Optimization of Autologous Hematopoietic Progenitor Stem Cell Apheresis Collection from Plerixafor-Mobilized Patients with Sickle Cell Disease

Introduction

Autologous hematopoietic progenitor cell (HPC)-based gene therapy and genome editing are emerging therapeutic strategies for patients with sickle cell disease (SCD). Given the high cell loss incurred during production of genetically modified HPCs, a large number of HPCs (10-15 x10 ⁶ cells/kg) are required. Obtaining sufficient autologous HPCs from patients with SCD is a critical rate limiting step for the success of these novel genetic therapies. Plerixafor, a CXCR4 antagonist, has been shown to be safe and efficient for HPC mobilization in patients with SCD. However, impaired red blood cell (RBC) rheology and chronic inflammation associated with SCD make HPC apheresis collection challenging. For example, HPCs can potentially form aggregates with sickled RBCs traveling deep in the buffy coat upon density-based separation. In addition, elevated levels of activated platelets can increase cell clumping during apheresis. Thus, autologous HPC collection using standard apheresis setting is generally less efficient in patients with SCD and often associated with clumping and unstable cell interfaces during apheresis. It has been reported that standard HPC apheresis collections yield <0.7 x 10 ⁶ CD34+ cells/kg per procedure in SCD patients despite adequate HPC mobilization (peak CD34 count 36-65/µL; Esrick, EB et al., 2018). To address these challenges, we modified our apheresis collection settings to improve the HPC collection efficiency and yield from plerixafor-mobilized patients with SCD.

Methods

Adult patients with SCD who were enrolled on two clinical trials (NCT03226691 and NCT04443907) at St. Jude Children's Research Hospital and the National Heart, Lung and Blood Institute at the National Institutes of Health were included. Hydroxyurea was discontinued at least 2 weeks and an RBC exchange was performed 3-7 days prior to HPC mobilization. The participants received 240 µg/kg of Plerixafor subcutaneously, and underwent HPC collection 2-4 hours later using the Spectra Optia continuous mononuclear cell collection. Acid-citrate-dextrose-solution A was used for anticoagulation and calcium gluconate was infused during apheresis to prevent citrate-induced hypocalcemia symptoms. Up to 4 total blood volumes were processed for each collection with an adjusted collection preference (CP) of 10-40 and AC/Inlet ratio of 6-12. Collection efficiency (CE) 1 was calculated as CD34+ cell yield / [(pre-CD34 count + post-CD34 count)/2 x whole blood volume processed]. CE 2 was calculated as CD34+ cell yield / (pre-CD34 count x whole blood volume processed).

Results

We performed 46 autologous HPC collections in 26 participants (age of 20 to 50 years). Mean (±1SD) pre-collection blood indices were: hematocrit (Hct) 27.4 ±3.8%, platelet count 226 ± 111 x10 ⁹/L, and hemoglobin S 25.7 ± 6.0%, WBC count 13.82 ± 6.0 x10 ⁹/L, and CD34+ cell count 62 ± 49 /µL. To address the altered HPC sedimentation and clumping associated with SCD, we targeted a deep buffy coat collection by lowing CP from standard 50 to 10-40, and used more anticoagulant by lowering Inlet/AC ratio from standard 12 to 6-12 respectively. The adjustments increased HPC yield, and 5.60 ± 5.48 x10 ⁶/kg CD34+ cells were collected per procedure. The CE1 was 49.21 ± 28.34 % and CE2 42.74 ± 26.24%, which are comparable to autologous HPC CEs for non-SCD individuals. A deep collection led to highly efficient collections, although this tended to increase RBC contamination of the products (product Hct 6.0 ±2.2% versus < 3% for standard collection). Nevertheless, the participants experienced minimal loss of RBCs with a post-collection Hct of 26.6 ± 2.9%. Platelet loss was also relatively low, as demonstrated by a post-collection platelet count of 152 ± 69 x 10 ⁹/L. No participants required platelet or blood transfusions, had bleeding or severe hypocalcemia-associated symptoms during and after the collections.

Conclusion

The adjusted apheresis settings with a low buffy coat collection and increased anticoagulant dosage allowed safe and efficient autologous HPC collection in the patients with SCD. Further optimization of apheresis settings is required to increase HPC collection efficiency.