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Open Access 01-12-2018 | Short Communication

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

Authors: Justin S. Antony, Ngadhnjim Latifi, A. K. M. Ashiqul Haque, Andrés Lamsfus-Calle, Alberto Daniel-Moreno, Sebastian Graeter, Praveen Baskaran, Petra Weinmann, Markus Mezger, Rupert Handgretinger, Michael S. D. Kormann

Published in: Molecular and Cellular Pediatrics | Issue 1/2018

Abstract

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.

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Title: Gene correction of HBB mutations in CD34+ hematopoietic stem cells using Cas9 mRNA and ssODN donors
Authors: Justin S. Antony
Ngadhnjim Latifi
A. K. M. Ashiqul Haque
Andrés Lamsfus-Calle
Alberto Daniel-Moreno
Sebastian Graeter
Praveen Baskaran
Petra Weinmann
Markus Mezger
Rupert Handgretinger
Michael S. D. Kormann
Publication date: 01-12-2018
Publisher: Springer Berlin Heidelberg
Published in: Molecular and Cellular Pediatrics / Issue 1/2018
Electronic ISSN: 2194-7791
DOI: https://doi.org/10.1186/s40348-018-0086-1

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

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

Abstract

Background

Methods

Results

Conclusion

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

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