Corrective Transduction of Human Epidermal Stem Cells in Laminin-5-Dependent Junctional Epidermolysis Bullosa

Abstract

Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses.

Gene Correction of Integrin β4-dependent Pyloric Atresia-Junctional Epidermolysis Bullosa Keratinocytes Establishes a Role for β4 Tyrosines 1422 and 1440 in Hemidesmosome Assembly

“…Informed consent was obtained from the parents. Keratinocytes were cultivated on a feeder layer of lethally irradiated 3T3-J2 cells as described (28,30,33) and passaged at the stage of subconfluence.…”

“…This said, the functional role of HD components in the proper assembly of mature HDs can in fact be studied in vitro because normal human primary keratinocytes can be cultivated in conditions that allow full epithelial differentiation (26 -28) and proper assembly of mature HDs (29,30). Keratinocytes can generate cohesive sheets of stratified epithelium that maintains virtually the same differentiation features and gene expression pattern of its in vivo counterpart so that it can be routinely transplanted in patients suffering from large skin or mucosal defects (31)(32)(33).…”

“…Keratinocytes can generate cohesive sheets of stratified epithelium that maintains virtually the same differentiation features and gene expression pattern of its in vivo counterpart so that it can be routinely transplanted in patients suffering from large skin or mucosal defects (31)(32)(33). When primary keratinocytes are seeded onto dead de-epidermized dermis in organotypic cultures (29), mature HDs are formed in vitro (30). Therefore, the availability of human ␤ 4 -deficient primary keratinocytes (see "Results"), the possibility of stably transducing primary keratinocytes with high efficiency (34), and the possibility of subcultivating stably transduced cells in conditions in which HDs are formed (30) provide a unique opportunity to clarify the above uncertainties and to investigate the role of ␤ 4 and of its potential TAM in the formation of mature HDs.…”

In vivo transduction of mouse epidermis with recombinant retroviral vectors: implications for cutaneous gene therapy

“…24 Transfer of the LAMB3 gene to cultured keratinocytes results in restoration of proper hemidesmosomal assembly and colony formation in vitro. 25 Long-term expression of a transgene in a rapidly renewing tissue such as epidermis is achieved only when the gene is stably transmitted to long-lived stem cells. RRV have been used with considerable success for transduction of epidermal stem cells in culture.…”

Thérapie génique cutanée

“…Dans le cas de l'approche ex vivo, il faut, bien entendu, maîtriser la culture des cellules appropriées (provenant de l'organe ou du tissu ciblé) et pouvoir corriger fonctionnellement et à long terme leur défaut génétique tout en préservant les perspectives de réimplantation chez le patient. L'épiderme, le tissu superficiel de la peau, occupe une place de premier plan dans le domaine de la théra-pie génique [1][2][3][4][5][6][7]. L'accessibilité du tissu cutané est un avantage évident.…”