Combination of lentiviral and genome editing technologies for the treatment of sickle cell disease

Mol Ther. 2022 Jan 5;30(1):145-163. doi: 10.1016/j.ymthe.2021.08.019. Epub 2021 Aug 19.


Sickle cell disease (SCD) is caused by a mutation in the β-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-sickling β-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 β-globin and increase the incorporation of an anti-sickling globin (AS3) in hemoglobin tetramers, or to induce the expression of anti-sickling fetal γ-globins. HSPCs from SCD patients were transduced with LVs expressing AS3 and a guide RNA either targeting the endogenous β-globin gene or regions involved in fetal hemoglobin silencing. Transfection 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.

Keywords: CRISPR-Cas9 nuclease; genome editing; lentiviral vectors; sickle cell disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anemia, Sickle Cell* / genetics
  • Anemia, Sickle Cell* / therapy
  • CRISPR-Associated Protein 9 / genetics
  • Fetal Hemoglobin / genetics
  • Gene Editing* / methods
  • Humans
  • beta-Globins / genetics


  • beta-Globins
  • Fetal Hemoglobin
  • CRISPR-Associated Protein 9