Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways

Abstract

RNA interference (RNAi) is a universal and evolutionarily conserved phenomenon of post-transcriptional gene silencing by means of sequence-specific mRNA degradation, triggered by small double-stranded RNAs. Because this mechanism can be efficiently induced in vivo by expressing target-complementary short hairpin RNA (shRNA) from non-viral and viral vectors, RNAi is attractive for functional genomics and human therapeutics. Here we systematically investigate the long-term effects of sustained high-level shRNA expression in livers of adult mice. Robust shRNA expression in all the hepatocytes after intravenous infusion was achieved with an optimized shRNA delivery vector based on duplex-DNA-containing adeno-associated virus type 8 (AAV8). An evaluation of 49 distinct AAV/shRNA vectors, unique in length and sequence and directed against six targets, showed that 36 resulted in dose-dependent liver injury, with 23 ultimately causing death. Morbidity was associated with the downregulation of liver-derived microRNAs (miRNAs), indicating possible competition of the latter with shRNAs for limiting cellular factors required for the processing of various small RNAs. In vitro and in vivo shRNA transfection studies implied that one such factor, shared by the shRNA/miRNA pathways and readily saturated, is the nuclear karyopherin exportin-5. Our findings have fundamental consequences for future RNAi-based strategies in animals and humans, because controlling intracellular shRNA expression levels will be imperative. However, the risk of oversaturating endogenous small RNA pathways can be minimized by optimizing shRNA dose and sequence, as exemplified here by our report of persistent and therapeutic RNAi against human hepatitis B virus in vivo.

Efficient in vivo microRNA targeting of liver metastasis

“…However, the pleiotropic effects of miRNAs may have unexpected or undesirable effects, which must be tested on an individual basis. Our data show that anti-miR therapy administered by intraperitoneal injection according to the mentioned schedules is generally safe, as opposed to the fatal liver toxicity observed with small hairpin RNA therapy (Grimm et al, 2006). The histological observation of hepatitis and the transaminitis detected on anti-miR treatment need to be further examined, possibly by using different treatment regimes or chemical modification patterns.…”

Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts

“…We show that the allelespecific siRNA silences the mutant transcript more efficiently than the wild-type transcript in comparison to the consistent silencing of both alleles by siR-atxn7. Although allele selectivity is lost at the highest dose of siRNA, the likelihood of saturation of the endogenous RNAi pathway at such doses 28 suggests that our allele-specific silencing approach may still have significant clinical relevance.…”

Lentiviral vector‐encoded microRNA‐based shRNA‐mediated gene knockdown of N‐methyl‐D‐aspartate receptors in skin reduces pain

“…However, excessive expression of siRNA driven by pol III promoters may result in off‐target disruption of gene expression and may induce cellular toxicity (Grimm et al., 2006; McBride et al., 2008). The gene knockdown effect of miRNA‐based shRNA whose expression is driven by pol II promoters is 12 times more potent than that of traditional shRNA whose expression is driven by pol III promoters.…”