539. Clinical LV Design Incorporating Human-Derived Cell-Fate Control (Suicide) Elements

We have previously reported on cell-fate control or ‘suicide’ transgenes based on the human enzymes deoxyCytidine Kinase (dCK) and Thymidylate Kinase (tmpK). These cell-fate control systems are based on modified human kinases, which convert exogenous non-toxic pro-drugs into toxic products. Modified dCK activates bromovinyldeoxyuridine (BVdU), l-deoxythymidine (LdT), l-deoxyuridine (LdU), etc., to become cytotoxic compounds that induce apoptosis. Modified tmpK specifically acts on metabolites of azidothymidine (AZT). It converts AZT-MP to AZT-DP. Subsequent conversion to AZT-TP results in inducible-programmed cell death of tmpK-transduced cells. Recently we have fused these ‘suicide’ transgenes to a cytoplasmic tail-truncated human LNGFR (CD271), a cell surface marker, enforcing a one-to-one correlation between components. Both the trLNGFRdCK and trLNGFRtmpK constructs follow an IRES element in bicistronic lentivectors (LVs) ensuring transfer of the therapeutic construct and the cell-fate control element into the same target cells. This system offers a robust method to eradicate transduced cells at the end of treatment or upon observation of adverse events. It also provides a facile method for vector tracking, and facilitates the evaluation of vector transduction efficiency and selection of expanded transduced cells ex vivo. Currently, we are engineering these cell-fate control elements into 3 independent LVs for clinical application: 1) in an IL-12-based construct to generate anti-tumor vaccines in AML, 2) to engineer overexpression of a lysosomal hydrolase to treat Fabry disease, and 3) to mitigate the graft-versus-host disease response resulting from adoptive T cell transfers. Pre-clinical data suggests significant expression of IL-12 in ex vivo transduced patient AML blasts and near complete AZT-responsive cell death. Our LV designed for treatment of Fabry disease restores stable alpha-galactosidase A activity in ex vivo-transduced CD34+HSCs and provides a mechanism for their death upon AZT addition if needed. The trLNGFRtmpK construct will provide a marker for ex vivo expansion of T cells carrying the cell-fate control cassette and allow for selective ablation of those cells if graft-versus-host disease occurs. Indeed, we have observed significant transduction frequencies and inducible death of transduced human T cells upon AZT addition. The trLNGFRdCK and trLNGFRtmpK constructs are novel human-derived cell-fate control (‘suicide’) elements for use in clinical vector design.