Oligonucleotide based-strategies for allergy with special reference to siRNA

Background: Allergic diseases are a significant global health care problem. Current pharmacological approaches address symptoms but do not alter the underlying immune dysregulation. Current allergen-specific immunotherapy has several drawbacks. Therefore, approaches that attenuate allergic responses safely and effectively at the level of upstream causative events are desirable. Oligonuleotide-based therapies [CpG DNA, antisense oligonucleotides, and small interfering RNA (siRNA)] are promising approaches. Objective/methods: We review developments in oligonucleotide-based therapies and the potential of siRNA for treating allergy. Results/conclusions: Strategies with oligonucleotides basically aim to reduce T helper type 2 (Th2) responses. It is controversial whether the reduction of Th2 responses does, in fact, attenuate allergic diseases. Increased understanding of allergic mechanisms will enhance the efficacy of oligonucleotide-based therapy     

Functional validation of the carbon dioxide receptor genes in Aedes aegypti mosquitoes using RNA interference

Carbon dioxide (CO(2)) is an important long-range chemosensory cue used by blood-feeding female mosquitoes to find their hosts. The CO(2) receptor in Drosophila melanogaster was previously determined to be a heterodimer comprised of two gustatory receptor (Gr) proteins, DmGr21a and DmGr63a. In the mosquito Aedes aegypti, two putative orthologous genes, AaGr1 and AaGr3, were identified in the genome database, along with an apparent paralogue of AaGr1, AaGr2. In this study, RNA interference (RNAi)-mediated gene knockdown of either AaGr1 or AaGr3 resulted in a loss of CO(2) sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer. RNAi-mediated knockdown of AaGr2 expression had no impact on CO(2) reception. All three Gr genes were expressed in the maxillary palps of both Ae. aegypti and the West Nile virus vector mosquito, Culex pipiens quinquefasciatus. Interestingly, expression of the two CO(2) receptor genes was not equivalent in the two sexes and the implications of differential sex expression of the CO(2) receptor in different species are discussed. The functional identification of the CO(2) receptor in a mosquito could prove invaluable in the strategic design of compounds that disrupt the mosquito's ability to find hosts.     

Short-hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells

Putative U6snRNA polymerase III (PolIII) promoters were cloned from the Anopheles gambiae and Aedes aegypti genomes. The PolIII promoters were tested for their ability to express short-hairpin RNA (shRNA) targeted to firefly luciferase and to mediate RNA interference (RNAi) knockdown of a co-transfected luciferase reporter gene vector in AG-55 Anopheles gambiae and ATC-10 Aedes aegypti cells. Promoters capable of silencing expression of the co-transfected luciferase plasmid by up to 95% in AG-55 cells and up to 75% in ATC-10 cells were identified. RNase protection experiments allowed detection of the 19 nt luciferase short-interfering RNA (siRNA) in transfected cells. These findings indicate that mosquito U6snRNA gene promoters can be used for production of shRNA to induce the RNAi response in mosquito cells.     

Evidence of multiple chromosomal inversions in Aedes aegypti formosus from Senegal

Chromosomal inversions are prevalent in mosquito species but polytene chromosomes are difficult to prepare and visualize in members of the tribe Aedinii and thus there exists only indirect evidence of inversions. We constructed an F₁ intercross family using a P₁ female from a laboratory strain of Aedes aegypti aegypti ( Aaa ) and a P₁ male Aedes aegypti formosus ( Aaf ) from a strain collected from south-eastern Senegal. Recombination rates in the F₂ offspring were severely reduced and genotype ratios suggested a deleterious recessive allele on chromosome 3. The F₂ linkage map was incongruent in most respects with the established map for Aaa . Furthermore, no increased recombination was detected in F₅ offspring. Recombination rates and gene order were consistent with the presence in Aaf of at least four large inversions on chromosome 1, a single small inversion on chromosome 2 and three inversions on chromosome 3.     

HLA-A2小干扰RNA的设计及沉默效果检测

背景：人类白细胞抗原（human leukocyte antigen,HLA）是人的主要组织相容性复合物,在移植排斥反应中起关键作用。降低HLA的表达,可以延长移植物的存活时间。目的：设计HLA-A2小干扰RNA,检测其对HLA-A2表达沉默的作用。方法：设计4种HLA-A2 siRNA靶点序列构建其慢病毒表达体系,体外感染HLA-A过表达的293T细胞,观察4种靶点的敲减效率并筛选出有效靶点序列。体外培养人胚肺成纤维细胞,并用携带目的干扰序列的慢病毒感染人胚肺成纤维细胞,荧光显微镜下观察感染效率,应用Western blot检测在人胚肺成纤维细胞中对HLA-A2表达沉默的作用。结果与结论：根据Genbank中HLA-A2的mRNA序列,设计合成了3个小干扰RNA,在体外能有效的沉默HLA-A2在293细胞中的表达,将基因序列亚克隆入干扰载体后感染人胚肺成纤维细胞,也能有效的沉默HLA-A2的表达,效率达80%左右。     

Anionic polymers for decreased toxicity and enhanced in vivo delivery of siRNA complexed with cationic liposomes

We recently reported a cationic lipid-based vector of siRNA, termed siRNA lipoplex that was very efficient in specific gene silencing, both in cell culture and in mouse disease models. To be more efficient, this vector included the addition of a plasmid DNA as an anionic “cargo.” Although this plasmid DNA was devoid of any eukaryotic expression cassette, we decided to replace it by an anionic polymer that would be more acceptable for clinical applications. We identified seven anionic polymers, regarded as non-toxic, biodegradable, of various characteristics and nature. The addition of polymers to siRNA lipoplexes led to the formation of particles with similar characteristics to crude siRNA lipoplexes, decreased cellular toxicity and variable in vitro gene silencing efficiency depending on the type of polymer used. Upon i.v. injection in mice, siRNA lipoplexes prepared with the best polymer, polyglutamate, led to significantly increased recovery of siRNA in liver and lung compared with lipoplexes without polymer.     

RNA-mediated gene silencing: mechanisms and its therapeutic applications

RNA interference, part of a complicated network of interconnected pathways for cellular defence, RNA surveillance and development, has become a powerful tool for the experimental manipulation of gene expression. It is the process by which double-stranded (dsRNA) silences specific gene expression through homology-dependent degradation of cognate mRNA. The dsRNA is converted into 21nt small interfering RNAs (siRNAs), which directs a complex ribonuclease system to substrate mRNA targets. The degradation of the target mRNA is initiated with the cleavage at a position corresponding to the centre of the siRNA. Dissecting individual cellular pathways to reveal the function of numerous proteins is an approach to drug discovery. Interfering RNA (RNAi) serves as a rapid and convenient tool, which works in various organisms. RNAi technology has the potential to facilitate our understanding of biological processes and potentially lead to exciting new drugs. Here we review various experimental approaches adopted with RNAi and possible therapeutic applications.     

Silencing of P‐glycoprotein increases mortality in temephos‐treated Aedes aegypti larvae

Re‐emergence of vector‐borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P‐glycoproteins (P‐gps) are ATP‐dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P‐glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P‐gps. The basal ATPase activity of ATP‐binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P‐gp (AaegP‐gp) gene expression was detected in temephos‐treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP‐gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)‐mediated silencing of the AaegP‐gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide‐induced P‐gp expression can be involved in the modulation of insecticide efflux.     

Structure of hermes integrations in the germline of the yellow fever mosquito, Aedes aegypti

The Hermes transposable element is derived from the house fly, Musca domestica, and can incorporate into the germline of the yellow fever mosquito, Aedes aegypti. Preliminary Southern analyses indicated that Hermes integrated along with the marker gene into the mosquito genomic DNA. Here we show that Hermes integrations are accompanied by the integration of the donor plasmid as well. In addition, breaks in the donor plasmid DNAs do not occur precisely, or at the end of the terminal inverted repeats, and are accompanied by small deletions in the plasmids. Furthermore, integrations do not cause the typical 8-bp duplications of the target site DNA. No integrations are observed in the absence of a source of Hermes transposase. The Hermes transposase clearly did not catalyse precise cut-and-paste transposition in these transformed lines. It may have integrated the transposon through general recombination or through a partial replicative transposition mechanism. The imprecision of Hermes integration may result from interactions of the transposase with an endogenous hAT-like element in the mosquito genome.     

RNA interference for treating haematological malignancies

RNA interference (RNAi) is a method for silencing gene expression. It is relatively gene-specific, potent, and minimally toxic. For these reasons, RNAi holds great promise for the treatment of haematological malignancies. Much has already been learned about RNAi in the laboratory, although many fundamental questions about its mechanisms remain to be elucidated. For human trials, major hurdles to be overcome include the induction of a nonspecific immune response to RNAi, the selection of the most appropriate targets, the design of more specific molecules, and the assurance of efficient delivery and safety in patients. Translational research efforts are currently well on their way to solving these problems, and will be reviewed here.     

Mosquito dopa decarboxylase cDNA characterization and blood-meal-induced ovarian expression

Dopa decarboxylase (DDC) functions in insect cate-cholamine biochemistry to produce materials essential for cross-linking reactions that result in tanning and/or melanitation, including tanning of the mosquito egg chorion and encapsulation of parasites. We have cloned Ddc from the mosquito, Aedes aegypti , and studied its expression in response to blood-feeding, which initiates events necessary for egg maturation in mosquitoes. The Ae. aegypti Ddc cDNA was isolated via heterologous screening using a clone from Drosophila melanogaster . A resulting 1.87 kilobase (kb) clone was sequenced to reveal an open reading frame of 1464 bp, as well as 5'- and 3'-untranslated segments. The inferred amino acid sequence of this clone shares 81% identity with the published Drosophila Ddc cDNA, including complete identity with twenty-four contiguous amino acids encompassing the pyridoxal-5-phosphate cofactor binding domain. Analysis of an F2 intercross population derived from a parental cross between two Ae. aegypti strains (Hamburg and Moyo-In-Dry) allowed us to map Ddc to a locus on linkage group 2. Expression studies demonstrated the presence of a 2.1 kb message, the majority of which occurs in the ovaries where Ddc-specific mRNA is up-regulated in response to Ingestion of a blood meal. The potential for egg-tanning in anautogenous mosquitoes as a model for understanding specific genetic events in the regulation of catecholamine metabolism is addressed.     

siRNA特异性抑制K562细胞Livin基因表达及其诱导凋亡作用

本研究探讨小分子RNA干扰技术抑制livin基因表达对白血病细胞系K562细胞凋亡的影响。设计合成livin特异性小干扰RNA(siRNA),核转染K562细胞,培养转染后的K562细胞,用RT-PCR检测livin mRNA的表达,Western blot检测Livin蛋白的表达。以未转染细胞作对照,同时转染带有增强型绿色荧光蛋白的载体作为阳性对照,用流式细胞术检测其细胞绿色荧光以确定转染效率。用膜联蛋白Ⅴ及碘化丙锭双染法检测细胞凋亡率。结果表明,电穿孔的转染效率可达50%。siRNA既可以抑制livin mRNA表达,也可以抑制livin蛋白表达。特异性siRNA转染细胞后48 h细胞凋亡率为(27.41±2.30)%,与对照组(9.63±0.89%)比较明显提高(P<0.05)。结论:SiRNA可以抑制livin基因的表达,并能抑制livin基因的抗凋亡作用。     

Differential responses of migratory locusts to systemic RNA interference via double‐stranded RNA injection and feeding

The migratory locust, Locusta migratoria, is one of the most destructive agricultural pests and has been widely used as a model system for insect physiology, neurobiology and behavioural research. In the present study, we investigated the effects of RNA interference (RNAi) using two delivery methods for double‐stranded RNA (dsRNA) molecules, namely, injection and feeding, to develop a potential new pest control strategy. Our results showed that locusts have a sensitive and systemic response to the injection of dsRNAs in a dose‐dependent manner, but do not respond to the feeding of dsRNAs. Further experiments suggested that the ineffectiveness of dsRNA feeding was attributable to the rapid degradation of dsRNA, which was probably induced by nuclease enzymes in the locust midgut. Moreover, we identified almost all the homologous genes involved in the endocytosis‐mediated dsRNA uptake from the locust genome, which provided possible clues regarding the dsRNA uptake mechanisms from the intestine to the midgut epithelium. These findings reveal the differential response models of fourth instar locust nymphs to dsRNA delivery methods, contribute to the current understanding of insect RNAi mechanisms and provide important information for the further application of RNAi as a genetic tool and pest control strategy.     

Specific developmental gene silencing in the honey bee using a homeobox motif

Manipulating the expression of genes in species that are not currently used as genetic models will provide comparative insights into the evolution of gene functions. However the experimental tools in doing so are limited in species that have not served as models for genetic studies. We have examined the effects of double stranded RNA (dsRNA) in the honey bee, an insect with considerably basic scientific interest. dsRNA derived from a 300 bp stretch of the E30 homeobox motif was injected into honey bee embryos at the anterior pole in the preblastoderm stage. We found that the dsRNA fragment successfully disrupted the protein expression of the target gene throughout the whole embryo. The disruption caused deficient phenotypes similar to known loss of function mutants of Drosophila engrailed, whereas embryos injected with nonsense dsRNA showed no abnormalities. We show that the large size of the honey bee egg (D: 0.3 mm, L: 1.6 mm) and the long preblastoderm stage (11-12 h) can be exploited to generate embryos with partial disruption of gene function, which may provide an elegant alternative to classical chimeric analyses. This is the first report of targeted disruption of gene function in the honey bee, and the results prove that the chosen target gene is a functional ortholog to engrailed in Drosophila.