Regeneration of the entire human epidermis using transgenic stem cells.
ABSTRACT: Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.
Project description:Junctional epidermolysis bullosa (JEB), a genetically heterogeneous group of blistering skin diseases, can be caused by mutations in the genes encoding laminin 5 or collagen XVII, which are components of the hemidesmosome-anchoring filament complex in the skin. Here, a family with severe nonlethal JEB and with mutations in genes for both proteins was identified. The index patient was compound heterozygous for the COL17A1 mutations L855X and R1226X and was heterozygous for the LAMB3 mutation R635X. As a consequence, two functionally related proteins were affected. Absence of collagen XVII and attenuated laminin 5 expression resulted in rudimentary hemidesmosome structure and separation of the epidermis from the basement membrane, with severe skin blistering as the clinical manifestation. In contrast, single heterozygotes carrying either (1) one or the other of the COL17A1 null alleles or (2) a double heterozygote for a COL17A1 and a LAMB3 null allele did not have a pathological skin phenotype. These observations indicate that the known allelic heterogeneity in JEB is further complicated by interactions between unlinked mutations. They also demonstrate that identification of one mutation in one gene is not sufficient for determination of the genetic basis of JEB in a given family.
Project description:Junctional epidermolysis bullosa (JEB) is a clinically and biologically heterogeneous genodermatosis, characterized by trauma-induced blistering and healing without scarring but sometimes with skin atrophy. We investigated three unrelated patients with different JEB phenotypes. Patients 1 and 2 had generalized atrophic benign epidermolysis bullosa (GABEB), with features including skin atrophy and alopecia. Patient 3 had the localisata variant of JEB, with predominantly acral blistering and normal hair. All patients carried novel homozygous point mutations (Q1016X, R1226X, and R1303Q) in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane component; and, therefore, not only GABEB but also the localisata JEB can be a collagen XVII disorder. The nonsense mutations led to drastically reduced collagen XVII mRNA and protein levels. In contrast, the missense mutation allowed expression of abnormal collagen XVII, and epidermal extracts from that patient contained polypeptides of normal size, as well as larger aggregates. The homozygous nonsense mutations in the COL17A1 gene were consistent with the absence of the collagen from the skin and with the GABEB phenotype, whereas homozygosity for the missense mutation resulted in expression of aberrant collagen XVII and, clinically, in localisata JEB.
Project description:In this study, we demonstrate the use of a genome-wide association mapping together with RNA-seq in a reduced number of samples, as an efficient approach to detect the causal mutation for a Mendelian disease. Junctional epidermolysis bullosa is a recessive genodermatosis that manifests with neonatal mechanical fragility of the skin, blistering confined to the lamina lucida of the basement membrane and severe alteration of the hemidesmosomal junctions. In Spanish Churra sheep, junctional epidermolysis bullosa (JEB) has been detected in two commercial flocks. The JEB locus was mapped to Ovis aries chromosome 11 by GWAS and subsequently fine-mapped to an 868-kb homozygous segment using the identical-by-descent method. The ITGB4, which is located within this region, was identified as the best positional and functional candidate gene. The RNA-seq variant analysis enabled us to discover a 4-bp deletion within exon 33 of the ITGB4 gene (c.4412_4415del). The c.4412_4415del mutation causes a frameshift resulting in a premature stop codon at position 1472 of the integrin ?4 protein. A functional analysis of this deletion revealed decreased levels of mRNA in JEB skin samples and the absence of integrin ?4 labeling in immunohistochemical assays. Genotyping of c.4412_4415del showed perfect concordance with the recessive mode of the disease phenotype. Selection against this causal mutation will now be used to solve the problem of JEB in flocks of Churra sheep. Furthermore, the identification of the ITGB4 mutation means that affected sheep can be used as a large mammal animal model for the human form of epidermolysis bullosa with aplasia cutis. Our approach evidences that RNA-seq offers cost-effective alternative to identify variants in the species in which high resolution exome-sequencing is not straightforward.
Project description:Mutations of the LAMB3 gene cause a lethal form of junctional epidermolysis bullosa (JEB). We hypothesized that early intra-amniotic gene transfer in a severe murine model of JEB would improve or correct the skin phenotype. Time-dated fetuses from heterozygous LAMB3(IAP) breeding pairs underwent ultrasound guided intra-amniotic injection of lentiviral vector encoding the murine LAMB3 gene at embryonic day 8 (E8). Gene expression was monitored by immunohistochemistry. The transgenic laminin-β3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-332 in the basement membrane zone of skin and mucosa. Ultrastructually, the restoration of ∼60% of hemidesmosomal structures was also noted. Although we could correct the skin phenotype in 11.9% of homozygous LAMB3(IAP) mice, none survived beyond 48 h. However, skin transplants from treated E18 homozygous LAMB3(IAP) fetuses maintained normal appearance for 6 months with persistence of normal assembly of laminin-332. These results demonstrate for the first time long-term phenotypic correction of the skin pathology in a severe model of JEB by in vivo prenatal gene transfer. Although survival remained limited due to the limitations of this mouse model, this study supports the potential for treatment of JEB by prenatal gene transfer.
Project description:There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy.
Project description:Epidermolysis bullosa (EB) is a class of intractable, rare, genetic disorders characterized by fragile skin and blister formation as a result of dermal-epidermal mechanical instability. EB presents with considerable clinical and molecular heterogeneity. Viable animal models of junctional EB (JEB), that both mimic the human disease and survive beyond the neonatal period, are needed. We identified a spontaneous, autosomal recessive mutation (Lamc2(jeb)) due to a murine leukemia virus long terminal repeat insertion in Lamc2 (laminin gamma2 gene) that results in a hypomorphic allele with reduced levels of LAMC2 protein. These mutant mice develop a progressive blistering disease validated at the gross and microscopic levels to closely resemble generalized non-Herlitz JEB. The Lamc2(jeb) mice display additional extracutaneous features such as loss of bone mineralization and abnormal teeth, as well as a respiratory phenotype that is recognized but not as well characterized in humans. This model faithfully recapitulates human JEB and provides an important preclinical tool to test therapeutic approaches.
Project description:In a highly inbred Australian Shepherd litter, three of the five puppies developed widespread ulcers of the skin, footpads, and oral mucosa within the first weeks of life. Histopathological examinations demonstrated clefting of the epidermis from the underlying dermis within or just below the basement membrane, which led to a tentative diagnosis of junctional epidermolysis bullosa (JEB) with autosomal recessive inheritance. Endoscopy in one affected dog also demonstrated separation between the epithelium and underlying tissue in the gastrointestinal tract. As a result of the severity of the clinical signs, all three dogs had to be euthanized. We sequenced the genome of one affected puppy and compared the data to 73 control genomes. A search for private variants in 37 known candidate genes for skin fragility phenotypes revealed a single protein-changing variant, LAMB3:c.1174T>C, or p.Cys392Arg. The variant was predicted to change a conserved cysteine in the laminin ?3 subunit of the heterotrimeric laminin-322, which mediates the binding of the epidermal basement membrane to the underlying dermis. Loss-of-function variants in the human LAMB3 gene lead to recessive forms of JEB. We confirmed the expected co-segregation of the genotypes in the Australian Shepherd family. The mutant allele was homozygous in two genotyped cases and heterozygous in three non-affected close relatives. It was not found in 242 other controls from the Australian Shepherd breed, nor in more than 600 other controls. These data suggest that LAMB3:c.1174T>C represents the causative variant. To the best of our knowledge, this study represents the first report of a LAMB3-related JEB in domestic animals.
Project description:Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitro adhesion advantage of laminin 332 production to positively select LAMB3-expressing keratinocytes. HDR and restored laminin 332 expression were evaluated at single-cell level. Notably, monoallelic-targeted integration of LAMB3 cDNA was sufficient to in vitro recapitulate the adhesive property, the colony formation typical of normal keratinocytes, as well as their cell growth. Grafting of genetically corrected skin equivalents onto immunodeficient mice showed a completely restored dermal-epidermal junction. This study provides evidence for efficient CRISPR/Cas9-mediated in situ restoration of LAMB3 expression, paving the way for ex vivo clinical application of this strategy to laminin 332 deficiency.
Project description:Amelogenesis imperfecta (AI) can be either isolated or part of a larger syndrome. Junctional epidermolysis bullosa (JEB) is a collection of autosomal-recessive disorders featuring AI associated with skin fragility and other symptoms. JEB is a recessive syndrome usually caused by mutations in both alleles of COL17A1, LAMA3, LAMB3, or LAMC2. In rare cases, heterozygous carriers in JEB kindreds display enamel malformations in the absence of skin fragility (isolated AI). We recruited two kindreds with autosomal-dominant amelogenesis imperfecta (ADAI) characterized by generalized severe enamel hypoplasia with deep linear grooves and pits. Whole-exome sequencing of both probands identified novel heterozygous mutations in the last exon of LAMB3 that likely truncated the protein. The mutations perfectly segregated with the enamel defects in both families. In Family 1, an 8-bp deletion (c.3446_3453del GACTGGAG) shifted the reading frame (p.Gly 1149Glufs*8). In Family 2, a single nucleotide substitution (c.C3431A) generated an in-frame translation termination codon (p.Ser1144*). We conclude that enamel formation is particularly sensitive to defects in hemidesmosome/basement-membrane complexes and that syndromic and non-syndromic forms of AI can be etiologically related.
Project description:Variably reduced expression of the basement membrane component laminin-332 (?3a?3?2) causes junctional epidermolysis bullosa generalized intermediate (JEB-GI), a skin fragility disorder with an increased susceptibility to squamous cell carcinoma (SCC) development in adulthood. Laminin-332 is highly expressed in several types of epithelial tumors and is central to signaling pathways that promote SCC tumorigenesis. However, laminin-332 mutations and expression in individuals affected by JEB-GI and suffering from recurrent SCCs have been poorly characterized. We studied a JEB-GI patient who developed over a hundred primary cutaneous SCCs. Molecular analysis combined with gene expression studies in patient skin and primary keratinocytes revealed that the patient is a functional hemizygous for the p.Cys1171* mutant allele which is transcribed in a stable mRNA encoding for a ?3 chain shortened of the last two C-terminal amino acids (Cys1171-Lys1172). The lack of the Cys1171 residue involved in the C-terminal disulphide bond to ?2 chain did not prevent assembly, secretion, and proteolytic processing of the heterotrimeric molecule. Immunohistochemistry of SCC specimens revealed accumulation of mutant laminin-332 at the epithelial-stromal interface of invasive front. We conclude that the C-terminal disulphide bond is a structural element crucial for laminin-332 adhesion function in-vivo. By saving laminin-332 amount, processing, and signaling role the p.Cys1171* mutation may allow intrinsic pro-tumorigenic properties of the protein to be conveyed, thus contributing to invasiveness and recurrence of SCCs in this patient.