Investigating Scaffold RNA Support of a Lead Hammerhead Ribozyme Therapeutic for RHO adRP

Abstract

Purpose : Hammerhead ribozymes (hhRz) have therapeutic potential as mRNA knockdown agents with high specificity relative to RNAi technology. Rational efforts to improve a lead RHO hhRz against human RHO mRNA in a larger viral scaffold lead us to hypothesize function in a small human tRNA scaffold. We investigated catalytic function without and with a tertiary accessory element (TAE) known to drive catalytic active state transitions in vivo. This hhRz is a candidate therapeutic for RHO mutations that cause autosomal dominant retinitis pigmentosa.

Methods : The 725 GUC↓ hhRz is embedded either in an engineered central domain of human adenoviral VAI RNA scaffold, or in an engineered anticodon domain of human tRNA-Lys3 scaffold. cDNAs for hhRzs or target hRHO fragment RNA (510 nt) were ligated in plasmids 3’ of T7 promoters. TAE was added upstream to the basic hhRz structure. hRHO fragment (510 nt) RNA harbors the 725 GUC↓ hhRz cleavage site. Controls are catalytic core hhRz mutations and scaffold only RNAs (no hhRz). In vitro hhRz cleavage reactions (Mg²⁺ ≈ 20 mM) used T7 RNA polymerase for co-transcription/cleavage of both hhRz and hRHO RNAs with analysis by denaturing PAGE and SYBR Gold fluorescence measures of substrate, enzyme, and product bands.

Results : Within the VAI scaffold 725 hhRz cleaved hRHO mRNA effectively, at the expected site, but not to completion (35% +/- 4% SEM). HhRz core mutation obviated cleavage, demonstrating RNA-mediated catalysis, and no cleavage occurred with scaffold alone. A TAE element upstream of the 725 hhRz in VAI scaffold did not enhance cleavage. Kinetic study of 725 HhRz in the VAI scaffold showed cleavage of hRHO substrate by 15 min and saturating over 1.5 hr with expected exponential character (t_1/2 = 54 min, p=4.5E-4). The 725 hhRz in the smaller tRNA-Lys3 scaffold efficiently cleaved target RHO mRNA in vitro without and with the TAE (40%) and control showed no cleavage.

Conclusions : Quantitative ribozyme assays are conducted in vitro with human mRNA targets. We identified a novel tRNA scaffold for our lead hhRz therapeutic which supports cleavage activity in the presence of a TAE structure that drives formation of the catalytically active state at intracellular Mg²⁺ levels (≤ 2mM). The small size of the therapeutic permits structural and biophysical studies to enhance efficacy. These candidate therapies are being tested in our fully humanized mouse model of adRP.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.