Journal
MOLECULAR THERAPY
Volume 30, Issue 1, Pages 145-163Publisher
CELL PRESS
DOI: 10.1016/j.ymthe.2021.08.019
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Funding
- Agence Nationale de la Recherche under Investissements d'avenir program [ANR-10-IAHU-01]
- Paris Ile de France Region under DIM Therapie gen-ique initiative
- Societe d'Acceleration du Transfert de Technolo-gies-SATT IDF Innov
- Fondation Maladies Rare
- Sanofi Innovation Award
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In this study, a novel therapeutic approach combining LV-based gene addition and CRISPR-Cas9 strategies was developed to treat Sickle Cell Disease. This versatile platform reduced the expression of sickle beta-globin and increased the expression of anti-sickling globins, resulting in improved treatment efficacy.
Sickle cell disease (SCD) is caused by a mutation in the beta-globin gene leading to polymerization of the sickle hemoglobin (HbS) and deformation of red blood cells. Autologous transplantation of hematopoietic stem/progenitor cells (HSPCs) genetically modified using lentiviral vectors (LVs) to express an anti-sick-ling b-globin leads to some clinical benefit in SCD patients, but it requires high-level transgene expression (i.e., high vector copy number [VCN]) to counteract HbS polymerization. Here, we developed therapeutic approaches combining LV-based gene addition and CRISPR-Cas9 strategies aimed to either knock down the sickle beta-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal gamma-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous beta-globin gene or regions involved in fetal hemoglobin silencing. Trans-fection of transduced cells with Cas9 protein resulted in high editing efficiency, elevated levels of anti-sickling hemoglobins, and rescue of the SCD phenotype at a significantly lower VCN compared to the conventional LV-based approach. This versatile platform can improve the efficacy of current gene addition approaches by combining different therapeutic strategies, thus reducing the vector amount required to achieve a therapeutic VCN and the associated genotoxicity risk.
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