A single epidermal stem cell strategy for safe ex vivo gene therapy
2015; Springer Nature; Volume: 7; Issue: 4 Linguagem: Inglês
10.15252/emmm.201404353
ISSN1757-4684
AutoresStéphanie Droz‐Georget Lathion, Ariane Rochat, Graham Knott, Alessandra Recchia, Danielle Martinet, Sara Benmohammed, Nicolas Grasset, Andrea Zaffalon, Nathalie Besuchet Schmutz, Emmanuelle Savioz-Dayer, J. Beckmann, Jacques Rougemont, Fulvio Mavilio, Yann Barrandon,
Tópico(s)Pluripotent Stem Cells Research
ResumoResearch Article27 February 2015Open Access A single epidermal stem cell strategy for safe ex vivo gene therapy Stéphanie Droz-Georget Lathion Stéphanie Droz-Georget Lathion Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Ariane Rochat Ariane Rochat Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Graham Knott Graham Knott Interdisciplinary Center for Electron Microscopy, Faculty of Life Sciences EPFL, Lausanne, Switzerland Search for more papers by this author Alessandra Recchia Alessandra Recchia Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Danielle Martinet Danielle Martinet Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland Search for more papers by this author Sara Benmohammed Sara Benmohammed Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland Search for more papers by this author Nicolas Grasset Nicolas Grasset Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Andrea Zaffalon Andrea Zaffalon Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Nathalie Besuchet Schmutz Nathalie Besuchet Schmutz Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland Search for more papers by this author Emmanuelle Savioz-Dayer Emmanuelle Savioz-Dayer Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Jacques Samuel Beckmann Jacques Samuel Beckmann Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland Search for more papers by this author Jacques Rougemont Jacques Rougemont Bioinformatics and Biostatistics Core Facility, Faculty of Life Sciences EPFL, Lausanne, Switzerland Search for more papers by this author Fulvio Mavilio Fulvio Mavilio Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Genethon, Evry, France Search for more papers by this author Yann Barrandon Corresponding Author Yann Barrandon Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Stéphanie Droz-Georget Lathion Stéphanie Droz-Georget Lathion Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Ariane Rochat Ariane Rochat Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Graham Knott Graham Knott Interdisciplinary Center for Electron Microscopy, Faculty of Life Sciences EPFL, Lausanne, Switzerland Search for more papers by this author Alessandra Recchia Alessandra Recchia Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Search for more papers by this author Danielle Martinet Danielle Martinet Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland Search for more papers by this author Sara Benmohammed Sara Benmohammed Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland Search for more papers by this author Nicolas Grasset Nicolas Grasset Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Andrea Zaffalon Andrea Zaffalon Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Nathalie Besuchet Schmutz Nathalie Besuchet Schmutz Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland Search for more papers by this author Emmanuelle Savioz-Dayer Emmanuelle Savioz-Dayer Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Jacques Samuel Beckmann Jacques Samuel Beckmann Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland Search for more papers by this author Jacques Rougemont Jacques Rougemont Bioinformatics and Biostatistics Core Facility, Faculty of Life Sciences EPFL, Lausanne, Switzerland Search for more papers by this author Fulvio Mavilio Fulvio Mavilio Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Genethon, Evry, France Search for more papers by this author Yann Barrandon Corresponding Author Yann Barrandon Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Author Information Stéphanie Droz-Georget Lathion1,2, Ariane Rochat1,2, Graham Knott3, Alessandra Recchia4, Danielle Martinet5, Sara Benmohammed6, Nicolas Grasset1,2, Andrea Zaffalon1,2, Nathalie Besuchet Schmutz5, Emmanuelle Savioz-Dayer1,2, Jacques Samuel Beckmann5,6, Jacques Rougemont7, Fulvio Mavilio4,8 and Yann Barrandon 1,2 1Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland 2Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 3Interdisciplinary Center for Electron Microscopy, Faculty of Life Sciences EPFL, Lausanne, Switzerland 4Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy 5Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland 6Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland 7Bioinformatics and Biostatistics Core Facility, Faculty of Life Sciences EPFL, Lausanne, Switzerland 8Genethon, Evry, France *Corresponding author. Tel: +41 21 314 24 61; Fax: +41 21 314 24 68; E-mail: [email protected] EMBO Mol Med (2015)7:380-393https://doi.org/10.15252/emmm.201404353 Correction added on 10 March 2015, after first online publication: author affiliations have been corrected. See also: JC Larsimont & C Blanpain (April 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract There is a widespread agreement from patient and professional organisations alike that the safety of stem cell therapeutics is of paramount importance, particularly for ex vivo autologous gene therapy. Yet current technology makes it difficult to thoroughly evaluate the behaviour of genetically corrected stem cells before they are transplanted. To address this, we have developed a strategy that permits transplantation of a clonal population of genetically corrected autologous stem cells that meet stringent selection criteria and the principle of precaution. As a proof of concept, we have stably transduced epidermal stem cells (holoclones) obtained from a patient suffering from recessive dystrophic epidermolysis bullosa. Holoclones were infected with self-inactivating retroviruses bearing a COL7A1 cDNA and cloned before the progeny of individual stem cells were characterised using a number of criteria. Clonal analysis revealed a great deal of heterogeneity among transduced stem cells in their capacity to produce functional type VII collagen (COLVII). Selected transduced stem cells transplanted onto immunodeficient mice regenerated a non-blistering epidermis for months and produced a functional COLVII. Safety was assessed by determining the sites of proviral integration, rearrangements and hit genes and by whole-genome sequencing. The progeny of the selected stem cells also had a diploid karyotype, was not tumorigenic and did not disseminate after long-term transplantation onto immunodeficient mice. In conclusion, a clonal strategy is a powerful and efficient means of by-passing the heterogeneity of a transduced stem cell population. It guarantees a safe and homogenous medicinal product, fulfilling the principle of precaution and the requirements of regulatory affairs. Furthermore, a clonal strategy makes it possible to envision exciting gene-editing technologies like zinc finger nucleases, TALENs and homologous recombination for next-generation gene therapy. Synopsis First time demonstration of a safe clonal strategy for ex vivo gene therapy before autologous transduced cells are transplanted into patients. Using recessive dystrophic epidermolysis bullosa (RDEB) COL7A1 corrected epidermal cloned stem cells as proof of principle, this strategy proves promising for clinical applications. Assessment of safety and efficacy of an ex vivo gene therapy product derived from a single epidermal stem cell transduced with a gene of interest. A clonal strategy was the best method to fulfil stringent safety regulatory requirements for ex vivo gene therapy. Long-term regeneration of anchoring fibrils from the progeny of a single genetically corrected epidermal stem cell from a patient with RDEB (type VII collagen deficiency) transplanted onto immunodeficient mice. Introduction Ex vivo gene therapy can permanently cure debilitating hereditary diseases (Hacein-Bey-Abina et al, 2002; Mavilio et al, 2006; Ott et al, 2006; Gargioli et al, 2008; Naldini, 2009; Mavilio, 2010; Tedesco et al, 2011; Aiuti et al, 2013; Biffi et al, 2013). Therapeutical success has been obtained in pioneer trials using genetically corrected human bone marrow stem cells to treat patients suffering from X-linked severe combined immunodeficiency (SCID) (Hacein-Bey-Abina et al, 2002), X-linked adrenoleukodystrophy (ALD) (Cartier et al, 2009) and SCID-adenosine deaminase (ADA-SCID) (Aiuti et al, 2009). However, unexpected complications like T-cell leukaemia have raised concerns (Hacein-Bey-Abina et al, 2003; Howe et al, 2008) about the safety of ex vivo gene therapy (Williams & Baum, 2003). Complications result from insertional mutagenesis together with clonal dominance (Hacein-Bey-Abina et al, 2008; Howe et al, 2008). Hence, the population of recombinant stem cells should be characterised before it is transplanted (Halme & Kessler, 2006; Fink, 2009). However, most tissue stem cells (e.g. hematopoietic and neural stem cells) cannot be efficiently expanded in culture by present technologies. To compensate for this limitation, integration sites have been documented in engrafted cells but it is only informative a posteriori (Aiuti et al, 2013). Human epidermal stem cells are privileged among adult (tissue) stem cells because they can be efficiently expanded ex vivo (Gallico et al, 1984; Rochat et al, 2013). The technology is based on the use of a lethally irradiated feeder layer of mouse 3T3-J2 cells that provides the necessary microenvironment to promote stem cell expansion (Rheinwald & Green, 1975; Barrandon & Green, 1987; Barrandon et al, 2012). Using this technology, it is possible to isolate enough epidermal stem cells from a small skin biopsy to generate large amounts of keratinocytes when cells are properly cultured; our laboratory is routinely using a strain of human diploid keratinocytes (YF29) isolated more than 25 years ago from the foreskin of a newborn. Most importantly, epidermal stem cells are used worldwide to treat extensive third-degree burn wounds to permanently restore epidermis (Gallico et al, 1984; Pellegrini et al, 1999; Ronfard et al, 2000; Chua et al, 2008; Cirodde et al, 2011). The use of autologous cultured epithelium is approved by FDA (Food and Drug Administration) as a humanitarian use device and is commercially available worldwide. Moreover, it is possible to efficiently clone epidermal stem cells and to obtain a large progeny from a single stem cell, a property that we have used to characterise growth capabilities and transplantability (Barrandon & Green, 1987; Rochat et al, 1994; Mathor et al, 1996; Claudinot et al, 2005; Majo et al, 2008; Bonfanti et al, 2010). Furthermore, human epidermal stem cells can be efficiently transduced by means of recombinant retroviruses to produce proteins of medical interest (Morgan et al, 1987; Mathor et al, 1996; Warrick et al, 2012). This was used to transplant a patient suffering from junctional epidermolysis bullosa with an engineered recombinant cultured epidermis producing laminin 5 (Mavilio et al, 2006; De Rosa et al, 2014). Taken together, these observations lead us to consider the feasibility of a single stem cell strategy for ex vivo gene therapy of debilitating hereditary skin disease while assessing its medical safety before clinical use. To demonstrate the feasibility of our strategy, we have selected severe generalised recessive dystrophic epidermolysis bullosa (Hallopeau-Siemens RDEB, OMIM 226600) as a model system for the following reasons. First, RDEB is a genodermatosis for which there is no curative treatment. RDEB is characterised by an extremely severe blistering due to poor adherence of epidermis to the dermis caused by deficient type VII collagen (COLVII), the major component of the anchoring fibrils (Bruckner-Tuderman et al, 1999; Fine et al, 2008). As a consequence, RDEB patients have extensive chronic wounds that can ultimately lead to death by invasive squamous cell carcinomas (Fine et al, 2009). Second, the severity of the disease has led to major therapeutic adventures like the transplantation of allogeneic bone marrow stem cells (Wagner et al, 2010), resulting in several patients' death (Tolar & Wagner, 2012, 2013) and other unconventional therapeutic alternatives (Woodley et al, 2007; Wong et al, 2008; Remington et al, 2009; Siprashvili et al, 2010; Itoh et al, 2011; Tolar et al, 2011). Third, we have demonstrated that a clone of human keratinocytes can produce COLVII and participate in the formation of anchoring fibrils (Regauer et al, 1990). Fourth, we have access to patients with well-characterised mutations, and among them a patient with a homozygous insertion–deletion resulting in a premature stop codon and absence of functional COLVII (Hilal et al, 1993; Hovnanian et al, 1997). Our strategy is inspired by the protocols and guidelines developed by the biotechnology industry and regulatory affairs to produce medicinal proteins by means of genetically engineered mammalian cells [http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q5D/Step4/Q5D_Guideline.pdf (ICH, 1997); http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q7/Step4/Q7_Guideline.pdf (ICH, 2000); http://www.isscr.org/docs/guidelines/isscrglclinicaltrans.pdf (ISSCR, 2008)]. The best clone following GLP (good laboratory practices) is first fully characterised and then transferred to GMP (good manufacturing practices) to prepare the master and working cell banks. The strategy for ex vivo gene therapy (Fig 1) is firstly isolation of epidermal stem cells from a patient's biopsy (step 1) and cultivation (step 2) before being permanently transduced by means of disease-specific viral shuttle vectors (step 3). Single cells are then isolated to obtain clones (step 4) that are expanded before they are individually frozen (step 5). In parallel, a small aliquot of each clone is expanded for further characterisation and validation (step 6). Once a clone fulfils the strict functionality and safety requirements described in Table 1, master and working cell banks are prepared in a GMP facility (step 7) in which genetically corrected autologous cultured epithelia (CEA) are also produced (step 8). These CEA are then transferred to the clinic and transplanted onto the patient (step 9). Our experiments have demonstrated that it is possible to produce enough genetically corrected autologous transplants from a single human epidermal stem cell for a pilot clinical trial fulfilling strict safety criteria. Table 1. Selection criteria for safety assessments of medicinal epidermal stem cells Selection criteria Assays Levels of confidence Mass culture Clonal culture Quality of medicinal product High growth potential Clonal analysis Low High Production of the protein of interest Western blotting/Immunocytochemistry Low High Long-term tissue regeneration In vivo transplantation onto immunodeficient mice Low High Long-term correction of the disease In vivo transplantation onto immunodeficient mice Low High Safety of medicinal product No immortalisation Serial passaging (cellular lifespan) Low High Western blotting (G1 checkpoint) Low High Karyotyping Low High No tumorigenic potential Subcutaneous injection into athymic mice Low High Determination of proviral integrations Ligation-mediated PCR Low Medium Fluorescence in situ hybridisation Low High Whole-genome sequencing Low High No dissemination of genetically modified human stem cells Organ analysis of transplanted immunodeficient mice Low High Selection criteria used to determine efficacy and safety of corrected stem cells before transplantation. These could be performed on mass culture or on single cell expansion. We determined the degree of reliability of each assay as low, medium and high. A clonal strategy gives a higher level of safety. Figure 1. Strategy to perform ex vivo gene therapy from a single epidermal stem cellSchematic strategy to produce a performant and safe gene therapy product from a single autologous epidermal stem cell. (1) A biopsy is obtained from the patient to isolate epidermal stem cells that are then expanded under appropriate conditions (2). An aliquot of the culture is infected with the ad hoc recombinant shuttle virus (3). Single cells are then isolated (4) and clones expanded to built a frozen stem cell bank (5). In parallel, an aliquot of each clone is expanded to select for a clone fulfilling the criteria described in Table 1. After validation (6), the approved clone is thawed and expanded to create master and working cell banks in a GMP facility (7). Genetically modified CEA are then produced (8) and grafts are transplanted onto the patient (9). MCB, master cell bank; WCB, working cell bank; CEA, cultured epidermal autografts; GLP, good laboratory practice; GMP, good manufacturing practice; GCP, good clinical practice. Download figure Download PowerPoint Results Identification of epidermal stem cells in the skin of an RDEB patient Recessive dystrophic epidermolysis bullosa keratinocytes were isolated from a small skin biopsy obtained from a 4-year-old patient with a homozygous insertion–deletion in the type VII collagen gene (COL7A1) leading to a premature stop codon in the fibronectin 5 domain and to the formation of severely truncated type VII collagen (Hilal et al, 1993). RDEB clonogenic keratinocytes were cultivated onto lethally irradiated 3T3-J2 cells according to standard procedures used for cell therapy of third-degree burn wounds (Pellegrini et al, 1999; Ronfard et al, 2000). No COLVII was detected in skin biopsies, nor in cultured fibroblasts and keratinocytes (Supplementary Fig S1). Because RDEB keratinocytes have been described as poor growers in the literature (Morley et al, 2003), we first determined the lifespan of RDEB clonogenic keratinocytes obtained from an early passage. Cells were subcultured once a week for 5 months (Fig 2A), and the percentage of growing colonies determined as it is the most reliable indicator of the growth capacity of a keratinocyte culture (Rochat et al, 2013). As expected, colony-forming efficiency (CFE) and the number of growing colonies decreased with time but at a similar rate to that of healthy keratinocytes, demonstrating that the growth potential of RDEB cells was not different from that of healthy cells of similar age. To further analyse the growth potential of RDEB clonogenic keratinocytes, we cloned individual RDEB keratinocytes that had already undergone five subcultures and at least 30 doublings (patient cells divided on average once a day) (Barrandon & Green, 1987). This experiment demonstrated the presence of holoclones (6% of clones) (Fig 2B), considered as the phenotype of human keratinocyte stem cells in culture (Barrandon & Green, 1987; Mathor et al, 1996; Rama et al, 2010; Rochat et al, 2013). Our data demonstrate that RDEB keratinocyte stem cells can be expanded in culture and cloned as are healthy keratinocytes. Figure 2. Extensive growth potential of recessive dystrophic epidermolysis bullosa (RDEB) epidermal keratinocytes Keratinocytes were isolated from the skin of a 4-year-old patient with severe-generalised RDEB linked to homozygous insertion–deletion in COL7A1 (Hilal et al, 1993). Cultured RDEB cells (blue line) were serially passaged for more than 4 months, displaying a growth potential similar to non-diseased control cells (YF29) isolated from the foreskin of a newborn (black line). To calculate the percentage of growing colonies, 100 to 1,000 cells were plated into indicator dishes at each passage. Cells were grown for 12 days, fixed and stained with rhodamine B. Colonies were scored as growing or aborted (Barrandon & Green, 1987). Clonal analysis demonstrated the presence of stem cells (holoclones) in a passage VII RDEB culture (95% of growing colonies). Download figure Download PowerPoint Genetic correction of RDEB epidermal stem cells RDEB keratinocytes were infected as a passage IV mass culture with a suspension of self-inactivating (SIN) retroviruses bearing a COL7A1 cDNA under the control of a minimal human elongation factor 1α (EF1α) promoter (Supplementary Fig S2) as previously described (Titeux et al, 2010). We first demonstrated that the infection procedure was compatible with stem cell maintenance (Supplementary Fig S3) and that on average thirty-five to forty-two per cent of the infected RDEB keratinocytes were positive for COLVII by immunostaining (Supplementary Fig S4). Next, we manually isolated one hundred and fifty single cells with a Pasteur pipette under an inverted microscope (Barrandon & Green, 1985). Sixty-seven clones were obtained, fifteen of which were obviously non-growing (paraclones). Each of the remaining fifty-two clones was transferred individually in several Petri dishes, first to expand the population, second to determine the clonal type (Barrandon & Green, 1987) and third to determine the production of COLVII. Three clones were classified as holoclones, forty-two as meroclones and three as paraclones; four clones were lost during cultivation for technical problems (Supplementary Table S1). COLVII was immunodetected in two holoclones (out of three), eighteen meroclones (out of forty-two) and three paraclones (all positive) (Supplementary Table S1). This demonstrated that keratinocytes with extensive or restricted growth potential were equally transduced and that COLVII expression was independent of clonal type. Next, we thoroughly characterised COLVII-positive clones (cl.6, cl.17, cl.22, cl.58 and cl.61) and COLVII-negative clones (cl.3, cl.24 and cl.54) (Fig 3A). qPCR experiments confirmed that the expression of COL7A1 was variable in different clones (Fig 3B), with levels of mRNAs varying from twofold to fiftyfold (clone 3 and clone 58, respectively) compared to uninfected RDEB keratinocytes. As expected, the lifespan of the individual clones was different, holoclones having a higher growth potential than meroclones (Supplementary Fig S5). We then showed that transduced keratinocytes expressed COLVII until the last subculture, eleven weeks after the start of the experiment (Supplementary Fig S6). Transduced COL7A1 cDNAs are known to frequently rearrange in contrast to other collagens (F. Mavilio, unpublished data); therefore, we performed Southern blots on genomic DNA obtained from several transduced clones using a COL7A1-specific probe (Fig 3C). The retroviral producer cloned line Flp293A-E1aColVII1 was used as a positive control. Bands corresponding to endogenous COL7A1 (16 kb) and proviral DNA (9.6 kb) were observed in control cells, whereas bands corresponding to the expected proviral DNA and rearranged proviral DNA were observed in clones 6 and 54. These rearrangements were not clearly detected in the infected cell pools from which clones 6 and 54 were isolated (lane 3); this does not mean that there was no rearrangement in the mass culture containing thousands of transduced stem cells, but rather that the use of a genetically homogenous population (clones) increased the threshold of detection. Next, the culture supernatants of transduced keratinocytes were analysed to determine whether COLVII was secreted. Only clone 6 correctly secreted COLVII while clone 54 did not (Fig 3D), further emphasising that a mass culture of transduced cells is vastly heterogeneous. This observation by itself justifies the clonal strategy. Figure 3. Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cellsSingle cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon & Green, 1987) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control. Download figure Download PowerPoint RDEB-corrected stem cells generate a functional self-renewing epidermis The first selection criterion for suitable gene therapy is the high growth potential of the COLVII-producing cells (corrected stem cells). This is a sine qua non condition to obtain a sufficient number of recombinant grafts to treat a patient. We thus performed serial transfer analysis of corrected clone 6 and compared it to COLVII non-producing clone 54 (uncorrected) (Fig 4A). Clones 6 and 54 had an extended lifespan as expected from their clonal type. Clone 6 could undergo eleven serial transfers from the day of cloning, which equals to fifty-nine population doublings, yielding a theoretical progeny of up to 5.7 × 1017 cells (Fig 4B). Collectively, these experiments demonstrated that a single transduced stem cell (holoclone) could generate a progeny large enough to produce medicinal CEA to treat wide areas of diseased skin. Figure 4. Long-term restoration of COLVII expression, generation of epidermis and anchoring fibrils by the progeny of a corrected recessive dystrop
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