ArticleBMC biotechnology2012
Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100.
Article in BMC biotechnology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 22 citations in OpenAlex.
- Genetic Alchemy unveiled: MicroRNA-mediated gene therapy as the Artisan craft in the battlefront against hepatocellular carcinoma-a comprehensive chronicle of strategies and innovations.Frontiers in genetics · 2024Review
- Artificial MicroRNAs Targeting C9orf72 Can Reduce Accumulation of Intra-nuclear Transcripts in ALS and FTD Patients.Molecular therapy. Nucleic acids · 2019Article
- Artificial MicroRNA-Mediated Inhibition of Japanese Encephalitis Virus Replication in Neuronal Cells.Nucleic acid therapeutics · 2018Article
- Translation of MicroRNA-Based Huntingtin-Lowering Therapies from Preclinical Studies to the Clinic.Molecular therapy : the journal of the American Society of Gene Therapy · 2018Review
- Targeting MicroRNAs in Cancer Gene Therapy.Genes · 2017Review
- Vector-delivered artificial miRNA effectively inhibited replication of Chikungunya virus.Antiviral research · 2016Article
- Guidelines for the optimal design of miRNA-based shRNAs.Methods (San Diego, Calif.) · 2016Review
- Design, Characterization, and Lead Selection of Therapeutic miRNAs Targeting Huntingtin for Development of Gene Therapy for Huntington's Disease.Molecular therapy. Nucleic acids · 2016Article
- Article
- Therapeutic expression of hairpins targeting apolipoprotein B100 induces phenotypic and transcriptome changes in murine liver.Gene therapy · 2014Article
- Artificial microRNAs can effectively inhibit replication of Venezuelan equine encephalitis virus.Antiviral research · 2013Article
- Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA.Avicenna journal of medical biotechnologyArticle
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Authors and funding
7 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundControlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.
resultsHighly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.
conclusionOur results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.
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