Precision treatment of viral pneumonia through macrophage-targeted lipid nanoparticle delivery.

Macrophages are integral components of the innate immune system, playing a dual role in host defense during infection and pathophysiological states. Macrophages contribute to immune responses and aid in combatting various infections, yet their production of abundant proinflammatory cytokines can lead to uncontrolled inflammation and worsened tissue damage. Therefore, reducing macrophage-derived proinflammatory cytokine release represents a promising approach for treating various acute and chronic inflammatory disorders. However, limited macrophage-specific delivery vehicles have hindered the development of macrophage-targeted therapies. In this study, we screened a pool of 112 lipid nanoparticles (LNPs) to identify an optimal LNP formulation for efficient siRNA delivery. Subsequently, by conjugating the macrophage-specific antibody F4/80 to the LNP surface, we constructed MacLNP, an enhanced LNP formulation designed for targeted macrophage delivery. In both in vitro and in vivo experiments, MacLNP demonstrated a significant enhancement in targeting macrophages. Specifically, delivery of siRNA targeting TAK1, a critical kinase upstream of multiple inflammatory pathways, effectively suppressed the phosphorylation/activation of NF-kB. LNP-mediated inhibition of NF-kB, a key upstream regulator in the classic inflammatory signaling pathway, in the murine macrophage cell line RAW264.7 significantly reduced the release of proinflammatory cytokines after stimulation with the viral RNA mimic Poly(I:C). Finally, intranasal administration of MacLNP-encapsulated TAK1 siRNA markedly ameliorated lung injury induced by influenza infection. In conclusion, our findings validate the potential of targeted macrophage interventions in attenuating inflammatory responses, reinforcing the potential of LNP-mediated macrophage targeting to treat pulmonary inflammatory disorders.

Cross-kingdom RNA interference mediated by insect salivary microRNAs may suppress plant immunity.

Communication between insects and plants relies on the exchange of bioactive molecules that traverse the species interface. Although proteinic effectors have been extensively studied, our knowledge of other molecules involved in this process remains limited. In this study, we investigate the role of salivary microRNAs (miRNAs) from the rice planthopper Nilaparvata lugens in suppressing plant immunity. A total of three miRNAs were confirmed to be secreted into host plants during insect feeding. Notably, the sequence-conserved miR-7-5P is specifically expressed in the salivary glands of N. lugens and is secreted into saliva, distinguishing it significantly from homologues found in other insects. Silencing miR-7-5P negatively affects N. lugens feeding on rice plants, but not on artificial diets. The impaired feeding performance of miR-7-5P-silenced insects can be rescued by transgenic plants overexpressing miR-7-5P. Through target prediction and experimental testing, we demonstrate that miR-7-5P targets multiple plant genes, including the immune-associated bZIP transcription factor 43 (OsbZIP43). Infestation of rice plants by miR-7-5P-silenced insects leads to the increased expression of OsbZIP43, while the presence of miR-7-5P counteracts this upregulation effect. Furthermore, overexpressing OsbZIP43 confers plant resistance against insects which can be subverted by miR-7-5P. Our findings suggest a mechanism by which herbivorous insects have evolved salivary miRNAs to suppress plant immunity, expanding our understanding of cross-kingdom RNA interference between interacting organisms.

Biocompatible hydroxyapatite-based nano vehicle bypasses viral transduction and enables sustained silencing of a pluripotency marker gene, demonstrating desired differentiation in mouse embryonic stem cells.

BACKGROUND: Differentiation of pluripotent stem cells into desired lineages is the key aspect of regenerative medicine and cell-based therapy. Although RNA interference (RNAi) technology is exploited extensively for this, methods for long term silencing of the target genes leading to differentiation remain a challenge. Sustained knockdown of the target gene by RNAi is often inefficient as a result of low delivery efficiencies, protocol induced toxicity and safety concerns related to viral vectors. Earlier, we established octa-arginine functionalized hydroxyapatite nano vehicles (R8HNPs) for delivery of small interfering RNA (siRNA) against a pluripotency marker gene in mouse embryonic stem cells. Although we demonstrated excellent knockdown efficiency of the target gene, sustained gene silencing leading to differentiation was yet to be achieved. METHODS: To establish a sustained non-viral gene silencing protocol using R8HNP, we investigated various methods of siRNA delivery: double delivery of adherent cells (Adh-D), suspension delivery followed by adherent delivery (Susp + Adh), single delivery in suspension (Susp-S) and multiple deliveries in suspension (Susp-R). Sustained knockdown of a pluripotent marker gene followed by differentiation was analysed by reverse transcriptase-PCR, fluoresence-activated cell sorting and immunofluorescence techniques. Impact on cell viability as a result of repeated exposure of the R8HNP was also tested. RESULTS: Amongst the protocols tested, the most efficient knockdown of the target gene for a prolonged period of time was obtained by repeated suspension delivery of the R8HNP-siRNA conjugate. The long-term silencing of a pluripotency marker gene resulted in differentiation of R1 ESCs predominantly towards the extra embryonic and ectodermal lineages. Cells displayed excellent tolerance to repeated exposures of R8HNPs. CONCLUSIONS: The results demonstrate that R8HNPs are promising, biocompatible, non-viral alternatives for prolonged gene silencing and obtaining differentiated cells for therapeutics.

Induction of male-like mandibles in XX individuals of a stag beetle by gene knockdown of a feminizer gene transformer.

Males and females share most of the genome, but many animals show different phenotypes between the sexes, known as sexual dimorphism. Many insect species show extreme sexual dimorphism, including beetles with "weapon traits" represented by extremely developed horns and mandibles. Existing studies of sex-specific development of beetle weapon traits suggest that sex-specific gene expression plays an important role. On the other hand, contributions of the Y-chromosome, which may potentially carry genes necessary for male development, to weapon trait expression have not been examined. In holometabolous insects, including beetles, the feminizing gene transformer (tra) is roughly conserved in its feminizing function. Only females express a functional isoform of Tra, which causes female differentiation. Knocking down tra in females leads to male tissue differentiation, enabling us to analyze male phenotypes in individuals lacking a Y-chromosome (XX-males). In this study, we investigate whether the Y-chromosome is necessary for stag beetles to express male-specific weapon traits by comparing tra-knockdown-induced XX-males with natural XY males. We show that XX-males could express weapons (enlarged mandibles) as in XY-males. These results suggest that the Y-chromosome does not have a major role in weapon trait expression in this species.

Injectable hydrogel encapsulating siMMP13 with anti-ROS and anti-apoptotic functions for osteoarthritis treatment.

BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease characterized by the progressive degeneration of articular cartilage, leading to pain, stiffness, and loss of joint function. The pathogenesis of OA involves multiple factors, including increased intracellular reactive oxygen species (ROS), enhanced chondrocyte apoptosis, and disturbances in cartilage matrix metabolism. These processes contribute to the breakdown of the extracellular matrix (ECM) and the loss of cartilage integrity, ultimately resulting in joint damage and dysfunction. RNA interference (RNAi) therapy has emerged as a promising approach for the treatment of various diseases, including hATTR and acute hepatic porphyria. By harnessing the natural cellular machinery for gene silencing, RNAi allows for the specific inhibition of target genes involved in disease pathogenesis. In the context of OA, targeting key molecules such as matrix metalloproteinase-13 (MMP13), which plays a critical role in cartilage degradation, holds great therapeutic potential. RESULTS: In this study, we developed an innovative therapeutic approach for OA using a combination of liposome-encapsulated siMMP13 and NG-Monomethyl-L-arginine Acetate (L-NMMA) to form an injectable hydrogel. The hydrogel served as a delivery vehicle for the siMMP13, allowing for sustained release and targeted delivery to the affected joint. Experiments conducted on destabilization of the medial meniscus (DMM) model mice demonstrated the therapeutic efficacy of this composite hydrogel. Treatment with the hydrogel significantly inhibited the degradation of cartilage matrix, as evidenced by histological analysis showing preserved cartilage structure and reduced loss of proteoglycans. Moreover, the hydrogel effectively suppressed intracellular ROS accumulation in chondrocytes, indicating its anti-oxidative properties. Furthermore, it attenuated chondrocyte apoptosis, as demonstrated by decreased levels of apoptotic markers. CONCLUSION: In summary, the injectable hydrogel containing siMMP13, endowed with anti-ROS and anti-apoptotic properties, may represent an effective therapeutic strategy for osteoarthritis in the future.

Expression profile and function analysis of MsCSP17 and MsCSP18 in the larval development of Mythimna separata.

Chemosensory proteins (CSPs) were necessary for insect sensory system to perform important processes such as feeding, mating, spawning, and avoiding natural enemies. However, their functions in non-olfactory organs have been poorly studied. To clarify the function of CSPs in the development of Mythimna separata (Walker) larvae, two CSP genes, MsCSP17 and MsCSP18, were identified from larval integument transcriptome dataset. Both of MsCSP17 and MsCSP18 contained four conserved cysteine sites (C × (6)-C × (18)-C × (2)-C), with a signal peptide at the N-terminal. RT-qPCR analysis showed that MsCSP17 and MsCSP18 have different expression patterns among different developmental stages and tissues. MsCSP17 was highly expressed in 1st-4th instar larvae, and MsCSP18 had high expression in adults. Both genes were expressed highly in larval head, thorax, integument and mandible. Moreover, both of MsCSP17 and MsCSP18 were lowly expressed in larval integuments when larvae molted for 6 h and 9 h from 3rd to 4th instar, but highly at the beginning and end phase during molting. After injection of dsMsCSP17 and dsMsCSP18, the expression levels of two genes decreased significantly, with the body weight of larvae decreased, the mortality increased, and the eclosion rate decreased. It was suggested that MsCSP17 and MsCSP18 contributed to the development of M. separata larvae.

Ovarian transcriptome and metabolic responses of RNAi-mediated farnesyl pyrophosphate synthase knockdown in Neocaridina denticulata sinensis.

Methyl farnesoate (MF) is considered the equivalent of JH in crustaceans and plays an essential role in many crucial physiological processes. It is believed that farnesyl pyrophosphate synthase (FPPS) plays an essential role in the biosynthesis of mevalonate, which is a branch of the JH/MF pathway. The full-length cDNA of FPPS (NdFPPS) from Neocaridina denticulata sinensis was isolated and characterized, and the deduced amino acid of NdFPPS contained a polyprenyl_synt domain. In addition to its ubiquitous tissue expression, NdFPPS was significantly expressed in the ovary. In vivo gene silencing with dsRNA interference was performed to further investigate the function of NdFPPS. An ovarian transcriptomic analysis of dsNdFPPS experimental and control groups was used to compare, annotate, and classify differentially expressed genes (DEGs). A total of 9230 DEGs were identified in the experimental and control groups based on FPKM values, of which 5082 were up-regulated genes and 4148 were down-regulated genes. 761 GO terms and 102 KEGG pathways were enriched for the DEGs. Our results suggest that NdFPPS might play an important role in ovarian development, however, further functional study is needed to elucidate physiological role of NdFPPS in reproduction.

Assessing the developing pharmacotherapeutic landscape in hepatitis B treatment: a spotlight on drugs in phase II clinical trials.

INTRODUCTION: Functional cure, defined as sustained HBsAg seroclearance, is associated with favorable outcomes in chronic hepatitis B (CHB). While nucleos(t)ide analogues (NAs) are effective in suppressing HBV replication, NAs are unable to induce functional cure at high rates. A range of novel HBV antivirals, aiming to induce functional cure, are currently under development. AREAS COVERED: This article covered novel hepatitis B virus (HBV) antivirals that have entered phase II trials. Virus-directing agents covered include entry inhibitors, transcription inhibitors, RNA silencers, core protein allosteric modulators, noncompetitive polymerase inhibitors, and viral protein export inhibitors. Immunomodulators covered include innate immune stimulators, T-cell modulators, therapeutic vaccines, and monoclonal antibodies. Upcoming developmental directions would also be discussed. EXPERT OPINION: Among novel HBV antivirals, RNA silencers, viral protein export inhibitors (with pegylated interferon), and entry inhibitors (with pegylated interferon) appear to be effective in suppressing HBsAg and may even induce functional cure. The other virus-targeting agents have variable effects on HBV DNA, HBsAg, HBeAg, and HBcrAg. Immunomodulators have modest effects on HBsAg but may have important roles in combination therapy. Upcoming trials will answer important questions on ideal dosing, long-term drug effects, and efficacy of combination regimens.

Efficient delivery of PKN3 shRNA for the treatment of breast cancer via lipid nanoparticles.

Protein kinase N3 (PKN3), an AGC-family member, is often overexpressed in breast tumor cells. RNAi therapy is a promising approach to inhibit tumor growth by reducing the expression of PKN3. In this report, lipid nanoparticles encapsulated with new shRNA PKN3 (SS-LNP/shPKN3) with redox-responsiveness were developed in order to specifically down-regulate the expression of PKN3 for breast cancer treatment. The SS-LNP/shPKN3 was prepared by microfluidic method using disulfide bonds based ionizable lipid as main component. The as-prepared SS-LNP/shPKN3 lipid nanoparticles were characterized via using dynamic light scattering (DLS) and transmission electron microscopy (TEM). The results indicated that the obtained SS-LNP/shPKN3 exhibited uniform particle size and regular spherical morphology. Moreover, glutathione (GSH) triggered release of shPKN3 confirmed the redox-responsiveness of the SS-LNP/shPKN3. Finally, the anti-tumor effect of SS-LNP/shPKN3 was evaluated against MDA-MB-231 cells and derived xenograft tumor bearing mice. It was found that the SS-LNP/shPKN3-2 had the highest PKN3 protein inhibition rate of 60.8% and tumor inhibition rate of 62.3%. Taken together, the SS-LNP/shPKN3 might be a potential therapeutic strategy for breast cancer.

Liver-directed drugs for transthyretin-mediated amyloidosis.

Transthyretin-mediated amyloidosis (ATTR) is a rare, under-recognized, progressively debilitating, fatal disease caused by the aggregation and extracellular deposition of amyloid transthyretin (TTR) fibrils in multiple organs and tissues throughout the body. TTR is predominantly synthesized by the liver and normally circulates as a homotetramer, while misfolded monomers aggregate to form amyloid fibrils. One strategy to treat ATTR amyloidosis is to reduce the amount of TTR produced by the liver using drugs that directly target the TTR mRNA or gene. This narrative review focuses on how TTR gene silencing tools act to reduce TTR production, describing strategies for improved targeted delivery of these agents to hepatocytes where TTR is preferentially expressed. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), termed RNA silencers, cause selective degradation of TTR mRNA, while a TTR gene editing tool reduces TTR expression by introducing nonsense mutations into the TTR gene. Two strategies to facilitate tissue-specific delivery of these nucleic acid-based drugs employ endogenous receptors expressed by hepatocytes. Lipid nanoparticles (LNPs) that recruit apolipoprotein E support low-density lipoprotein receptor-mediated uptake of unconjugated siRNA and are now used for CRISPR gene editing tools. Additionally, conjugating N-acetylgalactosamine (GalNAc) moieties to ASOs or siRNAs facilitates receptor-mediated uptake by the asialoglycoprotein receptor. In summary, ATTR is a progressive disease with various clinical manifestations due to TTR aggregation, deposition, and amyloid formation. Receptor-targeted ligands (eg, GalNAc) and nanoparticle encapsulation (eg, LNPs) are technologies to deliver ASOs, siRNAs, and gene editing tools to hepatocytes, the primary location of TTR synthesis.

Stealth oxime ether lipid vesicles promote delivery of functional DsiRNA in human lung cancer A549 tumor bearing mouse xenografts.

We previously reported that hydroxylated oxime ether lipids (OELs) efficiently deliver functional Dicer substrate siRNAs (DsiRNAs) in cells. Here, we explored in vivo utility of these OELs, using OEL4 as a prototype and report that surface modification of the OEL4 formulations was essential for their in vivo applications. These surface-modified OEL4 formulations were developed by inclusion of various PEGylated lipids. The vesicle stability and gene knock-down were dependent on the PEG chain length. OEL4 containing DSPE-PEG350 and DSPE-PEG1000 (surprisingly not DSPE2000) promoted gene silencing in cells. In vivo studies demonstrated that OEL4 vesicles formulated using 3 mol% DSPE-PEG350 accumulate in human lung cancer (A549-luc2) xenografts in mice and exhibit a significant increase in tumor to liver ratios. These vesicles also showed a statistically significant reduction of luciferase signal in tumors compared to untreated mice. Taken together, the scalable OEL4:DSPE-PEG350 formulation serves as a novel candidate for delivery of RNAi therapeutics.

Intercalation-Driven Formation of siRNA Nanogels for Cancer Therapy.

RNA interference (RNAi) is a powerful approach in the treatment of various diseases including cancers. The clinical translation of small interfering RNA (siRNA)-based therapy requires safe and efficient delivery vehicles. Here, we report a siRNA nanogels (NG)-based delivery vehicle, which is driven directly by the intercalation between nucleic acid bis-intercalator and siRNA molecules. The intercalation-based siRNA NG exhibits good physiological stability and can enter cells efficiently via different endocytosis pathways. Furthermore, the siRNA NG can not only silence the target genes in vitro but also significantly inhibit the tumor growth in vivo. Therefore, this study provides an intercalation-based strategy for the development of a siRNA delivery platform for cancer therapy. To the best of our knowledge, this is the first report of the intercalation-driven siRNA NG.

Molecular Mechanisms of Phenylpropane-Synthesis-Related Genes Regulating the Shoot Blight Resistance of Bambusa pervariabilis × Dendrocalamopsis grandis.

Bambusa pervariabilis × Dendrocalamopsis grandis shoot blight caused by Arthrinium phaeospermum is a fungal disease that has affected a large area in China in recent years. However, it is not clear which genes are responsible for the disease resistance of B. pervariabilis × D. grandis. Based on the analysis of transcriptome and proteome data, two genes, CCoAOMT2 and CAD5, which may be involved in disease resistance, were screened. Two gene expression-interfering varieties, COF RNAi and CAD RNAi were successfully obtained using RNAi technology. Quantitative real-time fluorescence (qRT-PCR) results showed that CCoAOMT2 gene, CAD5 gene and seven related genes expression was down-regulated in the transformed varieties. After inoculating pathogen spore suspension, the incidence and disease index of cof-RNAi and cad-RNAi transformed plants increased significantly. At the same time, it was found that the content of total lignin and flavonoids in the two transformed varieties were significantly lower than that of the wild-type. The subcellular localization results showed that both CCoAOMT2 and CAD5 were localized in the nucleus and cytoplasm. The above results confirm that the CCoAOMT2 and CAD5 genes are involved in the resistance of B. pervariabilis × D.grandis to shoot blight through regulating the synthesis of lignin and flavonoids.

Self-assembled fluorescent hybrid nanoparticles-mediated collaborative lncRNA CCAT1 silencing and curcumin delivery for synchronous colorectal cancer theranostics.

BACKGROUND: Cancer synergistic therapy strategy in combination with therapeutic gene and small molecule drug offers the possibility to amplify anticancer efficiency. Colon cancer-associated transcript-1 (CCAT1) is a well identified oncogenic long noncoding RNA (lncRNA) exerting tumorigenic effects in a variety of cancers including colorectal cancer (CRC). RESULTS: In the present work, curcumin (Cur) and small interfering RNA targeting lncRNA CCAT1(siCCAT1) were co-incorporated into polymeric hybrid nanoparticles (CSNP), which was constructed by self-assembling method with two amphiphilic copolymers, polyethyleneimine-poly (D, L-lactide) (PEI-PDLLA) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) (DSPE-mPEG). Owing to the multicolor fluorescence characteristics of PEI-PDLLA, the constructed CSNP could be served as a theranostic nanomedicine for synchronous therapy and imaging both in vitro and in vivo. Resultantly, proliferation and migration of HT-29 cells were efficiently inhibited, and the highest apoptosis ratio was induced by CSNP with coordination patterns. Effective knockdown of lncRNA CCAT1 and concurrent regulation of relevant downstream genes could be observed. Furthermore, CSNP triggered conspicuous anti-tumor efficacy in the HT-29 subcutaneous xenografts model with good biosafety and biocompatibility during the treatment. CONCLUSION: On the whole, our studies demonstrated that the collaborative lncRNA CCAT1 silencing and Cur delivery based on CSNP might emerge as a preferable and promising strategy for synergetic anti-CRC therapy.

Local delivery of RhoA siRNA by PgP nanocarrier reduces inflammatory response and improves neuronal cell survival in a rat TBI model.

Traumatic brain injury (TBI) is a leading cause of death and disability with complex pathophysiology including prolonged neuroinflammation, apoptosis, and glial scar formation. The upregulation of RhoA is a key factor in the pathological development of secondary injury following TBI. Previously, we developed a novel cationic, amphiphilic copolymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP), as a nanocarrier for delivery of therapeutic nucleic acids. In a rat compression spinal cord injury model, delivery of siRNA targeting RhoA (siRhoA) by PgP resulted in RhoA knockdown; reduced astrogliosis and inflammation; and promoted axonal regeneration/sparing. Here, we evaluated the effect of RhoA knockdown by PgP/siRhoA nanoplexes in a rat controlled cortical impact TBI model. A single intraparenchymal injection of PgP/siRhoA nanoplexes significantly reduced RhoA expression, lesion volume, neuroinflammation, and apoptosis, and increased neuronal survival in the ipsilateral cortex. These results suggest that PgP/siRhoA nanoplexes can efficiently knockdown RhoA expression in the injured brain and reduce secondary injury.

Tyrosine-modified linear PEIs for highly efficacious and biocompatible siRNA delivery in vitro and in vivo.

Therapeutic gene silencing by RNA interference relies on the safe and efficient in vivo delivery of small interfering RNAs (siRNAs). Polyethylenimines are among the most studied cationic polymers for gene delivery. For several reasons including superior tolerability, small linear PEIs would be preferable over branched PEIs, but they show poor siRNA complexation. Their chemical modification for siRNA formulation has not been extensively explored so far. We generated a set of small linear PEIs bearing tyrosine modifications (LPxY), leading to substantially enhanced siRNA delivery and knockdown efficacy in vitro in various cell lines, including hard-to-transfect cells. The tyrosine-modified linear 10 kDa PEI (LP10Y) is particularly powerful, associated with favorable physicochemical properties and very high biocompatibility. Systemically administered LP10Y/siRNA complexes reveal antitumor effects in mouse xenograft and patient-derived xenograft (PDX) models, and their direct application into the brain achieves therapeutic inhibition of orthotopic glioma xenografts. LP10Y is particularly interesting for therapeutic siRNA delivery.

Silencing of Ac45 Simultaneously Inhibits Osteoclast-Mediated Bone Resorption and Attenuates Dendritic Cell-Mediated Inflammation through Impairing Acidification and Cathepsin K Secretion.

Endodontic disease is characterized by inflammation and destruction of periapical tissues, leading to severe bone resorption and tooth loss. ATP6AP1 (Ac45) has been implicated in human immune diseases, yet the mechanism underlying how Ac45 regulates immune response and reaction in inflammatory diseases remains unknown. We generated endodontic disease mice through bacterial infection as an inflammatory disease model and used adeno-associated virus (AAV)-mediated Ac45 RNA interference knockdown to study the function of Ac45 in periapical inflammation and bone resorption. We demonstrated that the AAV small hairpin RNA targeting Ac45 (AAV-sh-Ac45) impaired cellular acidification, extracellular acidification, and bone resorption. Our results showed that local delivery of AAV-sh-Ac45 in periapical tissues in bacterium-induced inflammatory lesions largely reduced bone destruction, inhibited inflammation, and dramatically reduced mononuclear immune cells. T-cell, macrophage, and dendritic cell infiltration in the periapical lesion was dramatically reduced, and the periodontal ligament was protected from inflammation-induced destruction. Furthermore, AAV-sh-Ac45 significantly reduced osteoclast formation and the expression of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-10 (IL-10), IL-12, IL-1α, IL-6, and IL-17. Interestingly, AAV-sh-Ac45 impaired mature cathepsin K secretion more significantly than that by AAV-sh-C1 and AAV-sh-CtsK Unbiased genome-wide transcriptome sequencing analysis of Ctsk-/- dendritic cells stimulated with lipopolysaccharide demonstrated that the ablation of Ctsk dramatically reduced dendritic cell-mediated inflammatory signaling. Taken together, our results indicated that AAV-sh-Ac45 simultaneously inhibits osteoclast-mediated bone resorption and attenuates dendritic cell-mediated inflammation through impairing acidification and cathepsin K secretion. Thus, Ac45 may be a novel target for therapeutic approaches to attenuate inflammation and bone erosion in endodontic disease and other inflammation-related osteolytic diseases.

Development of RNAi methods to control the harlequin bug, Murgantia histrionica.

The harlequin bug (HB), Murgantia histrionica, is a major pest of cabbage family plants throughout its range in the United States. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism that is showing promise as a biopesticide due to the ability to target species-specific genes necessary for growth and/or survival with synthetic double-stranded RNA (dsRNA). In the present study, dsRNA stability assays revealed that nucleases present in the saliva of harlequin bugs did not rapidly degrade dsRNA. We tracked the movement and localization of radioactively labeled dsRNA in both mustard plant seedlings and harlequin bug nymphs that fed on treated host plants. Movement of 32 P-labeled-dsRNA from soil to plant and plant to insect was detected. The efficacy of RNAi in inducing mortality in harlequin bug adults and nymphs injected or fed with dsRNA targeting inhibitor of apoptosis (IAP), ATPase N2B (ATPase), serine/threonine-protein phosphatase PP1-ß catalytic subunit (PP1), signal recognition particle 54 kDa protein (SRP), and G protein-coupled receptor 161-like (GPCR) genes was evaluated. Injection of dsRNA targeting candidate genes into adults caused between 40% and 75% mortality and induced significant knockdown of target gene expression. Feeding dsRNA targeting the IAP gene to nymphs by plant-mediated and droplet feeding methods induced knockdown of the target gene and caused 40-55% mortality. These findings suggest that RNAi may be a viable approach for managing this pest.

Lysosomal Storage Disease-Associated Neuropathy: Targeting Stable Nucleic Acid Lipid Particle (SNALP)-Formulated siRNAs to the Brain as a Therapeutic Approach.

More than two thirds of Lysosomal Storage Diseases (LSDs) present central nervous system involvement. Nevertheless, only one of the currently approved therapies has an impact on neuropathology. Therefore, alternative approaches are under development, either addressing the underlying enzymatic defect or its downstream consequences. Also under study is the possibility to block substrate accumulation upstream, by promoting a decrease of its synthesis. This concept is known as substrate reduction therapy and may be triggered by several molecules, such as small interfering RNAs (siRNAs). siRNAs promote RNA interference, a naturally occurring sequence-specific post-transcriptional gene-silencing mechanism, and may target virtually any gene of interest, inhibiting its expression. Still, naked siRNAs have limited cellular uptake, low biological stability, and unfavorable pharmacokinetics. Thus, their translation into clinics requires proper delivery methods. One promising platform is a special class of liposomes called stable nucleic acid lipid particles (SNALPs), which are characterized by high cargo encapsulation efficiency and may be engineered to promote targeted delivery to specific receptors. Here, we review the concept of SNALPs, presenting a series of examples on their efficacy as siRNA nanodelivery systems. By doing so, we hope to unveil the therapeutic potential of these nanosystems for targeted brain delivery of siRNAs in LSDs.

Elucidation of the role of farnesoic acid O-methyltransferase (FAMeT) in the giant freshwater prawn, Macrobrachium rosenbergii: Possible functional correlation with ecdysteroid signaling.

Farnesoic acid O-methyltransferase (FAMeT) is a key enzyme involved in catalyzing the conversion of farnesoic acid (FA) to methylfarnesoate (MF) in the mandibular organ (MO) of crustaceans. In this study, a full-length cDNA encoding a 278-amino-acid FAMeT protein (MrFAMeT) was characterized from the giant freshwater prawn, Macrobrachium rosenbergii. Bioinformatics analysis revealed a high degree of conservation of FAMeT among crustaceans and a close phylogenetic relationship between MrFAMeT and that of Scylla paramamosain. The prokaryotic expressed MrFAMeT could catalyze the conversion of FA to MF in a radiochemical assay. Expression analysis by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that MrFAMeT mRNA was highly expressed in the muscle and the hepatopancreas of both females and males. During the molt cycle and the ovarian development, the mRNA expressions of MrFAMeT displayed stage-specific patterns in the muscle of both sexes and the female hepatopancreas, and the highest expressions were detected at intermolt and ovarian development stage V. Double stranded RNA (dsRNA)-mediated RNA interference (RNAi) of MrFAMeT increased expressions of myostatin in the muscle of both sexes and reduced expressions of vitellogenin (Vg) in the female hepatopancreas. Furthermore, both in the muscle and the hepatopancreas, silence of MrFAMeT downregulated the expression of ecydone receptor gene (MrEcR) and silence of MrEcR decreased the expression of MrFAMeT as well. Results in our study indicate that MrFAMeT is involved in prawn muscle growth and female vitellogenin biosynthesis and its function may be closely related with the ecdysteroid signaling.