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

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.

Gene editing for skin diseases: designer nucleases as tools for gene therapy of skin fragility disorders

“…; Droz‐Georget Lathion et al . ). This method was recently applied in a gene substitution treatment applied for JEB.…”

“…This can be achieved via correction of autologous stem cells and transplantation. A great advantage of targeting keratinocytes is the possibility of clonal selection and examination of modified stem cells upon treatment, thereby increasing the ability to assess the safety profile of the approach significantly (Larcher et al 2007;Duarte et al 2014;Droz-Georget Lathion et al 2015). This method was recently applied in a gene substitution treatment applied for JEB.…”

RNA‐based therapies for genodermatoses

“…Therefore, future efforts should focus on investigating off‐target events and further validating the safety of this approach. In the meantime, the most promising and safest strategy for a clinical application would be correction of stem cells followed by generation of skin grafts from single‐cell clones that have undergone careful safety profiling . Just as with AON‐mediated splicing modulation, reaching clinically relevant trans‐splicing efficiency may prove to be a hurdle.…”

“…larger than the viral vector capacity) are limitations that impede development of gene therapy approaches for skin diseases . At present, transplantation of gene‐corrected skin grafts seems to be the most promising approach . In addition, genome editing is emerging as a powerful tool for gene and cell therapy .…”

“…8,9 At present, transplantation of genecorrected skin grafts seems to be the most promising approach. [10][11][12][13][14] In addition, genome editing is emerging as a powerful tool for gene *Both authors contributed equally to this study. † These authors contributed equally to this study.…”