Abstract
Introduction: Sickle cell disease (SCD) arises from a single nucleotide mutation in the β-globin gene (HBB), producing hemoglobin S (HbS). Given its monogenic nature, SCD is an ideal candidate for gene editing. In 2023, the FDA approved the first CRISPR/Cas9 therapy to induce fetal hemoglobin (HbF) by targeting the BCL11A enhancer, thereby mitigating vaso-occlusive events (VOEs). In a groundbreaking milestone, a 39-year-old male treated with exagamglogene autotemcel (Casgevy) in 2020 became the first SCD individual to summit Mount Kilimanjaro (19,341 ft) on September 16, 2024, at 7:30 a.m. The ascent, lasting 5.5 days, involved exposure to extreme hypoxic conditions (10.1% effective oxygen at the summit), temperatures ranging from -10 to -20 degrees Celsius. In preparation for this feat, our team assessed blood function during baseline and under induced hypoxia to determine if these insights could predict his response to the extreme hypoxic environment and enable a successful summit.
Methods: Blood samples were transported in temperature-controlled containers, and processed within 24 hours. Collections occurred one month pre-ascent and three months post-summit. Flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA-WB-VCAM-1) and P-selectin (FA-WB-Psel) was quantified as previously described (Pittman et al., 2021). The Dynamic Sickling Assay (DSA) was used to measure red blood cell (RBC) sickling under enzymatically-induced hypoxia as previously described (Zaidi et al., 2023). p50 values were measured with a Hemox Analyzer (TCS Scientific). Mechanical Fragility Indices (MFI) were acquired as previously described to assess RBC membrane stability under shear-induced stress (Tarasev et al., 2022). Elongation indices (EI) and point of sickling (PoS) were determined using LoRRca ektacytometry (oxygenscan module). The percentage of F cells was quantified by flow cytometry as previously described (Davis et al., 2004). Hemoglobin fractions were analyzed via Sebia capillary zone electrophoresis.
Results: Hemoglobin profiles were stable pre- and post-summit: 45% HbS, 1% HbA₂, and 54% HbF. RBCs with detectable HbF were nearly 100% at both time points. DSA demonstrates the impact of induced hypoxia on sickling kinetics. The morphological point of sickling at 5% (mPoS@5%) was 3.4 min, a sickling rate of 16%/min, max sickling of 51%, and AUC 213%*min (range for unedited HbSS 2.2-4.5min, 19-75.9%/min, 48-98% and 265-584 %*min, respectively). Post-summit sickling kinetics revealed an mPoS@5% of 3.9 min,a sickling rate of 13%/min, max sickling of 44% and an AUC10 of 161%*min. Pre-ascent FA-WB-VCAM 72.6±5.4 cells/mm². Post-summit, FA-WB-VCAM was 156±38 cells/mm² (within normal ranges). FA-WB-Psel increased notably (from 10±2 to 163±54 cells/mm²), exceeding the critical threshold of 50 cells/mm². LoRRca oxygenscan post-summit showed EImin of 0.305, EImax of 0.485, and PoS of 17.91 mmHg. The p50 value was 21 mmHg. MFI-3 was 0.85 and MFI-10 was 6.11 (healthy ranges: 0.60-1.00 and 5.00-6.75 respectively).Post-summit complete blood count revealed leukopenia (white blood cell count: 3.14 x 103/µL]), elevated Hb (18.5 g/dL), increased mean corpuscular hemoglobin (MCH:34.1pg) and mean corpuscular hemoglobin concentration (MCHC: 38.4g/dL), with other parameters within normal limits.
Conclusions: This is the first assessment of RBC function to evaluate gene editing therapy's protective limits in an SCD patient before and after climbing Mt. Kilimanjaro. The pre-hike assessment predicted risk for sickling-mediated complications, which was confirmed by jaundice and hematuria at 15,100 ft. Supplemental oxygen was administered immediately, the pace of the hike was slowed, frequent examinations were performed by a pediatric critical care medicine physician and an adult hematologist who accompanied the individual on the hike. The Uhuru Peak (19,341 ft) was safely reached with no additional pain crises. Significant fatigue was reported even during the reduced pace, similar to that reported by healthy controls. This demonstrates the protective effect of HbF induced by gene editing, and shows the potential for DSA assessment to predict the limits of this protective effect. Future studies are underway to assess induced sickling on a population scale to better assess the predictive value of ex vivo induced sickling using the DSA and RBC health measurements.
