Problems Associated with RNAi Therapy − Rakyat Biologi

Although RNAi holds huge therapeutic promise, it might not be as effective as it first appeared. Problems associated with off-target effects have been observed, whereby different mRNAs with siRNA seed matches in their 3-UTR' ~~having similar sequences to the target gene~~ are bound and repressed by the siRNA/RISC complex [143]. Off-target effects can also result from aberrant loading of the passenger strand into the RISC complex, possibly targeting other mRNAs and leading to their knockdown [144]. These highlight the need for careful sxRNA design to avoid sequence similarity in other genes to the target mRNA. Algorithms have been developed to facilitate specific siRNA design to reduce the likelihood of these off-target effects [145]. Modifications to the passenger strand can also be used to this end [146, 147].

Exogenous RNA is known to induce cellular stress responses and cause toxicity in vivo [59]. For sxRNAs, toxicities can arise as a result of the saturation of endogenous RNAi pathways in cells, in which important natural miRNAs are outcompeted for interaction with limiting cellular factors. Several studies have reported hepatotoxic side effects of high shRNA expression in the liver of mice, which correlated with the downregulation of liver-derived miRNAs and the upregulation of miRNA-controlled hepatic genes [148, 149]. Further experiments revealed that the silencing capacity of both shRNAs and miRNAs can be improved by overexpressing exportin-5 in mice [148]. Exportin-5 is a protein of low abundance that is essential for the nuclear export of shRNAs, miRNAs and tRNAs, hence the data suggests it might be one limiting factor over which shRNAs and miRNAs compete to exert their silencing effects.

These studies underline the need for stringent control over sxRNA dosing for therapeutic applications in vivo. For viral shRNA delivery, the use of a moderate promoter and limited vector dosages should be considered to avoid saturating endogenous RNAi pathways and adverse effects in healthy cells. For non-viral siRNA delivery, as well as limiting dosages, the incorporation of PEG and tumour-targeting ligands could be considered to promote preferential siRNA uptake by cancer cells and mitigate uptake by healthy cells.