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 pLXSN␤3 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 ␣3␤1 in focal adhesions,14 and integrin ␣6␤4 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 (HK␤3) 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 HK␤3 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 HK␤3 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 appeared equimolar, which is indicative of a correct laminin ␤3 chain. This mutation is a deletion of two maturation of the recombinant laminin-5 protein. nucleotides (CA) at position 183 of the laminin ␤3 cDNA (GenBank accession number L25541), that generates a Enhanced adhesion of reverted H-JEB keratinocytes to premature termination codon causing decay of the the culture substrate aberrant ␤3 messenger RNA (not shown). As a conse- Expression of recombinant laminin-5 enhanced the quence, these H-JEB keratinocytes do not synthesize the adhesion capacity of HK␤3 keratinocytes that required laminin ␤3 chain and do not produce laminin-5. The longer exposure to trypsin than the parental H-JEB wild-type full-length cDNA for laminin ␤3 was then counterparts and HK-LXSN cells to be detached from the introduced into the H-JEB keratinocytes by retrovirus- plastic tissue culture support. The adhesion properties of mediated gene transfer. The vector was a pLXSN retrovi- HK␤3 cells seeded on different culture substrates were ral construct carrying a neomycin selectable marker therefore further investigated. Adhesion of HK␤3 kera- (neor) and the laminin ␤3 cDNA driven by the promotor- tinocytes to BSA, collagen IV, laminin-1 and fibronectin enhancer LTR sequences of MoMSV sequences (Figure 1). substrates was significantly higher (740, 300, 490 and 160, Corrective gene transfer in blistering skin diseases J Vailly et al 1324

Figure 2 Restored expression of laminin-5 in H-JEB keratinocytes infected with the retroviral vector pLXSN␤3. (A) Immunofluorescence analysis of HK-LXSN (a, c) and HK␤3 (b, d) cells with mAbs K140 (a, b) specific to the laminin ␤3 chain, and GB3 specific to native laminin-5 (c, d). (B) Immunoprecipitation analysis of culture medium conditioned by control human normal keratinocytes (lanes 1, 4, 7), and HK-LXSN (lanes 2, 5, 8) and HK␤3 (lanes 3, 6, 9) cells. Medium (1 ml) was immuno- precipitated using pAb SE144 (lanes 4, 5, 6) and mAb K140 (lanes 7, 8, 9) raised against the laminin ␥2 and ␤3 chains, respectively. Lanes 1, 2 and 3, control blank serum. The mass of the molecular markers is indicated on the left of the gel. Migration position of the laminin-5 polypeptides ␣3 (165 kDa), ␤3 (145 kDa), and ␥2 (155 and 105 kDa), and the laminin- 6 chains (190 and 210 kDa)37 are shown on the right of the gel. Unspecific slow-migrating bands are also visible.17 Immunoprecipitated complexes were fractionated on a 7.5% SDS-polyacrylamide gel. Exposure was 12 h on radiograph (Amersham, Les Ulis, France).

respectively) than that of the HK-LXSN cells, and similar 5 enhances adhesion of the HK␤3 keratinocytes and to that of control normal human keratinocytes influences their migration in vitro. (Figure 3a). Because it has been shown that the laminin-5-defective Relocalized distribution of hemidesmosomal H-JEB keratinocytes seeded on collagen type IV, display components in culture hypermotility compared with normal controls,17 we veri- HK␤3 keratinocytes displayed a flat and spreading shape fied whether expression of the recombinant laminin-5 contrasting with the small and rounded shape of the HK- influences motility of the HK␤3 cells. The migration LXSN cells and parental H-JEB keratinocytes. In vitro, capacity of the ‘cured’ H-JEB keratinocytes was evaluated normal keratinocytes synthesize adhesion structures by plating suspensions of HK␤3 cells on collagen IV sub- homologous to hemidesmosomes (SAC).14 Consistent strate coated with gold particles.17 Compared with HK- with the observation that laminin-5 induces formation of LXSN keratinocytes, which migrated leaving long tracks SAC that enhance cell adhesion and promote spreading,19 behind them, HK␤3 cells demonstrated reduced motility. absence of laminin-5 prevents formation of SAC in pri- Migration index value was 19.8 for HK-LXSN kera- mary H-JEB keratinocytes.17 To determine whether HK␤3 tinocytes, and 12.7 for HK␤3, representing a 35% keratinocytes express and assemble SAC, cell suspen- reduction of cell locomotion (Figure 3b). We therefore sions were seeded on to plastic dishes and were subjected concluded that re-expression of the recombinant laminin- to immunofluorescence analysis using antibodies specific Corrective gene transfer in blistering skin diseases J Vailly et al 1325 in the cytoplasm (Figure 4A). Examination by confocal immunofluorescence microscopy confirmed the basal localization of the hemidesmosome components in HK␤3 keratinocytes, and their non-polarized distribution in HK-LXSN cells (Figure 4B). We further enquired whether the extracellular matrix deposited by HK␤3 cells on the tissue culture support could induce nucleation of SAC in the parental H-JEB keratinocytes. Examination of cell cultures in which the transduced HK␤3 cells and the nontransduced parental H-JEB keratinocytes were mixed in the approximate ratio of 8:2, revealed that the H-JEB cells migrating on extra- cellular matrix deposited by the HK␤3 keratinocytes nucleated SAC. In addition, our results demonstrated that formation of SAC requires close contact of the basal plasma membrane with the laminin-5-containing deposit. Indeed, as illustrated in Figure 4C, the H-JEB kera- tinocytes nucleated hemidesmosome-like structures exclusively in the areas in contact with the tracks of extra- cellular matrix layered down by HK␤3 keratinocytes, whereas no SAC are detected in areas in contact with the plastic culture support.

Adhesion of a reconstructed epidermis in vitro and formation of hemidesmosomes To verify whether expression of laminin-5 enhances adhesion of artificial epidermis reconstructed in vitro using HK␤3 cells, suspensions of transduced H-JEB kera- tinocytes were seeded on to human dead de-epidermized dermis. At confluence, the keratinocyte cultures were exposed to air to obtain stratification into a multilayered epithelium. The organotypic cultures showed the hall- marks of complete differentiation into stratified epithelia presenting a well-organized and defined basal cell layer, a granular layer with cells rich in keratohyalin granules and a upper multilayered stratum corneum. Similar to the control normal human keratinocytes, the epithelia formed by the HK␤3 cells closely adhered to the dermis all along the dermal–epidermal junction (Figure 5A), while all the epithelia formed by the HK-LXSN and the parental H-JEB keratinocytes presented areas of large detachment from the underlying dermis and a fuzzy basal aspect all along the basement membrane zone. Figure 3 Adhesion and migration assays of reverted H-JEB keratinocytes. Since it could be argued that in the reconstructed epi- (a) Adhesion assay: a suspension of 3 × 105 cells per well was plated in dermis, separation of the epidermis from the matrix may 96-microtiter well plates coated with BSA, collagen IV, laminin-1 or fib- result from technical artifacts, we verified whether in epi- ronectin. After 12 h at 37°C, the cell layers were fixed, stained and their thelia formed with HK␤␤ keratinocytes the improved number evaluated as described in Materials and methods. Each experiment adhesion to DED correlated with assembly of HD. Elec- was repeated at least in triplicate. (b) Migration assay: 4 × 103 cells were seeded in 24-well culture plates coated with collagen IV (15 ␮g/ml) and tron microscopy analysis of the epidermis reconstructed colloidal gold particles.17 The phagokinetic tracks produced by individual with the transduced keratinocytes re-expressing laminin- cells were visualized 19 h after seeding by dark field microscopy, and 5 demonstrated that the basal cells assembled mature quantified by image analysis (BIOCOM, Les Ulis, France). The migration hemidesmosomes like the control epithelia obtained with index expresses the ratio (%) between the surface covered by the tracks normal human keratinocytes. The hemidesmosomes had and the total surface of the analyzed field. the external cytoplasmic plaque linked to the keratin intermediate filaments of the cytoskeleton, and a sub- basal dense plate connected to the anchoring filaments of the lamina lucida. In contrast, the parental H-JEB kera- to the major hemidesmosome proteins. All the known tinocytes and the HK-LXSN cells generated epithelia hemidesmosome components displayed a basal and dot- similar to H-JEB skin, with sparse and rudimentary hemi- like staining concentrated in the large ‘leopard skin’ desmosomes in the areas still adhering to the dermis. The patches characteristic of SAC formed in culture by nor- cytoplasmic plaque and the sub-basal dense plates were mal keratinocytes, while as expected in HK-LXSN kera- absent. The anchoring filaments were also reduced in tinocytes the fluorescence appeared diffusely distributed number (Figure 5B). Corrective gene transfer in blistering skin diseases J Vailly et al 1326

Figure 4 Relocalization of the hemidesmosomal components in the H-JEB reverted cells HK␤3. (A) Immunolocalization of integrin ␣6␤4 and BP180 in keratinocytes HK␤3. The cells were fixed 72 h after seeding, permeabilized 2 min with 0.1% Triton X100, and subjected to double immunofluorescence staining using mAb GoH3 directed against integrin ␣6 (a, b), 3E1 directed against integrin ␤4 (c, d), 233 specific to BP180 (e, f), and the anti-laminin ␤3 pAb ␤3B (not shown). In HK␤3 cells (b, d, f), staining displays the leopard skin-like pattern indicative of formation of SAC. In contrast, labeling of parental keratinocytes is diffusely distributed in the cytoplasm (a, c, e). (B) Immunolocalization of SAC by confocal scanning laser microscopy visualizes the rearranged distribution of integrin ␣6 (a, b), ␤4 (c, d) and BP180 (e, f) in HK-LXSN and HK␤3 keratinocytes. (C) Non-infected H-JEB cell migrating on a track of ECM deposited on the plastic tissue culture support by a HK␤3 keratinocyte. Immunofluorescence staining using mAb 233 (a), double staining with mAb 233 (yellow staining) and pAb ␤3B (red staining) (b). Bars, 50 ␮m.

Discussion tive transfer of a laminin-5 cDNA in these cells should therefore be easily monitored. H-JEB keratinocytes provide a promising model system As a first step towards developing a gene therapy for somatic gene therapy of heritable mechano-bullous approach to skin blistering diseases, we undertook stud- diseases. Lack of expression of laminin-5 causes the ies to determine whether expression of laminin-5 could severe complications of H-JEB. In the absence of this re-establish assembly of the complex dermal–epidermal epithelial adhesion ligand, hemidesmosomes are not attachment structures in H-JEB keratinocytes. In a pre- assembled. Consequently, anchoring of the keratinocyte vious work, we described the re-expression of laminin-5 to the extracellular matrix and association of the inter- in the immortalized H-JEB keratinocytes LSV5-R infected mediate filaments to the plasma membrane are severely with a retrovirus expressing a wild-type laminin ␥2 compromised. The positive dominant effect of a correc- chain.20 The transduced cells restructured focal Corrective gene transfer in blistering skin diseases J Vailly et al 1327

Figure 4 Continued. Corrective gene transfer in blistering skin diseases J Vailly et al 1328 C a b

Figure 4 Continued.

adhesions, modified their morphology and motility, and ration of the protein constitutes a key factor in hemides- improved their adhesion to the culture substrate, but mosome formation. Moreover, the results obtained in the failed to synthesize hemidesmosomal structures. These adhesion assays demonstrate that deposition of extra- results contrasted with the observation that immortalized cellular matrix containing laminin-5 markedly increases JEB keratinocytes deficient in the expression of integrin the adhesion of the reverted HK␤3 cells independently ␣6␤4 infected with retroviruses expressing the adequate from the nature of the culture substrate on which the cells wild-type transgene synthesize hemidesmosomal-like are grown. structures in vitro.21 A possible explanation of the partial Our results also show that parental H-JEB cells in con- phenotypic reversion observed with LSV5-R kera- tact with the extracellular matrix secreted by HK␤3 kera- tinocytes could reside in the reduced amounts of recom- tinocytes assemble SAC. This observation implies that the binant laminin-5 produced by these cells. Since normal recombinant laminin-5 exerts a paracrine effect on the keratinocytes down-regulate the synthesis of laminin-5 cells neighboring the cured H-JEB keratinocytes. Consist- along with culturing,17,22 we improved the culture con- ently, laminin-5 produced by the bladder epithelial cell ditions of primary H-JEB keratinocytes to obtain cell 804G was reported to exert a paracrine effect both on populations infected by retroviral vectors at a low num- adhesion of a variety of epithelial cell lines and on hemi- ber of passages in vitro. Based on immunoprecipitation desmosome preservation in the margins of human cornea analysis, the amount of recombinant laminin-5 synthe- explants.24 The paracrine effect of the recombinant lami- sized and secreted by the transduced HK␤3 keratinocytes nin-5 is of particular interest, because it implies that is similar to that produced by the wild-type primary efficient adhesion of the epidermis reconstituted with human keratinocytes. In addition, compared with the engineered keratinocytes re-expressing extracellular immortalized LSV5-R cells that deposit scant amounts of matrix components does not necessarily require a homo- mature laminin-5 on the culture substrate, the reverted geneous population of reverted cells. Our findings, there- HK␤3 keratinocytes efficiently process the recombinant fore, indicate that efficient gene transfer of diseased kera- laminin-5 into the mature 400 kDa form, which is readily tinocytes preserving a high proliferative potential may detected in the extracellular matrix. not need further enrichment of the transduced cell popu- Consistent with the notion that the total level of lations to produce self-renewing epithelia. This idea was expression of laminin-5 influences cell spreading and confirmed by the observation that organotypic cultures nucleation of SAC components in HaCaT keratinocytes,19 obtained with populations of keratinocytes containing and promotes adhesion of cultured JEB keratinocytes,23 approximately 80% of HK␤3 cells and 20% of parental H- HK␤3 cells display a flattened shape and assemble SAC JEB cells reconstituted epithelia in which the basal kera- in vitro. The changes in morphology of HK␤3 kera- tinocytes nucleated mature hemidesmosomes and tinocytes clearly indicate that the laminin-5 secreted by adhered tightly to the mesenchyme. However, it cannot these cells is biologically active and that efficient matu- be excluded that parental H-JEB keratinocytes unable to Corrective gene transfer in blistering skin diseases J Vailly et al 1329

Figure 5 Artificial epidermis reconstructed with reverted H-JEB keratinocytes HK␤3. (A) Histological sections of epidermis reconstituted with normal human control (a), parental H-JEB (b) and HK␤3 (c) keratinocytes. All along the dermal–epidermal junction of the artificial skin, basal HK␤3 and normal human keratinocytes adhere tightly to the dead de-epidermized dermis. Epithelia formed by parental H-JEB keratinocytes present areas of large detachment from the underlying dermis. (B) Ultrastructural examination of the dermal–epidermal junction of artificial epidermis generated with normal human control (a), parental H-JEB (b) and HK␤3 (c) keratinocytes. Similar to normal controls, basal HK␤3 keratinocytes nucleate mature hemidesmo- somes presenting an inner plaque associated with the keratin intermediate filaments (KIF). The outer plaque of the hemidesmosomes (OP), the anchoring filaments (AFl) of the lamina lucida (LL) and the sub-basal dense plate (SB) are clearly visible. The dermal anchoring fibrils (AFb) abutting the lamina densa (LD) are also detected. In contrast, in basal parental H-JEB keratinocytes, the hemidesmosomes are rudimentary, with no attachment plaque and sub-basal dense plate. The anchoring filaments are reduced in number. Bar, 200 nm. Corrective gene transfer in blistering skin diseases J Vailly et al 1330 synthesize hemidesmosomes are dislodged from the cation-defective retroviral vector pLXSN␤3. The plasmids basal layer by the proliferating HK␤3 keratinocytes and were amplified in E. coli XL1 blue, and purified using a persist within the multilayered epithelium. plasmid purification kit (Qiagen, Courtaboeuf, France). A paracrine effect of laminin-5 would also be in agree- High titer virus-producer cells were generated from ment with the observation that transplantation of wild- the ␺-CRIP packaging cell line using standard methods type primary muscle cells into dystrophia muscularis based on selection in the presence of G418. Fifteen clones (dy/dy) mice produce a gradient of diffusible laminin-2 were isolated and the one with the highest titer (5 × 105 accumulating around the proximal muscle fibers of the c.f.u./ml) was used to infect the H-JEB keratinocytes. laminin-2-defective host.25 In our organotypic skin cul- Integrity of retroviruses was verified by Southern blot ture model, diffusion of the recombinant laminin-5 analysis to detect the expected band in viral producer within the basement membrane zone of the reconstituted cells (not shown) after digestion with the restriction epithelia was not demonstrated, mainly because the dead enzyme EcoRI. For retroviral infection, subconfluent sec- de-epidermized dermis used as a culture support retains ondary keratinocyte cultures were trypsinized and a weak residual immunoreactivity to laminin-5 anti- seeded (5 × 103 cells per cm2) on a feeder-layer (3.5 × 104 bodies. Despite these limitations, our data clearly demon- cells per cm2) of lethally irradiated producer CRIP cells. strate that the reactive epitopes of laminin-5 detected on Three days later, the infected cultures were selected for the dead dermis do not induce nucleation of hemidesmo- neor in the presence of culture medium containing somes, because such anchoring structures are exclusively 400 ␮g/ml G418 (Sigma-Aldrich, St Louis, MO, USA). found in reconstructed epithelia with basal keratinocytes actively layering an extracellular matrix containing lami- Antibodies nin-5 on to the dermal support. Indeed, hemidesmosomal Synthesis of laminin-5 was monitored using mAb GB318 structures are not observed in artificial epithelia reconsti- that recognizes native laminin-5, mAb K14016 and poly- tuted with H-JEB keratinocytes that either produce no clonal antibody (pAb) ␤3B, specific to the laminin ␤3 laminin-5, or, like LSV5 cells, secrete scant amounts of chain; mAbs BM16531 and pAb SE85,32 specific to the lam- this adhesion ligand.17,20 In this system, the hemidesmo- inin ␣3 chain, and pAb SE144, specific to the laminin ␥2 somes reconstitute in correspondence to the anchoring chain33 were also used. Integrins ␣6 and ␤4, and BP180 fibrils of the de-epidermized dermis. Therefore, as in nor- were detected using mAbs GoH3,34 3E1 (Life mal skin, the laminin-5 deposited on the basement mem- Technologies) and 233,35 respectively. brane induces formation of the anchoring filaments and mediates their connection with the collagen VII mol- Immunoprecipitation and Western analysis ecules on the basement membrane.12 Since in JEB skin the Immunoprecipitation and Western analysis of cell papillary dermis assembles the anchoring fibrils,26 resur- extracts were performed using mAb K140 and pAb SE144 facing blister wounds with artificial epidermis obtained as already described.20 with reverted JEB keratinocytes is predicted to form the stable anchoring complexes that confer cohesion to the Immunofluorescence procedures integument. Immunofluorescence was performed as described pre- In conclusion, we have shown that transfer of a trans- viously,20 except that the second mAbs were conjugated gene restoring the expression of the major epithelial to Texas red (Dako, Glostrup, Denmark). To estimate the adhesion ligand in H-JEB keratinocytes regenerates arti- efficiency of infection of the cell cultures exposed to the ficial epithelia assembling the network of adhesion retroviral vector pLXSN␤3, the cell layers were double macromolecules present in the healthy skin. This is the stained using pAb ␤3B and mAb GoH3. The ratio first evidence that genetic treatment of inherited mech- between the number of cells expressing the transgene, ano-bullous conditions and extracellular matrix diseases stained by pAb ␤3B, and the total cell population, labeled is a realistic goal. by mAb GoH3, was estimated by image analysis of ran- domly chosen fields using a Biocom 500 image analysis system (Biocom, Les Ulis, France) coupled to a Zeiss Materials and methods Axiophot microscope.

Cell culture Cell adhesion and migration assays Human keratinocytes, obtained from a skin biopsy of a For adhesion assays, performed as previously H-JEB patient and a healthy control were cultivated on described,36 96-well plates were coated with 0.1% BSA in an irradiated feeder-layer of mouse 3T3-J2 cells in a 3:1 calcium- and magnesium-free phosphate-buffered saline mixture of Dulbecco’s modified eagle’s medium (DMEM) (PBS), 10 ␮g/ml collagen IV (Sigma-Aldrich) in 10 mm and Ham’s F-12 medium (Life Technologies, Cergy Pon- acetic acid, 10 ␮g/ml laminin-1 (Sigma-Aldrich) in PBS, toise, France) supplemented as described.27 or 10 ␮g/ml human fibronectin (Sigma-Aldrich) over- Swiss mouse 3T3-J2 cells,28 ␺-CRE and ␺-CRIP fibro- night at 4°C. Keratinocytes were plated (30 000 cells per blasts were grown in DMEM supplemented with 10% calf well) in medium in the absence of serum. Cells were serum in the case of 3T3-J2 cells or 10% fetal calf serum allowed to adhere for 12 h at 37°C. The cells in suspen- in the case of ␺-CRE and ␺-CRIP fibroblasts.29 sion were then removed by gentle washing with PBS. Adherent cells were fixed in 3% formaldehyde and Plasmids and retroviral infection stained with Crystal violet 0.5%, MetOH 20%, rinsed The full-length open reading frame of cDNA for the lami- three times with PBS. The dye was then eluted with 50% nin ␤ (GenBank accession number: L25541) was cloned EtOH/0.1 m sodium citrate, pH 4.2. Absorbance at 560 downstream of the mouse sarcoma virus long terminal nm was read in a microplate manager (Bio-Rad, Rich- repeat (LTR) of plasmid pLXSN30 to generate the repli- mond, CA, USA). Nonspecific adhesion was evaluated by Corrective gene transfer in blistering skin diseases J Vailly et al 1331 seeding keratinocytes either on glass or on bovine serum 7 Sullivan DM, Jensen TG, Taichman LB, Csaky KG. Ornithine-␦- albumin (0.1%). For HK␤3 cells, values were corrected by aminotransferase expression and ornithine metabolism in cul- the percentage of cells expressing the laminin-5. tured epidermal keratinocytes: towards metabolic sink therapy Cell migration assays were carried out using a phago- for gyrate atrophy. Gene Therapy 1997; 4: 1036–1044. kinetic track assay following a procedure detailed 8 Choate KA et al. Transglutaminase 1 delivery to lamellar ich- 17 tyosis keratinocytes. Hum Gene Ther 1996; 7: 2247–2253. previously. 9 Fine JD et al. Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. J Am Acad Dermatol 1991; 24: Artificial epidermis 119–135. Strips of human skin obtained from surgery were cut in 10 Christiano AM, Uitto J. Molecular complexity of the cutaneous 2-cm2 pieces, placed in calcium- and magnesium-free PBS basement membrane zone. Exp Dermatol 1996; 5: 1–11. for 10 days at 37°C. The epidermis was separated from 11 Borradori L, Sonnenberg A. Hemidesmosomes: roles in the dermis with fine forceps and the dermis was frozen adhesion, signaling and human diseases. Curr Opin Cell Biol and thawed 10 times. The resulting dead de-epidermized 1996; 8: 647–656. dermis (DED) was stored at −70°C until use. To recon- 12 Rousselle P et al. Laminin 5 binds the NC-1 domain of type VII × 4 collagen. J Cell Biol 1997; 138: 719–728. struct artificial epidermis, suspensions of 5 10 kera- 13 Burgeson RE et al. A new nomenclature for the laminins. Matrix tinocytes were seeded in stainless steel rings with a sur- Biol. 1994; 14: 209–211. 2 face of 1 cm laid on DED maintained by a grid and 14 Carter WG et al. Distinct functions for integrins alpha 3 beta 1 immersed in growth medium. The medium in the ring in focal adhesions and alpha 6 beta 4/bullous pemphigoid anti- was changed every day. A week later, the medium in gen in a new stable anchoring contact (SAC) of keratinocytes: the rings was removed and the keratinocyte culture was relation to hemidesmosomes. J Cell Biol 1990; 111: 3141–3154. brought at the air–liquid interface for 7 days. The 15 Stepp MA et al. ␣6␤4 Integrin heterodimer is a component of medium in contact with the dermal culture substrate was hemidesmosomes. Proc Natl Acad Sci USA 1990; 87: 8970–8974. changed every 2 days. 16 Marinkovich MP, Lunstrum GP, Burgeson RE. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem 1992; 267: 17900–17906. Ultrastructural analysis 17 Miquel C et al. Establishment and characterization of cell line Skin equivalents were fixed and embedded as previously LSV5 that retains the altered adhesive properties of human junc- 17 described. Ultrathin section were examined with a JEOL tional epidermolysis bullosa (H-JEB) keratinocytes. Exp Cell Res EM 1200 transmission electron microscope (Jeol, Croissy 1996; 224: 279–290. sur Seine, France). 18 Verrando P et al. Monoclonal antibody GB3, a new probe for the study of human basement membranes and hemidesmo- somes. Exp Cell Res 1987; 170: 116–128. Acknowledgements 19 Hormia M et al. Rapid spreading and mature hemidesmosome formation in HaCaT keratinocytes induced by incubation with We acknowledge A Spadafora, S Caillet, M Mari, A soluble laminin-5r. J Invest Dermatol 1995; 105: 557–561. Grima, C Minghelli for technical assistance and MS 20 Gagnoux-Palacios L et al. Functional re-expression of laminin-5 Campo for critical reading of the manuscript. We thank in laminin-␥2-deficient human keratinocytes modifies cell mor- R Burgeson for providing reagents, N Glaichenhaus for phology, motility and adhesion. J Biol Chem 1996; 271: 18437– ␺-CRIP and ␺-CRE cells, and W Hoeffler for the generous 18444. gift of antibody ␤3B. This work was supported by grants 21 Gagnoux-Palacios L, Gache Y, Ortonne JP, Meneguzzi G. Hemi- from: EEC BIOMED 2 (BMH4–97–2062), the Programme desmosome assembly assessed by expression of a wild-type Hospitalier de Recherche Clinique (France), the DEBRA integrin ␤4 cDNA in junctional epidermolysis bullosa kera- Foundation (UK), the Association Franc¸aise contre les tinocytes. Lab Invest 1997; 77: 1–10. Myopathies (France), the Ministe`re de l’Education 22 Matsui C et al. The assembly of laminin-5 subunits. J Biol Chem 1995; 270: 23496–23503. Nationale (ACCSV) and Ministe`re des Affaires Etran- 23 Coberly S et al. Exogenous laminin-5 protein therapy corrects ge`res (Programme Inte´gre´ Galile´e) France, Ministero JEB keratinocyte adhesive defects in vitro and basement mem- della Sanita` (Italy) and Telethon-Italy (grant A.106). brane defects in a JEB skin equivalent system. J Invest Dermatol 1997; 108: 549. 24 Baker SE et al. 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