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At least 73 records · Page 4Linked to original sources
PubMed2006

Intraperitoneal delivery of liposomal siRNA for therapy of advanced ovarian cancer.

PURPOSE: Intravenous (IV) delivery of siRNA incorporated into neutral liposomes allows efficient delivery to tumor tissue, and has therapeutic efficacy in preclinical proof-of-concept studies using EphA2-targeting siRNA. We sought to determine whether intraperitoneal (IP) delivery of these siRNA complexes was as effective at delivery and therapy as IV delivery. EXPERIMENTAL DESIGN: SiRNA was incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Alexa555-siRNA-DOPC was injected IP into nude mice bearing established ovarian tumors, and organs were collected for microscopic fluorescent examination. Subsequently, therapeutic efficacy of the IP versus IV routes was directly compared. RESULTS: Alexa555-siRNA in DOPC liposomes injected IP was diffusely distributed into intraperitoneal ovarian tumors. Delivery was also seen deeply into the liver and kidney parenchyma, suggesting that the predominant means of distribution was through the vasculature, rather than direct diffusion from the peritoneal cavity. In mice with orthotopic ovarian tumors, treatment with combined paclitaxel and IP EphA2-targeting siRNA-DOPC reduced tumor growth by 48-81% compared to paclitaxel/control siRNA-DOPC IP (HeyA8: 0.34 g v 0.66 g; SKOV3ip1: 0.04 v 0.21, p<0.01). This reduction was comparable to concurrently-treated mice with paclitaxel and EphA2 siRNA-DOPC injected IV, which showed a reduction in growth by 45-69% compared to paclitaxel/control siRNA-DOPC injected IV (HeyA8: 0.23g v. 0.42g; SKOV3ip1: 0.04 v. 0.13 g). CONCLUSIONS: IP injection of siRNA incorporated in DOPC allows intra-tumoral delivery and has therapeutic efficacy in orthotopic ovarian tumors. These findings may have therapeutic implications for siRNA-based strategies.

Animals↗
PubMed2006

Suppression of lung cancer with siRNA targeting PTTG.

Pituitary transforming gene (PTTG) is frequently expressed at high levels in malignant tumors. We report high levels of expression of PTTG in various lung tumors and tumor-derived cell lines. For a better understanding of its role in maintaining the cancer phenotype, we used RNA interference (RNAi) directed against PTTG. Transfection of H1299 cells with PTTG siRNA duplex (5'-UGG GAG AUC UCA AGU UUC A-3') to a final concentration of 100 nmol/l resulted in almost complete depletion of PTTG mRNA within 24 and 48 h when compared to expression in untransfected cells or cells transfected with control siRNA. Western blot analysis showed nearly a 60% reduction in PTTG protein within 48 h of transfection. In phenotype analysis, we investigated the effect of PTTG siRNA on colony formation on soft agar, and tumor development in nude mice. Transfection of H1299 cells with PTTG siRNA duplex significantly reduced colony formation compared to untransfected cells or cells transfected with control siRNA. Mice injected with H1299 cells transfected with PTTG siRNA followed by injection of siRNA developed no tumors within two weeks of injection, but developed small tumors (67.85+/-45.87 mg) within 4 weeks of injection, whereas untransfected cells, or cells transfected with control siRNA developed large tumors (232.12+/-102.78 and 231.57+/-83.76 mg respectively). Suppression of PTTG as well as Ki67, bFGF and CD34 was observed in H1299 tumors treated with PTTG siRNA compared to untreated and control-treated siRNA in nude mice. In conclusion, decreasing PTTG expression through PTTG siRNA inhibits tumor growth both in vitro and in vivo. Future studies are needed to test whether PTTG expression can be efficiently depleted by siRNA expressed from a DNA-based expression vector combined with a specific-promoter, such that RNAi can specifically target PTTG in cancer cells without affecting normal cells.

Animals↗
PubMed2006

Inhibition of RNAse A family enzymes prevents degradation and loss of silencing activity of siRNAs in serum.

Small interfering RNAs (siRNA), RNA duplexes of approximately 21 nucleotides, offer a promising approach to specifically degrade RNAs in target cells by a process termed RNA interference. Insufficient in vivo-stability is a major problem of a systemic application of siRNAs in humans. The present study demonstrated that RNAse A-like RNAses degraded siRNAs in serum. The susceptibility of siRNAs towards degradation in serum was strongly enhanced by local clustering of A/Us within the siRNA sequence, i.e. regions showing low thermal stability, most notably at the ends of the molecule, and by 3'-overhanging bases. Importantly, inhibition of RNAse A family enzymes prevented the degradation and loss of silencing activity of siRNAs in serum. Furthermore, the degradation of siRNAs was considerably faster in human than in mouse serum, suggesting that the degradation of siRNAs by RNAse A family enzymes might be a more challenging problem in a future therapeutic application of siRNAs in humans than in mouse models. Together, the present study indicates that siRNAs are degraded by RNAse A family enzymes in serum and that the kinetics of their degradation in serum depends on their sequence. These findings might be of great importance for a possible future human therapeutic application of siRNAs.

Base Sequence↗
PubMed2007

Batch RNAi selector: a standalone program to predict specific siRNA candidates in batches with enhanced sensitivity.

RNA interference (RNAi) is a popular and effective method for silencing gene expression. siRNAs should be gene-specific and effective to achieve specific and potent gene silencing. However, most currently available siRNA design programs are web-based programs that either require each sequence be submitted individually, making large-batch analyses difficult to conduct, or only provide limited options for searching off-target candidates (e.g. NCBI-BLAST). We have developed a stand-alone, enhanced RNAi design program that overcomes these shortcomings. We have implemented WU-BLAST, FASTA and SSEARCH homology searches for siRNA candidates to improve gene specific siRNA selection and to identity siRNA candidates that could lead to off-target gene silencing. We also included many new features such as siRNA score calculation and calculation of siRNA internal stability to help select highly potent siRNAs. This program is freely available for academic and commercial use (), and can be installed and run on any Linux machine. Our program automates the search for siRNAs and the resulting data files including a list of siRNA primers with scores and database search results for each siRNA candidate are stored locally for easy retrieval and inspection when needed.

Gene Expression↗
PubMed2005

SiRNA strategy against overexpression of extracellular matrix in diabetic retinopathy.

Increased synthesis of extracellular matrix (ECM) contributes to the development of vascular BM thickening, a prominent abnormality in diabetic retinopathy. RNA interference (RNAi) approach was used in this study to examine the effect of small interfering RNAs (siRNAs) for their ability to inhibit ECM-specific gene overexpression under high glucose condition in rat microvascular endothelial cells (RMECs). Four fibronectin (FN)-siRNAs, three collagen IV (Coll IV)-siRNAs, and four laminin (LM)-siRNAs, a total of 11 siRNAs were screened. RMECs were transfected with 10, 30, or 100 nm of each siRNAs in the presence of 8 microm lipofectin and subjected to analysis 72 hr after transfection. In long-term studies siRNA-transfected cells were examined after 12 days. Two FN siRNAs, two Coll IV siRNAs, and two LM siRNAs significantly reduced the respective target expressions. Findings from this study indicate that high glucose-induced abnormal expression of BM components may contribute to increased vascular permeability. SiRNA may be a useful tool in preventing excess vascular permeability, a characteristic feature of early diabetic retinopathy.

Animals↗
PubMed2006

siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene.

Development of RNA interference (RNAi) technology utilizing the short interfering RNA sequences (siRNA) based 'targeted' therapeutics has focused on creating methods for delivering siRNAs to cells and for enhancing siRNA stability in vitro and in vivo. Here, we describe a novel approach for siRNA cellular delivery using siRNA encapsulated into liposomes additionally bearing arginine octamer (R8) molecules attached to their outer surface (R8-liposomes). The R8-liposomal human double minute gene 2 (HDM2)-siRNA demonstrated a significant stability against degradation in the blood serum (siRNA-loaded R8-liposomes remained intact even after 24-h incubation), and higher transfection efficiency into all three tested lung tumor cell lines. siRNA delivery successfully proceeds in the presence of plasma proteins, and R8-liposomes demonstrate low non-specific toxicity. The mechanism of action of R8-liposome-encapsulated siRNA is associated with the RNAi-mediated degradation of the target mRNA. siRNA in R8-liposomes effectively inhibited the targeted gene and significantly reduced the proliferation of cancer cells. The approach offers the potential for siRNA delivery for various in vitro and in vivo applications.

Cell Line, Tumor↗
PubMed2006

PEG conjugated VEGF siRNA for anti-angiogenic gene therapy.

A novel siRNA delivery system based on polyelectrolyte complex (PEC) micelles was introduced in this study. Vascular endothelial growth factor (VEGF) siRNA was conjugated to poly(ethylene glycol) (PEG) via a disulfide linkage (siRNA-PEG). The siRNA-PEG conjugate could form PEC micelles by interacting with cationic polyethylenimine (PEI) as a core forming agent. The VEGF siRNA-PEG/PEI PEC micelles showed greater stability than naked VEGF siRNA against enzymatic degradation. Under a reductive condition similar to cytosolic environment, an intact form of siRNA was released from the siRNA-PEG conjugate by cleavage of the disulfide linkage. The VEGF siRNA-PEG/PEI PEC micelles effectively silenced VEGF gene expression in prostate carcinoma cells (PC-3) up to 96.5% under an optimized formulation condition. They also showed a far superior VEGF gene silencing effect than VEGF siRNA/PEI complexes even in the presence of serum. This study suggests that the siRNA delivery system using VEGF siRNA-PEG/PEI PEC micelles could be potentially applied to RNAi-based anti-angiogenic treatment of cancer in vivo.

Blood Proteins↗
PubMed2004

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus.

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects.

Adenosine Triphosphatases↗
PubMed2005

Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.

Targeted silencing of disease-associated genes by synthetic short interfering RNA (siRNA) holds considerable promise as a novel therapeutic strategy. However, unmodified siRNA can be potent triggers of the innate immune response, particularly when associated with delivery vehicles that facilitate intracellular uptake. This represents a significant barrier to the therapeutic development of siRNA due to toxicity and off-target gene effects associated with this inflammatory response. Here we show that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2'-O-methyl (2'OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex. These noninflammatory siRNA, containing less than 20% modified nucleotides, can be readily generated without disrupting their gene-silencing activity. We show that, coupled with an effective systemic delivery vehicle, 2'OMe-modified siRNA targeting apolipoprotein B (apoB) can mediate potent silencing of its target mRNA, causing significant decreases in serum apoB and cholesterol. This is achieved at therapeutically viable siRNA doses without cytokine induction, toxicity, or off-target effects associated with the use of unmodified siRNA. This approach to siRNA design and delivery should prove widely applicable and represents an important step in advancing synthetic siRNA into a broad range of therapeutic areas.

Animals↗
PubMed2003

Simultaneous inhibition of GSK3alpha and GSK3beta using hairpin siRNA expression vectors.

Short interfering RNAs (siRNAs) can mediate sequence-specific inhibition of gene expression in mammalian cells. We and others have recently developed expression vector-based systems for synthesizing siRNAs or hairpin siRNAs in mammalian cells. Expression vector-based RNA interference (RNAi) effectively suppresses expression of target genes and is likely to be a powerful tool for analysis of gene function. Here we compare inhibition by vectors expressing hairpin siRNA designs either with different loop sequences connecting the two siRNA strands, or with duplex regions of different lengths. Our results suggest that lengthening the 19-nucleotide duplex region of a relatively ineffective hairpin siRNA can increase inhibition, but increasing the length of an effective 19-nt hairpin siRNA does not increase inhibition. We also demonstrate that hairpin siRNA vectors can be used to inhibit two target genes simultaneously. We have targeted glycogen synthase kinase-3alpha (GSK-3alpha) and GSK-3beta, two related kinases involved in the regulation of a variety of cellular processes and also implicated in the pathogenesis of several human diseases. Inhibition of either GSK-3alpha or GSK-3beta by transfection of hairpin siRNA vectors leads to elevated expression of the GSK-3 target beta-catenin, whereas inhibition of both kinases further increases beta-catenin expression. Our results suggest that vector-based siRNA inhibition may be useful for dissecting the functional roles of GSK-3alpha and GSK-3beta in somatic cells. The ability to inhibit two or more genes simultaneously with hairpin siRNA expression vectors should facilitate studies of gene function in mammalian cells.

Animals↗
PubMed2005

RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo.

RNA interference (RNAi) represents a powerful, naturally occurring biological strategy for inhibition of gene expression. It is mediated through small interfering RNAs (siRNAs), which trigger specific mRNA degradation. In mammalian systems, however, the application of siRNAs is severely limited by the instability and poor delivery of unmodified siRNA molecules into the cells in vivo. In this study, we show that the noncovalent complexation of synthetic siRNAs with low molecular weight polyethylenimine (PEI) efficiently stabilizes siRNAs and delivers siRNAs into cells where they display full bioactivity at completely nontoxic concentrations. More importantly, in a subcutaneous mouse tumor model, the systemic (intraperitoneal, i.p.) administration of complexed, but not of naked siRNAs, leads to the delivery of the intact siRNAs into the tumors. The i.p. injection of PEI-complexed, but not of naked siRNAs targeting the c-erbB2/neu (HER-2) receptor results in a marked reduction of tumor growth through siRNA-mediated HER-2 downregulation. Hence, we establish a novel and simple system for the systemic in vivo application of siRNAs through PEI complexation as a powerful tool for future therapeutic use.

Animals↗
PubMed2005

A bi-functional siRNA construct induces RNA interference and also primes PCR amplification for its own quantification.

RNA interference (RNAi) is a process of post-transcriptional gene silencing initiated by double-stranded RNAs, including short interfering RNA (siRNA). Silencing is sequence-specific and RNAi has rapidly become central to the study of gene function. RNAi also carries promise for selective silencing of viral and endogenous genes causal for disease. To detect the very low levels of siRNA effective for RNAi we modified the 3' end of the sense strand of siRNA with a nuclease-resistant DNA hairpin. We show that the modified siRNA-DNA construct (termed 'crook' siRNA) functions as a primer for the PCR and describe a novel, yet simple PCR protocol for its quantification (amolar levels/cell). When transfected into mammalian cells, crook siRNA induces selective mRNA knock-down equivalent to its unmodified siRNA counterpart. This new bifunctional siRNA construct will enable future in vivo studies on the uptake, distribution and pharmacokinetics of siRNA, and is particularly important for the development of siRNA-based therapeutics. More generally, PCR-based detection of siRNA carries wide-ranging applications for RNAi reverse genetics.

Cell Line↗
PubMed2006

siRNA-resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations.

RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short-term cell culture. Our aim was to determine whether long-term RNAi might result in HCV-resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV-RNA was quantified by real-time RT-PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV-siRNA resulted in a 50% inhibition of HCV-RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV-RNA returned to 85% of pretreatment levels after cessation of HCV-siRNA treatment. Sequencing of the HCV-siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV-siRNA-treated cells during and after treatment had mutations. There were no mutations in the HCV-siRNA target region following control HBV-siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV-siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV-siRNA, HCV-RNA and HCV-NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.

Base Sequence↗
PubMed2006

RETRACTED: Secondary siRNAs result from unprimed RNA synthesis and form a distinct class.

In Caenorhabditis elegans, an effective RNA interference (RNAi) response requires the production of secondary short interfering RNAs (siRNAs) by RNA-directed RNA polymerases (RdRPs). We cloned secondary siRNAs from transgenic C. elegans lines expressing a single 22-nucleotide primary siRNA. Several secondary siRNAs start a few nucleotides downstream of the primary siRNA, indicating that non-RISC (RNA-induced silencing complex)-cleaved mRNAs are substrates for secondary siRNA production. In lines expressing primary siRNAs with single-nucleotide mismatches, secondary siRNAs do not carry the mismatch but contain the nucleotide complementary to the mRNA. We infer that RdRPs perform unprimed RNA synthesis. Secondary siRNAs are only of antisense polarity, carry 5' di- or triphosphates, and are only in the minority associated with RDE-1, the RNAi-specific Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. Their biogenesis depends on RdRPs, and we propose that each secondary siRNA is an individual RdRP product.

Animals↗
PubMed2006

p27kip1 siRNA induces proliferation in corneal endothelial cells from young but not older donors.

PURPOSE: To determine whether small interfering (si)RNA downregulation of the cyclin-dependent kinase inhibitor p27kip1 overcomes G(1)-phase arrest and promotes cell-cycle progression in human corneal endothelial cells (HCECs) from young (<30 years old) and older (>60 years old) donors. METHODS: Transfection of siRNA was confirmed by incubating confluent cultures of HCECs with FITC-labeled nonsilencing siRNA. Confluent cultures were transfected for 48 hours with p27kip1 siRNA (2.5, 5, 25, or 100 nM) or nonsilencing siRNA, with a lipid transfection reagent. As a comparison, cultures were also transfected for 48 hours with p27kip1 antisense (AS) or missense (MS) oligonucleotides (oligo). At various times after transfection, cells were fixed for immunocytochemical localization of p27kip1 or extracted for Western blot analysis to assess relative p27kip1 protein levels. Cultures were also prepared for ZO-1 immunolocalization, to assess the effect of transfection on the morphology of the monolayer. The number of cells was counted at 0, 48, 96, 144, and 192 hours after incubation, and a cell-viability assay was performed. RESULTS: A dose-dependent decrease in p27kip1 protein level was observed in Western blot analysis, and nuclear staining for p27kip1 was greatly reduced in HCECs incubated with p27kip1 siRNA. No change in p27kip1 levels or in nuclear staining was observed in the nonsilencing control. p27kip1 siRNA (25 nM) appeared to be quantitatively more efficient than antisense oligonucleotide (500 nM) in reducing p27kip1 protein levels. Viability was less affected by siRNA treatment than by AS oligo transfection. ZO-1 staining showed no effect on morphology of the monolayer. The number of HCECs from young donors (<30 years old) transfected with p27kip1 siRNA increased up to 144 hours after incubation, whereas no change in the number of cells was observed in HCECs transfected with nonsilencing siRNA. In contrast to the results from young donors, no change in the number of cells was observed at any time point tested in HCECs from older donors (>60 years old) after p27kip1 siRNA transfection. CONCLUSIONS: Transfection of p27kip1 siRNA was sufficient to promote proliferation in confluent cultures of HCECs from younger, but not older donors. These results suggest that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27kip1.

Adult↗
PubMed2006

Integrated siRNA design based on surveying of features associated with high RNAi effectiveness.

BACKGROUND: Short interfering RNAs have allowed the development of clean and easily regulated methods for disruption of gene expression. However, while these methods continue to grow in popularity, designing effective siRNA experiments can be challenging. The various existing siRNA design guidelines suffer from two problems: they differ considerably from each other, and they produce high levels of false-positive predictions when tested on data of independent origins. RESULTS: Using a distinctly large set of siRNA efficacy data assembled from a vast diversity of origins (the siRecords data, containing records of 3,277 siRNA experiments targeting 1,518 genes, derived from 1,417 independent studies), we conducted extensive analyses of all known features that have been implicated in increasing RNAi effectiveness. A number of features having positive impacts on siRNA efficacy were identified. By performing quantitative analyses on cooperative effects among these features, then applying a disjunctive rule merging (DRM) algorithm, we developed a bundle of siRNA design rule sets with the false positive problem well curbed. A comparison with 15 online siRNA design tools indicated that some of the rule sets we developed surpassed all of these design tools commonly used in siRNA design practice in positive predictive values (PPVs). CONCLUSION: The availability of the large and diverse siRNA dataset from siRecords and the approach we describe in this report have allowed the development of highly effective and generally applicable siRNA design rule sets. Together with ever improving RNAi lab techniques, these design rule sets are expected to make siRNAs a more useful tool for molecular genetics, functional genomics, and drug discovery studies.

Algorithms↗
PubMed2003

Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model.

PURPOSE: RNA interference mediated by small interfering RNAs (siRNAs) is a powerful technology allowing the silencing of mamalian genes with great specificity and potency. The purpose of this study was to demonstrate the feasibility of RNA interference mediated by siRNA in retinal cells in vitro and in the murine retina in vivo. METHODS: siRNAs specific for enhanced green fluorescent protein (EGFP) and murine and human vascular endothelial growth factor (VEGF) were designed. In vitro studies in human cell lines entailed modulation of endogenous VEGF levels through chemically induced hypoxia. Effects of siRNA treatment on these levels were measured by ELISA. In vivo studies evaluating effects of siRNA on levels of EGFP and VEGF were performed by co-injecting recombinant viruses carrying EGFP or hVEGF cDNAs along with the appropriate siRNAs subretinally in mice. Additional studies aimed at blocking production of endogenous mVEGF were performed using laser-induced choroidal neovascularization (CNV) in mice. Effects of in vivo treatments were evaluated ophthalmoscopically. Retinal/choroidal flat mounts were evaluated after perfusion with dextran-fluorescein. Alternatively, retinas were evaluated in histological sections or VEGF levels were measured in intact eyes using ELISA. RESULTS: Successful delivery of siRNA to the subretinal space was confirmed by observing significantly reduced levels of EGFP in eyes treated with Ad.CMV.EGFP plus EGFP-directed siRNA. siRNAs directed against hVEGF effectively and specifically inhibit hypoxia-induced VEGF levels in human cell lines and after adenoviral induced hVEGF transgene expression in vivo. In addition, subretinal delivery of siRNA directed against murine Vegf significantly inhibited CNV after laser photocoagulation. CONCLUSIONS: Delivery of siRNA can be used in vitro and in vivo to target specific RNAs and to reduce the levels of the specific protein product in the targeted cells. This work suggests that RNA interference has potential for application to studies of retinal biology and for the treatment of a variety of retinal diseases, including those involving abnormal blood vessel growth.

Adenoviruses, Human↗
PubMed2005

[Inhibition of hepatitis B virus by combined siRNAs in vitro].

Small interfering RNA (siRNA) technology is a powerful tool to knockdown gene expression in mammalian cells including genes of viral origin. To study the inhibition of hepatitis B virus (HBV) by combined siRNAs in vitro, HepG2 2.2.15, a human hepatoblastoma cell that constitutively produces HBV particles, was transfected with 12 siRNAs that were designed to target different regions on HBV genome. Enzyme-linked immunosorbent assay was used to measure the HBsAg and HBeAg levels in culture media, and HBV replication was determined by real-time quantitative PCR. The results showed that HBV replication could be inhibited by treatment with these siRNAs in a dose-dependent manner. The expression of HBsAg was inhibited by 80% and 90% with 80 nmol/L single siRNA treatment and 20 nmol/L combined siRNAs treatment, respectively. HBeAg was inhibited by about 60% with single siRNA treatment, and the inhibition didn't increase much in the case of combined siRNAs treatment. HBV replication was inhibited by 90% with 80 nmol/L single siRNA treatment or 20 nmol/L combined siRNAs treatment. These data provide solid evidence for developing new approaches in HBV treatment with combined siRNAs.

Enzyme-Linked Immunosorbent Assay↗