Crispr/cas9-based cellular engineering for targeted gene overexpression

Mark J. Osborn, Christopher J. Lees, Amber N. McElroy, Sarah C. Merkel, Cindy R. Eide, Wendy Mathews, Colby J. Feser, Madison Tschann, Ron T. McElmury, Beau R. Webber, Chong Jai Kim, Bruce R. Blazar, Jakub Tolar

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

Original languageEnglish (US)
Article number946
JournalInternational journal of molecular sciences
Volume19
Issue number4
DOIs
StatePublished - Apr 2018

Fingerprint

Cell Engineering
genes
Genes
engineering
T-cells
Genetic Therapy
Transcriptional Activation
gene therapy
Clustered Regularly Interspaced Short Palindromic Repeats
gene expression
T-Lymphocytes
Gene Expression
Inborn Genetic Diseases
blood
Feasibility Studies
Gene expression
delivery
Hematopoietic Stem Cells
T-Cell Antigen Receptor
Fetal Blood

Keywords

  • Adeno-associated virus
  • CRISPR/Cas9
  • Cord blood
  • Homology directed repair
  • Recessive dystrophic epidermolysis bullosa
  • T-cells
  • Transcriptional activation
  • Ubiquitous chromatin opening element

Cite this

Crispr/cas9-based cellular engineering for targeted gene overexpression. / Osborn, Mark J.; Lees, Christopher J.; McElroy, Amber N.; Merkel, Sarah C.; Eide, Cindy R.; Mathews, Wendy; Feser, Colby J.; Tschann, Madison; McElmury, Ron T.; Webber, Beau R.; Kim, Chong Jai; Blazar, Bruce R.; Tolar, Jakub.

In: International journal of molecular sciences, Vol. 19, No. 4, 946, 04.2018.

Research output: Contribution to journalArticle

@article{9d44c4effc6f4459b49486f2fd540565,
title = "Crispr/cas9-based cellular engineering for targeted gene overexpression",
abstract = "Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.",
keywords = "Adeno-associated virus, CRISPR/Cas9, Cord blood, Homology directed repair, Recessive dystrophic epidermolysis bullosa, T-cells, Transcriptional activation, Ubiquitous chromatin opening element",
author = "Osborn, {Mark J.} and Lees, {Christopher J.} and McElroy, {Amber N.} and Merkel, {Sarah C.} and Eide, {Cindy R.} and Wendy Mathews and Feser, {Colby J.} and Madison Tschann and McElmury, {Ron T.} and Webber, {Beau R.} and Kim, {Chong Jai} and Blazar, {Bruce R.} and Jakub Tolar",
year = "2018",
month = "4",
doi = "10.3390/ijms19040946",
language = "English (US)",
volume = "19",
journal = "International Journal of Molecular Sciences",
issn = "1661-6596",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "4",

}

TY - JOUR

T1 - Crispr/cas9-based cellular engineering for targeted gene overexpression

AU - Osborn, Mark J.

AU - Lees, Christopher J.

AU - McElroy, Amber N.

AU - Merkel, Sarah C.

AU - Eide, Cindy R.

AU - Mathews, Wendy

AU - Feser, Colby J.

AU - Tschann, Madison

AU - McElmury, Ron T.

AU - Webber, Beau R.

AU - Kim, Chong Jai

AU - Blazar, Bruce R.

AU - Tolar, Jakub

PY - 2018/4

Y1 - 2018/4

N2 - Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

AB - Gene and cellular therapies hold tremendous promise as agents for treating genetic disorders. However, the effective delivery of genes, particularly large ones, and expression at therapeutic levels can be challenging in cells of clinical relevance. To address this engineering hurdle, we sought to employ the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to insert powerful regulatory elements upstream of an endogenous gene. We achieved robust activation of the COL7A1 gene in primary human umbilical cord blood CD34+ hematopoietic stem cells and peripheral blood T-cells. CD34+ cells retained their colony forming potential and, in a second engineering step, we disrupted the T-cell receptor complex in T-cells. These cellular populations are of high translational impact due to their engraftment potential, broad circulatory properties, and favorable immune profile that supports delivery to multiple recipients. This study demonstrates the feasibility of targeted knock in of a ubiquitous chromatin opening element, promoter, and marker gene that doubles as a suicide gene for precision gene activation. This system merges the specificity of gene editing with the high level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression.

KW - Adeno-associated virus

KW - CRISPR/Cas9

KW - Cord blood

KW - Homology directed repair

KW - Recessive dystrophic epidermolysis bullosa

KW - T-cells

KW - Transcriptional activation

KW - Ubiquitous chromatin opening element

UR - http://www.scopus.com/inward/record.url?scp=85044479575&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85044479575&partnerID=8YFLogxK

U2 - 10.3390/ijms19040946

DO - 10.3390/ijms19040946

M3 - Article

C2 - 29565806

AN - SCOPUS:85044479575

VL - 19

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 4

M1 - 946

ER -