An improved ribozyme processing system that generates active RNA interference effector molecules from Pol II expression cassettes
Date
2009-05-28T08:19:10Z
Authors
Hean, Justin
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Abstract
RNA interference (RNAi) has been shown to be highly effective in targeted
gene knockdown and has the potential to be applied for therapy by silencing
pathology-causing genes. However, there remain several undesired properties
associated with the utilisation of RNAi for therapeutic purposes. These include:
innate immunostimulation, “off-target” cellular sequences and the possibility of
saturating the endogenous RNAi pathway, which is required for microRNA
biogenesis. RNA Polymerase III (Pol III) promoters have been used predominantly to
generate exogenous expressed RNAi precursors. Pol III promoters possess
constitutive activity in most tissue types and their transcripts can be easily tailored
into microRNA-like cellular precursor structures. Regulation of Pol III promoters is
however difficult to achieve, and their lack of tissue specificity and high activity are
responsible for the toxic saturating effects on the RNAi pathway. In contrast, RNA
polymerase II promoters express mRNAs which can be regulated and are
differentially expressed in specific tissues. However, Pol II-transcripts have additional sequences such as the 5’ 7-methyl guanosine cap and 3’ polyadenylation
sequence which make them unsuitable for the generation of important RNAi
precursors such as short hairpin RNAs (shRNAs). This study aimed to produce a
series of cytomegalovirus (CMV) promoter-controlled expression cassettes that
would generate shRNAs lacking unwanted flanking sequences. The precise hairpin
RNA strand was processed post-transcriptionally through the action of chimaeric ciscleaving
hammerhead ribozymes that are incorporated up- and down-stream of the shRNA. The hammerhead ribozymes were restored from a minimal state by inserting
additional extra-core elements allowing for intracellular activity. This design for
producing active RNAi effector sequences was termed a Ribozyme Processing
System (RyPS). To evaluate the inhibitory efficacy of RyPS, a previously
characterised shRNA targeted against the X open reading frame of the hepatitis B
virus was inserted into a RyPS expression cassette. In vitro co-transcription and
cleavage experiments demonstrated the processing potential of RyPS. This resulted in
the formation of 3 products; the upstream and downstream ribozymes and the
shRNA. Northern blot analysis of in vitro transcription products revealed the shRNA
and downstream ribozyme were smaller than anticipated. Using primer extension
analysis the precise ribozyme cleavage sites of the up- and downstream ribozymes
were mapped. The upstream ribozyme mapped the predicted site, however multiple cleavage sites were mapped for the downstream ribozyme. The aberrant cleavage of
the downstream ribozyme resulted in an shRNA cleaved within the antisense region,
a sequence which dictates the targeting and inhibitory potential of the shRNA.
Intracellular transfection of RyPS resulted in little to no inhibition of both live virus
and targeted reporter genes. It was noted however that the ribozymes maintained
intracellular activity according to a luciferase-based knockdown assay, in which
ribozyme activity resulted in luciferase mRNA destruction. By applying these results
to RNA folding algorithms, a model can be developed where atypical cleavage by the
flanking ribozymes is avoided, and further allow for the design of more stable RyPS
and individual ribozyme. Although the current design of the RyPS cassette was
shown to be ineffective at producing active shRNAs, possible optimisation would involve the substitution of the shRNA, or replacing the chimaeric hammerhead
ribozyme species with a naturally occurring species. With these changes further
developments of this post-transcriptional processing system may soon result in
effective Pol II-generated sequences for therapeutic RNAi.