Reichenbach
Fakultäten » Medizinische Fakultät » Kinderspital Zürich: Medizinische Klinik » Immunologie, Abteilung » Prof. Dr. Reinhard Seger (emeritiert) » Reichenbach
Completed research project
| Title / Titel | Gene therapy for chronic granulomatous disease: Optimization of retroviral gene expression in myeloid progenitors |
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| Abstract (PDF, 14 KB) | |||||
| Summary / Zusammenfassung | Chronic granulomatous disease (CGD) is a primary immunodeficiency that affects the oxidative microbial killing of phagocytic cells. The defect is due to absent or severely impaired activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of phagocytes, resulting in inadequate generation of reactive oxygen species (ROS), necessary for microbicidal activity within the phagosome. Approximately 60% of CGD cases are X-linked (X-CGD, OMIM #306400) and result from mutations in the gene encoding gp91phox (CYBB, Xp21.1, Gene Bank accession number X04011), which forms a heterodimer with p22phox to build membrane bound flavocytochrome b558, the terminal redox centre of the NADPH oxidase. The remaining 40% of CGD cases are inherited in an autosomal-recessive manner, caused by mutations in p22phox (CYBA, 16q24), and in the two cytoplasmic NADPH oxidase subunits, p47phox (NCF1, 7q11.23, OMIM #233700) and p67phox (NCF2, 1q25, OMIM #233710). Conventional treatment consists of prompt aggressive treatment of acute infections, and lifelong prophylaxis with antibiotics, antimycotics and human recombinant IFN-. In spite of this, morbidity is considerable and the annual rate of mortality is 2-5%, accounting for a need for more effective therapies. Curative treatment of CGD consists of human leukocyte antigen (HLA)-matched sibling allogeneic haematopoietic stem cell (HSC) transplantation. HSCT is limited by availability of suitable donors, the regimen-related toxicity of the transplant and the potential development of graft-versus host disease (GVHD). Currently HSCT is only performed when an HLA identical sibling or unrelated donor is found. CGD is a suitable disease for HSC-targeted gene therapy, as it arises from a single gene defect in HSCs, and as the four defective genes have been cloned. Reconstitution of NADPH oxidase activity in phagocytes by insertion of a functional copy of the affected gene into HSCs aims at a lifelong cure of the disease, providing a sufficient and stable number of gene-corrected neutrophils to overcome the infectious and granulomatous complications of CGD. Data from variant forms of CGD and from healthy female carriers of X-CGD with 10% normal neutrophils suggest that correction of the phenotype in a fraction of CGD cells could be sufficient to alleviate the symptoms of the disease. Our previous results on gene therapy for chronic granulomatous disease (CGD) have shown that a gene therapy approach for treatment of this devastating immunodeficiency is feasible and may substantially contribute to improve patient’s health (Ott et al. Nat Med. 12,401-9(2006)). The present project aims at improving efficacy and safety of this treatment by modifications in vector design together with restriction of transgene expression to the myeloid compartment. The goal is to determine the vector with best efficacy with selection based on criteria of functional correction for establishing the next generation of clinical protocols for gene therapy of CGD. The currently used vector, SF71gp91phox is a gammaretroviral vector in which gp91phox expression is driven by the long terminal repeat (LTR) of the Friend mink cell Spleen focus-forming virus (SFFV). Next generation vectors with safety improved properties are self-inactivating (SIN) gammaretroviral vectors which lack the potent enhancer elements present within the viral LTR. These vectors contain an internal promoter which drives transgene expression and allows for use of tissue(myeloid)-specific promoters, reducing the probability of transactivation in stem and progenitor cells. The present project aims at the selection of a future gene therapy vector, based on the lessons learned from the latest clinical gene therapy trial. The efficacy of three different vector systems will be compared, using the already established conditions for transduction and host conditioning. The minimal vector copy number and transduced cell doses for maximal therapeutic effect, i.e. reconstitution of phagocytic killing activity (NBT reduction, DHR 123 oxidation, and chemiluminescence assay) will be determined. O2- (superoxide anion) production will be quantified by cytochrome C assay in relation to gene dosage. Based on functional in vitro E. coli and A. fumigatus killing assays, the threshold of O2- production necessary for adequate killing activity will be evaluated. For each vector, the gene dosage, i.e. chimerism and transgene copy number per cell, necessary to achieve the limit of functional killing activity will be determined. Furthermore, we intend to establish an in vivo animal model in gp91phox-/- mice after bone marrow transplantation of gene transduced haematopoietic stem cells. In these animals local inflammation will be induced by subcutaneous injection of bioluminescent non-proliferating A. fumigatus and S. aureus. The transgene based reconstitution of functional in vivo pathogen killing/clearance will be monitored by the decline of luminescence derived from the bioluminescent pathogens and by measuring diminishing granuloma formation in gp91phox gene corrected X-CGD mice. Next generation clinical gene therapy study is intended to start in 2008. |
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| Publications / Publikationen | Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U, Glimm H, Kuhlcke K, Schilz A, Kunkel H, Naundorf S, Brinkmann A, Deichmann A, Fischer M, Ball C, Pilz I, Dunbar C, Du Y, Jenkins NA, Copeland NG, Luthi U, Hassan M, Thrasher AJ, Hoelzer D, von Kalle C, Seger R, Grez M. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med. (2006) 12: 401-9 | ||||
| Project leadership and contacts / Projektleitung und Kontakte |
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| Funding source(s) / Unterstützt durch |
Nachwuchsförderungskredit der Universität Zürich |
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| In collaboration with / In Zusammenarbeit mit |
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| Duration of Project / Projektdauer | Oct 2006 to Nov 2008 |