Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: Implications for the therapeutic development of RNAi

Abstract

Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.gene therapy ͉ Huntington's disease ͉ RNAi ͉ AAV

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.…”

RNAi-mediated gene silencing in tumour tissue using replication-competent retroviral vectors

“…18 MiRNA-adapted shRNA interferes to a lower extent with endogenous miRNA than conventional shRNAs, due to its more complex maturation process, which results in a lower steady-state level. 19 Moreover, the RCR vector guarantees a single copy delivery of the shRNA expression cassette per cell, which further prevents perturbation of the miRNA network.…”

“…This vector system encodes miRNA modified shRNA expression cassettes, which are more potent and less toxic. [15][16][17][18][19] We show efficient and stable knockdown of marker gene expression in vitro on the fibrosarcoma cell line HT1080 stably expressing GFP or firefly luciferase (luc). We further prove the applicability of this RNAi transfer system for gene function studies of tumour biology by suppressing polo-like kinase-1 (PLK1), which is tightly linked to cell proliferation and has been proposed as a diagnostic marker for several tumours 20 and matrix metalloprotease-14 (MMP14), a key target that promotes tumour cell invasion.…”

Epigenetic Mechanisms of Neurodegeneration in Huntington's Disease

“…Reduced toxicity compared with AAV-shRNA [123] N171-82Q transgenic mouse Improved behavior and prolonged lifespan [122] siRNA-Cholesterol mRNA/inhibition AAV1/8-HD400aa-100Q-injected mouse Less inclusion and behavior [125] ss-siRNA mRNA/inhibition HdhQ150 knock-in mouse model…”

“…Harper et al [117] showed that RNA interference using adeno-associated virus-small hairpin RNA (shRNA) improves motor deficits and neuropathological phenotypes in a transgenic (N171-82Q) mouse model of HD. Most RNA interference studies using adenovirus-shRNA, lentivirus-shRNA, adeno-associated virus-miRNA, and cholesterol-conjugated siRNA have shown a reduction of mthtt aggregates, improvement of motor behavior, and reduced neuropathological sequelae (Table 1) [118][119][120][121][122][123][124][125].…”