Efficient Delivery and Nuclear Uptake Is Not Sufficient to Detect Gene Editing in CD34+ Cells Directed by a Ribonucleoprotein Complex

CD34+ cells are prime targets for therapeutic strategies for gene editing, because modified progenitor cells have the capacity to differentiate through an erythropoietic lineage. Although experimental advances have been reported, the associated experimental protocols have largely been less than clea...

Full description

Saved in:
Bibliographic Details
Published in:Molecular therapy. Nucleic acids 2018-06, Vol.11 (C), p.116-129
Main Authors: Modarai, Shirin R., Man, Dula, Bialk, Pawel, Rivera-Torres, Natalia, Bloh, Kevin, Kmiec, Eric B.
Format: Article
Language:English
Subjects:
Biotechnology
CD34 antigen
CD34+ cells
Cell differentiation
Confocal microscopy
CRISPR
CRISPR/Cas9
Cytokines
Deoxyribonucleic acid
DNA
Efficiency
Experiments
Flow cytometry
gene editing
Genetic engineering
Genetic transformation
Genome editing
Genotypes
Laboratories
Mutation
nuclear uptake
Oligonucleotides
Progenitor cells
ribonucleoprotein
Ribonucleoproteins
Sickle cell disease
Stem cells
Transfection
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:CD34+ cells are prime targets for therapeutic strategies for gene editing, because modified progenitor cells have the capacity to differentiate through an erythropoietic lineage. Although experimental advances have been reported, the associated experimental protocols have largely been less than clear or robust. As such, we evaluated the relationships among cellular delivery; nuclear uptake, often viewed as the benchmark metric of successful gene editing; and single base repair. We took a combinatorial approach using single-stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild-type HBB into the sickle cell genotype by evaluating conditions for two common delivery strategies of gene editing tools into CD34+ cells. Confocal microscopy data show that the CRISPR/Cas9 ribonucleoprotein tends to accumulate at the outer membrane of the CD34+ cell nucleus when the Neon Transfection System is employed, while the ribonucleoproteins do pass into the cell nucleus when nucleofection is used. Despite the high efficiency of cellular transformation, and the traditional view of success in efficient nuclear uptake, neither delivery methodology enabled gene editing activity. Our results indicate that more stringent criteria must be established to facilitate the clinical translation and scientific robustness of gene editing for sickle cell disease.
ISSN:2162-2531
2162-2531
DOI:10.1016/j.omtn.2018.01.013