Gene Editing and Genotoxicity: Targeting the Off-Targets.

Gene editing technologies show great promise for application to human disease as a result of rapid developments in targeting tools notably based on ZFN, TALEN, and CRISPR-Cas systems. Precise modification of a DNA sequence is now possible in mature human somatic cells including stem and progenitor cells with increasing degrees of efficiency. At the same time new technologies are required to evaluate their safety and genotoxicity before widespread clinical application can be confidently implemented. A number of methodologies have now been developed in an attempt to predict expected and unexpected modifications occurring during gene editing. This review surveys the techniques currently available as state of the art, highlighting benefits and limitations, and discusses approaches that may achieve sufficient accuracy and predictability for application in clinical settings.

Suppression of tumor growth via IGFBP3 depletion as a potential treatment in glioma.

OBJECTIVE: Despite intensive medical treatment, patients with glioblastoma (grade IV glioma [GBM]) have a low 5-year survival rate of 5.5%. In this study, the authors tried to improve currently used therapies by identification of a therapeutic target, IGFBP3, for glioma treatment. METHODS: IGFBP3 RNA expression in 135 patients newly diagnosed with glioma was correlated with clinicopathological factors. Immunohistochemical analysis was performed to determine IGFBP3 protein expression in glioma specimens. The effect of IGFBP3 depletion on cell proliferation was examined using IGFBP3 knockdown glioma cells. Intracranial infusion of IGFBP3 siRNAs was performed to evaluate the effect of IGFBP3 depletion in mouse intracranial xenograft models. RESULTS: We demonstrated higher IGFBP3 expression in GBM than in tumor margin and grade II glioma. IGFBP3 expression was not only positively correlated with tumor grades but also associated with tumor histology and IDH1/2 mutation status. Additionally, higher IGFBP3 expression predicted shorter overall survival in glioma and GBM proneural subgroup patients. In vitro cell culture studies suggested IGFBP3 knockdown suppressed cell proliferation and induced cell cycle G2/M arrest as well as apoptosis in glioma cells. Also, accumulation of DNA double-strand breaks and γH2AX was observed in IGFBP3 knockdown cells. IGFBP3 knockdown delayed in vivo tumor growth in mouse subcutaneous xenograft models. Furthermore, convection-enhanced delivery of IGFBP3 siRNA to mouse brain suppressed intracranial tumor growth and prolonged survival of tumor-bearing mice. CONCLUSIONS: Our findings suggest IGFBP3 predicts poor outcome of glioma patients and is a potential therapeutic target for which depletion of its expression suppresses tumor growth through inducing apoptosis and accumulation of DNA damage in glioma cells.

Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell-like spheroids, and tumor xenografts through multiple cell signaling pathways.

OBJECTIVE Defects in the apoptotic machinery and augmented survival signals contribute to drug resistance in glioblastoma (GBM). Moreover, another complexity related to GBM treatment is the concept that GBM development and recurrence may arise from the expression of GBM stem cells (GSCs). Therefore, the use of a multifaceted approach or multitargeted agents that affect specific tumor cell characteristics will likely be necessary to successfully eradicate GBM. The objective of this study was to investigate the usefulness of sulforaphane (SFN)-a constituent of cruciferous vegetables with a multitargeted effect-as a therapeutic agent for GBM. METHODS The inhibitory effects of SFN on established cell lines, early primary cultures, CD133-positive GSCs, GSC-derived spheroids, and GBM xenografts were evaluated using various methods, including GSC isolation and the sphere-forming assay, analysis of reactive oxygen species (ROS) and apoptosis, cell growth inhibition assay, comet assays for assessing SFN-triggered DNA damage, confocal microscopy, Western blot analysis, and the determination of in vivo efficacy as assessed in human GBM xenograft models. RESULTS SFN triggered the significant inhibition of cell survival and induced apoptotic cell death, which was associated with caspase 3 and caspase 7 activation. Moreover, SFN triggered the formation of mitochondrial ROS, and SFN-triggered cell death was ROS dependent. Comet assays revealed that SFN increased single- and double-strand DNA breaks in GBM. Compared with the vehicle control cells, a significantly higher amount of γ-H2AX foci correlated with an increase in DNA double-strand breaks in the SFN-treated samples. Furthermore, SFN robustly inhibited the growth of GBM cell-induced cell death in established cell cultures and early-passage primary cultures and, most importantly, was effective in eliminating GSCs, which play a major role in drug resistance and disease recurrence. In vivo studies revealed that SFN administration at 100 mg/kg for 5-day cycles repeated for 3 weeks significantly decreased the growth of ectopic xenografts that were established from the early passage of primary cultures of GBM10. CONCLUSIONS These results suggest that SFN is a potent anti-GBM agent that targets several apoptosis and cell survival pathways and further preclinical and clinical studies may prove that SFN alone or in combination with other therapies may be potentially useful for GBM therapy.

The impact of cobalt-60 source age on biologically effective dose in high-dose functional Gamma Knife radiosurgery.

OBJECTIVE Functional Gamma Knife radiosurgery (GKRS) procedures have been increasingly used for treating patients with tremor, trigeminal neuralgia (TN), and refractory obsessive-compulsive disorder. Although its rates of toxicity are low, GKRS has been associated with some, if low, risks for serious sequelae, including hemiparesis and even death. Anecdotal reports have suggested that even with a standardized prescription dose, rates of functional GKRS toxicity increase after replacement of an old cobalt-60 source with a new source. Dose rate changes over the course of the useful lifespan of cobalt-60 are not routinely considered in the study of patients treated with functional GKRS, but these changes may be associated with significant variation in the biologically effective dose (BED) delivered to neural tissue. METHODS The authors constructed a linear-quadratic model of BED in functional GKRS with a dose-protraction factor to correct for intrafraction DNA-damage repair and used standard single-fraction doses for trigeminal nerve ablation for TN (85 Gy), thalamotomy for tremor (130 Gy), and capsulotomy for obsessive-compulsive disorder (180 Gy). Dose rate and treatment time for functional GKRS involving 4-mm collimators were derived from calibrations in the authors' department and from the cobalt-60 decay rate. Biologically plausible values for the ratio for radiosensitivity to fraction size (α/ß) and double-strand break (DSB) DNA repair halftimes (τ) were estimated from published experimental data. The biphasic characteristics of DSB repair in normal tissue were accounted for in deriving an effective τ1 halftime (fast repair) and τ2 halftime (slow repair). A sensitivity analysis was performed with a range of plausible parameter values. RESULTS After replacement of the cobalt-60 source, the functional GKRS dose rate rose from 1.48 to 2.99 Gy/min, treatment time fell, and estimated BED increased. Assuming the most biologically plausible parameters, source replacement resulted in an immediate relative BED increase of 11.7% for GKRS-based TN management with 85 Gy, 15.6% for thalamotomy with 130 Gy, and 18.6% for capsulotomy with 180 Gy. Over the course of the 63-month lifespan of the cobalt-60 source, BED decreased annually by 2.2% for TN management, 3.0% for thalamotomy, and 3.5% for capsulotomy. CONCLUSIONS Use of a new cobalt-60 source after replacement of an old source substantially increases the predicted BED for functional GKRS treatments for the same physical dose prescription. Source age, dose rate, and treatment time should be considered in the study of outcomes after high-dose functional GKRS treatments. Animal and clinical studies are needed to determine how this potential change in BED contributes to GKRS toxicity and whether technical adjustments should be made to reduce dose rates or prescription doses with newer cobalt-60 sources.