Institut for Biomedicin

Rasmus Bak

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Standard

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. / Charlesworth, Carsten T; Camarena, Joab; Cromer, M Kyle; Vaidyanathan, Sriram; Bak, Rasmus O; Carte, Jason M; Potter, Jason; Dever, Daniel P; Porteus, Matthew H.

I: Molecular Therapy - Nucleic Acids, Bind 12, 07.09.2018, s. 89-104.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Charlesworth, CT, Camarena, J, Cromer, MK, Vaidyanathan, S, Bak, RO, Carte, JM, Potter, J, Dever, DP & Porteus, MH 2018, 'Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting', Molecular Therapy - Nucleic Acids, bind 12, s. 89-104. https://doi.org/10.1016/j.omtn.2018.04.017

APA

Charlesworth, C. T., Camarena, J., Cromer, M. K., Vaidyanathan, S., Bak, R. O., Carte, J. M., ... Porteus, M. H. (2018). Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. Molecular Therapy - Nucleic Acids, 12, 89-104. https://doi.org/10.1016/j.omtn.2018.04.017

CBE

Charlesworth CT, Camarena J, Cromer MK, Vaidyanathan S, Bak RO, Carte JM, Potter J, Dever DP, Porteus MH. 2018. Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. Molecular Therapy - Nucleic Acids. 12:89-104. https://doi.org/10.1016/j.omtn.2018.04.017

MLA

Vancouver

Charlesworth CT, Camarena J, Cromer MK, Vaidyanathan S, Bak RO, Carte JM o.a. Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. Molecular Therapy - Nucleic Acids. 2018 sep 7;12:89-104. https://doi.org/10.1016/j.omtn.2018.04.017

Author

Charlesworth, Carsten T ; Camarena, Joab ; Cromer, M Kyle ; Vaidyanathan, Sriram ; Bak, Rasmus O ; Carte, Jason M ; Potter, Jason ; Dever, Daniel P ; Porteus, Matthew H. / Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. I: Molecular Therapy - Nucleic Acids. 2018 ; Bind 12. s. 89-104.

Bibtex

@article{07cde7385e614cea9d2415c999f3a427,
title = "Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting",
abstract = "Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70{\%} of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.",
author = "Charlesworth, {Carsten T} and Joab Camarena and Cromer, {M Kyle} and Sriram Vaidyanathan and Bak, {Rasmus O} and Carte, {Jason M} and Jason Potter and Dever, {Daniel P} and Porteus, {Matthew H}",
note = "Copyright {\circledC} 2018 The Authors. Published by Elsevier Inc. All rights reserved.",
year = "2018",
month = "9",
day = "7",
doi = "10.1016/j.omtn.2018.04.017",
language = "English",
volume = "12",
pages = "89--104",
journal = "Molecular Therapy - Nucleic Acids",
issn = "2162-2531",
publisher = "Nature Publishing Group",

}

RIS

TY - JOUR

T1 - Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting

AU - Charlesworth, Carsten T

AU - Camarena, Joab

AU - Cromer, M Kyle

AU - Vaidyanathan, Sriram

AU - Bak, Rasmus O

AU - Carte, Jason M

AU - Potter, Jason

AU - Dever, Daniel P

AU - Porteus, Matthew H

N1 - Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

PY - 2018/9/7

Y1 - 2018/9/7

N2 - Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

AB - Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

U2 - 10.1016/j.omtn.2018.04.017

DO - 10.1016/j.omtn.2018.04.017

M3 - Journal article

C2 - 30195800

VL - 12

SP - 89

EP - 104

JO - Molecular Therapy - Nucleic Acids

JF - Molecular Therapy - Nucleic Acids

SN - 2162-2531

ER -