3130 – OVEREXPRESSION OF CONSTITUTIVELY ACTIVE CXCR4 IS SUPERIOR TO WILD TYPE FOR EARLY HEMATOPOIETIC STEM AND PROGENITOR CELL HOMING AND ENGRAFTMENT

Abstract

<h3>BACKGROUND</h3> Genome engineering of human hematopoietic stem and progenitor cells (HSPCs) has the potential to cure many hematological diseases, but is limited by subtherapeutic levels of modified cells in vivo. Providing a selective homing and engraftment advantage to modified cells could overcome this, such as by increasing CXCR4 expression, a key adhesion molecule for stem cell homing. In vivo studies thus far utilized wild type (WT) CXCR4. Here we investigate overexpression of a constitutively active mutant (CAM) of CXCR4 in a novel competitive transplant model. The CAM is generated by a single amino acid substitution that activates intracellular signalling without ligand binding. <h3>METHODS</h3> G-CSF-mobilized human HSPCs were fluorophore-marked (VSVG lentiviral vector, GFP or mCherry) then electroporated with either WT or CAM CXCR4 mRNA. Equal numbers of WT and CAM CXCR4 treated cells were transplanted into NSG mice after sublethal irradiation in a double-cross competitive transplant experiment, and both short term bone marrow (BM) homing and long-term engraftment evaluated. <h3>RESULTS</h3> HSPCs overexpressing CAM CXCR4 showed improved early homing to BM compared to WT CXCR4 within 48 hours. By 18 weeks the two arms contributed equally to hematopoiesis. <h3>DISCUSSION</h3> In this pilot study, overexpression of CAM CXCR4 provided greater BM homing advantage than WT CXCR4. We successfully combined lentiviral transduction with mRNA transfection demonstrating that transient CXCR4 CAM overexpression is a strategy which can be successfully applied to genome engineered cells. This novel combinatorial competitive transplant model eliminates confounding experimental variables to allow direct comparison of 2 fluorophore-marked, mRNA-transfected arms within a single animal.