Rapid, cell-based toxicity screen of potentially therapeutic post-transcriptional gene silencing agents

Abstract

Post-transcriptional gene silencing (PTGS) agents such as antisense, ribozymes and RNA interference (RNAi) have great potential as therapeutics for a variety of eye diseases including retinal and macular degenerations, glaucoma, corneal degenerations, inflammatory and viral conditions. Despite their great potential and over thirty years of academic and corporate research only a single PTGS agent is currently approved for human therapy for a single disease. Substantial challenges exist to achieving both efficacious and safe PTGS agents. Efficacy, as measured in specific target mRNA and protein knockdown, depends upon a number of complex factors including the identification of rare regions of target mRNA accessibility, cellular colocalization of the PTGS agent in sufficient concentration with the target mRNA, and stability of the PTGS agent in the target cells in which it is delivered or expressed. Safety is commonly measured by lack of cytotoxicity or other deleterious cellular responses in cells in which the PTGS agent is delivered or expressed. To relieve major bottlenecks in RNA drug discovery novel, efficient, inexpensive, and rapid tools are needed to facilitate lead identification of the most efficacious PTGS agent, rational optimization of efficacy of the lead agent, and lead agent safety determinations. We have developed a technological platform using cell culture expression systems that permits lead identification and efficacy optimization of PTGS agents against arbitrary disease target mRNAs under relatively high throughput conditions. Here, we extend the technology platform to include PTGS safety determinations in cultured human cells that are expected to represent the common cellular housekeeping microenvironment. We developed a high throughput screening (HTS) cytotoxicity assay in 96-well plate format based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding to nuclear DNA. In this format we can test a number of PTGS agents for cellular toxicity relative to control elements. We also developed a HTS 96-well plate assay that allows us to assess the impact of any given PTGS agent on stimulating a variety of common cellular stress signaling pathways (e.g. CRE, SRE, AP-1, NFκB, Myc, and NFAT) that could indicate possible deleterious effects of PTGS agents either dependent or independent of base pairing complementarity with target mRNAs. To this end we exploited the secreted alkaline phosphatase (SEAP) Pathway Profiling System where the expression of the secreted reporter protein is coupled to transcriptional activation of a variety of promoter elements involved in common cell signaling pathways. We found that a variety of lead hammerhead ribozyme (hhRz) and short hairpin (shRNA) expression constructs did not exert cytotoxicity in human cells when driven by highly active RNA Pol-III promoters. We also found that most of the cell signaling pathways tested (CRE, SRE, Myc, and NFAT) did not significantly couple thro...

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

“…The high reliability of this approach to discriminate among differences in efficacy is a substantial advantage for RNA Drug Discovery. Other HTS approaches have been developed in this lab to further refine the lead PTGS agents once identified by this approach, and to evaluate PTGS agents for cellular toxicity (Kolniak and Sullivan, 2011). …”

Cell-Based Double-Screening Method to Identify a Reliable Candidate for Osteogenesis-Targeting Compounds

“…A screening strategy based on a large-scale compound library requires more efficient and accurate high-throughput screening systems. To date, several cell-based screening systems have been reported [ 11 ], which detect cell proliferation [ 32 ], cytotoxicity [ 33 ], and differentiation [ 34 ]. Most library screening systems for osteoblasts have used the ALP activity index of MSCs and MC3T3 cells [ 35 , 36 , 37 , 38 ].…”

Ultrahigh Resolution Mouse Optical Coherence Tomography to Aid Intraocular Injection in Retinal Gene Therapy Research

“…The extent of retinal coverage by a subretinal injection is typically not 100%, which may itself be toxic 31,32,33,34 . Hence, the ability to distinguish between transduced and nontransduced regions is critical to proper testing of hypotheses of rescue and toxicity for a given candidate therapeutic.…”