Development of CRISPR_SCD001, an Autologous Hematopoietic Stem Cell Gene Therapy for Sickle Cell Disease after CRISPR-Cas9 Mediated Correction.

We developed CRISPR_SCD001, an autologous cellular therapy in which the pathogenic HBB sickle allele is corrected by CRISPR-Cas9-mediated homology-directed repair using a single-stranded oligodeoxyribonucleotide as DNA donor template. The Drug Product (DP) from Plerixafor-mobilized peripheral-blood CD34+ cells from sickle and healthy donors were used for: 1) pharmacodynamic assessments to interrogate therapeutic potential; 2) evaluation of genotoxicity; and 3) in vivo characterization by a non-GLP toxicology study in NBSGW mice. Large-scale DP lots yielded 89 ± 5 % CD34+ purity with 84 ± 6 % viability. Mean gene correction at HBB was 22 ± 4 % of alleles, with 51 ± 6 % of allelic disruption. Edited erythroid cultures produced 39 ± 5 % adult hemoglobin and 40 ± 6 % fetal hemoglobin, reducing sickle hemoglobin from 89% to 22 ± 6 %. In NBSGW mice, CRISPR_SCD001 engrafted robustly (59 ± 25%), maintained editing frequencies comparable to injected cells (∼20% gene correction), showed polyclonal distribution of the edited cells, and no noteworthy treatment-related toxicity or pathologic evident were found by a blinded histopathology assessment. Our protocol generates a clinical-grade, cryopreserved CD34+ cell product that corrects the sickle mutation, restores anti-sickling hemoglobins, and meets pre-clinical safety criteria, establishing a reproducible manufacturing process suitable for clinical translation. These data support initiation of a first-in-human phase I trial of CRISPR_SCD001 for sickle patients presenting with severe pathology.