A single epidermal stem cell strategy for safe ex vivo gene therapy.
Bottom Line: 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.
Affiliation: Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.Show MeSH
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Mentions: 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 (Fig1) 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 Table1, 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.
Affiliation: Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.