Phenotypic Correction of Hemophilia A by Postnatal Intraperitoneal Transplantation of FVIII-Expressing MSC

Abstract 249 We recently re-established a line of sheep that accurately mimics the clinical symptoms and genetics of severe hemophilia A (HA). Herein, we tested the ability of a novel, postnatal, non-ablative transplant approach to correct the disease in 2 of the HA animals. Haploidentical (paternal) bone marrow mesenchymal stem cells (MSC) were transduced simultaneously with lentiviral vectors encoding a high expression porcine FVIII (pFVIII) or an eGFP transgene, to facilitate donor cell identification. Upon sedation, MSC were injected into the peritoneal cavity of the young adult animals by ultrasound-guided delivery, without any preconditioning. At the age of 4 months, the time of the first transplantation (Tx), animal #1 (HA#1) had received 32,200U of recombinant hFVIII in 22 treatments for muscular hematomas, rectal bleeding, and chronic, progressive, debilitating hemarthroses which had caused severe defects in posture and gait, rendering him nearly immobile. Low levels of hFVIII inhibitors (4-6) were detected by Bethesda assay at this time point. Tx of 30×10⋀6 transduced MSC resolved all existent hemarthroses, and resulted in a cessation of all spontaneous bleeding events. HA#19s damaged joints recovered fully; he regained normal posture and gait, and resumed normal physical activity. Despite HA#1 becoming and remaining factor-independent, however, a rise in Bethesda titer to ≂f625 was observed. At 5.5 months of age, a 2nd Tx with 120×10⋀6 transduced MSC was performed to test whether higher cell numbers would induce immune tolerance to FVIII; however, the levels of FVIII antibodies remained the same. At 7 months, HA#1 was euthanized after ingesting a sharp rock which caused a massive hematoma of the larynx, reducing his airway by >75%. Tissue analysis demonstrated large numbers of GFP+/FVIII+ MSC present within the synovium of the joints which had hemarthrosis at the time of the 1st Tx, suggesting the transplanted MSC homed to the sites of ongoing injury/inflammation and persisted for over 3 months, releasing FVIII locally within the joint, providing an explanation for the dramatic improvement we observed in this animal9s joints. In order to elucidate the mechanism whereby this transplant approach produced such pronounced systemic benefit, we performed PCR and confocal analysis on other tissues. PCR has demonstrated engraftment of transplanted MSC in all tissues thus far analyzed, including liver, lymph nodes, intestine, lung, kidney, omentum, and thymus. Confocal analysis revealed significant levels of engraftment within the lymph nodes, small intestine and the thymus. Analysis of other tissues is ongoing to fully define the sites of engraftment, and understand how these engrafted cells producing FVIII locally are exerting widespread clinical improvement. A 2nd HA animal (HA#2) was transplanted at 5 months of age with 120×10⋀6 transduced paternal MSC. In similarity to HA#1, hemarthroses present at the time of Tx resolved, and he resumed normal activity. However, despite having no detectable inhibitors prior to Tx, HA#2 developed titers of ≂f150 following this procedure. HA#2 is alive, has exhibited no spontaneous bleeds, and has only required FVIII treatment to resolve bleeding resulting from accidental trauma. His plasma is being assayed regularly to determine if the continued production of FVIII by the transplanted MSC will result in tolerance induction and a drop in inhibitor titer. In summary, this novel postnatal, nonmyeloablative MSC Tx successfully converted both animals from a severe, life-threatening phenotype to a moderate phenotype, devoid of spontaneous bleeds. Further mechanistic studies will be required to understand and ultimately overcome the formation of inhibitors resultant from this procedure since, in the absence of inhibitors, this approach could result in even more pronounced clinical improvement. Disclosures: No relevant conflicts of interest to declare.