It was 1948 when pediatrics Janet Watson postulated for the first time the importance of fetal hemoglobin (HbF;α2γ2), in sickle cell diseases. Meanwhile, it has been shown that the re-activation of fetal hemoglobin (HbF) expression prevents the polymerization of sickle hemoglobin and therefore might prevent or retard the resulting health issues.
BCL11A erythroid enhancer, is responsible for the repression of fetal hemoglobin and for the activation of beta-hemoglobin. Introduction of point mutations in the core sequences of the BCL11A erythroid enhancer region causes de-repression of HbF in adult cells and reduced expression of the sickle-cell beta-hemoglobin and formation of polymerized sickle-hemoglobin.
Using this approach allows to use CRISPR / Cas9 based NHEJ repair to introduce point mutations into the BCL11A enhancer, without the necessity of co-transfection of a DNA template as it would be required for a repair of the effected beta-globin gene
Via multiple optimization steps including testing of different sequences and sources of sgRNA, different Cas9 : sgRNA ratios a reduction of nearly 55% of BCl11A RNA-levels could be obtained with a potent induction of γ-globin expression in CD34+ HSC from beta-thalassemia patients in the range of 33 – 89%.
Further improvement of Indel frequency up to 98% was obtained by using a SpCas9 protein with 3NLS .
To test if gene-editing of BCL11A enhancer could indeed improve symptoms in patient cells, the authors edited CD34+ HSC from beta-thalassemia and SCD patients and they could show that:
- in vitro differentiated erythrocytes from SCD did resist sickling
- in vitro differentiated erythrocytes from beta-thalassemia patients showed higher frequency of enucleation, large size and circular shape erythroid cells due to improved globin chain balance.
Edited HSC from Beta-thalassemia and Sickle-cell donor did engraft into immunodeficient mice with robust induction of the gamma-globin expression in erythroid cells and were able to support secondary transplantation consistent with gene editing of self-renewing, long-term engrafting HSC.
Currently, several clinical trials using different strategies (BLC11A enhancer disruption, repair of beta-globin gene etc.) are ongoing and first promising results could be obtain. I am excited to hear about results of those clinical trials and further steps taken to improve the life of patients effected by those diseases.
Written by SST
