Dicer-substrate siRNAs Research Articles

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.

Rational design for controlled release of Dicer-substrate siRNA harbored in phi29 pRNA-based nanoparticles

Small interfering RNA (siRNA) for silencing genes and treating disease has been a dream since ranking as a top Breakthrough of the Year in 2002 by Science. With the recent FDA approval of four siRNA-based drugs, the potential of RNA therapeutics to become the third milestone in pharmaceutical drug development has become a reality. However, the field of RNA interference (RNAi) therapeutics still faces challenges such as specificity in targeting, intracellular processing, and endosome trapping after targeted delivery. Dicer-substrate siRNAs included onto RNA nanoparticles may be able to overcome these challenges. Here, we show that pRNA-based nanoparticles can be designed to efficiently harbor the Dicer-substrate siRNAs in vitro and in vivo to the cytosol of tumor cells and release the siRNA. The structure optimization and chemical modification for controlled release of Dicer-substrate siRNAs in tumor cells were also evaluated through molecular beacon analysis. Studies on the length requirement of the overhanging siRNA revealed that at least 23 nucleotides at the dweller’s arm were needed for dicer processing. The above sequence parameters and structure optimization were confirmed in recent studies demonstrating the release of functional Survivin siRNA from the pRNA-based nanoparticles for cancer inhibition in non-small-cell lung, breast, and prostate cancer animal models.

Cellular Delivery of siRNAs Using Bolaamphiphiles.

Discovery of RNA interference (RNAi) has opened up a new arena of therapeutic intervention for the treatment of cancerous as well as noncancerous diseases. The RNAi pathway utilizes RNAi inducers such as small interfering RNAs (siRNAs) to target and silence disease causing genes. However, efficient delivery of siRNAs for eliciting efficacious RNAi has remained a daunting challenge. Nonviral vectors such as lipids have shown great promise in delivering siRNAs. Recently, a novel class of cationic lipid molecules "bolaamphiphile lipids" or "bola lipids" has been shown to deliver siRNAs to cause effective gene silencing in cells. The present chapter showcases the ability of bola lipids to form micelles, bind with nucleic acids and protect nucleic acids against nucleases. Also, high in vitro transfection efficiency for silencing green fluorescent protein (GFP) using Dicer substrate siRNAs (dsiRNAs) designed against GFP at nontoxic dose in a human breast cancer model is demonstrated. Our results showed that these cationic bola lipids are promising siRNA delivery agents.

Abstract 3536: Targeting β-catenin with a Dicer-substrate siRNA (DsiRNA) in a sleeping beauty transposon-driven murine hepatoblastoma model

Abstract Hepatoblastoma (HB), the commonest pediatric liver tumor is frequently associated with an N-terminal deletion in the CTNNB1 gene, which encodes stable β-catenin and promotes nuclear localization and transactivation activity. In 80% of patients, nuclear localization of β-catenin and Yes Associated Protein (YAP), a Hippo-related transactivator, is seen together. Further, stable hepatic co-expression of Δ90-CTNNB1 and active-YAP in mice causes rapid and aggressive HB. Dicer-substrate siRNAs (DsiRNAs) are potent RNA interference (RNAi) triggers that that are efficacious in preclinical tumor models of diverse origin, and are currently under clinical evaluation. To determine if CTNNB1-targeting DsiRNAs have potential as a therapy in HB, we encapsulated this oligonucleotide payload into lipid nanoparticles (LNPs), and systemically administered into mice bearing CTNNB1/YAP-induced HB. The LNP platform used, termed EnCore (because of its specific Envelope and Core lipid components), enables high encapsulation efficiency, long-term stability, and consistent analytical criteria. Downstream of LNP-mediated DsiRNA delivery, both qPCR and in situ hybridization were used to visualize changes in CTNNB1 expression in both tumor and normal liver. Robust and specific silencing of CTNNB1 mRNA was achieved in the tumors. The distribution of mRNA knockdown within each tumor nodule suggests efficient extravasation and internalization of the LNP and its payload into the tumor parenchyma, in contrast to previous reports of vascular-channel limited nanoparticle accumulation in tumors. Intriguingly, a “liver-centric” LNP formulation, which delivers cargo efficiently to a normal liver due to rapid hepatic extraction and apolipoprotein-mediated internalization, was inactive in the tumors. Further evaluation demonstrated efficacy of β-catenin targeting in this model. In conclusion, we report a highly relevant modality of RNAi delivery in a mouse model of hepatoblastoma to target a classically-undruggable oncogene. Citation Format: Marc T. Abrams, Junyan Tao, Shanthi Ganesh, Wendy Cyr, Bo Ying, Martin Koser, Rokhand Arvan, Girish Chopda, Hank Dudek, Cheng Lai, Weimin Wang, Bob Brown, Satdarshan Monga. Targeting β-catenin with a Dicer-substrate siRNA (DsiRNA) in a sleeping beauty transposon-driven murine hepatoblastoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3536. doi:10.1158/1538-7445.AM2015-3536

Abstract 3533: Systemic delivery of CTNNB1 Dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin

Abstract The β-catenin/Wnt pathway is consistently activated in human tumors, including >50% of hepatocellular carcinomas (HCC) and >80% of colorectal cancers (CRC). This is often caused directly by tumorigenic mutations in the CTNNB1 gene, which encodes β-catenin protein. Preclinical evidence in genetic cancer models strongly suggests that inhibiting β-catenin function would yield therapeutic benefit to many cancer patients. Since β-catenin is very challenging to target via conventional modalities, no efficacious therapy targeting β-catenin has emerged despite decades of research. RNA interference (RNAi) technology has enabled the inhibition of previously undruggable targets at the mRNA level and has advanced to late-stage clinical development for several indications. However, the clinical application of RNAi has proven to be most successful for targeting mRNAs in the normal liver, and challenges remain with developing formulations that efficiently deliver to extra-hepatic tumors. Here we describe the development and characterization of a platform to treat tumors of diverse primary tissue origin and metastatic location with CTNNB1-targeting DsiRNA. Tumor types tested included xenograft tumors of colorectal origin (CRC), experimental melanoma lung metastases, and difficult-to-transfect leukemia cells which have infiltrated the spleen. In addition to strong silencing of CTNNB1 mRNA, >75% inhibition of tumor growth was observed for a CRC xenograft model. This was achieved by formulating the DisRNA payload in a lipid nanoparticle (LNP) platform termed EnCore (because of its specific Envelope and Core lipid components). EnCore activity against multiple tumors was further improved by optimizing critical cationic lipid and PEG-lipid components. Importantly, studies in knockout mice demonstrate that in vivo tumor activity is independent of Apolipoprotein E, which has been widely described as an essential endogenous ligand for internalization of LNPs into hepatocytes. This strongly suggests that Encore-mediated delivery to tumors in vivo is independent of the ApoE/LDLR system. DCR-MYC, an EnCore-delivered DsiRNA targeting MYC mRNA, is currently in Phase I clinical trials. Further evaluation of LNP-delivered DsiRNAs in PK/PD and efficacy models, including PDX and GEMM will guide a clinical development strategy for CTNNB1 DsiRNA. Citation Format: Shanthi Ganesh, Bo Ying, Martin Koser, Wendy Cyr, Girish Chopda, Hank Dudek, Cheng Lai, Weimin Wang, Bob Brown, Marc T. Abrams. Systemic delivery of CTNNB1 Dicer-substrate siRNAs (DsiRNAs) leads to efficient oncogene silencing in diverse tumor types of extra hepatic origin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3533. doi:10.1158/1538-7445.AM2015-3533

Effective inhibition of HIV-1 production by short hairpin RNAs and small interfering RNAs targeting a highly conserved site in HIV-1 Gag RNA is optimized by evaluating alternative length formats.

We have previously identified a target site in HIV-1 RNA that was particularly accessible to a ribozyme and a short hairpin RNA (shRNA). To design small interfering RNAs (siRNAs) targeting this site, we evaluated the effects of siRNAs with different lengths on HIV-1 production. The potency and efficacy of these siRNAs were dependent on the length of their intended sense strand with trends for symmetrical and asymmetrical formats that were similar. Although a typical canonical format with a 21-nucleotide (nt) sense strand was effective at inhibiting HIV-1 production, Dicer substrate siRNAs (dsiRNAs) with the longest lengths (27 to 29 nucleotides) were the most effective. Induction of double-stranded RNA immune responses and effects on cell viability were not detected in cells transfected with different siRNAs, suggesting that the differences observed were not related to indirect effects on HIV-1 production. For the corresponding shRNA designs, a different trend in potency and efficacy against HIV-1 production was observed, with the most effective shRNAs having stem lengths from 20 to 27 bp. Our results highlight the importance of evaluating different designs to identify the best siRNA and shRNA formats for any particular target site and provide a set of highly effective molecules for further development as drug and gene therapies for HIV-1 infection.

Combination siRNA therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro

Virulent biotypes of feline coronavirus (FCoV), commonly referred to as feline infectious peritonitis virus (FIPV), can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. We previously reported the successful in vitro inhibition of FIPV replication by synthetic siRNA mediated RNA interference (RNAi) in an immortalised cell line (McDonagh et al., 2011). A major challenge facing the development of any antiviral strategy is that of resistance, a problem which is particularly acute for RNAi based therapeutics due to the exquisite sequence specificity of the targeting mechanism. The development of resistance during treatment can be minimised using combination therapy to raise the genetic barrier or using highly potent compounds which result in a more rapid and pronounced reduction in the viral replication rate, thereby reducing the formation of mutant, and potentially resistant viruses. This study investigated the efficacy of combination siRNA therapy and its ability to delay or prevent viral escape. Virus serially passaged through cells treated with a single or dual siRNAs rapidly acquired resistance, with mutations identified in the siRNA target sites. Combination therapy with three siRNA prevented viral escape over the course of five passages. To identify more potent silencing molecules we also compared the efficacy, in terms of potency and duration of action, of canonical versus Dicer-substrate siRNAs for two previously identified effective viral motifs. Dicer-substrate siRNAs showed equivalent or better potency than canonical siRNAs for the target sites investigated, and may be a more appropriate molecule for in vivo use. Combined, these data inform the potential therapeutic application of antiviral RNAi against FIPV.

358 Knockdown of beta-catenin with dicer-substrate siRNAs down-regulates Wnt/beta-catenin pathway signaling

358 Knockdown of beta-catenin with dicer-substrate siRNAs down-regulates Wnt/beta-catenin pathway signaling

Targeted delivery of Dicer-substrate siRNAs using a dual targeting peptide decorated dendrimer delivery system

Small interfering RNAs (siRNA) are emerging as novel therapeutic agents, providing competent delivery systems that are available. Dendrimers, a special family of synthetic macromolecules, represent an exciting delivery platform by virtue of their well-defined dendritic structure and unique multivalency and cooperativity confined within a nanoscale volume. Here, we report a Dicer-substrate siRNA (dsiRNA) which, when delivered using a structurally flexible triethanolamine-core poly(amidoamine) dendrimer of generation 5 as the nanocarrier, gives rise to a much greater RNAi response than that produced with conventional siRNA. Further decoration of the dsiRNA/dendrimer complexes with a dual targeting peptide simultaneously promoted cancer cell targeting through interacting with integrins and cell penetration via the interaction with neuropilin-1 receptors, which led to improved gene silencing and anticancer activity. Altogether, our results disclosed here open a new avenue for therapeutic implementation of RNAi using dendrimer nanovector based targeted delivery. From the Clinical EditorThis study demonstrates superior therapeutic properties of siRNA when combined with a dendrimer-based targeted nano-delivery system. Similar approaches may eventually gain clinical utility following additional studies determining safety and efficacy.

Platelet Activating Factor Receptor Activation Improves siRNA Uptake and RNAi Responses in Well-differentiated Airway Epithelia.

Well-differentiated human airway epithelia present formidable barriers to efficient siRNA delivery. We previously reported that treatment of airway epithelia with specific small molecules improves oligonucleotide uptake and facilitates RNAi responses. Here, we exploited the platelet activating factor receptor (PAFR) pathway, utilized by specific bacteria to transcytose into epithelia, as a trigger for internalization of Dicer-substrate siRNAs (DsiRNA). PAFR is a G-protein coupled receptor which can be engaged and activated by phosphorylcholine residues on the lipooligosaccharide (LOS) of nontypeable Haemophilus influenzae and the teichoic acid of Streptococcus pneumoniae as well as by its natural ligand, platelet activating factor (PAF). When well-differentiated airway epithelia were simultaneously treated with either nontypeable Haemophilus influenzae LOS or PAF and transduced with DsiRNA formulated with the peptide transductin, we observed silencing of both endogenous and exogenous targets. PAF receptor antagonists prevented LOS or PAF-assisted DsiRNA silencing, demonstrating that ligand engagement of PAFR is essential for this process. Additionally, PAF-assisted DsiRNA transfection decreased CFTR protein expression and function and reduced exogenous viral protein levels and titer in human airway epithelia. Treatment with spiperone, a small molecule identified using the Connectivity map database to correlate gene expression changes in response to drug treatment with those associated with PAFR stimulation, also induced silencing. These results suggest that the signaling pathway activated by PAFR binding can be manipulated to facilitate siRNA entry and function in difficult to transfect well-differentiated airway epithelial cells.

In vivo comparison of local versus systemic delivery of immunostimulating siRNA in HPV‐driven tumours

Small interfering RNAs (siRNAs) to inhibit oncogene expression and also to activate innate immune responses via Toll-like receptor (TLR) recognition have been shown to be beneficial as anti-cancer therapy in certain cancer models. In this study, we investigated the effects of local versus systemic delivery of such immune-stimulating Dicer-substrate siRNAs (IS-DsiRNAs) on a human papillomavirus (HPV)-driven tumour model. Localized siRNA delivery using intratumour injection of siRNA was able to increase siRNA delivery to the tumour compared with intravenous (IV) delivery and potently activated innate immune responses. However, IV injection remained the more effective delivery route for reducing tumour growth. Although IS-DsiRNAs activated innate immune cells and required interferon-α (IFNα) for full effect on tumour growth, we found that potent silencing siRNA acting independently of IFNα were overall more effective at inhibiting TC-1 tumour growth. Other published work utilising IS-siRNAs have been carried out on tumour models with low levels of major histocompatibility complex (MHC)-class 1, a target of natural killer cells that are potently activated by IS-siRNA. As TC-1 cells used in our study express high levels of MHC-class I, the addition of the immunostimulatory motifs may not be as beneficial in this particular tumour model. Our data suggest that selection of siRNA profile and delivery method based on tumour environment is crucial to developing siRNA-based therapies.

Abstract B222: Dicer substrate siRNAs to MYC, B-catenin, and other target genes effectively induce in vivo target gene knockdown and tumor inhibition.

Abstract Although there are now multiple successful examples of targeted therapeutics in cancer, many key protein targets have remained largely ‘undruggable’, including transcription factors such as B-catenin (CTNNB1) and MYC. B-catenin mediates Wnt signaling, which is overactive in many cancers including hepatocarcinoma (HCC). RNAi offers a way to reach such undruggable targets by inhibiting their expression at the mRNA level. Dicer substrate siRNAs (“DsiRNAs”) can be particularly effective for gene silencing. DsiRNAs are longer than conventional siRNAs, and therefore are substrates for processing by Dicer, after which the product RNA duplexes are incorporated into RISC, leading to target mRNA knockdown. We have used DsiRNAs to target B-catenin, MYC, and other key genes in HCC and other cancers. Through large-scale DsiRNA screening, we have identified a series of high potency DsiRNAs with picomolar to sub-picomolar IC50 values for mRNA knockdown. Lead DsiRNAs fully tolerated extensive 2’-OMe modification, retaining high potency, and lacked detectable immunostimulatory activity. To test the effect of target gene knockdown on tumor growth in vivo, we used a lipid nanoparticle (LNP) delivery system. LNP/DsiRNA particles effectively delivered to tumors in mouse tumor models, leading to rapid knockdown of target gene mRNA and protein. Knockdown of B-catenin, MYC, and other target genes strongly inhibited tumor growth. B-catenin knockdown also strongly reduced expression of the B-catenin-regulated genes Axin2 and MYC, a potential mechanism for tumor inhibition. In summary, we have developed high potency DsiRNAs to cancer target genes, and effectively delivered these DsiRNAs to tumors in vivo, resulting in inhibition of target gene expression and inhibition of tumor growth. DsiRNA therapeutics show promise as novel agents for reaching traditionally undruggable target genes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B222. Citation Format: Hank Dudek, Kathleen Wortham, Rokhand Arvan, Anee Shah, Bo Ying, Wendy Cyr, Hailin Yang, Wei Zhou, Utsav Saxena, Yi Zhou, Rohan Diwanji, Ben Holmes, Ruchir Farkiwala, Aalok Shah, Bob Brown. Dicer substrate siRNAs to MYC, B-catenin, and other target genes effectively induce in vivo target gene knockdown and tumor inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B222.

Intracellular processing of immunostimulatory CpG–siRNA: Toll-like receptor 9 facilitates siRNA dicing and endosomal escape

Dicer-substrate siRNAs equipped with CpG oligodeoxyribonucleotides overcome the major hurdle in cell-specific siRNA delivery. The CpG–siRNA molecules are actively internalized by TLR9+ cells, without the need for transfection reagents, leading to RNA interference both in vitro and in vivo. Here, we elucidate the molecular mechanisms of CpG–siRNA processing in target cells. We show that shortly after uptake into early endosomes (EE), CpG and siRNA parts of the conjugate are uncoupled in the presence of Dicer endonuclease. Diced siRNA molecules are translocated from endosomes to endoplasmic reticulum, where they can interact with the RNA interference machinery. We previously observed that even though TLR9 is not involved in CpG–siRNA uptake, it is indispensable for induction of gene silencing. To explain the role of TLR9 in intracellular processing of CpG–siRNA, we used primary macrophages derived from wild-type and Tlr9-deficient mice. Macrophages lacking TLR9 showed extended endosomal colocalization of CpG and siRNA parts of the conjugate. However, Tlr9 ablation did not interfere with the interaction of CpG–siRNA with Dicer as shown by in situ proximity ligation assay. Using CpG–siRNA labeled with pH-sensitive dye, we finally identified that lack of TLR9 in macrophages resulted in significant retention of the siRNA in endosomes. Thus, TLR9 facilitates the critical step following CpG–siRNA uncoupling, which is cytoplasmic release of the diced siRNA. These findings suggest that the class of immunostimulatory siRNAs may benefit from activation of certain endosomal immune receptors, such as TLR9, in augmented gene silencing and therapeutic efficacy.

Functional In Vivo Delivery of Multiplexed Anti-HIV-1 siRNAs via a Chemically Synthesized Aptamer With a Sticky Bridge

One of the most formidable impediments to clinical translation of RNA interference (RNAi) is safe and effective delivery of the siRNAs to the desired target tissue at therapeutic doses. We previously described in vivo cell type-specific delivery of anti-HIV small-interfering RNAs (siRNAs) through covalent conjugation to an anti-gp120 aptamer. In order to improve the utility of aptamers as siRNA delivery vehicles, we chemically synthesized the gp120 aptamer with a 3′ 7-carbon linker (7C3), which in turn is attached to a 16-nucleotide 2′ OMe/2′ Fl GC-rich bridge sequence. This bridge facilitates the noncovalent binding and interchange of various siRNAs with the same aptamer. We show here that this aptamer-bridge-construct complexed with three different Dicer substrate siRNAs (DsiRNAs) results in effective delivery of the cocktail of DsiRNAs in vivo, resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication. Following cessation of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of DsiRNAs. This follow-up injection resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection. Collectively, these data demonstrate a facile, targeted approach for combinatorial delivery of antiviral and host DsiRNAs for HIV-1 therapy in vivo.

RNA interference in vitro and in vivo using DsiRNA targeting the nucleocapsid N mRNA of human metapneumovirus

Human metapneumovirus causes respiratory diseases with outcomes that can be severe in children, the immunocompromised, and the elderly. Synthetic small interfering RNAs (siRNAs) that silence targeted genes can be used as therapeutic agents. Currently, there is no specific therapy for hMPV. In this study, we designed Dicer-substrate siRNAs (DsiRNAs) that target metapneumovirus sequences on the mRNAs of the N, P, and L genes. In vitro, six DsiRNAs were shown to inhibit virus replication using cell proliferation tests. Of those, the DsiRNA that targets the most conserved mRNA sequence was then resynthesized in Evader™ format with heavy 2′-O-methyl modification of the guide strand. In a murine model, the prophylactic administration of this Evader™ DsiRNA was effective at partially inhibiting viral replication of hMPV (13×103 vs. 29×103PFU/g of lung; p<0.01), which was not the case for the control, a mismatched DsiRNA. Inhibition was achieved without inducing cytokines or off-target effects. Moreover, the specificity of the siRNA mechanism of action was demonstrated in vitro and in vivo using 5′-RACE methodology. This in vivo approach of using a DsiRNA against hMPV is an important step in the development of synthetic siRNA as a therapeutic agent for this virus.

Deep Sequencing Analyses of DsiRNAs Reveal the Influence of 3′ Terminal Overhangs on Dicing Polarity, Strand Selectivity, and RNA Editing of siRNAs

25/27 Base duplex RNAs that are substrates for Dicer have been demonstrated to enhance RNA interference (RNAi) potency and efficacy. Since the target sites are not always equally susceptible to suppression by small interfering RNA (siRNA), not all 27-mer duplexes that are processed into the corresponding conventional siRNAs show increased potency. Thus random designing of Dicer-substrate siRNAs (DsiRNAs) may generate siRNAs with poor RNAi due to unpredictable Dicer processing. Previous studies have demonstrated that the 3′-overhang affects dicing cleavage site and the orientation of Dicer entry. Moreover, an asymmetric 27-mer duplex having a 3′ two-nucleotide overhang and 3′-DNA residues on the blunt end has been rationally designed to obtain greater efficacy. This asymmetric structure directs dicing to predictably yield a single primary cleavage product. In the present study, we analyzed the in vitro and intracellular dicing patterns of chemically synthesized duplex RNAs with different 3′-overhangs. Consistent with previous studies, we observed that Dicer preferentially processes these RNAs at a site 21–22 nucleotide (nt) from the two-base 3′-overhangs. We also observed that the direction and ability of human Dicer to generate siRNAs can be partially or completely blocked by DNA residues at the 3′-termimi. To examine the effects of various 3′-end modifications on Dicer processing in cells, we employed Illumina Deep sequencing analyses to unravel the fates of the asymmetric 27-mer duplexes. To validate the strand selection process and knockdown capabilities we also conducted dual-luciferase psiCHECK reporter assays to monitor the RNAi potencies of both the “sense” (S) and “antisense” (AS) strands derived from these DsiRNAs. Consistent with our in vitro Dicer assays, the asymmetric duplexes were predictably processed into desired primary cleavage products of 21–22-mers in cells. We also observed the trimming of the 3′ end, especially when DNA residues were incorporated into the overhangs and this trimming ultimately influenced the Dicer-cleavage site and RNAi potency. Moreover, the observation that the most efficacious strand was the most abundant revealed that the relative frequencies of each “S” or “AS” strand are highly correlated with the silencing activity and strand selectivity. Collectively, our data demonstrate that even though the only differences between a family of DsiRNAs was the 3′ two-nuclotide overhang, dicing polarity and strand selectivity are distinct depending upon the sequence and chemical nature of this overhang. Thus, it is possible to predictably control dicing polarity and strand selectivity via simply changing the 3′-end overhangs without altering the original duplex sequence. These optimal design features of 3′-overhangs might provide a facile approach for rationally designing highly potent 25/27-mer DsiRNAs.

Single-pot enzymatic synthesis of Dicer-substrate siRNAs

We describe an inexpensive and efficient method for generating functional pools of Dicer-substrate small interfering RNAs (siRNAs) in a single reaction tube. The method exploits a highly active form of the enzyme Dicer from Giardia lamblia, which is capable of accurately processing double-stranded RNA (dsRNA) into 25–27 nt RNA pools during in vitro transcription. The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells. The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.

Systemic Administration of Combinatorial dsiRNAs via Nanoparticles Efficiently Suppresses HIV-1 Infection in Humanized Mice

We evaluated the in vivo efficacy of structurally flexible, cationic PAMAM dendrimers as a small interfering RNA (siRNA) delivery system in a Rag2(-)/-γc-/- (RAG-hu) humanized mouse model for HIV-1 infection. HIV-infected humanized Rag2-/-γc-/- mice (RAG-hu) were injected intravenously (i.v.) with dendrimer-siRNA nanoparticles consisting of a cocktail of dicer substrate siRNAs (dsiRNAs) targeting both viral and cellular transcripts. We report in this study that the dendrimer-dsiRNA treatment suppressed HIV-1 infection by several orders of magnitude and protected against viral induced CD4(+) T-cell depletion. We also demonstrated that follow-up injections of the dendrimer-cocktailed dsiRNAs following viral rebound resulted in complete inhibition of HIV-1 titers. Biodistribution studies demonstrate that the dendrimer-dsiRNAs preferentially accumulate in peripheral blood mononuclear cells (PBMCs) and liver and do not exhibit any discernable toxicity. These data demonstrate for the first time efficacious combinatorial delivery of anti-host and -viral siRNAs for HIV-1 treatment in vivo. The dendrimer delivery approach therefore represents a promising method for systemic delivery of combinations of siRNAs for treatment of HIV-1 infection.

Species specific inhibition of viral replication using dicer substrate siRNAs (DsiRNAs) targeting the viral nucleoprotein of the fish pathogenic rhabdovirus viral hemorrhagic septicemia virus (VHSV)

Gene knock down by the use of small interfering RNAs (siRNAs) is widely used as a method for reducing the expression of specific genes in eukaryotic cells via the RNA interference pathway. But, the effectivity of siRNA induced gene knock down in cells from fish has in several studies been questioned and the specificity seems to be a general problem in cells originating from both lower and higher vertebrates. Here we show that we are able to reduce the level of viral gene expression and replication specifically in fish cells in vitro. We do so by using 27/25-mer DsiRNAs acting as substrates for dicer for the generation of siRNAs targeting the nucleoprotein N gene of viral hemorrhagic septicemia virus (VHSV). This rhabdovirus infects salmonid fish and is responsible for large yearly losses in aquaculture production. Specificity of the DsiRNA is assured in two ways: first, by using the conventional method of testing a control DsiRNA which should not target the gene of interest. Second, by assuring that replication of a heterologous virus of the same genus as the target virus was not inhibited by the DsiRNA. Target controls are, as we have previously highlighted, essential for verification of the specificity of siRNA-induced interference with virus multiplication, but they are still not in general use.

Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

RNA interference (RNAi) pathway is a gene silencing process during which small double-stranded RNA (dsRNA) molecules trigger the degradation of homologous RNA targets. Small interfering RNAs (siRNAs) are the mediators of the RNAi that can be induced in vitro and in vivo by direct application of chemically synthesized siRNAs. Recently, promoter-targeted small non-coding RNAs have been described to be capable of regulating gene expression at the transcriptional level. In the present study we tested the hypothesis that Dicer-substrate siRNAs (D-siRNAs), which trigger gene silencing through intrinsic RNAi pathway and are therefore more potent in gene knockdown than traditionally used 21-23mer siRNAs, may also display regulatory effects at the transcriptional level. Synthetic 27mer D-siRNAs targeting the promoter/early coding region of the CDKN2A gene were designed and transfected into HeLa cells, and 48 h post-transfection the CDKN2A expression was analyzed in quantitative real-time PCR using the house-keeping G6PDH gene as reference. In comparison to the mutant version, the CDKN2A gene was effectively knocked-down by the D-siRNA, but no evidence of transcriptional regulation was found. We conclude that the D-siRNA-induced suppression is likely to occur after transcription.