Generation of novel self-inactivating retroviral vectors for gene therapy of Wiskott-Aldrich-Syndrome.

INES AVEDILLO DÍEZ; RICARDO A. DEWEY; AXEL SCHAMBACH; CHRISTOPHER BAUM; CHRISTOPH KLEIN

Wilsede, Germany

Jornada; XX. Jahrestagung der Kind-Philipp-Stiftung für Leukämieforschung. Zentrum für Frauen- Kinder- und Jugendmedizin Klinik für pädiatische Hämatologie und Onkologie.; 2007

Kind-Philipp-Stiftung für Leukämieforschung. Zentrum für Frauen- Kinder- und Jugendmedizin Klinik für pädiatische Hämatologie und Onkologie.

Hematopoietic stem cell gene therapy has been very effective in various monogenic immunodeficiency diseases. However, insertional mutagenesis remains an important concern. To develop retroviral vectors with improved safety features, we have generated a series of self-inactivating (SIN) constructs designed to express WASP in hematopoietic cells. We comparatively assessed four internal promoters derived from Spleen Focus Forming Virus (SF), Elongation Factor 1-á (EFS), Phosphoglycerate Kinase (PGK) and Wiskott-Aldrich Syndrome Protein (WASP), respectively. Upon retroviral gene transfer, we analyzed gene expression in various hematopoietic and non-hematopoietic cell lines, using GFP expression as a readout parameter. Among the four internal promoters tested, EFS and SF were found to induce the highest expression levels in all cell lines tested. As expected, the endogenous WASP promoter was only active in hematopoietic cells. For further studies, we focussed on the EFS promoter. EBV-LCL cell lines from WAS patients were transduced and shown to reconstitute their aberrant cytoskeleton. This reconstitution was comparable to cells corrected with a classical LTR-driven WASP vector. We are currently performing toxicity assays, gene transfer into human CD34+ cells, and in vivo reconstitution studies in the WASP-deficient mouse system to further assess the suitability of novel SIN vectors as a potential way to improve the safety profile of WASP-expressing vectors. Our data suggest that novel retroviral SIN vectors may be an alternative to conventional LTR-deriven retroviral vectors for future human gene therapy studies in WASP-deficient patients.