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1.  Embedding siRNA sequences targeting Apolipoprotein B100 in shRNA and miRNA scaffolds results in differential processing and in vivo efficacy 
Molecular Therapy  2012;21(1):217-227.
Overexpression of short hairpin RNA (shRNA) often causes cytotoxicity and using microRNA (miRNA) scaffolds can circumvent this problem. In this study, identically predicted small interfering RNA (siRNA) sequences targeting apolipoprotein B100 (siApoB) were embedded in shRNA (shApoB) or miRNA (miApoB) scaffolds and a direct comparison of the processing and long-term in vivo efficacy was performed. Next generation sequencing of small RNAs originating from shApoB- or miApoB-transfected cells revealed substantial differences in processing, resulting in different siApoB length, 5′ and 3′ cleavage sites and abundance of the guide or passenger strands. Murine liver transduction with adeno-associated virus (AAV) vectors expressing shApoB or miApoB resulted in high levels of siApoB expression associated with strong decrease of plasma ApoB protein and cholesterol. Expression of miApoB from the liver-specific LP1 promoter was restricted to the liver, while the H1 promoter-expressed shApoB was ectopically present. Delivery of 1 × 1011 genome copies AAV-shApoB or AAV-miApoB led to a gradual loss of ApoB and plasma cholesterol inhibition, which was circumvented by delivering a 20-fold lower vector dose. In conclusion, incorporating identical siRNA sequences in shRNA or miRNA scaffolds results in differential processing patterns and in vivo efficacy that may have serious consequences for future RNAi-based therapeutics.
PMCID: PMC3538299  PMID: 23089734
2.  Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100 
BMC Biotechnology  2012;12:42.
Controlling 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.
Highly 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.
Our 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.
PMCID: PMC3424168  PMID: 22827812
3.  In vivo knock-down of multidrug resistance transporters ABCC1 and ABCC2 by AAV-delivered shRNAs and by artificial miRNAs 
ABC transporters export clinically-relevant drugs and their over-expression causes multidrug resistance. In order to knock-down ABC transporters, ABCC1 and ABCC2, 13 shRNAs were developed. Four shRNA candidates were tested in vivo using self-complementary adeno-associated virus serotype 8. A strong, specific knock-down of Abbc2 was observed in mice liver, but at the cost of toxicity caused by oversaturation of the RNAi machinery due to high shRNA expression. Subsequent generation of artificial miRNAs showed better efficacy profile. These results demonstrate the feasibility of knocking down Abbc2 via AAV-delivered shRNAs to the liver, and encourage the use of miRNA in further therapeutics development.
PMCID: PMC3131674  PMID: 21769296
shRNA; miRNA; AAV; Abbc1; Abbc2; multidrug resistance; hepatocellular carcinoma
4.  Towards a reporter system to identify regulators of cross-talk between salicylate and jasmonate signaling pathways in Arabidopsis 
Plant Signaling & Behavior  2008;3(8):543-546.
The plant signaling hormones salicylic acid (SA) and jasmonic acid (JA) are regulators of inducible defenses that are activated upon pathogen or insect attack. Cross-talk between SA- and JA-dependent signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. SA-mediated suppression of the JA-responsive PDF1.2 promoter was exploited for setting up a genetic screen aiming at the isolation of signal transduction mutants that are impaired in this cross-talk mechanism. The PDF1.2 promoter was fused to the herbicide resistance gene BAR to allow for life/death screening of a population of mutagenized transgenic plants. Non-mutant plants should survive herbicide treatment when methyl jasmonate (MeJA) is applied, but suppression of the JA response by SA should be lethal in combination with the herbicide. Conversely, crucial SA/JA cross-talk mutants should survive the combination treatment. SA effectively suppressed the expression of the PDF1.2::BAR transgene. However, suppression of the BAR gene did not result in suppression of herbicide resistance. Hence, a screening method based on quantitative differences in the expression of a reporter gene may be better suited to identify SA/JA cross-talk mutants. Here, we demonstrate that the PDF1.2::GUS reporter will be excellently suited in this respect.
PMCID: PMC2634489  PMID: 19513248
plant defense; salicylic acid; jasmonic acid; cross-talk; mutant screen; Arabidopsis
5.  Histone modifications do not play a major role in salicylate-mediated suppression of jasmonate-induced PDF1.2 gene expression 
Cross-talk between salicylic acid (SA) and jasmonic acid (JA) defense signaling pathways allows a plant to finely tune its response to the attacker encountered. In Arabidopsis, pharmacological experiments revealed that SA exerts a strong antagonistic effect on JA-responsive genes, such as PDF1.2, indicating that the SA pathway can be prioritized over the JA pathway. We investigated the putative role of histone modifications in the regulation of SA-mediated suppression of PDF1.2 transcription. Chromatin immunoprecipitation analysis using an antibody directed against acetylated histone H3 revealed that SA does not affect the association of this histone modification at the PDF1.2 promoter, suggesting that chromatin remodeling does not play a major role in SA/JA cross-talk.
PMCID: PMC2686002  PMID: 19704873
Arabidopsis; chromatin remodeling; cross-talk; jasmonic acid; plant immune response; salicylic acid

Results 1-5 (5)