290. Baboon Envelope Pseudo Typed Lentiviral Vectors Mediate High-Level Gene Transfer in Human B Cells Allowing Secretion of FIX at Therapeutic Levels in NSG Mouse

B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction. In addition, long-lasting transgene expression in B cells is of particular interest for immunotherapy by its potential to induce specific immune activation or tolerance. Moreover, B cells are potent specialized protein secreting cells. One obstacle though is that primary B cells are poorly transduced by VSVG pseudotyped lentiviral vectors (LVs) even when stimulated through the BCR to induce proliferation. We demonstrated that this is due to the poor expression of the low density lipid (LDL) receptor, identified as the VSV receptor (Amirache et al, 2014).Since we recently showed that baboon retroviral envelope pseudotyped LVs (BaEV) outperform VSV-G-LVs for gene transfer into cytokine-stimulated and resting hematopoietic stem cells, we evaluated them here for gene transfer into primary human B cells. Upon B cell receptor stimulation, BaEV-LVs transduced up to 70% of the B cells. As expected, VSV-G-LVs were unable to transduce activated nor unstimulated resting human B cells efficiently, even at high vector doses (MOI= 100, VSV-G-LV < 5% transduction). Remarkably, BaEV-LVs permitted highly efficient transduction of 30% of resting B cells. Moreover, they transduced as well memory as naive B cells without inducing phenotypic changes. In addition, BaEV-LVs permitted up to 80% transduction of human plasmocytes. Adaptive transfer of mature BaEV-LV transduced human B cells into NSG mice allowed differentiation into plasmablasts and plasma B cells, both characterized by a high level gene marking in the lymphnodes, spleen and bone marrow. These results encouraged us to evaluate BaEV-LV mediated gene transfer of Factor IX into human B cells for treatment of heamophelia, a severe blood disorder. We produced high-titer BaEV-LVs carrying a codon optimized FIX with a hyperactivating FIX-R338L mutation. These BaEV-FIX-LVs efficiently transduced plasmocytic B cell lines leading to high-level FIX secretion (20-70% of normal levels in human serum). Transduction of primary human B cells followed by adaptive transfer into NSG mice resulted in therapeutic levels of FIX in the serum (7-25% of normal FIX levels) at 3 to 4 weeks of engraftment. Moreover, a faster coagulation of the blood proved that FIX was functional.Concluding, the BaEV-LVs might represent in the future valuable tools for therapeutic protein secretion from autologous B cells for treatment of heamophelia and other disorders in vivo.