Preclinical Gene Therapy of Fanconi Anemia with Transposon-Based Approaches

点击翻译按钮获取中文摘要

The Sleeping Beauty (SB) transposon constitutes a novel, integrative, non-viral vector system that may greatly innovate the field of gene therapy for three main reasons: 1) its characteristic and safer pattern of integration, as compared to integrating viral vectors, 2) the possibility of transfecting cells in vivo, as already shown in experimental models and 3) the simplicity and therefore the economy of vector production under GMP conditions. Significantly, the SB system has just entered clinical trials in the USA for the immunotherapy of cancer. The development of a hyperactive SB transposase/transposon system, with improved gene transfer properties has opened new perspectives for the treatment of rare diseases with non-viral vectors. In this proposal, we aim to develop this platform for gene therapy of Fanconi Anemia (FA), a genetic instability syndrome caused by mutations in any of the 18 genes described to date in the FA pathway that cooperate in the repair of DNA lesions. The disorder is associated with profound bone marrow failure due to the progressive depletion of hematopoietic progenitor and stem cells (HPSCs), with increased sensitivity to inflammatory cytokines and endogenous aldehydes. Therefore, upon the genetic correction of FA HPSCs, these cells will develop a marked proliferation advantage in comparison to uncorrected cells in vivo, which may rescue the patients from the bone marrow failure. In this project we will develop SB transposon vectors for the correction of Fanconi Anemia-A patients (the most prevalent subtype in FA), validate the performance of the vectors ex vivo in FA fibroblasts and HPSCs isolated from FA mice and FA patients, and thoroughly characterize the abilities of these gene-modified cells with respect to engraftment and hematopoietic reconstitution in recipient mice following transplantation. A comprehensive vector integration site analysis will be conducted to assess relative safety of the gene therapy approach. The project will establish, evaluate and refine clinically applicable protocols for transposon-mediated gene therapy of Fanconi Anemia in comparison to current approaches based on the use of lentiviral vectors.