Barcoding of Macaque Hematopoietic Stem and Progenitor Cells: A Robust Platform to Assess Vector Genotoxicity

Abstract

Gene therapies using integrating retroviral vectors to modify hematopoietic stem and progenitor cells have shown great promise for treatment of immune system and hematologic diseases. However, activation of proto-oncogenes via insertional mutagenesis has resulted in the development of leukemia. I have utilized cellular “barcoding” to investigate the impact of different vector designs on the clonal behavior of HSPC during in vivo expansion as a quantitative surrogate assay for genotoxicity in a non-human primate model with high relevance for human biology. I transplanted two rhesus macaques with autologous CD34+ HSPC transduced with three lentiviral vectors containing different promoters/enhancers of a predicted range of genotoxicities, each containing a high diversity barcode library that uniquely tags each individual transduced HSPC. Analysis of clonal output from thousands of individual HSPC transduced with these barcoded vectors revealed sustained clonal diversity, with no progressive dominance of clones containing any of the three vectors for up to almost three years post-transplantation. This data supports a low genotoxic risk for lentiviral vectors in HSPC, even those containing strong promoters/enhancers. Additionally, this flexible system can be used for testing of future vector designs.