Knockdown Efficacy Research Articles

Optimization of short hairpin RNA for lentiviral-mediated RNAi against WAS

The expression of short hairpin RNAs (shRNAs) with lentiviral vectors is useful to induce stable RNA interference, particularly in hematopoietic cells. Since primary cells integrate few copies of vector, we tested if several shRNA cassette modifications could improve knock-down efficacy. Using two shRNA sequences previously shown to inhibit the human WAS gene expression, we found that neither increasing the shRNA stem length from 19-nt to 29-nt, nor modifying the loop with 4-nt, 9-nt artificial loops or with the mir30 loop improved vector-induced shRNA efficacies. This cautions against extrapolating results obtained with synthetic molecules to shRNAs that are stably expressed from viral vectors. On the other hand, the duplication of the shRNA expression cassette resulted in twice as much knock-down per copy of integrated vector. This strategy allowed a strong suppression of WASp in CD34 + cells and will facilitate future studies on the role of WASp in human cells.

Combination of Active and Inactive siRNA Targeting the Mitotic Kinesin Eg5 Impairs Silencing Efficiency in Several Cancer Cell Lines

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.

Characterization of Antisense Oligonucleotides Comprising 2′‐Deoxy‐2′‐Fluoro‐β‐d‐Arabinonucleic Acid (FANA)

Antisense oligonucleotides (AON) are being developed for a wide array of therapeutic applications. Significant improvements in their serum stability, target affinity, and safety profile have been achieved with the development of chemically modified oligonucleotides. Here, we compared 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (FANA)-containing AONs with phosphorothioate oligodeoxynucleotides (PS-DNA), 2'-O-methyl-RNA/DNA chimeras and short interfering RNAs (siRNA) with respect to their target knockdown efficacy, duration of action and resistance to nuclease degradation. Results show that two different configurations of FANA/DNA chimeras (altimers and gapmers) were found to have potent antisense activity. Specific target inhibition was observed with both FANA configurations with an estimated EC50 value comparable to that of an siRNA but 20-to 100-fold lower than the other commonly used AONs. Moreover, the FANA/DNA chimeras showed increased serum stability that was correlated with sustained antisense activity for up to 4 days. Taken together, these results indicate that chimeric FANA/DNA AONs are promising new tools for therapeutic gene silencing when increased potency and duration of action are required.

The use of a pour-on and spray dip containing Amitraz to control ticks (Acari : Ixodidae) on cattle

Knockdown and persistence efficacies of a pour-on containing Amitraz 1% and Cypermethrin 1% and a spray dip containing 12.5% Amitraz were compared. Knock-down and persistence efficacies of the Amitraz spray dip against Rhipicephalus (Boophilus) decoloratus and Amblyomma hebraeum were significantly higher for the duration of the trial than those of the pour-on. In the case of Rhipicephalus appediculatus and Rhipicephalus evertsi evertsi, efficacy was significantly higher on Days 2 and 5. Resistance testing prior to the start of the trial indicated that R. (B.) decoloratus was resistant to both Cypermethrin and Amitraz, yet in the case of the spray dip excellent efficacy results were obtained. It is thought that the cattle's scruffy winter coat may have hindered the spread of the pour-on, but that the thorough wetting and especially the higher concentration of active ingredient applied via the spray dip allowed this formulation to be effective. These results show that under certain conditions a spray dip containing 12.5% Amitraz may be more effective than a pour-on containing 1% Amitraz and 1% Cypermethrin, despite apparent in vitro resistance.

Silencing of HIV-1 Subtype C Primary Isolates by Expressed Small Hairpin RNAs Targeted togag

Discovery of sequence-specific silencing by activating the RNA interference (RNAi) pathway has led to exciting new strategies for treating infection with human immunodeficiency virus type 1 (HIV-1). Of the HIV-1 subtypes, C is especially common in areas of the world that are worst affected. Although prone to mutation, genome plasticity of this subtype is limited in functionally important regions. We identified conserved sequences within the HIV-1 subtype C gag open reading frame and assessed whether they are suitable targets for inhibition of viral replication by RNA Pol III-driven small hairpin RNAs (shRNAs). Initially, the efficacy of each of a panel of 10 shRNAs against HIV-1 was determined using a reporter assay. shRNAs A and B, which targeted the 5 end of gag, were most effective and were used to assess inhibition of replication in cultured cells of two R5 isolates (Du151 and Du422) and one X4 virus (SW7). These shRNAs diminished intracellular HIV-1 gag RNA and HIV-1 protein concentrations as well as p24 secretion by up to 80% without inducing an interferon response. However, shRNA-mediated knockdown efficacy against each of these viral isolates varied slightly. These data show successful activation of RNAi to inhibit the replication of biologically distinct HIV-1 subtype C isolates. The effector shRNAs described here are potential candidates for gene therapy applications against the most common global subtype of HIV-1.

Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for position specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).

A new clothing impregnation method for personal protection against ticks and biting insects

The efficacy and residual activity of a factory-based, permethrin-impregnated military battle dress uniform (BDU) using a new polymer-coating technique has been evaluated by laboratory and field testing during deployment to Afghanistan and compared with two commercially available, widely used dipping methods. Residual permethrin concentrations and remaining contact toxicities on treated fabrics before laundering, after up to 100 launderings as well as after being worn-out during deployment were tested against Aedes aegypti (L.) and Ixodes ricinus (L.). The residual amount of permethrin was considerably higher with the polymer-coating technique with 280 mg/m 2 remaining after 100 launderings. Polymer-coated BDUs collected for disposal after being worn-out during military deployment showed equivalent or better residual knockdown efficacy against test arthropods when compared with the results obtained with the US Army IDA (Illinois Department of Agriculture)-Kit after 50 launderings, which represent the recommended baseline for re-impregantion or disposal of the impregnated fabric. BDUs impregnated by the polymer-coating method were found to be effective throughout the lifetime of the uniform, ensuring protection of soldiers in the field from arthropod vectors, while simultaneously decreasing logistical constraints and occupational health threats.

COMPARATIVE EFFICACY AND PERSISTENCY OF PERMETHRIN IN OLYSET®NET AND CONVENTIONALLY TREATED NET AGAINST AEDES AEGYPTI AND ANOPHELES STEPHENSI

The efficacy of the Olyset net was compared to a net treated conventionally with permethrin 10% emulsifiable concentrate at the World Health Organization recommended dose. The nets were assessed under laboratory conditions against the mosquito vectors Aedes aegypti and Anopheles stephensi. Knockdown efficacy for the Olyset net was assessed after the first 10 washings and then after the 15th and 20th washings. Data were not collected for the conventionally treated netting after 5 washings because the conventional treatment was no longer effective. The results of the study indicate permethrin effectively persisted on Olyset net for at least 20 washings, confirming the regeneration of pesticide after each wash.

Criteria for effective design, construction, and gene knockdown by shRNA vectors

BackgroundRNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized.ResultsTo determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings.ConclusionThe use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.

Uncoupling Proteins 2 and 3 Function in Concert to Augment Tolerance to Cardiac Ischemia

Transient cardiac ischemia activates cell survival signaling, conferring subsequent ischemia tolerance to the heart. This biological phenomenon, termed ischemic preconditioning, results in improved clinical outcome and attenuated infarct size following myocardial infarction. To explore genomic modifications underpinning this ischemia tolerance, we delineated the regulation and function of the cardiac enriched mitochondrial uncoupling proteins 2 and 3 during delayed ischemic preconditioning in the rat. Cardiac transcripts of genes encoding uncoupling proteins 2 and 3 are up-regulated in parallel with infarct size reduction in preconditioned hearts. Mitochondria isolated from preconditioned hearts exhibit an augmented inducible proton leak. In parallel, following anoxia-reoxygenation these mitochondria generate less hydrogen peroxide compared with non-preconditioned mitochondria. Preconditioning in rat cardiac derived myoblasts is abolished following uncoupling protein-2 depletion by RNA-interference. RNAi of uncoupling protein-3 partially attenuates the capacity to precondition these cells. Functional characterization of anoxia and reoxygenation tolerance following uncoupling protein 2 or 3 and combined 2 and 3 RNAi shows the largest reduction in viability follows depletion of both homologues. Uncoupling protein-2 depletion alone significantly attenuates anoxia-reoxygenation tolerance but uncoupling protein-3 depletion does not reduce anoxia tolerance. In parallel combined uncoupling protein depletion and isolated uncoupling protein-2 depletion augments ROS production in viable cardiomyocytes following anoxia-reoxygenation. Concurrent anti-oxidant administration ameliorates the uncoupling protein-depleted anoxia-susceptible phenotype. In conclusion, mitochondrial uncoupling proteins are necessary components of ischemia tolerance and function as components of the cellular antioxidant defense program. In the cytoprotective hierarchy, uncoupling protein-2 appears to play a greater role than uncoupling protein-3 in modulating ischemia/anoxia tolerance in heart-derived cells.

Long-term RNA interference from optimized siRNA expression constructs in adult mice

DNA constructs for small interfering RNA (siRNA) expression in mammalian cells have the potential for longer-term target gene knockdown than synthetic siRNAs. We compared in adult mice the efficacy and longevity of target gene knockdown from siRNA expression cassettes contained in plasmids, PCR-generated linear constructs or PCR constructs containing “dumbbell” ends using the hydrodynamic delivery method. Plasmid siRNA expression constructs were more effective than PCR constructs for target gene knockdown. The efficacy of the PCR constructs was improved by addition of short extensions beyond the transcription termination signal and greatly improved by addition of dumbbell ends. Constructs containing the H1 promoter were significantly less effective in mice than those containing the U6 promoter, whereas both promoters functioned equally well in cultured cells. Target gene knockdown perdured for at least 20 weeks in mice after delivery of either PCR or plasmid siRNA expression cassettes. These results will help guide RNAi vector design.

Blocking Mouse MMP-9 Production in Tumor Cells and Mouse Cornea by Short Hairpin (sh) RNA Encoding Plasmids

In this study, we assessed the efficacy of the specific knockdown of matrix metalloprotein-9 (MMP-9) in vitro and in vivo using short hairpin RNA (shRNA) against MMP-9. Two plasmids were generated encoding shRNA (pshRNA) targeted against two distinct MMP-9 gene sequences. Transfection of these pshMMP-9s could be shown to specifically inhibit MMP-9 expression both in vivo and in vitro. The effect occurred in vitro both in cells that endogenously produce MMP-9 and in cells exogenously transfected with an MMP-9-encoding plasmid. Using an in vivo transfection approach, the pshMMP- 9 was also effective at inhibiting MMP-9 protein expression in the mouse cornea. pshMMP-9s were also tested against herpes simplex virus (HSV) in the cornea. Delivery of the pshMMP-9 stopped angiogenesis and decreased the severity of herpetic keratitis. However, interferon-alpha (IFN-alpha) and IFN- beta induced by pshRNA might also contribute to inhibition of herpetic simplex keratitis (HSK) in the cornea.

Sequence characteristics of functional siRNAs

RNA interference in mammalian cells is actively used to conduct genetic screens, to identify and to validate targets, and to elucidate regulators and modifiers of cellular pathways. To ensure the specificity and efficacy of the active 21mer siRNA molecules, it is pertinent to develop a strategy for their rational design. Here we show that most functional siRNAs have characteristic sequence features. We tested 601 siRNAs targeting one exogenous and three endogenous genes. The efficacy of the siRNAs was determined at the protein level. Using a decision tree algorithm in combination with information analysis, our analyses revealed four sets of rules with a mean knockdown efficacy ranging from 60% to 73%. (To distinguish between percentages used to describe the quality of an siRNA and the percentages used to describe parts of data sets we underlined the former throughout this paper.) The best rule comprises an A/U at positions 10 and 19, a G/C at position 1, and more than three A/Us between positions 13 and 19, in the sense strand of the siRNA sequence. Using these rules, there is a 99.9% chance of designing an effective siRNA in a set of three with more than 50% knockdown efficiency in a biological readout.

Phorbol Ester-induced Apoptosis of C4-2 Cells Requires Both a Unique and a Redundant Protein Kinase C Signaling Pathway

Phorbol 12-myristate 13-acetate (PMA) potently induces apoptosis of LNCaP human prostate cancer cells. Here, we show that C4-2 cells, androgen-hypersensitive derivatives of LNCaP cells, also are sensitive to PMA-induced apoptosis. Previous reports have implicated activation of protein kinase C (PKC) isozymes alpha and delta in PMA-induced LNCaP apoptosis using overexpression, pharmacological inhibitors, and dominant-negative constructs, but have left unresolved if other isozymes are involved, if there are separate requirements for individual PKC isozymes, or if there is redundancy. We have resolved these questions in C4-2 cells using stable expression of short hairpin RNAs to knock down expression of specific PKC isozymes individually and in pairs. Partial knockdown of PKCdelta inhibited PMA-induced C4-2 cell death almost completely, whereas near-complete knockdown of PKCalpha had no effect. Knockdown of PKCepsilon alone had no effect, but simultaneous knockdown of both PKCalpha and PKCepsilon in C4-2 cells that continued to express normal levels of PKCdelta inhibited PMA-induced apoptosis. Thus, our data indicate that there is an absolute requirement for PKCdelta in PMA-induced C4-2 apoptosis but that the functions of PKCalpha and PKCepsilon in apoptosis induction are redundant, such that either one (but not both) is required. Investigation of PMA-induced events required for LNCaP and C4-2 apoptosis revealed that p38 activation is dependent on PKCdelta, whereas induction of retinoblastoma protein hypophosphorylation requires both PKC signaling pathways and is downstream of p38 activation in the PKCdelta pathway.

3,3',5'-Triiodo-L-Thyronine Reduces Efficiency of mRNA Knockdown by Antisense Oligodeoxynucleotides: A Study with Pyroglutamyl Aminopeptidase II in Adenohypophysis

The impact of hormones on the efficacy of antisense oligodeoxynucleotides (ASOs) is a poorly analyzed subject. We designed, based on the identification of potentially favorable local elements of mRNA secondary structure, eight phosphorothioate ASOs to knock down the expression of an ectopeptidase, pyroglutamyl aminopeptidase II (PPII), in primary cultures of adenohypophysis. Two of the PPII ASOs were very efficient, sequence-specific, and target-specific. Because the expression of PPII is upregulated by 3,3',5'-triiodo-L-thyronine (T3), we studied the impact of varying the protocol of PPII induction on the knockdown efficacy. Hormone removal at transfection increased markedly the ability of (1) PPII ASOs to reduce PPII mRNA levels or PPII activity in adenohypophyseal cells or in C6 rat glioma cells and (2) a thyrotropin-releasing hormone (TRH) receptor-1 (TRH-R1) ASO to reduce TRH-R1 mRNA levels in adenohypophyseal cells. There was no effect of hormone removal on transfection efficacy and no correlation between target mRNA levels and ASO efficacy. These data demonstrated that ASO efficacy could depend on T3 levels; this might be due to regulation of a step generally critical for ASO efficiency.

Differences in the Rapid Knockdown and Lethal Effects of Aerosol Formulations against German Cockroach (Blattaria, Blattellidae) Strains

ABSTRACT The knockdown and lethal efficacies of five aerosol formulations including Combat Speed® (AIs: 0.1 % imiprothrin and 0.3% cyphenothrin), Raid Power® (AIs: 1.0% pyrethrin and 0.2% permethrin), Home Keeper®, (AIs: 0.2% tetramethrin and 0.3% permethrin), Super Killer® (AIs: 0.32% tetramethrin and 0.08% bioresmethrin), and Perma Kill‐K® (AIs: 0.3% dichlorvos and 0.1% tetramethrin) against five strains of the German cockroach, Blattella germanica (L.) were assessed. The results show that the mean value of KT50 (5.4 sec.) of Combat Speed® was 4.5 and 3.1‐folds lower than those of Perma Kill‐K® and Home Keeper®, respectively. The mean value of KT90 (9.0 sec; slope = 10.02) of Combat Speed® was 3.8 to 5.8‐folds lower than Perma Kill‐K®, Supper Killer® and Home Keeper®. As lethal effects, the mean value of LT50 (17.3 sec.) of Combat Speed® was over 26 folds lower than Supper Killer® and Perma Kill‐K®. The mean value of LT90 (32.9 sec.) of Combat Speed® was 37.4 and 15.1‐folds lower than those of Supper Killer® and Perma Kill‐K®, respectively. In general, Combat Speed® and Raid Power® were considered the insecticide aerosols with faster knockdown and higher lethal effects than Supper Killer®, Perma Kill‐K®, and Home Keeper® against five strains of German cockroaches in Korea. Also, the knockdown and lethal effects of Supper Killer®, Perma Kill‐K®, and Home Keeper® were highly variable depends on the strains.

Laboratory testing of a lethal ovitrap for Aedes aegypti

Laboratory tests were conducted to determine the feasibility of making the mosquito ovitrap lethal to Aedes aegypti (L.) when they attempt to oviposit in the trap. Heavy-weight velour paper strips (2.54 x 11 cm) were used as an alternative to the wooden paddle normally provided as a substrate for mosquito oviposition. The paper strips were pretreated with insecticide solutions and allowed to dry before being used in oviposition cups of 473 ml capacity, filled with water initially to within 2.5 cm of the brim. Insecticides chosen for their quick knock-down efficacy were bendiocarb 76% WP (1.06 mg a.i./strip) and four pyrethroids: permethrin 25% WP (0.16 mg a.i./strip), deltamethin 4.75% SC (0.87 mg a.i./strip), cypermethrin 40% WP (2.81 mg a.i./strip), and cyfluthrin 20% WP (0.57 mg a.i./ strip). For experimental evaluation, two oviposition cups (one with an insecticide-treated strip and one with an untreated strip) were placed in cages (cubic 30 cm) with gravid female Ae. aegypti mosquitoes (aged 6-8 days) from a susceptible laboratory strain. Mortality-rates of female mosquitoes were 45% for bendiocarb, 47% for permethrin, 98% for deltamethrin, 100% for cypermethrin, and 100% for cyfluthrin. Young instar larvae added to the treated cups died within 2h. After water evaporation from the cups for 38 days, fresh mosquito females had access to previously submerged portions of the velour paper paddle, and mortality rates of 59% or more occurred. Cups that had water (360 ml) dripped into them, to simulate rain, produced female mosquito mortality rates of > 50% and all larvae died within 3 h of being added. These tests demonstrate that the ovitrap can be made lethal to both adults and larvae by insecticidal treatment of the ovistrip. Field efficacy trials are underway in Brazil to access the impact of this simple, low-cost, environmentally benign approach on populations of the dengue vector Ae. aegypti.

Comparative efficacy of fenthion, dichlorvos/fenitrothion and permethrin against the flea, Ctenocephahdes felis, on the dog

In a series of three replicated and controlled trials, nine dogs artificially infected with Ctenocephalides felis were treated with a systemically active ‘spot-on’ formulation of fenthion, six with a topical spray containing dichlorvos and fenitrothion and six with a surface active ‘spot-on’ preparation containing permethrin, all at recommended dose rates. Good knockdown efficacy was apparent at eight hours with the dichlorvos/fenitrothion combination and at 24 hours with the other formulations. Efficacy values in excess of 95 per cent persisted for at least 22 days in the case of the systemic fenthion preparation and the permethrin product and eight days for the dichlorvos and fenitrothion combination. Twenty-nine days after treatment there was still substantial protection in the fenthion- and permethrin-treated animals, indicated by an 88 and a 77 per cent reduction in flea counts in the two groups, respectively.

Efficacy of fenthion against the flea, Ctenocephalides felis, on the cat

ABSTRACTA controlled study was carried out in two replicated trials, each using three groups of domestic cats artificially infested with Ctenocephalides felis. In each trial three cats were treated with fenthion, three were treated with a dichlorvos/fenitrothion formulation, both at the recommended dose rate and the remainder acted as untreated controls. Good knockdown efficacy was evident 24 hours after both treatments. Efficacy values of 85 per cent or more were maintained for at least 15 days with fenthion and for less than eight days with dichlorvos/fenitrothion.

A New Trihaloimidazole Derivative S-377 with a High Fumigant Efficacy against the Pyrethroid-Resistant Colony of the German Cockroach, Blattella germanica(Dictyoptera:Blattellidae)

A new trihaloimidazole derivative S-377 [1-(4-chlorobutyloxymethyl)-2, 4, 5-trichloro-imidazole] showed high insecticidal activities against pyrethroid-resistant (O-colony) German cockroach Blattella germanica and physical and chemical properties appropriate for an active ingredient of heating fumigant. Consequently, this compound presented remarkable lethal and knockdown efficacy, comparable to that of methoxadiazone and permethrin, against the O-colony by three types of heating fumigant formulation in wider chambers. As for the mode of actions, S-377 showed low inhibitory effect on acertylcholine esterase in the German cockroach. According to electrophysiological study, this compound induced bursts of spontaneous firing on the abdominal nerve cord of German cockroaches, which continued for 60 min after the application. These results suggests that the central nervous system of German cockroaches is affected by this compound in a certain manner different from that of organophosphates, carbamates and pyrethroids. At least three possible modes of action have been suggested: (1) inhibitory actions on inhibitory systems, (2) inhibitory action on inactivating system of Na channels in a manner different from that of pyrethroids, and (3) facilitation of the excitatory system including the stimulation of excitatory synaptic transmission.