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CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression.

CRISPR/Cas9-Based Cellular Engineering for Targeted Gene Overexpression.
Author Information (click to view)

Osborn MJ, Lees CJ, McElroy AN, Merkel SC, Eide CR, Mathews W, Feser CJ, Tschann M, McElmury RT, Webber BR, Kim CJ, Blazar BR, Tolar J,


Osborn MJ, Lees CJ, McElroy AN, Merkel SC, Eide CR, Mathews W, Feser CJ, Tschann M, McElmury RT, Webber BR, Kim CJ, Blazar BR, Tolar J, (click to view)

Osborn MJ, Lees CJ, McElroy AN, Merkel SC, Eide CR, Mathews W, Feser CJ, Tschann M, McElmury RT, Webber BR, Kim CJ, Blazar BR, Tolar J,

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International journal of molecular sciences 2018 03 2219(4) pii 10.3390/ijms19040946

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 thegene 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.

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