title: Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model.
abstract: Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC) based gene therapy utilizing integrating viral vectors. To develop a pre-clinical model for the evaluation of vector-related genotoxicity directly in the relevant human target cells, cord blood CD34(+) HSCs were transplanted into immunodeficient NOD.SCID.IL2rg(-/-) (NSG) mice after transduction with an LTR-driven gammaretroviral vector (GV). Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance. Clonality of human hematopoiesis in NSG mice was assessed by high throughput insertion site analyses and validated by insertion site-specific PCR depicting a GV typical integration profile with insertion sites resembling to 25% those of clinical studies. No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels. While these data underscore the potential value of the NSG model, our studies also identified short-comings such as overall low numbers of engrafted HSCs, limited in vivo observation time, and the challenges of in-depth insertion site analyses by low contribution of gene modified hematopoiesis.
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Research Group Reprogramming and Gene Therapy, Rebirth Cluster-of-Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Haemmerle
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Research Group Reprogramming and Gene Therapy, Rebirth Cluster-of-Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Ruhi
authors.initials: R
authors.lastname: Phaltane
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Rothe
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Schröder
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Research Group Reprogramming and Gene Therapy, Rebirth Cluster-of-Excellence, Hannover Medical School, Hannover, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Moritz
authors.affiliation: 1] Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany [2] Research Group for Gene Modification in Stem Cells, LOEWE Centre for Cell and Gene Therapy Frankfurt/Main and the Paul-Ehrlich-Institute, Langen, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Modlich