Health Science Reviews

by Gemma Gauntlett

Background

Pre-birth, individuals have fetal hemoglobin (α2 γ 2), which consists of 2 alpha-globin (α2) subunits and 2 gamma-globin (γ2) subunits. Around the time of birth, y-globin gene (HBG1 and HBG2) production switches to B-globin (HBB), causing the level of fetal hemoglobin (α2 γ2) to decrease and adult haemoglobin (α2 β2) levels to increase (Figure 1a¹). Sickle cell disease (SCD) is an autosomal recessive disorder caused by mutations in the HBB gene, with the most common SCD mutation causing a homozygous p.Glu6Val substitution, resulting in the production of sickle haemoglobin (α2 βS2)². Sickle haemoglobin polymerizes at low oxygen concentrations, causing red blood cells to become sickle-shaped and rigid which leads to vascular occlusion, haemolysis, and inflammation (Figure 1b³). Consequently, patients with SCD have chronic anaemia, severe pain episodes, progressive organ damage, and have an increased risk of early death².

Figure 1: a) The fetal-to-adult haemoglobin switch. The top illustration demonstrates the level of globin synthesis (%) corresponding to the various developmental stages. The bottom illustration depicts the B-Globin locus. b) Sickle haemoglobin genetic and phenotypic mutations.

The Problem

All currently available medical therapies to treat SCD are only partially effective, with the most effective treatment, a stem-cell transplant, only being available for less than 20% of patients! It has previously been proven that increased levels of fetal hemoglobin in red blood cells protects against the complications of SCD². Therefore, the researchers set out to determine whether CRISPR-Cas9 disruption of the HBG1 and HBG2 gene promoters was an effective and safe strategy to elevate fetal hemoglobin levels to reduce the symptoms associated with SCD.

Methodology  

The researchers described the outcomes of a study on the use of a gene-editing therapy called OTQ923 by assessing the outcomes in the first three participants with SCD who received OTQ923 in-fusions. OTQ923 is a product that is createdby editing CD34+ hematopoietic stem cells using the CRISPR-Cas9 technology to repress the HBG1 promoter, the HBG2 promoter, or both, caused by a ribonucleoprotein complex consisting of Streptococcus genes Cas9 protein-single guide RNA (gRNA).

First, the researchers performed a CRISPR-Cas9 screen of the HBG1 and HBG2 promoters to identify the gRNA that resulted in the highest level of red cells expressing fetal haemoglobin (F cells). To do this, they electroporated CD34+ cells from healthy donors with Cas9 complexed with one of 72 gRNAs across the HBG1 and HBG2 promoters.

The gRNA resulting in the highest level of F cells (gRNA-68 in this case) was selected for clinical development. CD34+ HSCs were then collected from participants with severe SCD and their CD34+ cells were enriched for by immunomagnetic selection and electroporated with CRISPR-Cas9-gRNA-68 to produce autologous QTQ923 (whereby the donor and recipient is the same person). Participants then received the autologous QTQ923 infusion and the adverse-effects, the level of fetal haemoglobin expression and the percentage of fetal haemoglobin was monitored.

Results

The transcriptional repressor element in the HBG1 and HBG2 promoter gRNA-68 was selected as editing with gRNA-68 increased the percentage of F cells to levels higher than that achieved with other gRNAs. In the study, three participants received the autologous QTQ923 and notably, all three patients showed stable induction of fetal haemoglobin at the end of the 18 month follow-up period (having % fetal haemoglobin of 19-26.8% and % F cells of 69.7-87.8%) as shown in Figure 2 (note purple line plot and green bars). With great impact, all SCD associated symptoms decreased during the follow up period.

Figure 2: The total haemoglobin level and haemoglobin fractionation over time in the three participants.

Conclusions and take-home message

Disruption of the HBG1 and HBG2 gene promoters using CRISPR-Cas9 showed to be an effective strategy to induce fetal haemoglobin production and autologous QTQ923 infusion with severe SCD resulted in stable induction of F cell and fetal haemoglobin induction and partial improvement of SCD symptoms in all patients. Despite associated toxicity being observed, this approach demonstrates great potential as a future therapy for patients suffering with severe SCD.

Bibliography

1. Sankaran, V.G., and Orkin, S.H. (2013). The switch from fetal to adult hemoglobin. Cold Spring Harbor perspectives in medicine 3. 10.1101/CSHPERSPECT.A011643.
2. Sharma, A., Boelens, J.-J., Cancio, M., Hankins, J.S., Bhad, P., Azizy, M., Lewandowski, A., Zhao, X., Chitnis, S., Peddinti, R., et al. (2023). CRISPR-Cas9 Editing of the HBG1 and HBG2 Promoters to Treat Sickle Cell Disease. The New England Journal of Medicine 389, 820–832. 10.1056/NEJMOA2215643/SUPPL_FILE/NEJMOA2215643_DATA-SHARING.PDF.
3. What Causes Sickle Cell Disease? https://sickle-cell.com/causes.