Corrective Gene Transfer of Keratinocytes from Patients with Junctional Epidermolysis Bullosa Restores Assembly of Hemidesmosomes in Reconstructed Epithelia
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Gene Therapy (1998) 5, 1322–1332 1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia J Vailly1, L Gagnoux-Palacios1, E Dell’Ambra2, C Rome´ro1, M Pinola3, G Zambruno3, M De Luca2,3 J-P Ortonne1,4 and G Meneguzzi1 1U385 INSERM, Faculte´ de Me´decine, Nice; 4Service de Dermatologie, Hoˆpital L’Archet, Nice, France; Laboratories of 2Tissue Engineering and 3Molecular and Cell Biology, Istituto Dermopatico dell’Immacolata, Rome, Italy Herlitz junctional epidermolysis bullosa (H-JEB) provides deposited into the extracellular matrix. Re-expression of a promising model for somatic gene therapy of heritable laminin-5 induced cell spreading, nucleation of hemides- mechano-bullous disorders. This genodermatosis is mosomal-like structures and enhanced adhesion to the cul- caused by the lack of laminin-5 that results in absence of ture substrate. Organotypic cultures performed with the hemidesmosomes (HD) and defective adhesion of squam- transduced keratinocytes, reconstituted epidermis closely ous epithelia. To establish whether re-expression of lami- adhering to the mesenchyme and presenting mature hemi- nin-5 can restore assembly of the dermal-epidermal attach- desmosomes, bridging the cytoplasmic intermediate fila- ment 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 evi- laminin-5 in human H-JEB keratinocytes by transfer of a dence of phenotypic reversion of JEB keratinocytes by laminin 3 transgene. The transduced keratinocytes syn- somatic gene therapy and demonstrate that genetic treat- thesized 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 bio- inherited extracellular matrix pathologies is a realistic goal. logically active laminin-5 that was secreted, processed and Keywords: laminin-5; cell adhesion; extracellular matrix; basement membrane; artificial epidermis Introduction circulation,7 and recently the transglutaminase 1 cDNA has been transduced into keratinocytes from patients The epidermis presents a significant potential for somatic presenting with lamellar ichthyosis to recover enzymatic gene therapy in the treatment of inherited and acquired activity and restore the cutaneous barrier function.8 1 skin diseases. Skin keratinocytes are easily accessible Whether the epidermis may constitute a target tissue for and expanded in culture, and retain the ability for con- gene therapy of genetic disorders affecting structural pro- tinuous self-renewal through the proliferative potential teins and components of the extracellular matrix, how- 2,3 of a cell stem population. Grafting of in vitro reconsti- ever, remains an open question. tuted epithelia is routinely used to regenerate epidermis In the skin, perturbed expression of the structural pro- in patients with burn injuries and chronic ulcers. The risk teins crucial for the integrity of the dermal–epidermal of complications is low, because implants are easily adhesion zone results in epidermolysis bullosa (EB), a monitored and excised if needed. group of heritable blistering disorders that comprises The ability of transduced human keratinocytes to syn- clinical manifestations amenable to treatment by gene thesize and secrete biologically active recombinant pro- therapy. Among them, the mild junctional forms of EB teins has been demonstrated. Human growth hormone, (JEB) appear particularly suitable, because these con- apolipoprotein E and the coagulation cascade factor IX ditions do not require treatment of the whole integu- are successfully delivered by genetically modified kera- ment.9 JEB is a recessively inherited genodermatosis 4–6 ␦ tinocytes. Expression of ornithine- -aminotransferase characterized by continuous blistering, consequent to by engineered keratinocytes has also been used to mechanical trauma, with tissue separation within the develop a metabolic system for clearing ornithine from lamina lucida of the basement membrane of the dermal– epidermal junction. The various clinical forms of JEB have been associated with mutations in the genes enco- Correspondence: G Meneguzzi, U385 INSERM, Faculte´ de Me´decine, ding the hemidesmosome components and laminin-5, the 06107 Nice cedex 2, France major adhesion ligand of squamous and transitional Received 8 January 1998; accepted 20 April 1998 epithelia.10 Corrective gene transfer in blistering skin diseases J Vailly et al 1323 Hemidesmosomes are specialized junctions that main- tain strong adhesion of epithelia to basement membrane and connective tissue.11 Ultrastructurally, hemidesmo- somes appear as dense thickenings of the basal plasma membrane of the basal keratinocytes that connect the intermediate filaments of the cytoskeleton to the anchor- ing filaments. The anchoring filaments extend through  the lamina lucida to the lamina densa of the basement Figure 1 Schematic structure of the retroviral vector pLXSN 3 express- ing the laminin 3 chain. Plasmid pLXSN3 comprises the 5′ and 3′ membrane zone, and bridge the hemidesmosomes with Moloney mouse sarcoma virus LTRs, the full-length laminin 3 cDNA 12 the anchoring fibrils of the upper dermis. These (3.9 kb), and a neor gene driven by the SV40 promoter. The cloning adhesion complexes are thought to mediate signaling restriction sites of the laminin 3 cDNA sequences are indicated. between the cell and the extracellular matrix. Laminin-5 is a heterotrimeric glycoprotein comprising an ␣3, 3 and ␥2 chain which is encoded by distinct genes.13 Synthesized and secreted by the basal epithelial cells, laminin-5 mediates epithelial cell adhesion via inte- Controls were parental H-JEB keratinocytes before and grin ␣31 in focal adhesions,14 and integrin ␣64 in hemi- after infection with the retrovirus vector pLXSN express- desmosomes.15 In the skin, it colocalizes with the anchor- ing the neor gene (HK-LXSN cells). ing filaments, and provides a specific substrate for The transduced cultures were enriched in infected cells adhesion of proliferating and migrating cells. In the by partial selection for neomycin resistance in the pres- extracellular matrix, laminin-5 is found in two predomi- ence of G418. After 10 days, the number of keratinocytes nant forms comprising a species with a molecular mass expressing the transgene (HK3) was estimated by image of 440 kDa which is processed into a form of 400 kDa by analysis of randomly chosen areas of exponentially grow- proteolytic cleavage of the ␥2 chain.16 ing cell cultures double-labeled with pAb 3B and mAb Genetic mutations blocking the expression of laminin- GoH3, directed against the laminin 3 and integrin ␣6 5 result in the lethal, generalized form of JEB (H-JEB), polypeptides, respectively, that react with non-differen- a condition characterized by extensive blistering of the tiated keratinocytes. All the keratinocytes were stained epithelia and synthesis of rudimentary hemidesmosomes. by mAb GoH3, while 80% of them was also reactive to In vitro, H-JEB keratinocytes do not assemble hemides- pAb 3B, which indicated that the remaining cells (20%) mosomal structures (defined as SAC, for stable anchoring were non-infected parental H-JEB keratinocytes. contacts), and adhere poorly to the tissue culture sup- Expression of the recombinant laminin 3 chain was port.17 These cells, therefore, constitute a useful model further monitored by immunofluorescence analysis of the system to evaluate the capacity of JEB keratinocytes recipient cells using mAb K140, that recognizes the lami- treated by somatic gene therapy to re-express biologically nin 3 polypeptide,16 and mAb GB3, which reacts with active adhesion proteins and restore the assembly of the the native laminin-5 heterotrimer.18 The intense cytoplas- supramolecular cell adhesion structures that assure the mic staining and labeling of the extracellular matrix cohesion of the integument. deposited on the tissue culture substrate by the HK3 In this work, we demonstrate that corrective gene cells confirmed that the synthesis and secretion of a transfer of H-JEB keratinocytes leads to the full pheno- native recombinant laminin-5 had taken place in all the typic reversion of the diseased cells. The cured kera- keratinocytes expressing the recombinant 3 polypeptide tinocytes generate artificial epithelia assembling mature (Figure 2a). No immunoreactivity was noted with the stable anchoring complexes. parental H-JEB keratinocytes. Immunoprecipitation analysis of conditioned medium from HK3 cell cultures using antibodies specific to each Results distinct chain of laminin-5 identified polypeptides that in SDS-PAGE electrophoresis migrated with an apparent Synthesis of recombinant laminin-5 by transduced molecular mass of 165, 155, 145 and 105 kDa, identical to keratinocytes from a H-JEB patient that of the polypeptides that constitute the precursor and To revert phenotypically adhesion-defective epithelial processed forms of extracellular laminin-5 (Figure 2B).16 cells, we used secondary cultures of keratinocytes The ratio between the amount of the processed (105 kDa) obtained from a H-JEB patient carrying a homozygous and the unprocessed (155 kDa) laminin ␥2 chain mutation (183delCA) in the gene (LAMB3) coding for the