Siler
Fakultäten » Medizinische Fakultät » Kinderspital Zürich: Medizinische Klinik » Immunologie, Abteilung » Prof. Dr. Reinhard Seger » Siler
Completed research project
| Title / Titel | Development of a gammaretroviral vector for gene replacement therapy of the p47phox-deficient form of Chronic Granulomatous Disease (CGD) | ||||||
|---|---|---|---|---|---|---|---|
| Abstract (PDF, 14 KB) | |||||||
| Summary / Zusammenfassung | Chronic Granulomatous Disease (CGD) comprises a group of primary immunodeficiencies characterized by the failure of respiratory burst and an impaired antimicrobial activity. The impaired potential to kill pathogens is caused by an inactive phagocyte NADPH oxidase complex consisting of two membrane spanning subunits, gp91phox and p22phox, and three cytosolic components p47phox, p67phox and p40phox. Approximately 60% of all cases result from mutations in the X-linked (X-CGD) gp91phox gene and 30% are caused by p47phox gene mutations. The first successful clinical study aimed at the correction of X-CGD (gp91phox deficiency) was conducted in 2005 by the University Children’s Hospital Zürich, Division of Immunology/Hematology/ BMT headed by Prof. R. Seger in collaboration with our partners in Germany (Ott et al. Nat Med. 12, 401-9 (2006)). This study has shown that gammaretroviral correction of CGD is possible. This therapy represents an alternative to bone marrow transplantation, which is associated with unacceptable high rates of morbidity, mortality and graft failure, except in very selected cases with an HLA-identical donor available. For the p47phox-deficient form of CGD, bone marrow transplantation still is the only curative therapy. Therefore, the intended project aims to offer the possibility of correcting the p47phox-CGD phenotype by gammaretroviral gene therapy. As in our clinical X-CGD gene therapy trial transactivation of proto-oncogenes adjacent to viral integration sites could be identified as serious side effect of gene therapy we intend to reduce the transactivation potential of our new viral construct by the use of a next generation gammaretroviral self-inactivating (SIN) vector. The p47phox transgene expression is aimed to be controlled by an internal myeloid-specific promoter restricting the p47phox expression and thereby potential transactivation to terminally differentiated granulocytes, the population with natural p47phox expression in peripheral blood. First we tested a variety of promoter candidates for their potential to induce GFP expression in PLB-985 cells upon granulocytic differentiation. The best promoter candidates were cloned in gammaretrociral SIN vectors together with the p47phox transgene. Transduced primary murine hematopoietic stem cells were differentiated to granulocytes in liquid cell culture and the induction of p47phox expression as well as the reconstitution of respiratory burst activity was analyzed. These screens revealed three myelospecific promoter candidates which all were able to restore the respiratory burst activity in granulocytes. In parallel, we were able to improve the virus production by modifying our protocol increasing our virus titers by one log. The best promoter candidate judged by virus titers and by the p47phox expression in granulocytes was tested in vivo in p47phox-/- CGD mice. Six weeks after autologous bone marrow transplantation with gene modified stem cells, the p47phox expression was analyzed on blood, spleen and bone marrow. The analysis revealed a nearly exclusive expression in granulocytes. Additionally, the vector was able to reconstitute the respiratory burst activity in p47phox-/- CGD mice in vivo. The restricted expression pattern of the new vector prevents transactivation events as it was seen in our recent GT trial. Beside the mentioned transactivation events, we observed a decrease in transgene expression over time by promoter methylation (silencing) in the mentioned GT trial. Therefore, an additional activity in our laboratory aims to prevent the transgene silencing. Our approach to prevent silencing is to separate the transgene expression unit from the surrounding chromatin by incorporating cHS4 insulator elements 5’ and 3’ of the transgene. Additionally, the insulated DNA segment needs to be kept in an open conformation. The UCOE (ubiquitous chromatin opening element) element is reported to facilitate chromatin opening activity. Therefore, this element is currently introduced into our vector. The inhibitory effect of the UCOE element on silencing activity will be assayed in F9 cells and NIH3T3 cells which are reported to possess strong and no silencing activity. |
||||||
| 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: 401-9Stein S, Siler U, Ott MG, Seger R, Grez M. Gene therapy for chronic granulomatous disease. Curr Opin Mol Ther. (2006) 8: 415-22 | ||||||
| Keywords / Suchbegriffe | CGD, gene therapy | ||||||
| Project leadership and contacts / Projektleitung und Kontakte |
|
||||||
| Funding source(s) / Unterstützt durch |
SNF (Personen- und Projektförderung), Others Novartis Stiftung für Medizinisch-Biologische Forschung |
||||||
| In collaboration with / In Zusammenarbeit mit |
|
||||||
| Duration of Project / Projektdauer | Nov 2008 to Nov 2010 |