Abstract
Sickle cell disease (SCD) can lead to acute complications, chronic organ damage, and early mortality. The only cure for SCD is a hematopoietic cell transplant (HCT). Traditionally, young patients with an HLA-matched donor received myeloablative conditioning (MAC) followed by allogeneic HCT. The recently FDA-approved gene therapies Lyfgenia and Casgevy also employ MAC. Unfortunately, most adults with SCD who have overt organ damage cannot tolerate MAC; in addition, less than 15% of patients have an HLA-matched sibling donor. We and others have adapted a non-myeloablative (NMA) haploidentical HCT approach aiming to achieve mixed donor and recipient chimerism since we have reported that 20% donor myeloid chimerism (DMC) is sufficient to reverse the SCD phenotype. Still, two major concerns face NMA haploidentical HCT: graft failure and the persistence of patient-derived preleukemic clones. Following conditioning and graft failure, these clones might acquire additional mutations, leading to the development of hematologic malignancies. Therefore, our aim has shifted towards maximizing donor chimerism levels. This work explores non-genotoxic antibody-based conditioning in a mismatched murine HCT model for SCD.
We targeted two cell surface receptors: CD45, a pan-hematopoietic cell marker, or CD117, a receptor expressed mainly on hematopoietic stem and progenitor cells. Saporin (sap), the drug payload, is released intracellularly, halting protein synthesis and inducing cell death. We first investigated the efficiency of CD45- and CD117-sap as conditioning agents compared to classical total body irradiation (TBI) and busulfan. Our base conditioning regimen also included post-transplant cyclophosphamide (PT-Cy) and sirolimus. We utilized an allogeneic murine HCT model in which female donor Balb/c bone marrow (BM) cells were transplanted into female C57Bl/6J recipients. Peripheral blood (PB) analysis showed that conjugated antibodies successfully achieved a state of mixed chimerism, while the naked version failed. Long-term follow-up (24 weeks) showed that only CD45-sap exhibited an upward trajectory of PB total, B cell, T cell chimerism, and stable DMC. In contrast, all other groups showed diminishing levels of chimerism.
Next, we adapted an additional lymphocyte-depleting approach using anti-mouse thy1.2 (thy1.2). Unlike CD45-sap, PB analysis showed synergy between CD117-sap and thy1.2, leading to significantly higher chimerism levels than mice receiving CD117-sap and 50cGy TBI (DMC; 96.27±1.08% vs 54.65±0.35%, respectively, p<0.0001). Interestingly, the falling donor chimerism observed in mice that received CD117-sap alone was reversed with the addition of thy1.2. Mice that received the combined CD117-sap and thy1.2 regimen experienced fatal anemia within one week of HCT, but this could be reversed by a single blood transfusion.
HbSS Townes mice are an established model of SCD, with a steady state hemoglobin (Hb) level of 9.96±0.22 g/dl. Consequently, three blood transfusions were administered the week before conditioning. Hb levels were restored to the normal range (12.05±0.12 g/dl), and mice were conditioned with 200cGy TBI or CD117-sap, along with thy1.2. Hb analysis showed a severe drop at day 2 pre- and day 6 post-HCT, followed by restoration of the normal levels by day 13 post-HCT in both groups. Chimerism analysis at 24 weeks post-HCT showed that Townes mice conditioned with CD117-sap showed significantly higher DMC levels compared to mice that received 200cGy TBI (73.60±5.28% vs 40.07±0.77%, respectively, p<0.0001). Further, Hb levels were significantly higher in CD117-sap-conditioned mice (13.40±0.59 vs 8.67±0.15 g/dl, p<0.0001). HPLC analysis at 20 weeks post-HCT showed that recipients conditioned with CD117-sap exhibited donor-derived Hb, while recipients conditioned with 200cGy TBI showed mixed donor/recipient Hb. Furthermore, BM analysis at 24 weeks post-HCT also showed a significant increase in the frequencies of donor-derived short- (52.50±5.67% vs 4.80±2.40%, p=0.0010) and long-term (43.28±5.79% vs 0.79±0.79%, p=0.0016) hematopoietic stem cells in CD117-sap conditioned recipients, which further confirms the successful long-term engraftment of CD117-sap conditioned recipients.In summary, CD117-sap, in combination with anti-thy1.2, PT-Cy, and sirolimus, is a promising novel NMC regimen that can achieve high levels of donor chimerism in the Townes SCD mouse model, capable of reversing SCD.
