Disease Correction of a Diamond-Blackfan Anemia Mouse Model Using Non-Genotoxic Conditioning and Hematopoietic Stem Cell Transplantation

Background: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome that presents during early childhood and is characterized by congenital malformations, macrocytic anemia, and predisposition to cancer. More than 90% of patients are diagnosed during their first year of life due to severe anemia and require standard treatment with frequent red blood cell transfusions and/or corticosteroid therapy. These treatments often lead to iron overload, infections, and other side effects. Hematopoietic stem cell transplantation (HSCT) is the only curative treatment for DBA, though with high morbidity and mortality, which is primarily due to genotoxic myeloablative chemotherapy and/or irradiation pre-conditioning. Non-genotoxic antibody-based conditioning has been pioneered by our group as an alternative treatment approach by targeting the HSC cell surface receptor CD117 which has shown clinical success in several different disease indications. Here, we report preliminary studies using a CD117-antibody-drug-conjugate (CD117-ADC) as HSCT pre-conditioning in our previously reported DBA mouse model with haploinsufficiency of Rpl11 (Rpl11^+/Δ), which recapitulates the hematological features of DBA including severe macrocytic anemia at a young age.

Methods: For CD117-ADC conditioning, Rpl11^+/Δ mice (n=4) and wildtype (WT) littermates (n=4) were treated with CD117-ADC (2B8-SAP) at a dose of 1.5mg/kg 8 days prior to HSCT. These mice were compared with unconditioned (n=5) and sub-lethal total body irradiation (TBI) with 6Gy conditioned (n=3) controls. All mice were transplanted with 10×10⁶ whole bone marrow cells from WT B6 CD45.1 donors and compared to nontransplanted controls (n=3). Depletion of LT HSCs (Lin⁻Sca-1⁺CD117⁺CD150⁺CD48⁻) in the bone marrow (BM) was assessed by a BM aspirate immediately prior to HSCT. Peripheral blood was monitored every 4 weeks (wks) for up to 24 wks post HSCT for donor engraftment (%CD45.1) by flow cytometry and blood counts were obtained.

Results: Treatment with non-genotoxic CD117-ADC led to host HSC depletion in Rpl11^+/Δ mice and enhanced donor engraftment after HSCT. Untreated Rpl11^+/Δ mice had 22% decreased LT-HSC frequencies compared to WT mice, which were further depleted 5-fold post CD117-ADC treatment immediately prior to HSCT. Multi-lineage donor engraftment was observed as early as 4 wks post HSCT in all transplanted mice, which increased over time. After CD117-ADC-conditioned HSCT, we observed increasing total donor chimerism of up to 61% in the peripheral blood of Rpl11^+/Δ mice at 24-wks. Total donor chimerism in CD117-ADC-conditioning group (61%) was significantly more compared to the unconditioned group (5%), although it was less than that in the TBI group (92%). Importantly, red blood cell counts (Rbc) were rapidly corrected from 3.6 to 9.7 M/uL at 4 wks post-HSCT in CD117-ADC treated diseased mice, and hemoglobin (Hgb) normalized from 7.3 to 14.1 g/dL, which was comparable to Rpl11^+/Δ mice with TBI conditioning. While there were no differences in restored Rbc and Hgb levels between CD117-ADC and unconditioned HSCT groups, analysis of erythropoiesis showed that the unconditioned mice had significantly more immature cells in early erythroid populations (I and II) during differentiation and more diseased (CD45.2+) BM cells than in the conditioned groups, which could be due to stress on the rare engrafted hematopoietic cells. In contrast, CD117-ADC HSCT treated mice had comparable donor (CD45.1+) erythroid progenitor cells as the TBI-conditioned Rpl11^+/Δ HSCT recipients.

Conclusion/Implications: Our data suggest that antibody-based treatment prior to HSCT could be potentially non-genotoxic conditioning for DBA patients. While our preliminary results show efficacy without toxicity of this approach, the long-term impact of CD117-ADC on DBA mice is still under investigation. Moreover, these transplants were initially conducted with syngeneic cells representative of autologous gene therapy. Parallel experiments with allogeneic donors are being performed. These studies will enable pediatric DBA patients to receive HSCT without the side effects of chemotherapy and irradiation, thereby potentially expanding the use of HSCT to more patients with DBA and other diseases.