Tiffany A. Kolniak

Bottlenecks in development of retinal therapeutic post-transcriptional gene silencing agents

Development of post-transcriptional gene silencing (PTGS) agents for therapeutic purposes is an immense challenge in modern biology. Established technologies used to knockdown a specific target RNA and its cognate protein: antisense, ribozyme, RNAi, all conditionally depend upon an initial, critical annealing event of the PTGS ligand to a target RNA. In this review we address the nature of the bottlenecks, emphasizing the biocomplexity of target RNA structure, that currently limit PTGS therapeutic development. We briefly review existing and emerging technologies designed to release these constraints to realize the potential of PTGS agents in gene based therapies.

Relieving Bottlenecks in RNA Drug Discovery for Retinal Diseases

The development of efficacious and safe post transcriptional gene silencing (PTGS) agents is a challenging scientific endeavor that embraces “biocomplexity” at many levels. The target mRNA exhibits a level of structural complexity that profoundly limits annealing of PTGS agents. PTGS agents are macromolecular RNAs that must be designed to fold into catalytically active structures able to cleave the target mRNA. Pushing into and beyond the biological complexity requires new technologies for high throughput screening to efficiently and rapidly assess a set of biological and experimental variables engaged in RNA drug discovery.

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

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 co-localization 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 an 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 through upregulation to expression of the set of PTGS agents tested. AP-1 and NFκB upregulation both appear to couple to the expression of some PTGS agents which likely reflect the known properties of these pathways to be stimulated by abundant small structured RNAs.

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

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.