542. A Novel In Vitro/Vivo Selection/Suicide Strategy for Lentiviral Vectors

Abstract

Genetic engineering of hematopoietic progenitor cells (HPC) by lentiviral-based vectors suffers from their low transduction rate with ~20% when aiming a single integration event. This amount of modified cells may not be sufficient for the correction of a number of diseases and therefore enrichment of transduced cells would be needed. We propose a novel in vitro/in vivo selection/suicide system based on the expression of the human reduced folate carrier 1 (hRFC1) and the human ribonucleoside-diphosphate reductase subunit M2 (RRM2). These genes are encoding for proteins which are responsible for drug resistances/sensitivities.The vector design is based on a bicistronic self-inactivating vector, meaning that the vector comprises the ubiquitin promoter (UBI) which drives the expression of EGFP in order to track transduced cells. The PGK promoter drives the expression of hRFC1 and the RRM2, which are connected through the cleavage peptide T2A.The hRFC1 is the main transport protein for the uptake of folates which are indispensable for purine synthesis and therefore for DNA synthesis. A greater expression level of hRFC1 should render cells more sensitive to the antifolate Methotrexate (suicide), which mainly enters the cells via the hRFC1. In contrast, it is possible to enrich for the transduced cells with Trimetrexate, which is an analog of Methotrexate. Its very lipophilic nature allows Trimetrexate to passively diffuse through cell membranes. As a result, transduced cells can escape the toxic effects from Trimetrexate due to their augmented transport of folic acid trough the overexpressed hRFC.In contrast, non-transduced cells are expected to show the opposite effects with higher resistance to Methotrexate but lower threshold killing to Trimetrexate.The second resistance gene RRM2 catalyzes the reduction of ribonucleotides into desoxyribonucleotides and its overexpression should lead to resistance to the drug hydroxyurea. When applying both selection drugs (Trimetrexate and Hydroxyurea) we expect an additive or even synergistic toxic effect on non-transduced cells while the transduced cells experience lower side effects.We are currently examining the concept proposed in vitro using various cell lines. Providing promising results, we would like to use the vector on CD34+ hematopoietic stem cells derived from umbilical cord blood. In addition, we would like to transplant immunocompromised NSG mice with a mixture of transduced and non-transduced CD34+ cells and then apply the Trimetrexate/Hydroxyurea in vivo to select and then Methotrexate in order to remove the transduced cell from the circulation.