Abstract
Purpose: :
To use bioinformatics and experimental approaches to investigate the extent to which animal model disease mRNAs are suitable targets for RNA drug development (e.g. ribozyme, RNAi) intended for human therapy.
Methods: :
A platform of validated bioinformatics tools was used to evaluate the sterical and energetic landscapes of target RNA folding in silico. Transfection of RNA drugs and disease target expression plasmids into HEK293 cells were used to experimentally test for differences in RNA drug efficacy between animal and human disease targets.
Results: :
Rod rhodopsin (RHO) is a highly conserved protein across all vertebrates, and its mRNA is well conserved in primary RNA sequence homology not only in the coding but also the 5’UT and 3’UT regions. RHO is a disease target for retinitis pigmentosa and RNA drug agents have been prepared against mammalian versions of this disease mRNA. Despite local primary mRNA sequence conservation, the global secondary structures of the full length and isolated coding regions of all mammalian targets tested (human, monkey, pig, dog, mouse, rat) are markedly different. However, at the local level there are more uniform estimates of accessibility across mammalian targets that are generally not statistically significant (p > 0.05). One highly conserved target site in the coding region of human RHOmRNA was previously proven to be highly accessible to RNA drug annealing by bioinformatics and experimental testing, and highly potent RNA drug agents (≈90% mRNA knockdown) were developed to target this site. Local differences in estimated mean accessibility between mammalian targets at this site were not statistically significant. Experimental efficacy of the potent RNA drug agents directed to the identical annealing site of human and mouse RHO mRNAs lead to statistically significant differences (p < 0.05) in mean RHOmRNA levels, with the human mRNA knockdown being more substantial.
Conclusions: :
This study shows that significant differences in efficacy of a single RNA drug can occur when tested against different mammalian disease mRNAs, despite complete conservation of primary RNA sequence. Local and possibly global secondary and tertiary structures influence accessibility to annealing and the resultant efficacy. Critically, proven efficacy results of RNA drugs developed and tested against an animal mRNA may not translate equivalently to a human clinical target and vice versa. Fully humanized animal models of disease appear indicated to properly evaluate RNA drugs in translationally-directed preclinical studies.
Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • age-related macular degeneration