β-hemoglobinopathies are amongst the most common inherited diseases in the world with devastating prospects for the affected individuals if untreated. The discovery that high fetal hemoglobin (HbF) levels are beneficial for patients has been one of the key drivers of hemoglobin research. Naturally occurring single nucleotide variants in the promoter region of fetal globin result in the continued expression of HbF into adulthood - a benign condition known as Hereditary Persistence of Fetal Hemoglobin (HPFH). Individuals with HPFH have fetal hemoglobin levels between 3 % and 40 % whilst the normal adult only produces about 1 % of HbF. The high HbF levels in individuals with HPFH are sufficient to ameliorate the symptoms in individuals with b‑hemoglobinopathies such as b‑thalassemia and sickle cell anemia. The aim of our research is to explore reactivation of fetal globin expression in adult life as a therapeutic strategy by developing mechanistic understanding of these advantageous HPFH mutations and by introducing these mutations into cell models in proof-of-principal studies.

Here we introduce a naturally occurring T to C substitution 198bp upstream of the fetal globin gene in an erythroid cell model using CRISPR/Cas9-mediated genome editing. With the help of our cell model we were able to uncover the molecular mechanism underlying ‑198 T>C HPFH. Through in vitro and in vivo binding studies we demonstrate that the T to C substitution at position ‑198 creates a de novo binding site for the erythroid specific activator KLF1.

We found that introduction of this mutation substantially elevated expression of HbF in these cells. Interestingly we also observed that the ‑198 T>C mutation integrates with a much higher frequency than other HPFH mutations in this cell type. Thus, we propose that introducing beneficial mutations by gene therapy could represent an effective approach to ameliorate symptoms in patients with β‑hemoglobinopathies.