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

Abstract

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.

A Discovery with Potential to Revitalize Hammerhead Ribozyme Therapeutics for Treatment of Inherited Retinal Degenerations

“…By inhibiting toxic protein production, the disease state could be treated. HhRzs are optimal PTGS agents because they are capable of multiturnover reactions, do not rely on endogenous cell machinery, and have low potential for off-target effects due to high annealing specificity (Sullivan et al 2011). Researchers have developed hhRzs for many conditions including HIV, cancer, and ocular disease with successes in vitro (Birikh et al 1997;Persidis 1997).…”

“…Over 200 mutations have been reported in RHO that lead to adRP (Froebel et al 2017). A knockdown-reconstitution strategy can be employed which targets both wild-type and mutant RHO with only one hhRz, creating a single therapeutic for the diverse set of mutations (Sullivan et al 2011). RHO is then reconstituted, to avoid haploinsufficiency, in a form that creates WT protein but is immune to cleavage.…”

Systematic Screening, Rational Development, and Initial Optimization of Efficacious RNA Silencing Agents for Human Rod Opsin Therapeutics

“…The sequence-specific knockdown of target disease mRNAs through posttranscriptional gene silencing (PTGS) agents offers attractive therapeutic strategies for the treatment of autosomal dominant (ad) hereditary retinal diseases. A mutation-independent (MI) approach has gained favor in gene therapy strategies using a PTGS agent, which suppresses the endogenous expression of both the wild-type (WT) and the mutation-containing transcripts, which is combined with an expression construct that produces a WT transcript that is unaffected by the specific gene silencing agent 1–9. This approach addresses the profound genotypic heterogeneity that exists for many ad retinal degenerations.…”