291. CD26 Inhibition Enhances Chimerism of Lentivirally Transduced Hematopoietic Stem Cells in a Non-Myeloablative Setting

Despite the progress of gene therapy for monogenic diseases over the last years, the engrafting capacity of gene-modified cells, as well as constant and high-level gene expression in vivo, require further optimization, especially when a reduced intensity conditioning is mostly preferred. Culture conditions applied for effective gene transfer lead to significant loss of long-term repopulating cells and ultimately, impaired engraftment. The ex vivo inhibition of CD26 peptidase activity with Diprotin A was shown to improve homing and engraftment of unmanipulated hematopoietic stem cells (HSCs). We here, sought to assess the homing and engrafting capacity of lentivirally GFP-transduced murine bone marrow (BM) cells under competitive niche settings generated by a non-myeloablative conditioning. Following CD26 inhibition with 5mM Diprotin A of GFP-transduced murine HSCs, we assessed migration towards SDF-1 in transwell systems and the engraftment capacity after partial myeloablation (Busulfan 100mg/kg, equivalent to 8mg/kg in humans) in a syngeneic mouse model, allowing for donor chimera detection (C57Bl6-CD45.2+ donors/PepBoy-CD45.1+ recipients). We also investigated the possible effects of Diprotin A on gene transfer efficiency and transgene expression in bulk and clonogenic cultures of HSCs. In vitro, Diprotin A significantly increased the expression of the CXCR4 receptor (25.6±0.75 vs 16.4±1.02, P=0.02), as well as the %migration of gene-modified HSCs towards SDF-1 over untreated transduced cells (71.78±3.79% vs 55.71±5.34%, P=0.03), implying a potentially enhanced engrafting dynamic in the BM. Indeed, in vivo, the Diprotin A-treated transduced HSCs exhibited faster hematologic reconstitution by, at least, one week (P=0.03) and both superior long-term engraftment and GFP expression in all hematopoietic tissues (peripheral blood, BM, spleen) of the recipients, over non-Diprotin A-treated transduced cells (blood 4th month post-transplant, %CD45.2+: 77.26±5.26 vs 13.11±11.69, P=0.0002; %GFP+: 30.21±6.86 vs 3.9±2.33, P=0.03). The increased GFP expression observed in vivo in the Diprotin A cohort reflected the enhanced engraftment rates, since Diprotin A per se did not affect gene transfer efficiency or transgene expression prior to transplantation (%transduction with Diprotin A: 93±3.05 vs without Diprotin A: 89.9±7.47, P=0.73; %GFP in pools of colonies with Diprotin A: 74.8±7.14 vs without Diprotin A: 72.1±8.45, P=0.81). Upon sacrifice, the Diprotin A animals displayed sustained gene marking with ~1 vector copy in all hematopoietic tissues, as opposed to almost undetectable vector copy number (VCN) in the non-Diprotin A cohort. Likewise, individual colonies from the chimeric BM, demonstrated significantly higher VCN in the Diprotin A mice (2.8±0.48 vs 0.37±0.37, P=0.01). Overall, the ex vivo treatment with Diprotin A seems to abrogate the negative impact of culture conditions, allowing for enhanced donor chimerism under partial myeloablation and consequently, increased gene marking in vivo. This ex vivo, easily applicable approach may serve to overcome major constraints for the clinical implementation of gene therapy should the data be confirmed with human CD34+ cells.