratin network defects and decreased resilience in ΔN28-KLHL24 cells. The KLHL24-mediated degradation of foetal keratins could contribute to congenital skin defects in EBS-KLHL24. Furthermore, we observed that primary keratinocytes from EBS-KLHL24 patients undergo accelerated clonal conversion with reduced colony forming efficiency (CFE) and early replicative senescence. Finally, our findings pointed out a reduced CFE in ΔN28-KLHL24-transduced foetal keratinocytes as compared to controls, suggesting that mutant KLHL24 contributes to patients' keratinocyte clonogenicity impairment.

Keratins as an Inflammation Trigger Point in Epidermolysis Bullosa Simplex.

Epidermolysis bullosa simplex (EBS) is a group of inherited keratinopathies that, in most cases, arise due to mutations in keratins and lead to intraepidermal ruptures. The cellular pathology of most EBS subtypes is associated with the fragility of the intermediate filament network, cytolysis of the basal layer of the epidermis, or attenuation of hemidesmosomal/desmosomal components. Mutations in keratins 5/14 or in other genes that encode associated proteins induce structural disarrangements of different strengths depending on their locations in the genes. Keratin aggregates display impaired dynamics of assembly and diminished solubility and appear to be the trigger for endoplasmic reticulum (ER) stress upon being phosphorylated by MAPKs. Global changes in cellular signaling mainly occur in cases of severe dominant EBS mutations. The spectrum of changes initiated by phosphorylation includes the inhibition of proteasome degradation, TNF-α signaling activation, deregulated proliferation, abnormal cell migration, and impaired adherence of keratinocytes. ER stress also leads to the release of proinflammatory danger-associated molecular pattern (DAMP) molecules, which enhance avalanche-like inflammation. Many instances of positive feedback in the course of cellular stress and the development of sterile inflammation led to systemic chronic inflammation in EBS. This highlights the role of keratin in the maintenance of epidermal and immune homeostasis.

Identifying Plectin Isoform Functions through Animal Models.

Plectin, a high-molecular-weight cytoskeletal linker protein, binds with high affinity to intermediate filaments of all types and connects them to junctional complexes, organelles, and inner membrane systems. In addition, it interacts with actomyosin structures and microtubules. As a multifunctional protein, plectin has been implicated in several multisystemic diseases, the most common of which is epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). A great part of our knowledge about plectin's functional diversity has been gained through the analysis of a unique collection of transgenic mice that includes a full (null) knockout (KO), several tissue-restricted and isoform-specific KOs, three double KOs, and two knock-in lines. The key molecular features and pathological phenotypes of these mice will be discussed in this review. In summary, the analysis of the different genetic models indicated that a functional plectin is required for the proper function of striated and simple epithelia, cardiac and skeletal muscle, the neuromuscular junction, and the vascular endothelium, recapitulating the symptoms of humans carrying plectin mutations. The plectin-null line showed severe skin and muscle phenotypes reflecting the importance of plectin for hemidesmosome and sarcomere integrity; whereas the ablation of individual isoforms caused a specific phenotype in myofibers, basal keratinocytes, or neurons. Tissue-restricted ablation of plectin rendered the targeted cells less resilient to mechanical stress. Studies based on animal models other than the mouse, such as zebrafish and C. elegans, will be discussed as well.

Muscle-Related Plectinopathies.

Plectin is a giant cytoskeletal crosslinker and intermediate filament stabilizing protein. Mutations in the human plectin gene (PLEC) cause several rare diseases that are grouped under the term plectinopathies. The most common disorder is autosomal recessive disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), which is characterized by skin blistering and progressive muscle weakness. Besides EBS-MD, PLEC mutations lead to EBS with nail dystrophy, EBS-MD with a myasthenic syndrome, EBS with pyloric atresia, limb-girdle muscular dystrophy type R17, or EBS-Ogna. In this review, we focus on the clinical and pathological manifestations caused by PLEC mutations on skeletal and cardiac muscle. Skeletal muscle biopsies from EBS-MD patients and plectin-deficient mice revealed severe dystrophic features with variation in fiber size, degenerative myofibrillar changes, mitochondrial alterations, and pathological desmin-positive protein aggregates. Ultrastructurally, PLEC mutations lead to a disorganization of myofibrils and sarcomeres, Z- and I-band alterations, autophagic vacuoles and cytoplasmic bodies, and misplaced and degenerating mitochondria. We also summarize a variety of genetically manipulated mouse and cell models, which are either plectin-deficient or that specifically lack a skeletal muscle-expressed plectin isoform. These models are powerful tools to study functional and molecular consequences of PLEC defects and their downstream effects on the skeletal muscle organization.

Keratin Dynamics and Spatial Distribution in Wild-Type and K14 R125P Mutant Cells-A Computational Model.

Keratins are one of the most abundant proteins in epithelial cells. They form a cytoskeletal filament network whose structural organization seriously conditions its function. Dynamic keratin particles and aggregates are often observed at the periphery of mutant keratinocytes related to the hereditary skin disorder epidermolysis bullosa simplex, which is due to mutations in keratins 5 and 14. To account for their emergence in mutant cells, we extended an existing mathematical model of keratin turnover in wild-type cells and developed a novel 2D phase-field model to predict the keratin distribution inside the cell. This model includes the turnover between soluble, particulate and filamentous keratin forms. We assumed that the mutation causes a slowdown in the assembly of an intermediate keratin phase into filaments, and demonstrated that this change is enough to account for the loss of keratin filaments in the cell's interior and the emergence of keratin particles at its periphery. The developed mathematical model is also particularly tailored to model the spatial distribution of keratins as the cell changes its shape.

Biallelic KRT5 mutations in autosomal recessive epidermolysis bullosa simplex, including a complete human keratin 5 "knock-out".

Epidermolysis bullosa simplex (EBS) is usually inherited as an autosomal dominant disease due to monoallelic gain-of-function mutations in KRT5 or KRT14. Although autosomal recessive forms of EBS have been associated with mutations in at least 10 genes, recessive EBS due to homozygous biallelic KRT5 mutations has not been reported previously; it has been hypothesized that it would result in prenatal lethality. We sought the genetic causes of EB in a cohort of 512 distinct EB families by performing whole exome sequencing (WES) and using an EB-targeting next-generation sequencing (NGS) panel of 21 genes. The pathogenicity and consequences of the mutations were determined by expression profiling and at tissue and ultrastructural levels. Two pathogenic, homozygous missense variants of KRT5 in two patients with generalized EBS and a homozygous null mutation in a patient who died as a neonate from complications of EB were found. The two missense mutations disrupted keratin 5 expression on immunofluorescence microscopy, and the human "knock-out" of KRT5 showed no RNA and protein expression. Collectively, these findings identify biallelic KRT5 mutations with a phenotypic spectrum varying from mild, localized and generalized to perinatal lethal, expanding the genotypic profile of autosomal recessive EBS.

Threonine 150 Phosphorylation of Keratin 5 Is Linked to Epidermolysis Bullosa Simplex and Regulates Filament Assembly and Cell Viability.

A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII-/-) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.

Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases.

With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessively and dominantly inherited monogenic disorders. In this study, we have corrected a causal hotspot mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double-nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-delivery into EBS keratinocytes of a Cas9 D10A nickase (Cas9n), a predicted single guide RNA pair specific for intron 7, and a minicircle donor vector harboring the homology donor template resulted in a recombination efficiency of >30% and correction of the mutant KRT14 allele. Phenotypic correction of EBS-gen sev keratinocytes was demonstrated by immunofluorescence analysis, revealing the absence of disease-associated K14 aggregates within the cytoplasm. We achieved a promising safety profile for the CRISPR/Cas9 double-nicking approach, with no detectable off-target activity for a set of predicted off-target genes as confirmed by next generation sequencing. In conclusion, we demonstrate a highly efficient and specific gene-editing approach for KRT14, offering a causal treatment option for EBS.