Soutenance mémoire
L’épidermolyse bulleuse dystrophique récessive
Une peau reconstruite avec des kératinocytes et des fibroblastes génétiquement modifiés pour le traitement de l’épidermolyse bulleuse dystrophique récessive
Résumé
L’épidermolyse bulleuse dystrophique récessive (EBDR) est une maladie génétique rare causée par des mutations du gène COL7A1, codant pour le collagène de type VII (COLVII) et qui est sécrétée par les kératinocytes et les fibroblastes cutanés. L’absence du COLVII empêche la formation des fibrilles d’ancrage à la jonction dermo-épidermique. Nous proposons de traiter les patients EBDR avec des greffes de peau reconstruites par le génie tissulaire formées à partir des fibroblastes et des kératinocytes génétiquement corrigées par thérapie génique. D’abord, l’efficacité du transfert de l’ADNc normal du gène COL7A1 des vecteurs rétroviraux pseudotypés avec Ampho, Baev, Galv, et RD114 a été testée. Le vecteur Ampho était le meilleur pour transduire les fibroblastes. De plus, les facilitateurs de transduction polybrène et le peptide EF-C ont été testés et ce dernier s’est avéré plus efficace. Ainsi, en utilisant le vecteur Ampho et le peptide EF-C, le gène COL7A1 portant l’étiquette HA a été transduit dans 19% des fibroblastes et 36% des kératinocytes. Dans les peaux reconstruites produites avec ces cellules, nous avons observé un dépôt continu du HA-COLVII à la jonction dermo-épidermique, ainsi que des cellules souches épidermiques transduites. Cette approche est prometteuse pour offrir un traitement aux patients souffrant d’EBDR.
Manuscrit à être soumis pour publication
A TISSUE-ENGINEERED SKIN WITH GENETICALLY ENGINEERED KERATINOCYTES AND FIBROBLASTS FOR THE TREATMENT OF RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA
Angela Dakiw Piaceski1, Danielle Larouche1, Karim Ghani2, Francis Bisson1, Sergio Cortez Ghio1, Sebastien Larochelle1, Véronique Moulin1, Manuel Caruso2 and Lucie Germain1
1Centre de Recherche en organogénèse expérimentale de l’Université Laval/LOEX, Québec, Canada
2Centre de Recherche sur le cancer de l’Université Laval, Québec, Canada
1,2Centre de Recherche du CHU de Québec-Université Laval, Québec, Canada
*Address correspondence to:
E-mail: lucie.germain@fmed.ulaval.ca; manuel.caruso@crhdq.ulaval.ca
Short title: Self-assembled skin substitute for the treatment of RDEB
Keywords: epidermolysis bullosa; tissue-engineering; gene therapy; cell therapy; retroviral vector; stem cells, reconstructed skin.
Abstract
Recessive Dystrophic Epidermolysis Bullosa (RDEB) is a rare genetic disease caused by mutations in the COL7A1 gene that encodes type VII collagen (COLVII). This disease is characterized by intense blistering, fragility of the skin and mucous membranes, and many other debilitating symptoms. There is no cure for RDEB, and only palliative treatments are offered to patients. Collagen VII that is normally expressed by epidermal keratinocytes and dermal fibroblasts forms anchoring fibrils at the dermal-epidermal junction (DEJ). One logical approach that has been proposed to correct the defect is the transfer of a normal COL7A1 cDNA in keratinocytes. In this study, we are setting-up the base of a strategy for RDEB that includes gene therapy and tissue engineering. We propose to treat RDEB patients with grafts of tissue-engineered skin (TES) that comprise dermal and epidermal layers made with genetically corrected fibroblasts and keratinocytes. First, gene transfer efficacy of different retroviral vectors pseudotyped with the Ampho, Baev, Galv and RD114 envelopes was tested. We found that the Ampho vector was superior for gene delivery into fibroblasts. Most importantly, we report that the EF-C peptide was a much better transduction enhancer than polybrene that is commonly used to facilitate gene transfer. A 3 to 4-fold increase in fibroblasts and at least a 2-fold increase in keratinocytes was obtained. By using these conditions, a 19% gene transfer of a HA-tagged COL7A1 gene was achieved in fibroblasts and 36% in keratinocytes. In TES made with the transduced cells, we observed a continuous and intense deposit of HA-collagen VII at the DEJ. Furthermore, transduced epidermal stem cells and the characteristics of a skin graft are preserved in this model. This approach is promising to offer a treatment to patients suffering from RDEB.
Introduction
Epidermolysis bullosa (EB) is a genetic disease comprising a heterogeneous group of rare inherited skin disorders in which minor mechanical stress in the skin and mucous membranes causes the formation of blisters and erosions. There are four major types of EB depending on the location of the target proteins and the level of blisters: EB simplex (EBS; epidermolytic), junctional EB (JEB; lucidolytic), dystrophic EB (DEB; dermolytic), and Kindler syndrome (mixed levels of blistering) (1, 12). Blister formation occurs beneath the lamina densa within the papillary dermis in the skin of patients with DEB. Mutations in the COL7A1 gene are responsible for DEB, which can be autosomal dominant or recessive (RDEB) depending on the type of mutations (21). The COL7A1 gene encodes type VII collagen (COLVII) that is naturally secreted into the extracellular space by dermal fibroblasts and epidermal keratinocytes. These collagen molecules then assemble into the anchoring fibrils, a major component of the dermal-epidermal junction (DEJ) responsible for the adhesion between the epidermis and the dermis (7). DEB is characterized by abnormalities of anchoring fibrils, which are absent, reduced in number or malformed. Patients with RDEB can display other symptoms than blistering and fragility of the skin and mucous membranes; their hands and feet can be deformed, and they can suffer from anemia, malnutrition and growth retardation. The development of squamous cell carcinoma in areas subjected to intense blistering is a common trait in RDEB patients after their third decade. There is no cure for RDEB, and only palliative treatments restricted to individual wounds are currently offered to patients (1).
Several experimental strategies for RDEB have been tested in preclinical studies and in patients that include protein therapy, cell therapy (fibroblasts, mesenchymal stromal cells and hematopoietic stem cells), and gene therapy (9, 10, 13, 28, 31, 32, 36, 40-42). The latter approach was also used to treat successfully one JEB patient; keratinocytes obtained from the patient own skin were cultured in vitro and transduced with a retroviral vector containing the LAMB3 gene that encodes a subunit of laminin 332. After expansion of the corrected cells, cultured epidermal sheets were successfully transplanted on the patient’s legs. Absence of blistering was observed long-term, suggesting that this strategy could be applied to other EB types, like RDEB (10, 28).
Compared to the LAMB3 cDNA, the large size of the COL7A1 cDNA (8.9 kb) has limited the generation of high-titer retroviral vectors from stable retrovirus producer cells (8, 17, 37). Recombinant retroviruses can incorporate heterologous envelope glycoproteins at their surface, and this process known as pseudotyping is used to increase viral infectivity and/or modify vector tropism. High-titer retroviral packaging cell lines derived from 293Vec cells have been recently constructed in our laboratory. They release viral particles pseudotyped with the amphotropic (Ampho), the baboon endogenous virus (Baev), the gibbon ape leukemia virus (Galv) and the RD114 envelopes (15, 16). In this study, we have investigated the potential of GFP and COL7A1 vectors produced from these new packaging cell lines for the transduction of human keratinocytes and fibroblasts. We have also tested in this setting a 12-amino acid peptide, EF-C, derived from the HIV-1 envelope glycoprotein that was shown to be more potent than the commonly used polycation polybrene for increasing the transduction of fibroblasts and macrophages (29, 43).
A bilayered skin substitute with both, a dermis and an epidermis, could significantly improve the outcome of a graft in RDEB patients in term of resistance and mechanical properties. Furthermore, if the COL7A1 gene is introduced in fibroblasts as well as in keratinocytes, COLVII production at the DEJ should be optimal. These skin substitutes can be produced with a variety of methods based on the addition of biomaterials, synthetic materials or exogenous extracellular matrix (3, 4, 6, 38). In our laboratory, we have developed a bilayered tissue-engineered skin (TES) produced by the self-assembly approach in which the cells produce and organize their own extracellular matrix (24, 33, 34). TES have been successfully used to treat ulcers and burn patients (11, 27).
In this study, we have investigated the use of retroviral vectors produced from the 293Vec packaging cell lines for their ability to transduce fibroblasts and keratinocytes in presence of the peptide EF-C. We have also investigated for the first time the potential of transduced cells to produce and organize COLVII at the DEJ of TESs produced by the self-assembly approach of tissue-engineering.
Material and Methods
The study was approved by the institutional animal care and use committee and by the institutional committee for the protection of human subjects. The procedures followed were in accordance with the Helsinki Declaration of 1975.
Plasmids
The GFP retroviral plasmid was derived from GFP3 and contained a hygromycin resistance gene (Hygror) followed by an encephalomyocarditis virus internal ribosomal entry site sequence linked to the GFP gene. Both genes were under the control of the Moloney murine leukemia virus (MoMLV) long terminal repeat sequences (LTR) (35). The pLZRS-HACOL7A1 retroviral plasmid containing a human influenza hemagglutinin (HA)-tagged full-length human COL7A1 gene under the control of the MoMLV LTR was kindly provided by Alfred Lane (37).
Cell culture
HT-1080 cells, 293T cells, the retrovirus parental packaging cell lines 293Vec-Ampho, 293Vec-Baev, 293Vec-Galv and 293Vec-RD114 (15, 16), and their derivatives containing the GFP vector and the HA-COL7A1 vector were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wisent, St-Bruno, Canada) supplemented with 10% fetal calf serum (FCS, HyClone, Logan, UT) and 1% penicillin/streptomycin. The retrovirus producer cell lines were generated by infection with VSV-G pseudotyped recombinant retroviruses produced by transient transfection of 293T cells. The 293Vec-Ampho-HA-COL7A1 cells were 20% positive as assessed by immunofluorescence. Producer cells infected with the GFP virus were subsequently selected in hygromycine at 200 µg/ml, and the retrovirus producer cells were 99% GFP positive as assessed by the fluorescence-activated cell sorting (FACS) analysis.
Human fibroblasts and keratinocytes were obtained from mammary surgery of healthy adult donors after informed consent. Fibroblasts and keratinocytes were isolated by the two-step thermolysin and trypsin method and were cryopreserved as described previously (14, 26). Fibroblasts were thawed and cultured at passage 3 to 4 in DMEM containing 10% fetal calf serum and antibiotics. Keratinocytes were thawed and cultured at passage 1 in keratinocyte medium (3:1 mixture of the DMEM and Ham’s F12 medium (Invitrogen, Burlington, Canada) supplemented with 5% FCS, insulin, hydrocortisone, isoproterenol (Sandoz Canada Inc.), epidermal growth factor (EGF), and antibiotics. For all experiences, keratinocytes were co-cultured with irradiated human feeder layers (iHFL) as previously described (5). At each passage, cell number, and the average cell size were analyzed with a Beckman Coulter. Growth rates were calculated using the following formula (26):
37
( / )
=
Retroviral infection
GFP viral titers were determined by scoring positive target cells by FACS analysis. Briefly, HT-1080 cells were inoculated at a density of 105 cells per well in 24-well plates and cultured in 1 ml of medium overnight. The medium from each well was replaced with 1 ml of serial dilutions of virus supernatants in a 1 ml final volume containing 8 µg/ml polybrene. Forty-eight hours later, cells were trypsinized and analyzed for GFP fluorescence by FACS. For the HA-COL7A1 vector, HT-1080 cells were infected in 6-well plates and positive cells for HA were measured by immunofluorescence. Vector titers were calculated as follows: = ! ×! »#!$× , where F is the percentage of GFP or HA positive cells, determined by flow cytometry or immunofluorescence; Cinf is the total number of target cells at the time of infection; V is the viral volume applied; and D is the virus dilution factor. Infections resulting in 2-20% of GFP or HA positive cells were considered for titer calculation based on the linear range of the assay. Titers obtained for the 4 pseudotyped GFP viruses were 2 x 107 infectious viral particles (ivp)/ml. The titer for the 293Vec-Ampho-HA-COL7A1 was 1.8 x 105 ivp/ml.
The infectivity of the 4 GFP pseudotyped retroviruses in presence of polybrene and EF-C (Genscript, Piscataway Township, NJ) was assessed by infecting normal fibroblasts or keratinocytes plated at a density of 13,000 cells/cm2 in 12-well plates. Fibroblasts were incubated for two hours and keratinocytes for four hours at 37oC at 8% CO2. Then, fibroblasts were transduced at a multiplicity of infection (MOI) of 0.3 and keratinocytes at a MOI of 1 in the presence of 8 µg/ml of polybrene or 10 µg/ml of EF-C peptide. Transduction efficiency was evaluated by FACS analysis. The transduction with the HA-COL7A1 vector was done similarly to the GFP viruses but in T75 cm2 flask at a MOI of 0.36. The expression of HA-COL7A1 was assessed by immunofluorescence.
Tissue-engineered skin
The tissue-engineered skin method used here derives from the self-assembled skin substitute method referred to as “SASS-4” in Larouche et al (23). TES were produced with different combinations of transduced cells at MOI 0.36 and untransduced fibroblasts and keratinocytes. Briefly, 12,000 fibroblasts/cm2 at P5 transduced or not were cultured for 26 days in a fibroblast medium supplemented with 50 mg/ml ascorbic acid (Sigma Chemicals) until the production of a tissue sheet. Passage 2 keratinocytes transduced or not were seeded at a density of 200,000 cells/cm2 on one of the two sheets and were submerged in the keratinocyte medium supplemented with 50 mg/ml ascorbic acid for four days. Then, keratinocytes/fibroblasts sheets were superimposed over the fibroblast-derived tissue sheet to produce the TES. TES were cultured at the air–liquid interface in keratinocyte medium 50 mg/ml ascorbic acid without EGF for 21 days.
Fluorescence-activated cell sorting, immunofluorescence and histological analysis
GFP expression in transduced cultured cells was assessed by FACS analysis. HT-1080 cells, fibroblasts and keratinocytes were trypsinized 72 hrs after infection and analyzed for GFP expression. Fluorescence was measured with a Coulter EPICS XL-MCL flow cytometer and Expo32 software (Beckman Coulter, Brea, CA) or with a FACSCalibur (Becton Dickinson, San Jose, CA).
HA-COLVII expression in transduced cultured cells and in retrovirus producer cells was assessed by immunofluorescence. HT-1080 cells, 293Vec-Ampho-HA-COL7A1 cells, keratinocytes and fibroblasts were cultured in 6-well plates until they reached sub-confluence. Cells were fixed with methanol, or 4% paraformaldehyde followed by 0.2% Triton X-100. Next, cells were incubated with a primary mouse monoclonal antibody raised against HA (1:500) (clone HA.11; Biolegend, San Diego, CA) followed by an Alexa488-conjugated goat anti-mouse (1:1000) (Invitrogen) and nuclei were colored with Hoechst reagent 33258 (Sigma Chemicals).
For immunodetection in TES, biopsies were embedded in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. Immunofluorescence assays for transgene expression and markers were performed on 5-mm thick cryosections permeabilized with cold acetone with the following antibodies: mouse monoclonal anti-HA (clone HA.11), rabbit polyclonal anti-human collagen IV (1:400) (Abcam, Cambridge, MA), mouse monoclonal anti-K10 (1:200) (clone RKSE60; Cedarlane, Canada), rabbit polyclonal anti-K14 (1:1600) (Cedarlane, Canada), mouse anti-human Ki67 (1:800) (BD Biosciences, Canada), rabbit anti-human-loricrin (1:240) (Covance, Dedham, MA) and rabbit anti-human K19 (1:200) (LOEX, Canada). The following secondary antibodies were used: Alexa-488-conjugated goat anti-mouse and Alexa-594-conjugated goat anti-rabbit.
For histological analysis, biopsies from TES were fixed in Histochoice (Amresco, Solon, OH) and embedded in paraffin. Microtome sections (5-mm-thick) were stained with Haematoxylin and Eosin.
Statistical analysis
All analyses were performed in R (v3.3.0; RStudio v0.99.465)(44). The level of significance for all tests was set at p<0.05. Results were compared in the same experimental plan. Models were fitted using the nlme package(45). For doubling rate of controls, data was analyzed with one-way linear mixed models with treatment as a fixed factor and cell population as a random factor. Percentage of GFP-positive cells was analyzed with two-way linear mixed models with both pseudotyped vectors and transduction enhancers as fixed factors and cell population as a random factor. Normality of residuals (verified by visual assessment of qq-plots) was achieved by data transformation. Homoscedasticity (verified by visual assessment of fitted data ~ residuals) was accounted for by the addition of appropriate weights when necessary. Once assumptions were met, the best fit as described by the smallest Akaike criterion was selected. When models were significant (p<0.05), Tukey post-hoc tests were performed (multcomp package, version v1.4-0)(46) and significant pair-wise comparisons (p<0.05) were reported. Statistical power was calculated with the nlmeU package (v0.70-3)(47).
Results
Efficient transduction of human fibroblasts and keratinocytes with vectors produced from 293Vec packaging cell lines in presence of the EF-C peptide
It is admitted that retroviral vectors pseudotyped with the RD114 and Galv envelopes are highly efficient to transduce cells from hematopoietic origins (16, 20, 22). In this study, one of our objectives was to discriminate which envelope between Ampho, Baev, Galv and RD114 would be the most efficient for the transduction of human fibroblasts and keratinocytes. In addition to these four different envelopes, we have investigated the potential of the EF-C peptide as a transduction enhancer of fibroblasts and keratinocytes. High-titer recombinant GFP retroviruses produced from the 4 stable packaging cell lines were used to infect fibroblasts at a MOI of 0.3 and keratinocytes at a MOI of 1 in the presence of EF-C or polybrene. Fibroblasts were better transduced with the Ampho vector as compared to the 3 other pseudotyped viruses. In the presence of polybrene, the percentage of GFP positive cells was 10.1% with the Ampho vector, and 7.9%, 6.4% and 6.1% with the Baev, Galv and RD114 viruses, respectively. A similar trend was observed with the EF-C peptide with 32.3% transduction achieved with the Ampho vector as compared to 25.3%, 22.3% and 23.4% with the Baev, Galv and RD114 viruses, respectively (Fig. 2.1A). For the keratinocytes, the Baev pseudotyped was slightly less efficient than the other vectors. In presence of polybrene, 32.6% of keratinocytes were transduced with the Baev vector compared to 44.7%, 40.9% and 42.5% with the Ampho, Galv and RD114 vectors, respectively. With the EF-C peptide, 47.5% of keratinocytes were GFP positive with the Baev vector and 58.4%, 54.8% and 55.9% with the Ampho, Galv and RD114 vectors, respectively (Fig. 2.1B).
The most important results of these experiments are that the EF-C peptide was a much better transduction enhancer than polybrene. EF-C was able to increase the transduction efficiency of fibroblasts with all 4 pseudotyped by 3 to 4 folds. For the keratinocytes, the transduction rate was increased by 30 to 45% with EF-C over the polybrene condition. We could then conclude that the EF-C peptide outperformed polybrene as a transduction enhancer.
|
Table des matières
Chapitre 1 : Introduction
1.1 La peau
1.1.1 Le collagène de type VII
1.2 L’épidermolyse bulleuse
1.2.1 L’épidermolyse bulleuse dystrophique récessive
1.3 Les thérapies en développement pour traiter l’épidermolyse bulleuse dystrophique récessive
1.3.1 La thérapie par remplacement de protéines
1.3.2 La thérapie cellulaire
1.3.3 La thérapie génique
1.3.3.1 Vecteurs utilisés en thérapie génique pour traiter l’épidermolyse bulleuse dystrophique récessive
1.3.3.2 L’édition génique : l’édition ou la correction des séquences génomiques mutées
1.4 Le génie tissulaire pour la formation des peaux reconstruites
1.5 La thérapie génique combinée au génie tissulaire pour traiter l’épidermolyse bulleuse dystrophique récessive
1.6 Quelles cellules utiliser pour traiter l’épidermolyse bulleuse dystrophique récessive à l’aide de la thérapie génique et du génie tissulaire
1.7 Les essais cliniques associant la thérapie génique ex vivo au génie tissulaire
1.8 Hypothèse et objectifs
Chapitre 2 : Une peau reconstruite avec des kératinocytes et des fibroblastes génétiquement modifiés pour le traitement de l’épidermolyse bulleuse dystrophique récessive
2.1 Résumé
2.2 Manuscrit à être soumis pour publication
2.2.1 Abstract
2.2.2 Introduction
2.2.3 Material and Methods
2.2.3.1 Plasmids
2.2.3.2 Cell culture
2.2.3.3 Retroviral infection
2.2.3.4 Tissue-engineered skin
2.2.3.5 Fluorescence-activated cell sorting, immunofluorescence and histological analysis
2.2.3.6 Statistical analysis
2.2.4 Results
2.2.4.1 Efficient transduction of human fibroblasts and keratinocytes with vectors produced from 293Vec packaging cell lines in presence of the EF-C peptide
2.2.4.2 Efficient retroviral delivery of the COL7A1 gene to human fibroblasts and keratinocytes in the presence of EF-C
2.2.4.3 Transduced fibroblasts and keratinocytes self-assemble in tissue-engineered skin substitutes and express the exogenous collagen VII
2.2.4.4 Transduced keratinocyte stem cells are preserved in TES
2.2.4.5 The epidermal proliferation and differentiation profiles are conserved in TES made with transduced keratinocytes
2.2.5 Discussion
2.2.6 Acknowledgments
2.2.7 Conflicts of Interest
2.2.8 References
Chapitre 3 : Discussion, perspectives et conclusion
Références
Appendice 1. Strategies integrating gene therapy and tissue engineering into the development of treatments for Recessive Dystrophic Epidermolysis Bullosa
Télécharger le rapport complet
