The Nontoxic Cell Cycle Modulator Indirubin Augments Transduction of Adeno-Associated Viral Vectors and Zinc-Finger Nuclease-Mediated Gene Targeting

Abstract

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.

Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells

“…It has been shown that small molecule compounds can effectively activate or block certain DNA repair pathways (Hollick et al, 2003; Rahman et al, 2013; Srivastava et al, 2012). However, it remains unclear whether small molecules could be used to modulate CRISPRinduced genome editing and DNA repair via the HDR pathway.…”

Engineered Viruses as Genome Editing Devices

“…In what the latter genome editing approaches are concerned, these experiments involved the targeting of both reporter and endogenous loci after the delivery of ZFNs and gene correcting templates into a diverse panel of human cell types. These different cell types included, U2OS osteosarcoma cells, 47 , 48 HEK 293 cells, 46 HeLa cervix carcinoma cells, 48 HT-1080 fibrosarcoma cells, 48 and bona fide human embryonic stem cells (ESCs) as well as iPSCs. 49 Noticeably, due to the very diverse range of tools, experimental models and conditions, the gene-targeting frequencies in both absolute and relative terms ( i.e.…”

Adeno-associated Virus as a Mammalian DNA Vector

“…Single-strand DNA vector genomes are uncoated within the nucleus and form discrete foci whose abundance directly correlates to the transduction efficiency (128, 147). The generation of a DNA molecule competent for transcription is dependent upon the genome concentration within the nucleus.…”

“…In particular, the role of intact cell cycle checkpoints, and the growth phase in general, also appear to affect transduction and furthermore, confound the direct implication of cell cycle regulators, such as ATM and p53, as controllers of AAV vector transduction. As mentioned above, several genotoxic stresses that also arrest the cell cycle result in increased transduction (9, 147). The mechanism of this enhancement is not completely understood but could be explained by the sequestration of repair factors that are reported to inhibit rAAV transduction away from the AAV genome (such as the MRN complex).…”