Recessive Dystrophic Epidermolysis Bullosa Patient Research Articles

Twin Prime Editing Mediated Exon Skipping/Reinsertion for Restored Collagen VII Expression in Recessive Dystrophic Epidermolysis Bullosa

Gene editing nucleases, base editors, and prime editors are potential locus specific genetic treatment strategies for recessive dystrophic epidermolysis bullosa (RDEB); however, many RDEB COL7A1 mutations are unique, making the development of personalized editing reagents challenging. 270 of the ∼320 COL7A1 EB mutations reside in exons that can be skipped, and antisense oligonucleotides (ASO) and gene editing nucleases have been used to create in-frame deletions. ASOs are transient and nucleases generate deleterious double stranded DNA breaks (DSB) and uncontrolled mixtures of allele products. We developed a twin prime editing (twinPE) strategy using the PEmax and recently evolved PE6 prime editors and dual prime editing guide RNAs flanking COL7A1 exon five. Prime editing-mediated deletion of exon 5 with a homozygous premature stop codon was achieved in RDEB fibroblasts, keratinocytes, and iPSC with minimal DSBs, and collagen type VII (C7) protein was restored. TwinPE can replace the target exon with recombinase attachment sequences, and we exploited this to re-insert a normal copy of exon 5 using the Bxb1 recombinase. These findings demonstrate that twinPE can facilitate locus-specific, predictable, in-frame deletions and sequence replacement with few DSBs as a strategy that may enable a single therapeutic agent to treat multiple RDEB patient cohorts.

Splicing Modulation via Antisense Oligonucleotides in Recessive Dystrophic Epidermolysis Bullosa.

Antisense oligonucleotides (ASOs) represent an emerging therapeutic platform for targeting genetic diseases by influencing various aspects of (pre-)mRNA biology, such as splicing, stability, and translation. In this study, we investigated the potential of modulating the splicing pattern in recessive dystrophic epidermolysis bullosa (RDEB) patient cells carrying a frequent genomic variant (c.425A > G) that disrupts splicing in the COL7A1 gene by using short 2'-O-(2-Methoxyethyl) oligoribo-nucleotides (2'-MOE ASOs). COL7A1-encoded type VII collagen (C7) forms the anchoring fibrils within the skin that are essential for the attachment of the epidermis to the underlying dermis. As such, gene variants of COL7A1 leading to functionally impaired or absent C7 manifest in the form of extensive blistering and wounding. The severity of the disease pattern warrants the development of novel therapies for patients. The c.425A > G variant at the COL7A1 exon 3/intron 3 junction lowers the efficiency of splicing at this junction, resulting in non-functional C7 transcripts. However, we found that correct splicing still occurs, albeit at a very low level, highlighting an opportunity for intervention by modulating the splicing reaction. We therefore screened 2'-MOE ASOs that bind along the COL7A1 target region ranging from exon 3 to the intron 3/exon 4 junction for their ability to modulate splicing. We identified ASOs capable of increasing the relative levels of correctly spliced COL7A1 transcripts by RT-PCR, sqRT-PCR, and ddPCR. Furthermore, RDEB-derived skin equivalents treated with one of the most promising ASOs exhibited an increase in full-length C7 expression and its accurate deposition along the basement membrane zone (BMZ).

CRISPR-Cas9-based non-viral gene editing therapy for topical treatment of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is an autosomal monogenic skin disease caused by mutations in COL7A1 gene and lack of functional type VII collagen (C7). Currently, there is no cure for RDEB, and most of the gene therapies under development have been designed as exvivo strategies because of the shortage of efficient and safe carriers for gene delivery. Herein, we designed, synthesized, and screened a new group of highly branched poly(β amino ester)s (HPAEs) as non-viral carriers for the delivery of plasmids encoding dual single-guide RNA (sgRNA)-guided CRISPR-Cas9 machinery to delete COL7A1 exon 80 containing the c.6527dupC mutation. The selected HPAEs (named PTTA-DATOD) showed robust transfection efficiency, comparable with or surpassing that of leading commercial gene transfection reagents such as Lipofectamine 3000, Xfect, and jetPEI, while maintaining negligible cytotoxicity. Furthermore, CRISPR-Cas9 plasmids delivered by PTTA-DATOD achieved efficient targeted deletion and restored bulk C7 production in RDEB patient keratinocyte polyclones. The non-viral CRISPR-Cas9-based COL7A1 exon deletion approach developed here has great potential to be used as a topical treatment for RDEB patients with mutations in COL7A1 exon 80. Besides, this therapeutic strategy can easily be adapted for mutations in other COL7A1 exons, other epidermolysis bullosa subtypes, and other genetic diseases.

593 Preventing fibrosis in patients with recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare skin disorder characterized by defects in collagen VII leading to a range of pathologies dominated by painful blister formation. Aberrant wound healing leads to fibrosis that contributes to life threatening cancers. We have developed an in vitro model of fibrotic extracellular matrix (ECM) produced by primary RDEB patient dermal fibroblasts, in which we use detachment of the cells’ matrix from plastic as a surrogate for fibrosis. This study first sought to determine the molecular mechanisms of matrix detachment, using Atomic Force Microscopy, total collagen assays, and measurements of gene and protein expression, and then assessed application of chemical compounds to reduce fibrosis.

ABE8e adenine base editor precisely and efficiently corrects a recurrent COL7A1 nonsense mutation

Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB patient fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C > T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Off-target DNA analysis did not detect off-target editing in treated patient-derived fibroblasts and there was no detectable increase in A-to-I changes in the RNA. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.

Preclinical model for phenotypic correction of dystrophic epidermolysis bullosa by in vivo CRISPR-Cas9 delivery using adenoviral vectors

Recessive dystrophic epidermolysis bullosa, a devastating skin fragility disease characterized by recurrent skin blistering, scarring, and a high risk of developing squamous cell carcinoma is caused by mutations in COL7A1, the gene encoding type VII collagen, which is the major component of the anchoring fibrils that bind the dermis and epidermis. Ex vivo correction of COL7A1 by gene editing in patients’ cells has been achieved before. However, in vivo editing approaches are necessary to address the direct treatment of the blistering lesions characteristic of this disease. We have now generated adenoviral vectors for CRISPR-Cas9 delivery to remove exon 80 of COL7A1, which contains a highly prevalent frameshift mutation in Spanish patients. For in vivo testing, a humanized skin mouse model was used. Efficient viral transduction of skin was observed after excisional wounds generated with a surgical punch on regenerated patient skin grafts were filled with the adenoviral vectors embedded in a fibrin gel. Type VII collagen deposition in the basement membrane zone of the wounded areas treated with the vectors correlated with restoration of dermal-epidermal adhesion, demonstrating that recessive dystrophic epidermolysis bullosa (RDEB) patient skin lesions can be directly treated by CRISPR-Cas9 delivery in vivo.

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.

Generation of the induced pluripotent stem cell line (ZSPHARi001-A) from a patient with recessive dystrophic epidermolysis bullosa carrying compound heterozygous mutation in the COL7A1 gene

The COL7A1 gene mutation causes type VII collagen dysfunction, which subsequently leads to recessive dystrophic epidermolysis bullosa (RDEB). Patients who suffer from RDEB experience severe blisters and chronic trauma, which can eventually result in serious infection and the development of fatal squamous cell carcinoma. In our study, peripheral blood mononuclear cells (PBMCs) from an RDEB patient with the COL7A1 compound heterozygous mutation were collected and then reprogrammed into induced pluripotent stem cells (iPSC). The RDEB iPSC line can provide a cellular resource for the study of pathogenesis and drug screening.

Ancestral patterns of recessive dystrophic epidermolysis bullosa mutations in Hispanic populations suggest sephardic ancestry.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genodermatosis caused by mutations in the gene coding for type VII collagen (COL7A1). More than 800 different pathogenic mutations in COL7A1 have been described to date; however, the ancestral origins of many of these mutations have not been precisely identified. In this study, 32 RDEB patient samples from the Southwestern United States, Mexico, Chile, and Colombia carrying common mutations in the COL7A1 gene were investigated to determine the origins of these mutations and the extent to which shared ancestry contributes to disease prevalence. The results demonstrate both shared European and American origins of RDEB mutations in distinct populations in the Americas and suggest the influence of Sephardic ancestry in at least some RDEB mutations of European origins. Knowledge of ancestry and relatedness among RDEB patient populations will be crucial for the development of future clinical trials and the advancement of novel therapeutics.

Improvement with Fractional Ablative Laser and Topical Poly-L-Lactic Acid in an RDEB Patient

Recessive dystrophic epidermolysis bullosa (RDEB) results from mutations in the COL7A1 gene for type VII collagen. Affected patients demonstrate skin fragility and blistering with scarring. Associated morbidity is great, and there are limited therapeutic options. Fractional carbon dioxide (CO2) ablative laser (FABL) treatments have been shown to promote collagen remodeling and demonstrate synergy with topical poly-l-lactic acid (PLLA) applications. We report the case of a young woman with RDEB who experienced decreased blistering, decreased healing time, and improved quality of life in a local skin area treated with FABL followed by topical PLLA. In comparing biopsy specimens from treated and untreated skin, improved collagen organization and collagen maturity were observed by histopathological examination including with Herovici staining. Moreover, type VII collagen, absent in untreated skin, was detected in treated affected skin by immunostaining. This report illustrates a treatment response to FABL and topical PLLA in a patient with RDEB including enhanced healing time, collagen remodeling, and type VII collagen expression. The findings are promising and, with further study, may have broader implications for treating this disorder.

Improved Double-Nicking Strategies for COL7A1-Editing by Homologous Recombination

Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of 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.

395 Collagen VII is required in both fibroblasts and keratinocytes for anchoring fibril formation in bilayer skin substitutes

The blistering disease Recessive Dystrophic Epidermolysis Bullosa (RDEB) is caused by mutations in 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 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 anchoring fibril restoration. Hypothetically, COL7 expression in both dermis and epidermis of a bilayer skin substitute will be required to enable functional anchoring fibril formation. To test this, engineered skin substitutes (ESS) were prepared using four combinations of normal + RDEB cells: (1) RDEB fibroblasts + keratinocytes; (2) RDEB fibroblasts + normal keratinocytes; (3) normal fibroblasts + RDEB keratinocytes; and (4) normal fibroblasts + keratinocytes. ESS were incubated in vitro at the air-liquid interface for 10 days prior to grafting to full-thickness wounds in immunodeficient mice. Biopsies were analyzed in vitro and at 1, 2, and 3 wks after grafting. COL7 expression was not detected by immunohistochemistry in ESS prepared with all RDEB cells (group 1), and gross blistering was observed by 2 wks after grafting. COL7 was expressed, in vitro and in vivo, in ESS prepared using combinations of normal + RDEB cells (groups 2 & 3) or all normal cells (group 4). However, TEM revealed structurally normal anchoring fibrils, in vitro and in vivo, only in ESS prepared using all normal cells (group 4). The results suggest that although bilayer skin substitutes enable formation of anchoring fibrils in vitro, prior to grafting, this requires expression of COL7 in both fibroblasts and keratinocytes.

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

Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.

Efficient Gene Reframing Therapy for Recessive Dystrophic Epidermolysis Bullosa with CRISPR/Cas9

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system induces site-specific double-strand breaks, which stimulate cellular DNA repair through either the homologous recombination or non-homologous end-joining pathways. The non-homologous end-joining pathway, which is activated more frequently than homologous recombination, is prone to introducing small insertions and/or deletions at the double-strand break site, leading to changes in the reading frame. We hypothesized that the non-homologous end-joining pathway is applicable to genetic diseases caused by a frameshift mutation through restoration of the reading frame. Recessive dystrophic epidermolysis bullosa is a hereditary skin disorder caused by mutations in COL7A1. In this study, we applied gene reframing therapy to a recurrent frameshift mutation, c.5819delC, in COL7A1, which results in a premature termination codon. CRISPR/Cas9 targeting this specific mutation site was delivered to recessive dystrophic epidermolysis bullosa patient fibroblasts. After genotyping a large collection of gene-edited fibroblast clones, we identified a significant number (17/50) of clones in which the frameshift in COL7A1 was restored. The reframed COL7 was functional, as shown by triple-helix formation assay invitro, and was correctly distributed in the basement membrane zone in mice. Our data suggest that mutation site-specific non-homologous end-joining might be a highly efficient gene therapy for inherited disorders caused by frameshift mutations.

Thrombospondin-1 Is a Major Activator of TGF-β Signaling in Recessive Dystrophic Epidermolysis Bullosa Fibroblasts

Mutations in the gene encoding collagen VII cause the devastating blistering disease recessive dystrophic epidermolysis bullosa (RDEB). RDEB is characterized by severe skin fragility and nonhealing wounds aggravated by scarring and fibrosis. We previously showed that TSP1 is increased in RDEB fibroblasts. Because transforming growth factor-β (TGF-β) signaling is also increased in RDEB, and TSP1 is known to activate TGF-β, we investigated the role of TSP1 in TGF-β signaling in RDEB patient cells. Knockdown of TSP1 reduced phosphorylation of smad3 (a downstream target of TGF-β signaling) in RDEB primary fibroblasts, whereas overexpression of collagen VII reduced phosphorylation of smad3. Furthermore, inhibition of TSP1 binding to the LAP/TGF-β complex decreased fibrosis in engineered extracellular matrix formed by RDEB fibroblasts, as evaluated by picrosirius red staining and analyses of birefringent collagen fibrillar deposits. We show that collagen VII binds TSP1, which could potentially limit TSP1-LAP association and subsequent TGF-β activation. Our study suggests a previously unreported mechanism for increased TGF-β signaling in the absence of collagen VII in RDEB patient skin. Moreover, these data identify TSP1 as a possible target for reducing fibrosis in the tumor-promoting dermal microenvironment of RDEB patients.

Generation of transgene-free induced pluripotent stem cells from keratinocytes of recessive dystrophic epidermolysis bullosa patient

Generation of transgene-free induced pluripotent stem cells from keratinocytes of recessive dystrophic epidermolysis bullosa patient

Squamous-cell carcinoma in a female patient suffering from recessive dystrophic epidermolysis bullosa

The article describes a case of squamous-cell carcinoma in a female patient aged 30 suffering from a rare inherited disease -recessive dystrophic epidermolysis bullosa (RDEB). RDEB is characterized by a high risk of squamous cell carcinoma in young patients. The most frequent form is a highly differentiated form of cancer characterized by an aggressive course with the early development of metastases and fast progression, which is the most frequent cause of death in RDEB patients. The described case of squamous cell carcinoma in a young female RDEB patient emphasizes the role of early tumor diagnostics.

401 Dermal fibroblasts with loss of TSC2 express increased galectin-3, a potential biomarker for tuberous sclerosis complex

400 Amlexanox: Efficacy and mechanism for potential therapy for RDEB V Atanasova, Q Jiang, J Uitto and A South Thomas Jefferson University, Philadelphia, PA Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (ColVII). ColVII is the main, if not exclusive, component of anchoring fibrils that adhere the epidermis to the dermal layer of the skin. Mutations in COL7A1 lead to defective anchoring fibrils and subsequent perturbed adhesion between the two skin layers resulting in severe skin fragility and constant wounding without ability to heal. A subgroup of RDEB patients harbors nonsense mutations in COL7A1 which create premature termination codons (PTCs) of translation in the mRNA molecule. This leads to mRNA instability and lack of functional protein synthesized. A group of drugs have been reported for their ability to induce “readthrough” and allow for insertion of an amino acid at the site of PTC, thus producing fulllength protein. The mechanism by which these drugs act is currently not known. Here we focus on a PTC read-through drug, amlexanox, previously approved by the FDA for mouth ulcers. Our data show that amlexanox is able to induce full-length synthesis of ColVII in HpV immortalized RDEB patient derived keratinocytes and fibroblasts, as shown by western blotting. After 48 hours of treatment keratinocytes showed up to 14 fold increase in full-length ColVII protein synthesis as compared to non-treated controls. The same treatment in HpV fibroblasts led up to 3 fold increase in ColVII as compared to non-treated controls, or 25% of normal human fibroblasts. While the mechanism by which amlexanox is able to recover protein synthesis from the PTC containing COL7A1 gene is currently unclear, activation of UPF1 by its phosphorylation has been shown to be required for assembly of mRNA degradation machinery. Our data show that amlexanox treatment increases UPF1 phosphorylation, which may be part of the PTC read-through mechanism. In conclusion, we present data to support amlexanox as a read-through agent for the treatment of RDEB in cases with PTC in COL7A1. 401 Dermal fibroblasts with loss of TSC2 express increased galectin-3, a potential biomarker for tuberous sclerosis complex P Klover, R Thangapazham, J Kato, J Wang, S Li, C Dalgard, M Wilkerson, E Mccart, J Moss and T Darling 1 Dermatology, Uniformed Services University of the Health Sciences, Bethesda, MD, 2 Cardiovascular and Pulmonary Branch, NIH/NHLBI, Bethesda, MD and 3 Anatomy, Physiology, and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD Tuberous sclerosis complex (TSC) is an autosomal genetic disorder in which tumors form in multiple organs due to biallelic inactivating mutations in TSC1 or TSC2 and increased mTORC1 signaling. In order to discover biomarkers for TSC, we studied our mouse model of TSC with conditional deletion of Tsc2 in dermal mesenchyme (Tsc2cKO), testing the potential clinical relevance of findings using human TSC skin tumors. RNA-sequencing analysis of cultured Tsc2-null and wild-type mouse dermal fibroblasts identified galectin-3 as overexpressed by the mutant cells. Levels of both intracellular and secreted galectin-3 protein were approximately 3-fold greater in Tsc2-null fibroblasts than wild-type fibroblasts. Tsc2-null cells incubated in the presence of 20 nM sirolimus, an mTORC1-inhibitor, showed decreased galectin-3 expression but not to the level of wild-type fibroblasts, suggesting that overexpression of galectin-3 is only partially mTORC1-dependent. Serum levels of galectin-3 were 68% greater (p1⁄40.0015) in Tsc2 cKO than wild-type mice and sirolimus treatment for 4 weeks decreased galectin-3 serum levels 25% (p1⁄40.036) in the Tsc2cKO. Fibroblast-like cells grown from human TSC skin tumors, including angiofibromas, fibrous cephalic plaque, and periungual fibromas, showed greater intracellular and secreted galectin-3 levels than patient normal fibroblasts. Immunohistochemical analysis showed abundant galectin-3 positive dermal cells in TSC skin tumors but nearly absent dermal staining in paired samples of patient normal-appearing skin. Our results demonstrate that loss of TSC2 in dermal fibroblasts is associated with increased galectin-3 and suggest that this molecule should be investigated as a biomarker for TSC and possibly other diseases associated with increased mTORC1 activation.

Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.

Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.