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T. A. Kolniak, E. H. Yau, J. M. Sullivan; Modifications to Adenoviral VAI RNA as a Cytoplasmic Carrier for Post-Transcriptional Gene Silencing Agents. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3018.
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To determine if modifications to an adenoviral VAI-hammerhead ribozyme (VAI-hhRz) chimera improve intracellular cleavage efficiency. The activity of post-transcriptional gene silencing (PTGS) agents is dependent upon co-localization with the target as well as efficient annealing to a single-stranded target region. Improvement of hhRzs was tested by modifying the adenoviral VAI cytoplasmic carrier as well as adding the RNA helicase-attracting constitutive transport element (CTE).
hhRz cDNA sequences targeting regions of determined accessibility and inaccessibility in human rod opsin mRNA (RHO mRNA) were cloned into a Pol-III vector that drives high cytoplasmic expression of either a full-length adenoviral VAI-hhRz chimera (pUC-VAI), or a modified VAI-hhRz chimera in which nucleotide substitutions were made in the VAI central domain and part of stem 4 (Prislei-VAI). To generate VAI-hhRz-CTE expression vectors we inserted the CTE from the Mason-Pfizer monkey virus in both chimeras. HhRz chimera plasmids, with and without the CTE, were co-transfected with RHO plasmid into human embryonic kidney (HEK293) cells. Knockdown in RHO mRNA was measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
The hhRz targeting the known accessible region (725) in RHO mRNA demonstrated the most significant reduction oftarget mRNA (88%) in the Prislei-VAI chimera compared to the pUC-VAI chimera (50% reduction, p<0.01). Addition of CTE to Prislei-VAI-725-HH16 and pUC-VAI-725-HH16 chimeras promoted only a 10% and 21% reduction of RHO, respectively, which were not significantly different (p>0.05). Reduction in RHO mRNA by pUC-CTE-725-HH16 was not significantly different from the reduction produced by the same VAI-hhRz chimera lacking the CTE, pUC-VAI-725-HH16 (p>0.05).
Modifications to the central domain of adenoviral VAI produced the most efficacious VAI-hhRz chimera, Prislei-VAI-725-HH16. Addition of the CTE did not increase hhRz efficiency indicating that the RHOmRNA 725 target site is already fully accessible for molecular recognition. The significant decrease in the amount of target reduction indicates that the CTE may antagonize hhRz activity. The structure of the hhRz within the VAI RNA chimera plays a major role in efficacy. Other approaches being tested to enhance RHO hhRz activity include a smaller adenoviral VAI-type cytoplasmic carrier that is also expected to co-localize with target mRNAs.
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