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T. Kolniak, E. H. Yau, M. C. Butler, J. M. Sullivan; Cellular Toxicity Screening of Candidate Therapeutic Ribozyme Chimeras. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1699. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the effects of VAI-hammerhead ribozyme (VAI-hhRz) chimeras on key signal transduction pathways and cell death in human cultured cells. Post transcriptional gene silencing (PTGS) agents may have unforeseen effects on signal transduction pathways in vivo which could overshadow therapeutic benefits. Using a cell viability assay and the secreted alkaline phosphatase (SEAP) Pathway Profiling System we were able to evaluate the effects of VAI-hhRz chimeras on key cellular pathways in a high throughput screen before testing the PTGS agents in an animal model.
HhRz cDNA sequences targeting regions of determined accessibility in human rod opsin mRNA (Rho mRNA) were cloned into a Pol-III vector that drives high level cytoplasmic expression of adenoviral VA1-hhRz RNA chimeras. Cytotoxicity was assessed using SYTOX Green nuclear stain in human embryonic kidney (HEK293) cells transfected with VAI-hhRz chimeras or controls. Activation of enhancer elements, CRE, SRE, AP-1, NFB, Myc, and NFAT was evaluated by co-transfection of corresponding reporter plasmids from the SEAP Pathway Profiling System with the VAI-hhRz chimeras or controls and assaying for SEAP expression by a high throughput screening 96-well fluorescence assay.
Three VAI-hhRz chimeras tested did not produce any significant cell death as detected by SYTOX Green staining when transfected into HEK293 cells compared to controls. There was no significant induction of SEAP under the control of CRE, NF?B, Myc, NFAT, or SRE by the VAI-hhRz chimeras, but there was significant induction of SEAP under the control of AP-1 by the VAI control vector and VAI-hhRz chimeras.
The SEAP Pathway Profiling System is a useful toxicity screen for PTGS agents but will require preclinical correlative testing. VAI-hhRzs appear to be relatively nontoxic to human cells when probed by housekeeping signal transduction pathways. The mechanism of upregulation of AP-1 signaling, almost 4-fold relative to the control, by the chimeric hhRz expression vectors is currently unclear but similar events can occur, for example, by viral infections. This approach is readily extended to other PTGS modalities (e.g. RNAi).
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