Enhancing Fetal Hemoglobin Expression: The Development of EDIT-301 for Sickle Cell Disease Treatment

Sickle cell disease (SCD) is a genetic blood disorder impacting around 100,000 people in the U.S. Elevated levels of fetal hemoglobin (HbF) can mitigate the severity of SCD. A study found that individuals with a combination of sickle hemoglobin (HbS) and hereditary persistence of fetal hemoglobin (HPFH) traits, who had about 30% HbF, did not exhibit SCD symptoms. Consequently, the development of EDIT-301 is underway, an experimental cell therapy that uses genetically engineered CD34+ cells with a Cas12a ribonucleoprotein to enhance HbF levels for treating SCD.

Multiple HPFH mutations have been observed at the HBG gene locus. Notably, alterations in the distal CCAAT-box region of the HBG1/2 promoters were linked with increased HbF levels, indicating its potential as a genome editing target for SCD treatment. Analysis of genetic variations in this region revealed those responsible for increased HbF expression. Larger deletions and insertions (indels) were typically associated with higher HbF levels, whereas smaller indels had less or no effect.

Experiments with different RNP configurations, using either SpCas9 or Cas12a, showed that Cas12a induced larger deletions and a higher frequency of effective indels compared to SpCas9. The latter predominantly relied on the microhomology-mediated end joining (MMEJ) repair mechanism, which is less common in hematopoietic stem cells (HSC), suggesting lower in vivo editing efficiency. In contrast, Cas12a produced more effective indels at the HBG1/2 promoters, regardless of the DNA repair mechanism.

Based on these findings, it's hypothesized that Cas12a editing of the distal CCAAT-box region could support the long-term maintenance of effective indels and sustained HbF expression. Consistent with this, 80-90% editing was achieved in mobilized peripheral blood CD34+ cells from healthy donors using Cas12a targeting the HBG1/2 promoters, leading to roughly 40% HbF expression in their erythroid progeny. When these cells were infused into NBSGW mice, they showed long-term multilineage reconstitution at 16 weeks post-infusion, with no loss in editing levels. A diverse on-target editing profile was observed, indicating polyclonal engraftment. Erythroid cells from the bone marrow of treated animals showed significant HbF expression, averaging 40-50%, compared to about 5% in the control group.

The off-target activity of Cas12a was also assessed using various methods to identify potential off-target sites, followed by targeted PCR-NGS analysis in electroporated CD34+ cells. No off-target editing was detected, highlighting the specificity of the Cas12a RNP.

In summary, a specific Cas12a RNP was identified that efficiently edits the distal CCAAT-box region of the HBG1/2 promoters in CD34+ cells, leading to long-term polyclonal multilineage engraftment and therapeutically significant HbF levels of 40-50% in vivo. As a result, steps have been taken to prepare for an Investigational New Drug (IND) application for EDIT-301, an experimental autologous cell therapy using Cas12a-RNP modified CD34+ cells for the potential treatment of SCD.