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Gamma‐glutamylcysteine synthetase‐based selection strategy for gene therapy of chronic granulomatous disease and graft‐vs.‐host disease
Author(s) -
Rappa Germana,
Anzanello Fabio,
Alexeyev Mikhail,
Fodstad Oystein,
Lorico Aurelio
Publication year - 2007
Publication title -
european journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.904
H-Index - 84
eISSN - 1600-0609
pISSN - 0902-4441
DOI - 10.1111/j.1600-0609.2007.00833.x
Subject(s) - genetic enhancement , thymidine kinase , biology , transduction (biophysics) , ex vivo , viral vector , haematopoiesis , progenitor cell , suicide gene , in vivo , bone marrow , immunology , cancer research , virology , stem cell , microbiology and biotechnology , gene , herpes simplex virus , virus , genetics , biochemistry , recombinant dna
Efficient ex vivo / in vivo selection of genetically modified hematopoietic stem/progenitor cells (HPCs) and T lymphocytes could greatly improve several gene therapy strategies. We have previously reported that primary murine HPCs, transduced with a bicistronic retroviral vector, co‐expressing the catalytic subunit of gamma‐glutamylcysteine synthetase ( γ ‐GCSh) and eGFP, could be selected by l ‐buthionine‐ S , R ‐sulfoximine (BSO). Upon ex vivo transduction with a low, defined gene dosage and BSO selection, HPCs were able to repopulate the bone marrow of syngeneic myeloablated hosts, showing multi‐lineage expression [ Hum Gene Ther , 16 (2005), 711]. We now provide ‘proof‐of‐principle’ that the same strategy can be applied to the gene therapy of graft‐vs.‐host disease (GVHD) subsequent to allogeneic bone marrow transplantation (ABMT), and of chromosome X‐associated chronic granulomatous disease (CGD). Transfer of the herpes simplex virus‐thymidine kinase (HSV‐Tk) ‘suicide’ gene into donor T lymphocytes is a potential method to control GVHD after ABMT. However, an efficient selection system is required to eliminate non‐HSV‐Tk‐expressing T lymphocytes before administration to the patient. We now report that, upon transduction with a retroviral vector, co‐expressing γ ‐GCSh and eGFP, and subsequent selection by BSO, over 95% human T lymphocytes were found to express eGFP; moreover, upon transduction with a novel retroviral vector co‐expressing γ ‐GCSh and HSV‐Tk, and subsequent BSO treatment, over 95% of T lymphocytes could be eliminated by ganciclovir. The efficacy of the γ ‐GCSh‐BSO selection strategy was then tested on an in vitro model of CGD. Upon transduction of gp91 (phox)‐deficient PLBKO cells with a novel bicistronic retroviral vector co‐expressing human gp91 (phox) and γ ‐GCSh, exposure to BSO for 48 h eliminated most non‐transduced cells, resulting in selection of gp91 (phox)‐expressing cells, and reconstitution of NADPH oxidase activity.