Co-Treatment of Erythroid Cells from β-Thalassemia Patients with CRISPR-Cas9-Based β039-Globin Gene Editing and Induction of Fetal Hemoglobin

Abstract

Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF.

The Long Scientific Journey of Sirolimus (Rapamycin): From the Soil of Easter Island (Rapa Nui) to Applied Research and Clinical Trials on β-Thalassemia and Other Hemoglobinopathies

“…The combined treatments should also include novel genomic approaches, including gene therapy and genomic editing (GE). For instance, Cosenza et al were recently able to demonstrate that the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing [ 111 ] can be combined with rapamycin-mediated HbF induction [ 102 ] ( Figure 3 ).…”

“…The data obtained by Cosenza et al [ 106 ] demonstrated that the maximum level of production of HbA and HbF was obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from the studied β 0 39-thalassemia patients [ 102 ].…”

“…The indicated research activities have been taken from the following studies: Sehgal, et al, 1975 [ 16 ], Vézina et al, 1975 [ 17 ], Mischiati et al, 2004 [ 89 ], Zuccato et al, 2007 [ 90 ], Fibach et al, 2006 [ 91 ], Pecoraro et al, 2015 [ 92 ], Zhang, X. et al, 2014 [ 93 ]; Khaibullina, A. et al, 2015 [ 94 ], Wang, J. et al, 2016 [ 95 ], Lechauve, C. et al, 2019 [ 96 ], Gamberini et al, 2021 [ 97 ], Zuccato et al, 2022 [ 98 ], Gaudre, N. et al, 2017 [ 99 ]; Al-Khatti, A.A. et al, 2019 [ 100 ], Zuccato C. et al, 2022 [ 101 ], Cosenza L.C. et al, 2022 [ 102 ].…”

“…Demonstration that in (GE + RAPA)-treated cells HbF is increased, together with the “de novo” production of HbA. Modified from Cosenza et al, with permission (copyright can be found at , accessed on 3 July 2023) [ 102 ].…”

Combined approaches for increasing fetal hemoglobin (HbF) and de novo production of adult hemoglobin (HbA) in erythroid cells from β-thalassemia patients: treatment with HbF inducers and CRISPR-Cas9 based genome editing

“…The first example of combined treatment using HbF inducers and gene editing was recently reported by Cosenza et al (2022) , who previously developed a protocol for CRISPR-Cas9-based gene correction of the β 0 39-thalassemia mutation, one of the most frequent in the Mediterranean area ( Origa, 2017 ). The study was aimed at determining whether pharmacologic induction of HbF could be combined with de novo production of HbA obtained by the correction of the β 0 -thalassemia mutation using the developed CRISPR-Cas9 protocol ( Cosenza et al, 2022 ) ( Figure 3 ). As an inducer of HbF rapamycin (also known as sirolimus) ( Sehgal, 2003 ), was selected.…”