Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa.

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-ß3 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.

Establishment and characterization of cell line LSV5 that retains the altered adhesive properties of human junctional epidermolysis bullosa keratinocytes.

Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by hampered expression of the adhesion ligand laminin-5. Thus far, analysis of the processes underlying the epithelial-mesenchymal dysadhesion marking this disease has been limited by the reduced growth and adhesive capabilities of the epithelial cells derived from H-JEB patients. To overcome these difficulties, we used SV40 virus to immortalize H-JEB keratinocytes with a homozygous nonsense mutation in the gene that encodes the gamma2 chain of laminin-5. Cell lines (LSV) derived from infected keratinocytes maintain a stable karyotype, grow independent of 3T3 feeder layers and are not tumorigenic. Further analysis of clone LSV5 showed an increased secretion of laminin-6 and fibronectin compared to normal keratinocytes. Similar to parental H-JEB keratinocytes, these cells regenerate stratified epidermis in vitro and, in in vivo models, they synthesize a basement membrane lacking laminin-5. LSV cells show hypermotility and reduced adhesive properties resulting from an incomplete association with the underlying culture substrate. These results demonstrate that LSV5 cells retain the pathologic phenotype of H-JEB keratinocytes and can serve as a model system to study the adhesion processes mediated by laminin-5.