Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

Zulema Romero; Anastasia Lomova; Suzanne Said; Alexandra Miggelbrink; Caroline Y. Kuo; Beatriz Campo-Fernandez; Megan D. Hoban; Katelyn E. Masiuk; Danielle N. Clark; Joseph Long; Julie M. Sanchez; Miriam Velez; Eric Miyahira; Ruixue Zhang; Devin Brown; Xiaoyan Wang; Yerbol Kurmangaliyev; Roger P. Hollis; Donald B. Kohn

doi:10.1016/j.ymthe.2019.05.014

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Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

Journal article

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Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

Zulema Romero, Anastasia Lomova, Suzanne Said, Alexandra Miggelbrink, Caroline Y. Kuo, Beatriz Campo-Fernandez, Megan D. Hoban, Katelyn E. Masiuk, Danielle N. Clark, Joseph Long, …

Molecular therapy, Vol.27(8), pp.1389-1406

08/07/2019

DOI: https://doi.org/10.1016/j.ymthe.2019.05.014

PMID: 31178391

Abstract

adeno-associated virus 6

CRISPR/Cas9

gene editing

hematopoietic stem cells

homologous recombination

non-homologous end joining

sickle cell disease

zinc finger nuclease

Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs. [Display omitted] Different endonucleases and methods to deliver homologous donor sequences to human hematopoietic stem and progenitor cells (HSPCs) were compared to correct the canonical beta-globin gene mutation of sickle cell disease. Gene correction was higher when analyzed in vitro than in vivo, indicating lower efficiency in engrafting stem cells.

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Title: Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates
Creators: Zulema Romero - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Anastasia Lomova - Department of Molecular and Medical Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA
Suzanne Said - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Alexandra Miggelbrink - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Caroline Y. Kuo - Division of Allergy & Immunology, Department of Pediatrics, University of California, Los Angeles, Los Angeles, CA, USA
Beatriz Campo-Fernandez - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Megan D. Hoban - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Katelyn E. Masiuk - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Danielle N. Clark - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Joseph Long - Division of Allergy & Immunology, Department of Pediatrics, University of California, Los Angeles, Los Angeles, CA, USA
Julie M. Sanchez - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Miriam Velez - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Eric Miyahira - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Ruixue Zhang - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Devin Brown - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Xiaoyan Wang - Department of Medicine Statistics Core, University of California, Los Angeles, Los Angeles, CA, USA
Yerbol Kurmangaliyev
Roger P. Hollis - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Donald B. Kohn - Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, USA
Publication Details: Molecular therapy, Vol.27(8), pp.1389-1406
Publisher: Elsevier Inc
Identifiers: 9924209575401921
Academic Unit: Department of Biology
Language: English
Resource Type: Journal article

Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates

Abstract

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