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.
Highlights
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)
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)
The strategy described here aims at narrowing the risk associated with ex vivo gene therapy as the medicinal product is thoroughly characterised before its use in the clinic
Summary
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-BeyAbina et al, 2002), X-linked adrenoleukodystrophy (ALD) (Cartier et al, 2009) and SCID-adenosine deaminase (ADA-SCID) (Aiuti et al, 2009). 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). Most tissue stem cells (e.g. hematopoietic and neural stem cells) cannot be efficiently expanded in culture by
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