AbstractsBiology & Animal Science

Comprehensive integration site analysis for monitoring the clonal dynamics in clinical gene therapy has revealed that the insertion of the therapeutic retroviral vector (RV) can deregulate and even substantially activate neighboring genes leading to selection advantage and clonal outgrowth. Strikingly, 7 out of 10 Wiskott Aldrich Syndrome (WAS) gene therapy patients developed acute leukemia driven by gene corrected cell clones aberrantly expressing LMO2, MDS1 or MN1. This indicates that RV-mediated activation of adjacent regions cannot only influence the fate of hematopoietic stem cells but also cause clonal dominance up to leukemia. To identify novel regulators of benign hematopoiesis we established a systematic selection strategy using the total genomic integration site dataset of normal and highly polyclonal clinical blood and bone marrow samples. In this thesis, the unique integration site (IS) dataset within a cohort of 10 WAS gene therapy patients was systematically analyzed to select for candidate genes involved in the regulation of hematopoiesis. Initially, a total of 12.887 unique IS in vicinity of 3.267 genes were identified. Next, we selected all genes with at least 10 different IS within a 200 kb window around the gene (n=588). To enrich for genes with increased probability of transcriptional activation we then chose those genes with at least 10 IS within a 50kb window around the transcriptional start site (n=424). After stringent exclusion of all genes located within gene clusters 32 candidate genes were identified. To evaluate the hematopoietic activity of gene corrected cell clones, their contribution to blood cell formation within four years post gene therapy was monitored. We observed that these clones were detectable 15 to 93 times in a total of 102 individually analyzed patient samples, demonstrating long term activity of these hematopoietic stem cell clones. Interestingly, 20 out of the 32 highest ranked genes such as EVI1, CCND2 and LMO2 are known hematopoietic key regulators, strongly validating our selection strategy. After identification of 12 novel hematopoietic regulatory candidate genes, the top five ranked genes, ZNF217, LRRC33, PLCB4, EVL and IRF2BPL were chosen for further functional analysis in murine hematopoietic primary cells. To evaluate the endogenous expression of candidate genes in murine hematopoietic stem and progenitor cells global transcriptome datasets from purified populations were evaluated. We observed that all five selected candidate genes were expressed in at least one out of the five analyzed hematopoietic stem and progenitor cell populations which may point to an important role in the respective cell fraction. In order to validate our selection II strategy and to further investigate whether the chosen candidate genes play roles in hematopoiesis, we performed various in vitro and in vivo assays. We tested the effect of the selected candidate genes on proliferation, differentiation, and cytokine independency as well as for their influence on long term multilineage…