Fig. 7

A The CTX001 molecular approach and preclinical studies. Panel A illustrates the shift from fetal hemoglobin (HbF) to adult hemoglobin (HbA) shortly after birth and the role of the transcription factor BCL11A in suppressing γ-globin, a component of fetal hemoglobin. When fetal hemoglobin levels decrease approximately 3 months after birth, individuals who cannot produce enough functional β-globin experience symptoms. This has implications for conditions like sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT). Moving to Panel B, it showcases the specific editing site targeted by the single guide RNA (sgRNA) that guides CRISPR-Cas9 to the erythroid-specific enhancer region of BCL11A. The five BCL11A exons are represented as gold boxes, and GATA1 is the binding site for the GATA1 transcription factor. PAM, the protospacer adjacent motif (NGG), is a specific DNA sequence required immediately following the Cas9 target DNA sequence. Panel C displays preclinical data that reveals the percentage of fetal hemoglobin as a portion of total hemoglobin after editing and the differentiation of erythroid cells. This data was obtained from samples taken from 10 healthy donors, with error bars indicating the standard deviation. Finally, Panel D presents the results of an off-target evaluation. GUIDE-seq (genomewide unbiased identification of double-strand breaks enabled by sequencing) was independently performed on three CD34 + HSPC (hematopoietic stem and progenitor cell) healthy donor samples to nominate sites. Subsequently, hybrid capture was conducted on four CD34 + HSPC healthy donor samples to confirm these sites. The on-target allelic editing was confirmed in each experiment with an average of 57%, and no detectable off-target editing was observed at any of the sites identified by GUIDE-seq and sequence homology. Panel A was adapted with permission from Canver and Orkin. B The data related to hemoglobin fractionation, F-cell levels, and transfusion events in the two groups of patients under study. Panel A depicts the results of CRISPR-Cas9 treatment for transfusion-dependent β-thalassemia in Patient 1, while Panel D presents data for Patient 2, who received treatment for sickle cell disease, showcasing various hemoglobin adducts and variants. The changes in F-cell percentages over time can be observed in Panel B for Patient 1 and in Panel E for Patient 2. Baseline levels of hemoglobin and F-cells were established during the initial assessment prior to treatment. Additionally, Panel C shows the progression of transfusion events over time in Patient 1, and Panel F displays vaso-occlusive crises (VOCs) or episodes and transfusion events in Patient 2. It's worth noting that exchange transfusions performed according to the study protocol before the infusion of CTX001 during the on-study period are not included in the figures. Reprinted from [152] with permission from the New England Journal of Medicine