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

Droz-Georget Lathion S, Rochat A, Knott G, Recchia A, Martinet D, Benmohammed S, Grasset N, Zaffalon A, Besuchet Schmutz N, Savioz-Dayer E, Beckmann JS, Rougemont J, Mavilio F, Barrandon Y - EMBO Mol Med (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery, Lausanne University Hospital (CHUV), Lausanne, Switzerland Laboratory of Stem Cell Dynamics, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

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Strategy to perform ex vivo gene therapy from a single epidermal stem cellSchematic strategy to produce a performant and safe gene therapy product from a single autologous epidermal stem cell. (1) A biopsy is obtained from the patient to isolate epidermal stem cells that are then expanded under appropriate conditions (2). An aliquot of the culture is infected with the ad hoc recombinant shuttle virus (3). Single cells are then isolated (4) and clones expanded to built a frozen stem cell bank (5). In parallel, an aliquot of each clone is expanded to select for a clone fulfilling the criteria described in Table1. After validation (6), the approved clone is thawed and expanded to create master and working cell banks in a GMP facility (7). Genetically modified CEA are then produced (8) and grafts are transplanted onto the patient (9). MCB, master cell bank; WCB, working cell bank; CEA, cultured epidermal autografts; GLP, good laboratory practice; GMP, good manufacturing practice; GCP, good clinical practice.
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fig01: Strategy to perform ex vivo gene therapy from a single epidermal stem cellSchematic strategy to produce a performant and safe gene therapy product from a single autologous epidermal stem cell. (1) A biopsy is obtained from the patient to isolate epidermal stem cells that are then expanded under appropriate conditions (2). An aliquot of the culture is infected with the ad hoc recombinant shuttle virus (3). Single cells are then isolated (4) and clones expanded to built a frozen stem cell bank (5). In parallel, an aliquot of each clone is expanded to select for a clone fulfilling the criteria described in Table1. After validation (6), the approved clone is thawed and expanded to create master and working cell banks in a GMP facility (7). Genetically modified CEA are then produced (8) and grafts are transplanted onto the patient (9). MCB, master cell bank; WCB, working cell bank; CEA, cultured epidermal autografts; GLP, good laboratory practice; GMP, good manufacturing practice; GCP, good clinical practice.

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.


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

Droz-Georget Lathion S, Rochat A, Knott G, Recchia A, Martinet D, Benmohammed S, Grasset N, Zaffalon A, Besuchet Schmutz N, Savioz-Dayer E, Beckmann JS, Rougemont J, Mavilio F, Barrandon Y - EMBO Mol Med (2015)

Strategy to perform ex vivo gene therapy from a single epidermal stem cellSchematic strategy to produce a performant and safe gene therapy product from a single autologous epidermal stem cell. (1) A biopsy is obtained from the patient to isolate epidermal stem cells that are then expanded under appropriate conditions (2). An aliquot of the culture is infected with the ad hoc recombinant shuttle virus (3). Single cells are then isolated (4) and clones expanded to built a frozen stem cell bank (5). In parallel, an aliquot of each clone is expanded to select for a clone fulfilling the criteria described in Table1. After validation (6), the approved clone is thawed and expanded to create master and working cell banks in a GMP facility (7). Genetically modified CEA are then produced (8) and grafts are transplanted onto the patient (9). MCB, master cell bank; WCB, working cell bank; CEA, cultured epidermal autografts; GLP, good laboratory practice; GMP, good manufacturing practice; GCP, good clinical practice.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403041&req=5

fig01: Strategy to perform ex vivo gene therapy from a single epidermal stem cellSchematic strategy to produce a performant and safe gene therapy product from a single autologous epidermal stem cell. (1) A biopsy is obtained from the patient to isolate epidermal stem cells that are then expanded under appropriate conditions (2). An aliquot of the culture is infected with the ad hoc recombinant shuttle virus (3). Single cells are then isolated (4) and clones expanded to built a frozen stem cell bank (5). In parallel, an aliquot of each clone is expanded to select for a clone fulfilling the criteria described in Table1. After validation (6), the approved clone is thawed and expanded to create master and working cell banks in a GMP facility (7). Genetically modified CEA are then produced (8) and grafts are transplanted onto the patient (9). MCB, master cell bank; WCB, working cell bank; CEA, cultured epidermal autografts; GLP, good laboratory practice; GMP, good manufacturing practice; GCP, good clinical practice.
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.

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.

View Article: PubMed Central - PubMed

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
Related in: MedlinePlus