Promising New Gene Editing Techniques for Sickle Cell

Varun Katta

New data presented at the American Society of Gene & Cell Therapy (ASGCT) Virtual Meeting shows how genome editing techniques could transform care for sickle cell disease (SCD) patients.

Sickle cell is characterized by pain crises, chronic anemia, multi-organ dysfunction, and early mortality. In addition, patients who co-inherit sickle cell disease mutations and genetic variations that cause hereditary persistence of fetal hemoglobin (HPFH) are generally asymptomatic.

This leads researchers to believe inducing fetal hemoglobin (HbF, α2γ2) to replace abnormal sickle adult hemoglobin (HbS, α2βS 2) can be a promising genome editing strategy.

Researchers recently found the disruption of an erythroid-specific enhancer of BCL11A to elevate HbF (α2γ2) was an effective strategy in early phase trials.

Another approach is to disrupt repressor-binding motifs for BCL11A or ZBTB7A proteins in the γ-globin gene promoters in HSCs.

A team, led by Varun Katta, Hematology, St Jude Children’s Research Hospital, compared the efficiency of editing BCL11A and ZBTB7A binding sites in the γ-globin gene (HBG1 and HBG2) promoters and associated levels of HbF (α2γ2) induction.

In the study, the researchers electroporated human primary CD34+ hematopoietic stem and progenitor cells (HSPCs) with Cas9-3xNLS ribonucleoproteins (RNPs).

There were high editing efficiencies, 83.8-97.9% indels, as well as transplanted edited HSPCs into immunodeficient NBSGW mice.

The researchers also found all hematopoietic lineages derived from RNP-treated donor HSPCs exhibited 63.5-92.7% indel mutations at the γ-globin promoter ZBTZ7A or BCL11A binding sites, indicating consistent, high-level editing of repopulating hematopoietic stem cells (HSCs) 17 weeks post-transplantation.

By exiting the BCL11A binding site, HbF induction was up to 31.8% in erythroid progeny, compared to less than 2% in erythroid progeny from unedited HSCs.

“Disruption of the ZBTB7A binding site at similar frequencies also resulted in erythroid HbF induction, although to a lesser extent (13-18%),” the authors wrote.

To assess this approach specifically for sickle cell patients, the research team edited plerixafor-mobilized CD34+ HSPCs from 1 healthy donor and 3 adult individuals with SCD using Cas9-3xNLS RNPs targeting the BCL11A binding site in the γ-globin promoter.

There was consistently high indel rates, which ranged from 80.6-94.5% in both CD34+/CD90- progenitor and CD34+/CD90+ HSC-enriched populations and 17 weeks following xenotransplantation of edited cells, the researchers observed persistent high-level editing (49.3-91.5%) of all HSC-derived lineages with HbF levels of 18.3-34.3% in human erythroid progeny compared to less than 5% in unedited controls.

Compared to controls (<6%), single cell western blot revealed broad HbF induction, where 49-58% of edited erythroblasts showed γ-globin expression.

However, a concern following the study was that Cas9- directed double strand breaks in the γ-globin promoters could result in the deletion of the intervening 4.9-kb region.

Using digital droplet PCR, the researchers observed 20-25% frequencies of this 4.9 kb deletion in HSPCs prior to xenotransplantation and at 17 weeks post-engraftment.

The investigators are currently validating unintended genome wide activities identified by CHANGE-seq and in silico methods.

“In conclusion, our preclinical data suggests that ex vivo modification of autologous HSPCs via CRISPRCas mediated disruption of the BCL11A repressor binding site in the gamma-globin promoter genes can induce HbF to therapeutically relevant levels, and therefore, represents a promising genome editing cell therapy for SCD,” the authors wrote.

The study, “CRISPR-Cas9 Genome Editing of Human CD34+ Cells at Gamma-globin Promoter to Induce Fetal Hemoglobin as Sickle Cell Disease Therapy,” was published online in Molecular Therapy.