Bleeding Phenotype of Murine Bernard Soulier Syndrome Is Potentially Corrected by Non-Myeloablative Hematopoietic Stem Cell Transplantation
Authors: Kanaji Sachiko , Fahs Scot A. , Ware Jerry , Montgomery Robert R. , Shi Qizhen 
Published: October 2019

Abstract
Abstract 3340
Bernard Soulier Syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP)Ib/IX complex. Platelet transfusion is the primary treatment for hemorrhage but is often limited because of the potential for provoking allo-immunization and refractoriness. We have previously shown that the macrothrombocytopenia and prolonged bleeding of murine BSS (GPIbαnull) can be corrected by myeloablative total body irradiation (TBI) and transplantation of hematopoietic stem cells (HSCs) transduced with a lentivirus that expresses hGPIbα under integrin αIIb promoter control (2bIbαLV). For application of this gene therapy approach to the treatment of human patients, it is important to minimize treatment-related adverse effects. In this study, we established a transgenic mouse that expresses hGPIbα at levels similar to the average levels observed in 2bIbαLV transduced HSC recipients that can be used as a consistent source of bone marrow HSCs (hGPIbαtg+). We undertook two approaches for evaluation of HSC transplantation using hGPIbαtg+ mouse bone marrow (BM) cells; 1) determine the percentage of hGPIbαtg+ HSCs necessary for therapeutic benefit and 2) determine if non-myeloablative strategies can achieve hGPIbα expression levels sufficient to correct the bleeding phenotype of GPIbαnullmice.
In order to determine the percentage of HSCs required to achieve therapeutic effects, mixed BM chimeras were generated by reconstituting lethally irradiated GPIbαnull mice with variable mixtures (5% - 100%) of BM cells isolated from hGPIbαtg+ and GPIbαnull mice. Tail bleeding time assays performed after BM reconstitution demonstrated that 10% hGPIbαtg+ BM mononuclear cells mixed with 90% GPIbαnull BM mononuclear cells were sufficient to correct the tail bleeding time (n=5). Platelet analysis showed that the bleeding phenotype was rescued in recipients having higher than 40 × 103/μl of hGPIbαtg+ platelets, which corresponds to 6.5% of wild type mouse platelet counts (630 ± 57 × 103/μl, n = 8). These results suggest therapeutic potential for non-myeloablative conditioning regimens for the treatment of murine BSS.
Thus we next tested the transplantation of hGPIbαtg+ mouse BM cells into GPIbαnull recipients conditioned with two non-myeloablative doses of busulfan (25 mg/kg on days -2 and -1). Analysis of platelets by flow cytometry showed that, as expected, the percentage of hGPIbα-positive platelets in busulfan-conditioned recipients was lower than those transplanted after lethal TBI conditioning (63.2% ±36.6%, n = 15 vs. 95.2% ±1.7%, n = 8). However, tail bleeding time assays showed that bleeding times of busulfan-conditioned recipients were corrected and not significantly different from lethally irradiated recipients (2.7 ± 3.1 minutes, n = 15 vs 1.9 ± 1.5 minutes, n = 8), while no untreated GPIbαnull mice (n=8) stopped bleeding within 10 minutes. Platelet counts were also significantly increased in busulfan-conditioned recipients compared to untreated GPIbαnull mice (381 ± 132 × 103/μl, n = 11 vs. 181 ± 35 × 103/μl, n = 11). Thus non-myeloablative doses of busulfan conditioning and transplantation of hGPIbαtg+mouse BM cells were shown to achieve relevant levels of engraftment in murine BSS with significantly corrected bleeding times and platelet counts.
Antibody response to hGPIbα and immune-mediated thrombocytopenia was documented in 3 of 15 recipient mice, suggesting immunogenicity of hGPIbα transgene protein in GPIbαnullmice partially immunosuppressed with busulfan. However, immunotolerance was identified without treatment and antibody disappeared in all three mice 3 to 7 months after BM transplantation.
In conclusion, non-myeloablative doses of busulfan conditioning may be a reasonable alternative to TBI for the BMT-based treatment of BSS and could be utilized in non-myeloablative autologous gene therapy in human BSS.

Disclosures:
No relevant conflicts of interest to declare.