A single epidermal stem cell strategy for safe ex vivo gene therapy

EMBO Molecular Medicine

Stéphanie Droz-Georget Lathion⁵  Ariane Rochat⁵  Graham Knott⁶  Alessandra Recchia²  Danielle Martinet⁴  Sara Benmohammed³  Nicolas Grasset⁵  Andrea Zaffalon⁵  Nathalie Besuchet Schmutz⁴  Emmanuelle Savioz-Dayer⁵  Jacques Samuel Beckmann⁴  Jacques Rougemont¹  Fulvio Mavilio² 

[1] Bioinformatics and Biostatistics Core Facility, Faculty of Life Sciences EPFL, Lausanne, Switzerland;Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy;Department of Medical Genetics, Université de Lausanne, Lausanne, Switzerland;Service de Génétique Médicale, Lausanne University Hospital (CHUV), Lausanne, Switzerland;Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland;Interdisciplinary Center for Electron Microscopy, Faculty of Life Sciences EPFL, Lausanne, Switzerland

关键词: cell therapy;    regulatory affairs;    stem cells;    wound healing;   

DOI  :  10.15252/emmm.201404353

来源: Wiley

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 The Authors. Published under the terms of the CC BY 4.0 license

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