Gene correction of HBB mutations in CD34+ hematopoietic stem cells using Cas9 mRNA and ssODN donors.

Background: β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control.

Methods: We have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common β-thalassemia splicing variant HBB^IVS1-110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34⁺ hematopoietic stem cells (HSCs).

Results: Our results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBB^IVS1-110 mutation showed indels in both K562 cells (up to 77%) and CD34⁺ hematopoietic stem cells-HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34⁺ HSCs.

Conclusion: Our approach provides guidance on non-viral gene correction in CD34⁺ HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for β-thalassemia.