A single epidermal stem cell strategy for safe ex vivo gene therapy.
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.
Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Stéphanie
S
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.
Ariane
A
Rochat
Interdisciplinary Center for Electron Microscopy, Faculty of Life Sciences EPFL, Lausanne, Switzerland.
Graham
G
Knott
Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
Alessandra
A
Recchia
Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland.
Danielle
D
Martinet
Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland.
Sara
S
Benmohammed
Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Nicolas
N
Grasset
Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Andrea
A
Zaffalon
Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland.
Nathalie
N
Besuchet Schmutz
Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Emmanuelle
E
Savioz-Dayer
Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland.
Jacques Samuel
JS
Beckmann
Bioinformatics and Biostatistics Core Facility, Faculty of Life Sciences EPFL, Lausanne, Switzerland.
Jacques
J
Rougemont
Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy Genethon, Evry, France.
Fulvio
F
Mavilio
Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland yann.barrandon@epfl.ch.
Yann
Y
Barrandon
D000328:Adult; D000818:Animals; D002478:Cells, Cultured; D024143:Collagen Type VII / Q000096:biosynthesis / Q000235:genetics; D004817:Epidermis; D016108:Epidermolysis Bullosa Dystrophica / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D064593:Heterografts; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D051379:Mice; D016513:Mice, SCID; D012190:Retroviridae / Q000235:genetics; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000378:metabolism* / Q000473:pathology; D014161:Transduction, Genetic
C569053:COL7A1 protein, human; D024143:Collagen Type VII
cell therapy; regulatory affairs; stem cells; wound healing