ALBERT

Description: Dystrophic epidermolysis bullosa (EB) is a disorder of incurable skin fragility, blistering and perturbations in anchoring fibrils caused by mutations in the type VII collagen gene (COL7A1). Since bone marrow (BM) includes cells with capacity to differentiate into diverse cell types and can seed various organs, including skin, we hypothesized that if type VII collagen producing cells are present in BM, BM infusions could be a beneficial therapy for EB. To test this hypothesis this in vivo, we assessed multiple candidate populations for cellular therapy of murine recessive dystrophic EB (RDEB, col7a1 −/−). As the col7a1 −/− newborn mice develop blisters and do not survive after two weeks of life, we can readily identify the mutant pups and the lethality provided a robust readout for the cellular interventions. The following cell populations were tested: epidermal stem cells and transit amplifying cells (from epidermis); total BM; BM derived multipotent adult progenitor cells and mesenchymal stem cells; fetal liver cells; and lineage positive and lineage negative BM fractions. None of these cell populations, however, provided any correction in col7a1 −/− recipients (N=223), even when the infusion regimen was varied by cell dose, age of donor, in utero and postnatal age at the time of infusion, and intravenous versus intrahepatic infusion. In a search for alternatives, we used CD150+ and CD48− BM cells, since signaling molecule SLAM family receptor positive populations have been shown to have significant pluripotentiality. We used several doses and infused them early postnatally into unconditioned col7a1 −/− mutant animals. One of these conditions, namely CD150+/CD48− BM population from C57Bl/6 GFP transgenic mice at a dose of 8 million cells per animal administered on day 3 or 4 after birth intravenously, resulted in survival of 3 out of 13 (23%) animals in two independent experiments. One of them was harvested at postnatal day 55; two of them were electively harvested at postnatal day 70. The animals were confirmed to be mutants by genotyping using PCR. The weights of these three animals were lower than that of their normal litter mates. Donor engraftment was up to 3% in peripheral blood and up to 12% in BM. Strikingly, the skin blisters characteristic of EB healed. Tissue analyses showed that the adoptive transfer of donor SLAM CD150+ selected BM resulted in skin engraftment of GFP+ donor cells and in the production of VII collagen mRNA in the skin, as assessed by RT- PCR. BM cells explanted from the 3 surviving recipients showed collagen VII producing cells. Ultrastructurally, we were able to identify anchoring fibrils, structures in the skin that are comprised exclusively of type VII collagen protein. This is consistent with the hypothesis that adoptive transfer of enriched wild-type BM cells resulted in, at least partial, functional correction of RDEB. Experiments are ongoing to identify the type VII collagen producing cells in CD150+ and CD48− BM population, and, in parallel, to identify immature BM cells that could be induced into becoming a skin stem cell. Collectively, these data demonstrate proof-of-principle of BM transfer for correction of the basement membrane zone defect in col7a1 −/− animals that may offer a valuable approach for treatment of human RDEB.

Description: Abstract 660 Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable, often fatal mucocutaneous blistering skin disease caused by mutations in the type VII collagen (C7) gene, COL7A1. These pathogenic mutations result in severely diminished expression of C7, a collagen localized at the dermal-epidermal junction (DEJ), and absence of anchoring fibrils (AFs) which are C7 containing structures that tether the epidermal basement membrane to the dermal matrix. From birth on, children with RDEB develop painful erosions and blisters on mucosal membranes and skin often resulting in esophageal strictures, mutilating scarring, joint contractures, fusion of fingers and toes and, aggressive squamous cell carcinomas. After first demonstrating that a stem cell enriched fraction of bone marrow (BM) rescued a proportion of RDEB mice from lethality and resulted in a) expression of C7 in skin and mucosal membranes, b) formation of new AFs, and c) resistance to blistering, a ‘first-in-human' phase I-II clinical trial was initiated in October 2007. To date, 7 patients have been treated with stem cells from BM from an HLA matched sibling donor (n=6) or unrelated cord blood (CB) donor (n=1). Follow-up data are reported through August 18, 2009. Conditioning consisted of busulfan 0.8 mg/kg per dose every 6 hours on days–9 to–6, fludarabine 25 mg/m2/day on days–5 to–3, and cyclophosphamide 50 mg/kg/day on days–5 to–2. After infusion of stem cells on day 0, immunoprophylaxis consisted of cyclosporine and mycophenolate mofetil. Patient and graft characteristics are shown in Table 1. Of the 4 patients with adequate follow-up, a progressive increase in C7 deposition by immunofluorescence (IF) at the DEJ, AFs or AF-like structures by electron microscopy, and wound healing with marked reduction in blister formation were documented. Unexpectedly, all patients had substantial chimerism in the skin (11-93%) that persisted over time. In 2 patients with a sex mismatched donor, perivascular localization of the donor cells in the dermis could be discerned using probes to the centromere regions of chromosomes X and Y. In summary, this is the first demonstration that the infusion of BM can ameliorate the severe systemic mucocutaneous manifestations of RDEB and sets the stage for using marrow stem cells in the treatment of a broad spectrum of extracellular matrix disorders. PtDonor (cell dose: NC × 108/kg)Transplant Related ToxicitiesC7 AssessmentAnchoring Fibril AssessmentClinical OutcomeSurvival Days1 15 mo maleHLA 8/8 male sibling BM/CB (3.04; 0.66)NoneIncreased by IF↑ Rudimentary AFsImproved but no change in use of dressingsAlive day 6592 9 mo femaleHLA 8/8 male sibling BMCardiomyo-pathyNot evaluableNot evaluableNot evaluableDied day 03 5.9 maleHLA 5/6 female unrelated CB (0.55)Graft rejectionIncreased by IF and Western↑ Rudimentary AFsNot evaluableDied day 1834 6.3 yo maleHLA 8/8 female sibling BM (3.76)Transient Dialysis ARDSNo change by IF° but WesternNormal AFs observedMarked reduction in blisters and dressingsAlive day 2475 6.2 yo femaleHLA 8/8 male sibling BM (3.07)Transient DialysisIncreased by IF and Western↑ Rudimentary AFsMarked reduction in blisters and dressingsAlive day 1286 6.0 yo femaleHLA 8/8 female sibling BM (3.11)EpistaxispendingpendingEarly reduction in blisteringAlive day 567 14.5 yo femaleHLA 8/8 female sibling BMToo early to evaluatependingpendingToo early to evaluateAlive day -9 Disclosures: No relevant conflicts of interest to declare.

Description: The recessive dystrophic form of epidermolysis bullosa (RDEB) is a disorder of incurable skin fragility and blistering caused by mutations in the type VII collagen gene (Col7a1). The absence of type VII collagen production leads to the loss of adhesion at the basement membrane zone due to the absence of anchoring fibrils, which are composed of type VII collagen. We report that wild-type, congenic bone marrow cells homed to damaged skin, produced type VII collagen protein and anchoring fibrils, ameliorated skin fragility, and reduced lethality in the murine model of RDEB generated by targeted Col7a1 disruption. These data provide the first evidence that a population of marrow cells can correct the basement membrane zone defect found in mice with RDEB and offer a potentially valuable approach for treatment of human RDEB and other extracellular matrix disorders.