Abstract
Purpose :
We previously discovered that hammerhead ribozymes can be engineered with enhanced kinetic turnover rates (EhhRzs) to target structured mRNA in cis, in trans, and within the intracellular environment. Here, plasmids (bicistronic, fusion RNA) are developed to incorporate the endogenous cleavage site in human rhodopsin mRNA (hRHO) target or multiple cleavage sites embedded in a mCherry reporter to allow efficient quantitative assays to test therapeutic potential.
Methods :
T7pol transcribed full-length WT (CUC↓) or Hardened (CUG) hRHO for in trans cleavage assays with a 41nt synthetic EhhRz targeting position 266. In vitro T7pol co-synthesis/cleavage reactions had WT or Hard hRHO along with a fusion RNA mCherry reporter containing 4 hRHO structured targets (embedding the 266 cleavage site (CUC↓)) and 4 downstream EhhRzs in cis. A bicistronic plasmid expressed hRHO downstream of the CMV promoter and an IRES element preceding a mCherry reporter. In vitro cleavage reactions were analyzed by PAGE urea gels. In vivo functional assays in HEK293 cells measured suppression of fluorescence of mCherry and hRHO (Alexa647-1D4 mAb) by quantitative imaging (Keyence). RT-PCR measured mRNAs by the ΔΔCt method. Controls were catalytically-inactivated EhhRzs and hardened mRNA targets that prevent cleavage. Statistical analysis was in Origin.
Results :
EhhRzs cleave greater than 50% of WT hRHO accessible 266 (CUC↓) in trans under physiological Mg2+ (1mM) while Hard 266 prevents cleavage (1.5 hr). Co-synthesis of the mCherry-EhhRz with the independent WT hRHO leads to a robust 266 nt product indicating that EhhRzs self-cleaving from the in cis target are able to encounter and cleave an endogenous structured hRHO target element in trans. Transfection of the bicistronic plasmid revealed the active cis-liberated EhhRz cleaves the endogenous hRHO 266 (CUC↓) in trans with 67% hRHO protein suppression (p<<0.05). Comparable knockdown levels of mCherry FL were observed at 59% (p<<0.05). RT-PCR analysis revealed 96% hRHO (p<<0.05) and 97% mCherry (p<<0.05) drop in mRNA levels compared to inactive EhhRz.
Conclusions :
A 41nt synthetic EhhRz cleaves WT hRHO at accessible 266 (CUC↓) in trans. Cleavage of endogenous 266 in hRHO and target elements in UTR regions of a mCherry fusion RNA reporter or bicistronic mRNA strongly suppress target expression. These experiments further develop EhhRz therapeutics for ocular diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.