725. Correction of Laminin-5-Deficient Junctional Epidermolysis Bullosa by Transplantation of Genetically Modified Epidermal Stem Cells. A Phase-I Clinical Trial

Mutations in genes encoding the laminin-5 heterotrimer, a key component of the epidermal-dermal junction, cause junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Epidermal stem cells isolated from patients affected by |[beta]|3 chain-deficient JEB were transduced with a retroviral vector expressing a |[beta]|3 cDNA, and used to generate uniformly transduced cultured skin implants. The transgene was steadily expressed for >160 cell doublings in culture, leading to restoration of normal laminin 5 levels, assembly of functional hemidesmosomes, and full phenotypic correction. Cloning and sequencing of vector integrations showed that <20 stem cells are responsible for long-term maintenance of a transplantable skin culture. A phase-I clinical trial started in October 2005, aimed at proving the safety of the transduction/transplantation procedure, and analyzing persistence of transgene expression and long-term survival of transduced stem cells. The first patient was a 30-yr-old male affected by non-lethal JEB, carrying a null mutation in one LAMB3 allele and a point mutation (E212K) in the other one. The mutation affects the assembly of the laminin-5 heterotrimer, present at residual levels (<5%) in vitro and in vivo. Six genetically modified, cultured epidermal sheets of 100 sq cm were transplanted on both legs after removal of the outer skin layer using a minimally invasive technique. The procedure was well tolerated, and the patient discharged after five days. Engraftment was completed after 10 days, and transplanted skin remained stable on both legs in the absence of blistering or inflammation for the duration of the follow-up (4 months at the time of writing). 3-mm punch biopsies were taken 1 and 3 months after transplantation, and analyzed for vector presence by quantitative PCR and for protein expression by immunohistochemistry. A vector signal compatible with full transduction of the transplanted epidermis was observed at both time points. Synthesis and assembly of normal levels of heterotrimeric laminin-5 and |[alpha]|6|[beta]|4 integrin was observed at the level of the basal lamina in all biopsies, together with the development of a firmly adherent, fully differentiated epidermis. Epidermal stem cells (p64+) were detected in the basal layer of the transplanted skin in normal numbers. These data show that gene therapy of JEB by transplantation of genetically corrected stem cells is feasible, and leads to full phenotypic correction of the adhesion defect in vivo. Safety studies are under way, which include detection or humoral or cytotoxic immune responses against laminin-5, and ex vivo cloning and sequencing of the integrated proviruses.