Bone Marrow Characterization in Sickle Cell Disease: Inflammation and Stress Erythropoiesis Lead to Suboptimal CD34 Recovery Compared to Normal Volunteer Bone Marrow

Introduction Gene therapy for sickle cell disease (SCD) requires modification of a high number of long term engrafting hematopoietic stem cells (LT-HSCs) sufficient to sustain production of the gene of interest at levels capable of overcoming the pathogenic HbSS phenotype. Unlike β-Thalassemia, the inflammatory bone marrow (BM) environment and stress erythropoiesis associated with SCD may have significant impacts on HSC quality and yield necessary for disease amelioration. Important work to optimize gene therapy through improvement in gene transfer efficiency, editing strategies, or transplant conditioning can only improve gene therapy in SCD if enough autologous HSCs are LT-HSCs, thus characterization of SCD BM and CD34+ HSCs is required. Collection type, storage, and delays in processing may further impact CD34+ recovery and should be investigated as a strategy to maximize LT-HSC recovery. Methods Twenty milliliters of BM from subjects with SCD (HbSS genotype) and normal volunteers was collected in different anticoagulants (Heparin, ACD-A) and processed immediately(day 0) or stored at 40C and processed the following day(day 1). After isolation via Ficoll density gradient centrifugation, the mononuclear (MN) layer was stained with antibodies against inflammatory markers (CD36, CD35, CD11b, CD62L, CD62P), non-MN cells (GPA, CD66b, CD41/61), or processed for CD34+ selection using a magnetic microbead CD34+ selection kit and stained for CD34, CD45, and GPA expression. Data were analyzed by conventional and imaging flow cytometry, the latter confirming post-CD34+ selection flow data and demonstrating antibody intensity as a characterization of HSC heterogeneity and progenitor lineage. Complete blood count and hemoglobin (Hb) electrophoresis were obtained at the time of BM collection. Statistical analyses were performed using unpaired t-tests. Results BM was collected from 18 subjects (16 with SCD; 11M; age 21-41 years). Median Hb (8.6 vs. 13.5 gm/dL, p<0.01) and white blood cell count (8.8 vs. 4.2 K/mcL, p<0.05) differed significantly between SCD and non-SCD subjects. Median percent sickle Hb in SCD subjects was 62%. Inflammatory markers and contamination with red cell and platelet markers in the post-Ficoll MN layer were higher in SCD vs. non-SCD BM regardless of anticoagulant (CD35 24% vs. 13%, p<0.05; CD36 22% vs. 11%, p<0.05; CD62P 16% vs. 3%, p<0.05; GPA 16% vs. 4%, p<0.05; CD41/61 19% vs. 3%, p<0.05), and trended higher on day 1 in SCD BM in both anticoagulants, significantly in Heparin (GPA 23% vs. 33% on day 1, p<0.05). Total CD45 expression was lower in SCD vs. non-SCD BM in both anticoagulants (p<0.05) and on day 0 (p<0.05) and 1 (p<0.01), with Amnis data confirming a higher CD34+CD45- population in SCD BM (4 ± 2% vs. 0.5 ± 0.3%, p<0.05). While there was no significant difference in total CD34+ cell count between SCD and non-SCD BM after selection post-Ficoll, there was a trend for lower CD34+ count in SCD in both anticoagulants (2.6x10^5 vs. 4.7x10^5, p=0.1). SCD CD34+ cells were characterized by higher GPA expression (28 ± 5% vs. 13 ± 3% in non-SCD BM, p<0.01) that worsened in Heparin on day 1 (22 ± 6.3% vs. 35 ± 12.4%, p<0.05). Image cytometry confirmed a majority of GPA expression in SCD BM is from single cell CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs in addition to red cell aggregates, with an increase in CD34+CD45-GPA+ HSCs on day 1 (10 ± 5% vs. 0.6 ± 0.2 % on day 0, p<0.05). Furthermore, the percentage of CD34hi HSCs was lower in SCD vs. non-SCD BM, with >50% SCD HSCs characterized as CD34dim (56% vs. 4% in non-SCD BM, p<0.001). Lastly, the purity of CD34+ selection worsened from day 0 to day 1 in SCD BM in heparin (94% vs. 68 ± 8%, p<0.05) and ACD-A (88% vs. 68 ± 0.7%, p<0.05). Conclusions SCD BM is characterized by increased inflammation and cell contamination in the MN layer regardless of anticoagulant that worsens over time in Heparin more significantly than in ACD-A. Compared to non-SCD BM, CD34+ HSC yield post-Ficoll is lower in SCD subjects, and is characterized by a larger proportion of CD34+CD45+GPA+ and CD34+CD45-GPA+ HSCs that rise with delays in processing. This indication of early differentiation along the erythroid lineage, with more than 50% of HSCs losing CD34+ intensity suggesting they are not LT-HSCs, suggests suppression of inflammation and stress erythropoiesis, combined with early cell processing may be critical for maximal HSC recovery necessary for successful gene therapy in SCD. Disclosures Luo: bluebird bio Inc.: Employment. Pierciey: bluebird bio: Employment.