[3] Development of stable cell lines expressing high levels of point mutants of human opsin for biochemical and biophysical studies

Experimental Eye Research

Butler

Sullivan

2016

Self Cite

Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N=G,C,A,U; H=C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization.

Section: Detailed Methodsmentioning

confidence: 99%

A cellular high-throughput screening approach for therapeutic trans-cleaving ribozymes and RNAi against arbitrary mRNA disease targets

Experimental Eye Research

Butler

Sullivan

2016

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“…The human RHO expression vector used in this study contains a cDNA harvested from plasmid pCIS-hRHO (Nathans and Hogness, 1984; Nathans et al, 1989) and cloned downstream of the CMV promoter in pCDNA3 to form pCDNA3-WT- RHO (Sullivan and Satchwell, 2000). This construct expresses abundant WT or mutant opsin proteins in HEK293S cells (Sullivan and Satchwell, 2000).…”

Section: Methodsmentioning

confidence: 99%

“…This construct expresses abundant WT or mutant opsin proteins in HEK293S cells (Sullivan and Satchwell, 2000). In this construct the first 74 nt (of 95 nt) of the 5' UTR are replaced with vector sequence.…”

Section: Methodsmentioning

confidence: 99%

“…Resuspended HEK293 cells were quantified by hemocytometer-calibrated OD 800 measurements (Mohler et al, 1996). Equivalent numbers of cells (6 × 10 6 cells) were grown in 10 cm plates at 37°C in DMEM/F12 supplemented with 10% (v/v) heat inactivated calf serum, 2 mM L-glutamine, and penicillin/streptomycin (Sullivan and Satchwell, 2000). Cells were cotransfected at 60–70% confluence with pCDNA3-human rod opsin cDNA (7181 bp) (2 μg, 4.21 × 10 −13 moles plasmid, 1.28 × 10 13 moles of RHO expression construct), pGVAL-hhRz (3533 bp) (5 μg, 2.14 × 10 −12 moles plasmid, 1.62 × 10–13 moles of VAI-hhRz expression construct) (or 5 μg pGVAL, control) and pEGFP (2 μg) expression plasmids by Lipofectamine Plus (InVitrogen).…”

Section: Methodsmentioning

confidence: 99%

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Development of lead hammerhead ribozyme candidates against human rod opsin mRNA for retinal degeneration therapy

Abdelmaksoud

Experimental Eye Research

Zuker

et al. 2009

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To identify lead candidate allele-independent hammerhead ribozymes (hhRz) for the treatment of autosomal dominant mutations in the human rod opsin (RHO) gene, we tested a series of hhRzs for potential to significantly knockdown human RHO gene expression in a human cell expression system. Multiple computational criteria were used to select target mRNA regions likely to be single stranded and accessible to hhRz annealing and cleavage. Target regions are tested for accessibility in a human cell culture expression system where the hhRz RNA and target mRNA and protein are coexpressed. The hhRz RNA is embedded in an adenoviral VAI RNA chimeric RNA of established structure and properties which are critical to the experimental paradigm. The chimeric hhRz-VAI RNA is abundantly transcribed so that the hhRzs are expected to be in great excess over substrate mRNA. HhRz-VAI traffics predominantly to the cytoplasm to colocalize with the RHO mRNA target. Colocalization is essential for second-order annealing reactions. The VAI chimera protects the hhRz RNA from degradation and provides for a long half life. With cell lines chosen for high transfection efficiency and a molar excess of hhRz plasmid over target plasmid, the conditions of this experimental paradigm are specifically designed to evaluate for regions of accessibility of the target mRNA in cellulo. Western analysis was used to measure the impact of hhRz expression on RHO protein expression. Three lead candidate hhRz designs were identified that significantly knockdown target protein expression relative to control (p < 0.05). Successful lead candidates (hhRz CUC↓ 266, hhRz CUC↓ 1411, hhRz AUA↓ 1414) targeted regions of human RHO mRNA that were predicted to be accessible by a bioinformatics approach, whereas regions predicted to be inaccessible supported no knockdown. The maximum opsin protein level knockdown is approximately 30% over a 48 hr paradigm of testing. These results validate a rigorous computational bioinformatics approach to detect accessible regions of target mRNAs in cellulo. The opsin knockdown effect could prove to be clinically significant when integrated over longer periods in photoreceptors. Further optimization and animal testing is the next step in this stratified RNA drug discovery program. A recently developed novel and efficient screening assay based upon expression of a dicistronic mRNA (RHO-IRES-SEAP) containing both RHO and reporter (SEAP) cDNAs was used to compare the hhRz 266 lead candidate to another agent (Rz525/hhRz485) already known to partially rescue retinal degeneration in a rodent model. Lead hhRz 266 CUC↓ proved more efficacious than Rz525/hhRz485 which infers viability for rescue of retinal degeneration in appropriate preclinical models of disease.

“…Also, expression of the mutant and normal proteins will be needed to compare functional profiles, if adequate assays are available (e.g. [208, 209]). A cellular expression to test for cell localization of the mutant versus WT protein clearly is indicated to insure that the variant protein has appropriate WT trafficking phenotype.…”

Section: Strategies and Approaches For Ptgs Therapymentioning

confidence: 99%

Variables and Strategies in Development of Therapeutic Post-Transcriptional Gene Silencing Agents

Sullivan

Journal of Ophthalmology

Kolniak

et al. 2011

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Post-transcriptional gene silencing (PTGS) agents such as ribozymes, RNAi and antisense have substantial potential for gene therapy of human retinal degenerations. These technologies are used to knockdown a specific target RNA and its cognate protein. The disease target mRNA may be a mutant mRNA causing an autosomal dominant retinal degeneration or a normal mRNA that is overexpressed in certain diseases. All PTGS technologies depend upon the initial critical annealing event of the PTGS ligand to the target RNA. This event requires that the PTGS agent is in a conformational state able to support hybridization and that the target have a large and accessible single-stranded platform to allow rapid annealing, although such platforms are rare. We address the biocomplexity that currently limits PTGS therapeutic development with particular emphasis on biophysical variables that influence cellular performance. We address the different strategies that can be used for development of PTGS agents intended for therapeutic translation. These issues apply generally to the development of PTGS agents for retinal, ocular, or systemic diseases. This review should assist the interested reader to rapidly appreciate critical variables in PTGS development and facilitate initial design and testing of such agents against new targets of clinical interest.