Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia

Abstract

Herlitz junctional epidermolysis bullosa (H-JEB) providesdeposited into the extracellular matrix. Re-expression of a promising model for somatic gene therapy of heritable laminin-5 induced cell spreading, nucleation of hemidesmechano-bullous disorders. This genodermatosis is mosomal-like structures and enhanced adhesion to the culcaused by the lack of laminin-5 that results in absence of ture substrate. Organotypic cultures performed with the hemidesmosomes (HD) and defective adhesion of squamtransduced keratinocytes, reconstituted epidermis closely ous epithelia. To establish whether re-expression of lamiadhering to the mesenchyme and presenting mature heminin-5 can restore assembly of the dermal-epidermal attachdesmosomes, bridging the cytoplasmic intermediate filament structures lacking in the H-JEB skin, we corrected the ments of the basal cells to the anchoring filaments of the genetic mutation hindering expression of the ␤3 chain of basement membrane. Our results provide the first evilaminin-5 in human H-JEB keratinocytes by transfer of a dence of phenotypic reversion of JEB keratinocytes by laminin ␤3 transgene. The transduced keratinocytes synsomatic gene therapy and demonstrate that genetic treatthesized a recombinant ␤3 polypeptide that assembled ment of the mild forms of skin blistering diseases and other with the endogenous laminin ␣3 and ␥2 chains into a bioinherited extracellular matrix pathologies is a realistic goal. logically active laminin-5 that was secreted, processed and

Confluence switch signaling regulates ECM composition and plasmin proteolytic cascade in keratinocytes

“…To assess whether absence of laminin secretion by HKs inhibits perlecan deposition, and thereby deregulates PAI-1 ECM accumulation, perlecan deposition was investigated in Lm-332 null-keratinocytes. Lm-332-null keratinocytes were isolated from patients affected by junctional epidermolysis bullosa (JEB) caused by absent expression of laminin b3 subunit (Vailly et al, 1998). Slot-blot analysis of ECM extracts failed to detect perlecan in post-confluent JEB keratinocyte cell cultures despite presence of perlecan in the spent culture medium (Fig.…”

Genética Molecular das Epidermólises Bolhosas

“…Já há relatos da manipulação ex vivo de queratinócitos de portadores de EBJ, incapazes de produzir a cadeia ß3 da laminina 5, os quais, após transferência gênica, se mostraram capazes -ainda que transitoriamente -de sintetizá-la, abrindo novas perspectivas terapêuticas para esse grupo de genodermatoses. 54 Modelo animal com ratos transgênicos, simulando a doença humana, tem acrescentado informações relevantes na pesquisa das EB. 10,12 Alguns autores consideram que as correlações entre genótipo e fenótipo estejam apenas começando 34 e que a expansão dos bancos de dados sobre as alterações gênicas seja de extrema importância, pois permitirá, cada vez mais, que se melhore essa correlação e talvez até se reclassifique, com base em aspectos moleculares, parte das genodermatoses.…”

Tissue-specific expression and long-term deposition of human collagen VII in the skin of transgenic mice: implications for gene therapy

“…27,28 Retrovirus-mediated gene transfer of the LAMB3 gene to cultured keratinocytes from patients with junctional EB (JEB) resulted in correct synthesis, processing and deposition of laminin 5 and restoration of assembly of the hemidesmosomes missing in JEB skin. 29 In another ex vivo study, the COL17A1 cDNA was used to restore BP180/collagen XVII protein synthesis in primary JEB keratinocytes. 30 Grafted keratinocytes regenerated skin in an immunodeficient mouse model.…”

“…The successful restoration of hemidesmosomes in JEB keratinocytes 29 suggests that resurfacing blister wounds of DEB patients with artificial epidermis obtained from DEB keratinocytes stably transfected with a genomic COL7A1 vector could result in the formation of stable anchoring complexes. Alternatively, direct in vivo approaches by way of either intradermal injection 49,50 of genomic COL7A1 vector or topical administration of naked DNA to skin via hair follicles 51 may result in adsorption and expression in the epidermis.…”