Lentivirus-Mediated Platelet Gene Therapy Corrects Bleeding Diathesis and Induces Immune Tolerance in Murine Hemophilia B Mice

Abstract 1101

While data from the clinical trials using AAV vector expression FIX in hemophilia B gene therapy in humans are very encouraging, for individuals with severe liver disease or neutralizing antibodies to AAV, an alternative gene therapy approach might be desired. Our previous studies have demonstrated that lentivirus-mediated platelet gene therapy can correct murine hemophilia A phenotype, but this approach has not been explored for hemophilia B. In the current study, we developed a clinical translatable approach for platelet gene therapy of hemophilia B. Platelet-FIX (2bF9) expression in hemophilia B (FIX^null) mice was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). The recipients were analyzed beginning at 3 weeks after bone marrow (BM) transplantation. Expression of the 2bF9 product was detected by PCR in all recipients that received 2bF9 LV-transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF9 LV transfer vector. The expression of the hFIX transgene protein in the transduced platelets was confirmed by immunofluorescent confocal microscopy. Flow cytometry showed that there were 20.8 ± 12.1% (n = 7) and 14.8 ± 10.7% (n = 6) 2bF9 LV-transduced platelets respectively in the recipients preconditioned with 1100 cGy or 660 cGy. The antigen levels of FIX (FIX:Ag) were 2.89 ± 1.75 mU/10⁸ platelets (n = 9) in the recipients preconditioned with 1100 cGy and 1.87 ± 1.30 mU/10⁸ platelets (n = 7) in the 660 cGy group, while the activity (FIX:C) levels were 1.67 ± 1.15 and 1.13 ± 0.85 mU/10⁸ platelets respectively. There was a small amount of FIX detected in the 2bF9 LV-transduced recipient plasma with the average levels of 2.22 mU/ml in 1100 cGy group and 1.44 mU/ml in 660 cGy group. To analyze the distribution of the FIX between platelets and plasma, we normalized FIX levels to total whole blood FIX content. The results demonstrated that 90% to 95% of whole blood FIX was stored in platelets. The tail clip survival test demonstrated that 15 out of 16 mice that received 2bF9 LV-transduced HSCs survived the tail clip challenge, while 8 out of 10 FIX^null control mice died after tail clipping. Nine months after transplantation, sequential transplantation was performed on some of the primary recipients. Platelet-hFIX expression in the secondary recipients was sustained, leading to phenotypic correction and confirming that long-term engrafting HSCs were successfully transduced with 2bF9 LV. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. To investigate whether immune tolerance was induced in 2bF9 LV-transduced recipients, we challenged the recipients with recombinant human FIX (rhFIX) in the presence of adjuvant. Only 1 out of 9 2bF9 LV-transduced recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) as measured by a modified Bethesda assay. In contrast, all of the FIX^null controls developed inhibitory antibodies ranging from 17 – 37 BU/ml after the same challenge (n = 5). To ensure that the immune system was not defective in the 2bF9 LV-transduced recipients and that the tolerance induction is FIX antigen-specific, we further challenged the animals with ovalbumin (OVA) absorbed on Alum. Both the 2bF9 LV-transduced and FIX^null control mice developed high-titer of anti-OVA antibodies. The levels of anti-OVA IgG in the 2bF9 transduced recipients were not significantly different from FIX^null mice after the OVA immunization, confirming that tolerance induction in 2bF9 LV-transduced mice is FIX-specific. Taken together, our data suggest that lentivirus-mediated bone marrow transduction and transplantation can not only provide sustained phenotypic correction, but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans.