The nontoxic cell cycle modulator indirubin augments transduction of adeno-associated viral vectors and zinc-finger nuclease-mediated gene targeting.
Parameters that regulate or affect the cell cycle or the DNA repair choice between non-homologous end-joining and homology-directed repair (HDR) are excellent targets to enhance therapeutic gene targeting. Here, we have evaluated the impact of five cell-cycle modulating drugs on targeted genome engineering mediated by DNA double-strand break (DSB)-inducing nucleases, such as zinc-finger nucleases (ZFNs). For a side-by-side comparison, we have established four reporter cell lines by integrating a mutated EGFP gene into either three transformed human cell lines or primary umbilical cord-derived mesenchymal stromal cells (UC-MSCs). After treatment with different cytostatic drugs, cells were transduced with adeno-associated virus (AAV) vectors that encode a nuclease or a repair donor to rescue EGFP expression through DSB-induced HDR. We show that transient cell-cycle arrest increased AAV transduction and AAV-mediated HDR up to six-fold in human cell lines and ten-fold in UC-MSCs, respectively. Targeted gene correction was observed in up to 34% of transduced cells. Both the absolute and the relative gene-targeting frequencies were dependent on the cell type, the cytostatic drug, the vector dose, and the nuclease. Treatment of cells with the cyclin-dependent kinase inhibitor indirubin-3'-monoxime was especially promising as this compound combined high stimulatory effects with minimal cytotoxicity. In conclusion, indirubin-3'-monoxime significantly improved AAV transduction and the efficiency of AAV/ZFN-mediated gene targeting and may thus represent a promising compound to enhance DSB-mediated genome engineering in human stem cells, such as UC-MSCs, which hold great promise for future clinical applications.
Laboratory of Cell and Gene Therapy, Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79108 Freiburg, Germany. toni.cathomen@uniklinik-freiburg.de
Shamim H
SH
Rahman
Sylwia
S
Bobis-Wozowicz
Debanjana
D
Chatterjee
Katharina
K
Gellhaus
Kaweh
K
Pars
Regine
R
Heilbronn
Roland
R
Jacobs
Toni
T
Cathomen
D015153:Blotting, Western; D059447:Cell Cycle Checkpoints / Q000502:physiology; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004260:DNA Repair / Q000502:physiology; D003851:Deoxyribonucleases / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000627:therapeutic use*; D059630:Mesenchymal Stem Cells; D010091:Oximes / Q000627:therapeutic use*; D060888:Real-Time Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods*
D017931:DNA Primers; D007211:Indoles; D010091:Oximes; C485184:enhanced green fluorescent protein; C474021:indirubin-3'-monoxime; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases