Recessive Dystrophic Epidermolysis Bullosa Fibroblasts Research Articles

Highly efficient CRISPR/Cas9‐mediated exon skipping for recessive dystrophic epidermolysis bullosa

AbstractGene therapy based on the CRISPR/Cas9 system has emerged as a promising strategy for treating the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB). With this approach problematic wounds could be grafted with gene edited, patient‐specific skin equivalents. Precise gene editing using homology‐directed repair (HDR) is the ultimate goal, however low efficiencies have hindered progress. Reframing strategies based on highly efficient non‐homologous end joining (NHEJ) repair aimed at excising dispensable, mutation‐harboring exons offer a promising alternative approach for restoring the COL7A1 open reading frame. To this end, we employed an exon skipping strategy using dual single guide RNA (sgRNA)/Cas9 ribonucleoproteins (RNPs) targeted at three novel COL7A1 exons (31, 68, and 109) containing pathogenic heterozygous mutations, and achieved exon deletion rates of up to 95%. Deletion of exon 31 in both primary human RDEB keratinocytes and fibroblasts resulted in the restoration of type VII collagen (C7), leading to increased cellular adhesion in vitro and accurate C7 deposition at the dermal‐epidermal junction in a 3D skin model. Taken together, we extend the list of COL7A1 exons amenable to therapeutic deletion. As an incidental finding, we find that long‐read Nanopore sequencing detected large on‐target structural variants comprised of deletions up to >5 kb at a frequency of ~10%. Although this frequency may be acceptable given the high rates of intended editing outcomes, our data demonstrate that standard short‐read sequencing may underestimate the full range of unexpected Cas9‐mediated editing events.

Gamma-Secretase Inhibitors Downregulate the Profibrotic NOTCH Signaling Pathway in Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare skin fragility disorder caused by mutations in COL7A1. RDEB is hallmarked by trauma-induced unremitting blistering, chronic wounds with inflammation and progressive fibrosis, leading to severe disease complications. There is currently no cure for RDEB-associated fibrosis. Our previous studies and increasing evidence highlighted the pro-fibrotic role of Notch pathway in different skin disorders, including RDEB. Here, we further investigated the role of Notch signaling in RDEB pathogenesis and explored the effects of its inhibition by γ-secretase inhibitors DAPT and PF-03084014 (nirogacestat). Our analyses demonstrated that JAG1 and cleaved NOTCH1 are up-regulated in primary RDEB fibroblasts (RDEB-FBs) as compared with controls, and their protein levels are further increased by TGF-β1 stimulation. Functional assays unveiled the involvement of JAG1/NOTCH1 axis in RDEB fibrosis and demonstrated that its blockade counteracts a variety of fibrotic traits. In particular, RDEB-FBs treated with PF-03084014 showed: (i) a significant reduction of contractility; (ii) a diminished secretion of TGF-β1 and collagens, and (iii) the down-regulation of several fibrotic proteins. Although less marked as compared to PF-03084014-treated cells, RDEB-FBs exhibited a reduction of fibrotic traits also upon DAPT treatment. This study provides new potential therapeutic strategies to antagonize RDEB fibrosis onset and progression.

Highly efficient biallelic correction of homozygous COL7A1 mutation using ABE8e adenine base editor.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic skin disorder characterized by severe skin blistering. With no cure at present, we have pursued a gene repair approach using base editing of COL7A1. Following on from our previous report of highly efficient base editing using the novel base editor, ABE8e, here we explore the use of lipid nanoparticle (LNP)-based delivery systems instead of electroporation to effect base editing in RDEB fibroblasts and correct a missense G > A mutation. We demonstrate increasingly high editing efficiencies, averaging 90%, which vary with the ABE8e dosage. We then demonstrate the viability of topical delivery of mRNA encapsulated in an LNP. Our work underscores the translational potential of this therapeutic route for DEB.

Highly Efficient Ex Vivo Correction of COL7A1 through Ribonucleoprotein-Based CRISPR/Cas9 and Homology-Directed Repair to Treat Recessive Dystrophic Epidermolysis Bullosa

Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare and severe genetic skin disease responsible for blistering of the skin and mucosa after minor trauma. RDEB is caused by a wide variety of mutations in COL7A1 encoding C7, the major component of anchoring fibrils (AFs) which form key attachment structures for dermal-epidermal adherence. Here, we achieved highly efficient COL7A1 editing in primary RDEB keratinocytes (RDEB-K) and fibroblasts (RDEB-F) from two patients homozygous for the c.6508C>T (p.Gln2170*) mutation through CRISPR/Cas9-mediated Homology-Directed Repair. Three gRNAs targeting the c.6508C>T mutation or harboring sequences, were delivered together with hfCas9 as a ribonucleoprotein complex (RNP). Among them, one achieved 73% cleavage activity in primary RDEB-K and RDEB-F. Then, we treated RDEB-K and RDEB-F with this specific RNP and the corresponding donor template delivered as ssODN and achieved up to 58% of genetic correction as well as C7 rescue. Finally, grafting of corrected 3D skin onto nude mice induced re-expression and normal localization of C7 as well as AFs formation at the DEJ at five and ten weeks post-grafting. With this promising non-viral approach, we achieved therapeutically relevant specific gene editing which could be applicable to all mutations in exon 80 of COL7A1 in primary RDEB cells.

Inflammation-mediated fibroblast activation and immune dysregulation in collagen VII-deficient skin.

Inflammation is known to play a critical role in all stages of tumorigenesis; however, less is known about how it predisposes the tissue microenvironment preceding tumor formation. Recessive dystrophic epidermolysis bullosa (RDEB), a skin-blistering disease secondary to COL7A1 mutations and associated with chronic wounding, inflammation, fibrosis, and cutaneous squamous cell carcinoma (cSCC), models this dynamic. Here, we used single-cell RNA sequencing (scRNAseq) to analyze gene expression patterns in skin cells from a mouse model of RDEB. We uncovered a complex landscape within the RDEB dermal microenvironment that exhibited altered metabolism, enhanced angiogenesis, hyperproliferative keratinocytes, infiltration and activation of immune cell populations, and inflammatory fibroblast priming. We demonstrated the presence of activated neutrophil and Langerhans cell subpopulations and elevated expression of PD-1 and PD-L1 in T cells and antigen-presenting cells, respectively. Unsupervised clustering within the fibroblast population further revealed two differentiation pathways in RDEB fibroblasts, one toward myofibroblasts and the other toward a phenotype that shares the characteristics of inflammatory fibroblast subsets in other inflammatory diseases as well as the IL-1-induced inflammatory cancer-associated fibroblasts (iCAFs) reported in various cancer types. Quantitation of inflammatory cytokines indicated dynamic waves of IL-1α, TGF-β1, TNF, IL-6, and IFN-γ concentrations, along with dermal NF-κB activation preceding JAK/STAT signaling. We further demonstrated the divergent and overlapping roles of these cytokines in inducing inflammatory phenotypes in RDEB patients as well as RDEB mouse-derived fibroblasts together with their healthy controls. In summary, our data have suggested a potential role of inflammation, driven by the chronic release of inflammatory cytokines such as IL-1, in creating an immune-suppressed dermal microenvironment that underlies RDEB disease progression.

P07 Optimizing DNA specificity and applicability of base and prime editors on COL7A1 variants causing recessive dystrophic epidermolysis bullosa

Abstract Base and prime editors are novel CRISPR variants that reduce the risk of off-target effects by avoiding double-stranded DNA cleavage, bypassing the need for homology-directed repair. These tools offer great therapeutic potential for inherited skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB). Biallelic loss-of-function mutations in COL7A1 cause RDEB, resulting from loss of functional type VII collagen (C7) in the basement membrane zone. Current therapies for RDEB are limited, but ex vivo and in vivo COL7A1 base or prime editing therapies can potentially restore normal protein production with a single intervention treatment. This study compared DNA editing specificity, safety and efficiency on RDEB fibroblasts harbouring a homozygous c.4448 G>A (p.Gly1483Asp) mutation in COL7A1. Firstly, transfected adenine base editor (ABE8e) mRNA together with single guide RNA (sgRNA) were introduced into patient primary fibroblasts with Lipofectamine MessengerMax. By varying concentrations and ratios of guide RNA and ABE8e mRNA, we have achieved 100% biallelic correction without introducing any bystander mutations using 200 ng ABE8e and 28 ng sgRNA by transfecting 100 000 cells. Initial prime editing experiments are underway with PEmax plasmid and engineered prime editing guide RNAs (epegRNA) to optimize the guide design and to compare PE2max and PE4max editing systems. The functionality of the 100% biallelic correction using ABE8e is being evaluated with Western blot and in three-dimensional skin models. These early experiments demonstrate the potential value of base editing as a gene-editing tool in RDEB therapeutics; however, more functional work and off-target analyses are needed.

COL7A1 Editing via RNA Trans-Splicing in RDEB-Derived Skin Equivalents

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin's basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3'-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3'-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3'-RTMS6m repair molecule.

838 Rete ridges improve epidermal adhesion in engineered skin substitutes containing recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes and normal or RDEB fibroblasts

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe, hereditary blistering disease due to mutations in collagen VII (COL7A1 gene), which forms anchoring fibrils that attach the epidermis to the dermis. Engineered epidermal grafts containing COL7A1-expressing keratinocytes have shown promise in clinical trials, but preclinical studies suggest that engineered skin substitutes (ESS) containing both COL7A1-expressing fibroblasts and keratinocytes might provide greater benefit. Rete ridges are interdigitations of the epidermis and dermis that enhance tissue mechanics, increase epidermal adhesion, and improve shear resistance.

Collagen VII maintains proteostasis in dermal fibroblasts by scaffolding TANGO1 cargo

Lack of type VII collagen (C7) disrupts cellular proteostasis yet the mechanism remains undescribed. By studying the relationship between C7 and the extracellular matrix (ECM)-associated proteins thrombospondin-1 (TSP1), type XII collagen (C12) and tissue transglutaminase (TGM2) in primary human dermal fibroblasts from multiple donors with or without the genetic disease recessive dystrophic epidermolysis bullosa (RDEB) (n=31), we demonstrate that secretion of each of these proteins is increased in the presence of C7. In dermal fibroblasts isolated from patients with RDEB, where C7 is absent or defective, association with the COPII outer coat protein SEC31 and ultimately secretion of each of these ECM-associated proteins is reduced and intracellular levels are increased. In RDEB fibroblasts, overall collagen secretion (as determined by the levels of hydroxyproline in the media) is unchanged while traffic from the ER to Golgi of TSP1, C12 and TGM2 occurs in a type I collagen (C1) dependent manner. In normal fibroblasts association of TSP1, C12 and TGM2 with the ER exit site transmembrane protein Transport ANd Golgi Organization-1 (TANGO1) as determined by proximity ligation assays, requires C7. In the absence of wild-type C7, or when ECM-associated proteins are overexpressed, C1 proximity and intracellular levels increase resulting in elevated cellular stress responses and elevated TGFβ signaling. Collectively, these data demonstrate a role for C7 in loading COPII vesicle cargo and provides a mechanism for disrupted proteostasis, elevated cellular stress and increased TGFβ signaling in patients with RDEB. Furthermore, our data point to a threshold of cargo loading that can be exceeded with increased protein levels leading to pathological outcomes in otherwise normal cells.

Mechanistic interrogation of mutation-independent disease modulators of RDEB identifies the small leucine-rich proteoglycan PRELP as a TGF-β antagonist and inhibitor of fibrosis

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic extracellular matrix disease caused by deficiency in type VII collagen (Col VII). The disease manifests with devastating mucocutaneous fragility leading to progressive fibrosis and metastatic squamous cell carcinomas. Although Col VII abundance is considered the main predictor of symptom course, previous studies have revealed the existence of mutation-independent mechanisms that control disease progression. Here, to investigate and validate new molecular modifiers of wound healing and fibrosis in a natural human setting, and toward development of disease-modulating treatment of RDEB, we performed gene expression profiling of primary fibroblast from RDEB siblings with marked phenotypic variations, despite having equal COL7A1 genotype. Gene enrichment analysis suggested that severe RDEB was associated with enhanced response to TGF-β stimulus, oxidoreductase activity, and cell contraction. Consistently, we found an increased response to TGF-β, higher levels of basal and induced reactive oxygen species (ROS), and greater contractile ability in collagen lattices in RDEB fibroblasts (RDEBFs) from donors with severe RDEB vs mild RDEB. Treatment with antioxidants allowed a reduction of the pro-fibrotic and contractile phenotype. Importantly, our analyses revealed higher expression and deposition in skin of the relatively uncharacterized small leucine-rich extracellular proteoglycan PRELP/prolargin associated with milder RDEB manifestations. Mechanistic investigations showed that PRELP effectively attenuated fibroblasts' response to TGF-β1 stimulus and cell contractile capacity. Moreover, PRELP overexpression in RDEBFs enhanced RDEB keratinocyte attachment to fibroblast-derived extracellular matrix in the absence of Col VII. Our results highlight the clinical relevance of pro-oxidant status and hyper-responsiveness to TGF-β in RDEB severity and progression. Of note, our study also reveals PRELP as a novel and natural TGF-β antagonist with a likely dermo-epidermal pro-adhesive capacity.

Therapeutic base editing and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). For fibroblasts from two patients (Pat1 and Pat2), we applied adenine base editors (ABEs) to correct the pathogenic mutation of COL7A1 or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors(PEs) to correct the COL7A1 mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that transfer of edited patient-derived skin equivalents (i.e., RDEB keratinocytes and PE-corrected RDEB fibroblasts from the RDEB patient) intothe skin of immunodeficient mice led to C7 deposition and anchoring fibril formation within the dermal-epidermal junction, suggesting that base editing and prime editing could be feasible strategies for exvivo gene editing to treat RDEB.

In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain–severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

Increased abundance of Cbl E3 ligases alters PDGFR signaling in recessive dystrophic epidermolysis bullosa

In recessive dystrophic epidermolysis bullosa (RDEB), loss of collagen VII, the main component of anchoring fibrils critical for epidermal-dermal cohesion, affects several intracellular signaling pathways and leads to impaired wound healing and fibrosis. In skin fibroblasts, wound healing is also affected by platelet-derived growth factor receptor (PDGFR) signaling. To study a potential effect of loss of collagen VII on PDGFR signaling we performed unbiased disease phosphoproteomics. Whereas RDEB fibroblasts exhibited an overall weaker response to PDGF, Cbl E3 ubiquitin ligases, negative regulators of growth factor signaling, were stronger phosphorylated. This increase in phosphorylation was linked to higher Cbl mRNA and protein levels due to increased TGFβ signaling in RDEB. In turn, increased Cbl levels led to increased PDGFR ubiquitination, internalization, and degradation negatively affecting MAPK and AKT downstream signaling pathways. Thus, our results indicate that elevated TGFβ signaling leads to an attenuated response to growth factors, which contributes to impaired dermal wound healing in RDEB.

Fludarabine modulates expression of type VII collagen during haematopoietic stem cell transplantation for recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe, complicated inherited blistering skin disease with few treatment options currently available. Recently, haematopoietic stem cell transplantation (HCT) has been used as an alternative therapy that can improve skin integrity, but it is not known if the preparative HCT regimen also contributes to the therapeutic response. To determine whether chemotherapy drugs used in the HCT preparative regimen influence type VII collagen (C7) expression, which is inherently reduced or absent in RDEB skin, and to explore the pathomechanisms of such responses, if present. Drugs from the HCT preparative regimen (busulfan, cyclophosphamide, ciclosporin A, fludarabine and mycophenolate) with inhibitors (PD98059, U0126, LY294002, SR11302, SIS3 and N-acetyl-l-cysteine) were added to normal human dermal and human RDEB fibroblasts. C7 expression was measured using reversetranscription polymerase chain reaction and immunoblotting. We uncovered a previously unknown consequence of fludarabine whereby dermal fibroblasts exposed to fludarabine upregulate C7. This effect is mediated, in part, through activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/protein kinase B and transforming growth factor-β pathways. Activation of these pathways leads to activation of downstream transcription factors, including activator protein 1 (AP-1) and SMAD. Subsequently, both AP-1 and SMAD bind the COL7A1 promoter and increase COL7A1 expression. Fludarabine influences the production of type VII collagen in RDEB fibroblasts.

Impaired Mitochondrial and Metabolic Function of Fibroblasts Derived from Patients with Recessive Dystrophic and Junctional Epidermolysis Bullosa

Background: Recessive dystrophic epidermolysis bullosa (RDEB) and junctional EB (JEB) are inherited disorders characterised by fragility and blistering of epithelial tissues leading to pain, pruritus, and adherent scarring. The severity and chronic nature of the resultant skin wounds significantly reduces quality and length of life. Current therapies primarily consist of protective bandaging and nutritional supplementation; there is no cure for these disorders. Although the skin fragility results from a lack of C7 protein (RDEB) and laminin-332 (JEB), other serious aspects of these disorders, such as inflammation that interferes with healing and aggressive squamous cell carcinoma, have not been completely elucidated. Recent research has suggested that mitochondrial function plays a significant role in skin healing. Objective: To evaluate how mitochondrial function differs in patients with RDEB and JEB. Method: The energy status of RDEB and JEB patient-derived fibroblasts was determined by Seahorse analysis and metabolite production. The energetics and overall morphology of RDEB and JEB patient-derived fibroblasts were assayed as a measure of metabolic stress. Results: EB patient-derived fibroblasts showed impaired oxidative phosphorylation with concomitant compensation by glycolysis. Morphological parameters were altered in RDEB and JEB fibroblasts compared with controls. Conclusion: This is the first study to describe changes in mitochondrial energy metabolism, metabolic profile, and mitochondrial morphology of EB patients.

Diversity of Mechanisms Underlying Latent TGF-β Activation in Recessive Dystrophic Epidermolysis Bullosa

Injury- and inflammation-driven progressive dermal fibrosis is a severe manifestation of recessive dystrophic epidermolysis bullosa-a genetic skin blistering disease caused by mutations in COL7A1. TGF-β activation plays a prominent part in progressing dermal fibrosis. However, the underlying mechanisms are not fully elucidated. TGF-β is secreted in a latent form, which has to be activated for its biological functions. In this study, we determined that recessive dystrophic epidermolysis bullosa fibroblasts have an enhanced capacity to activate the latent form. Mechanistic and functional assessment demonstrated that this process depends on multiple latent TGF-β activators, including TSP-1, RGD-binding integrins, matrix metalloproteinases, and ROS, which act in concert, in a self-perpetuating feedback loop to progress fibrosis. Importantly, our study also disclosed keratinocytes as prominent facilitators of fibrosis in recessive dystrophic epidermolysis bullosa. They stimulate microenvironmental latent TGF-β activation through enhanced production of the above mediators. Collectively, our study provides data on the molecular mechanism behind dysregulated TGF-β signaling in recessive dystrophic epidermolysis bullosa, which are much needed for the development of evidence-based fibrosis-delaying treatments.

631 Oxidative stress imbalance as contributing factor in the establishment of fibrosis in recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare skin fragility disorder characterized by deficient dermo-epidermal adhesion, due to mutations in the gene encoding collagen VII. Patients with RDEB suffer chronic wounds and inflammation that ultimately trigger fibrosis and highly aggressive squamous cell carcinoma development. We previously reported a clinical case of two siblings carrying in homozygosis the same null mutation (c.6527insC) in the COL7A1 gene, but with marked phenotypic differences with respect to the extent of mucocutaneous involvement, skin fragility and fibrosis. Transcriptomic analysis (RNA-Seq) of primary fibroblasts revealed a significant downregulation in the gene encoding the antioxidant enzyme ALDH1A1 in all RDEB patients, but not in the sibling with mild phenotype (M-RDEB) whose expression levels remain at those found in fibroblasts from healthy volunteers. In this study, flow cytometry analysis showed higher levels of basal and induced reactive oxygen species (ROS) in RDEB fibroblasts, compared to healthy controls and M-RDEB, suggesting redox imbalance as a possible new disease-severity modulator. Thus, treatment of RDEB fibroblasts with N-acetylcysteine (NAC) as an antioxidant allowed a reduction of the fibrotic and activated phenotype, possibly through interaction with the TGF-β pathway. This study proposes a contributing role of oxidative stress in RDEB pathophysiology, opening new therapeutic opportunities to treat the fibrotic and pro-tumorigenic dermal stroma of these patients.

475 Targeting the JAK/STAT3 pathway with ruxolitinib for RDEB-cSCC therapy

The most lethal complication of recessive dystrophic epidermolysis bullosa (RDEB) is the development of aggressive cutaneous squamous cell carcinoma (cSCCs), which lead the high mortality rates in these patients. Elevated levels of phospho-STAT3 have been found in two RDEB-derived cSCC cell lines, demonstrating that constitutive activation and dysregulation of the JAK/STAT pathway may play a role in RDEB-cSCC pathogenesis. Ruxolitininb is an FDA approved JAK1/2 inhibitor that has efficacy in myelofibroblast, human head, and neck squamous cell carcinomas, as well as lung and breast carcinomas. However, its therapeutic effect has not been elucidated in RDEB-cSCC. Here, we investigated the in vivo pharmacological activity of ruxolitinib for locally advanced RDEB-cSCC treatment in a novel mouse xenograft model of RDEB-cSCC. We first built 3D cSCC skin constructs (SCs) using RDEB-cSCC and RDEB fibroblasts (FB), which were then grafted onto immunocompromised mice. The animals developed tumors within 4 weeks post-grafting, with characteristic morphological features of human RDEB-cSCC. We used this model to test ruxolitinib for its in vivo activity to reverse the accelerated tumor growth by oral and topical route of administration. We found that daily oral administration of 70 mg/kg ruxolitinib for 4 weeks has an inhibitory effect on tumor growth. Next, we exploited the ability of ruxolitinib to penetrate skin and its potency for inhibiting tumor growth via topical application. We observed that topical application of 2% ruxolitinib for 4 weeks has an inhibitory effect on smaller than 4 mm in dimeter tumor growth. Moreover, no toxicity or loss of body weight was observed during the treatment period. Taken together, we showed in vivo evidence of the therapeutic potential of ruxolitinib in RDEB-cSCC, thereby making ruxolitinib a promising anti-cancer drug against cSCC development and progression.

Base Editor Correction of COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Patient-Derived Fibroblasts and iPSCs

Genome editing represents a promising strategy for the therapeutic correction of COL7A1 mutations that cause recessive dystrophic epidermolysis bullosa (RDEB). DNA cleavage followed by homology-directed repair (HDR) using an exogenous template has previously been used to correct COL7A1 mutations. HDR rates can be modest, and the double-strand DNA breaks that initiate HDR commonly result in accompanying undesired insertions and deletions (indels). To overcome these limitations, we applied an A•T→G•C adenine base editor (ABE) to correct two different COL7A1 mutations in primary fibroblasts derived from RDEB patients. ABE enabled higher COL7A1 correction efficiencies than previously reported HDR efforts. Moreover, ABE obviated the need for a repair template, and minimal indels or editing at off-target sites was detected. Base editing restored the endogenous type VII collagen expression and function invitro. We also treated induced pluripotent stem cells (iPSCs) derived from RDEB fibroblasts with ABE. The edited iPSCs were differentiated into mesenchymal stromal cells, a cell population with therapeutic potential for RDEB. In a mouse teratoma model, the skin derived from ABE-treated iPSCs showed the proper deposition of C7 at the dermal-epidermal junction invivo. These demonstrate that base editing provides an efficient and precise genome editing method for autologous cell engineering for RDEB.

Collagen VII Expression Is Required in Both Keratinocytes and Fibroblasts for Anchoring Fibril Formation in Bilayer Engineered Skin Substitutes

The blistering disease recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in the gene encoding collagen VII (COL7), which forms anchoring fibrils that attach the epidermis to the dermis. Cutaneous gene therapy to restore COL7 expression in RDEB patient cells has been proposed, and cultured epithelial autograft containing COL7-modified keratinocytes was previously tested in clinical trials. Because COL7 in normal skin is expressed in both fibroblasts and keratinocytes, cutaneous gene therapy using a bilayer skin substitute may enable faster restoration of anchoring fibrils. Hypothetically, COL7 expression in either dermal fibroblasts or epidermal keratinocytes might be sufficient for functional anchoring fibril formation in a bilayer skin substitute. To test this, engineered skin substitutes (ESS) were prepared using four combinations of normal + RDEB cells: (1) RDEB fibroblasts + RDEB keratinocytes; (2) RDEB fibroblasts + normal keratinocytes; (3) normal fibroblasts + RDEB keratinocytes; and (4) normal fibroblasts + normal keratinocytes. ESS were incubated in vitro for 2 weeks prior to grafting to full-thickness wounds in immunodeficient mice. Biopsies were analyzed in vitro and at 1, 2, or 3 weeks after grafting. COL7 was undetectable in ESS prepared using all RDEB cells (group 1), and macroscopic blistering was observed by 2 weeks after grafting in ESS containing RDEB cells. COL7 was expressed, in vitro and in vivo, in ESS prepared using combinations of normal + RDEB cells (groups 2 and 3) or all normal cells (group 4). However, transmission electron microscopy revealed structurally normal anchoring fibrils, in vitro and by week 2 in vivo, only in ESS prepared using all normal cells (group 4). The results suggest that although COL7 protein is produced in engineered skin when cells in only one layer express the COL7 gene, formation of structurally normal anchoring fibrils appears to require expression of COL7 in both dermal fibroblasts and epidermal keratinocytes.