Abstract
Purpose :
Develop a hammerhead ribozyme (hhRz) with enhanced catalytic activity at low molar ratios of hhRz to substrate RNA and physiological conditions of MgCl2 (0.5-1mM) to cleave human rod rhodopsin (hRHO) mRNA. The optimized hhRz may provide a novel gene therapy agent for Stargardt/dry AMD/adRP by suppression of hRHO mRNA.
Methods :
Ribozyme and hRHO (including 5` and 3`UTRs) cDNAs were cloned into plasmids with T7 promoter to drive in vitro transcription. H1 pol III promoter and CMV promoter drove expression of therapeutic RNAs and hRHO target mRNA in HEK293S cells after transfection by lipofectamine 3000. Real time RT-PCR for hRHO was performed by comparative CT method (ΔΔCT method) using primers spanning exon 1 and 2 and an internal probe containing a fluorescent dye (FAM) at the 5` end with a quenching dye (BHQ1) at the 3` end. Human b-Actin mRNA was the internal control. Alexa-647-labeled 1D4 monoclonal antibody measures rhodopsin protein in cells using our Quantitative Imaging platform (QIP).
Results :
An H1 driven transcript from a commercially available plasmid (pSUPER) revealed a marked drop in hRHO protein with hRHO mRNA levels unaffected. Computational modelling and experimental approaches revealed a pSUPER RNA element interaction site and potential hhRz target site around nt 266 in hRHO. The pSUPER RNA element was added downstream of a 266 targeting hhRz. In vitro cleavage activity revealed Super-hhRz-266 was efficacious in high Mg2+ levels (10 mM) with ~50% cleavage at a (0.25:1) substrate excess, compared to the cleavage extent at a (10:1) hhRz excess. At physiological free Mg2+ levels, cleavage activity was ~57% under substrate RNA excess (0.25:1), compared to the cleavage efficiency at a (10:1) hhRz excess. Super-hhRz-266 exerted significant knockdown of hRHO protein (p=5.79E-12) and mRNA (p=0.003) which reversed with hhRz catalytic core mutation, to indicate knockdown specific to hhRz function.
Conclusions :
Our new Super-hhRz-266 candidate cleaves at physiological levels of free Mg2+ and under Michaelis-Menten conditions in which the substrate is in excess. These properties enhance turnover and minimize toxicity, which is essential for gene therapeutics in vivo. We are optimizing this new lead agent for preclinical therapeutic intervention.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.