RNAi Trigger Research Articles

A Comparative Analysis of RNAi Trigger Uptake and Distribution in Mosquito Vectors of Disease.

In mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to a gene of interest. However, RNAi in mosquitoes is often hampered by inconsistencies in target gene knockdown between experimental setups. While the core RNAi pathway is known to function in most mosquito strains, the uptake and biodistribution of dsRNAs across different mosquito species and life stages have yet to be extensively explored as a source of variation in RNAi experiments. To better understand mosquito-RNAi dynamics, the biodistribution of a dsRNA to a heterologous gene, LacZ (iLacZ), was tracked following various routes of exposure in the larval and adult stages of Aedes aegypti, Anopheles gambiae, and Culex pipiens. iLacZ was largely limited to the gut lumen when exposed per os, or to the cuticle when topically applied, but spread through the hemocoel when injected. Uptake of dsRNA was noted in a subset of cells including: hemocytes, pericardial cells of the dorsal vessel, ovarian follicles, and ganglia of the ventral nerve cord. These cell types are all known to undergo phagocytosis, pinocytosis, or both, and as such may actively take up RNAi triggers. In Ae. aegypti, iLacZ was detected for up to one week post exposure by Northern blotting, but uptake and degradation drastically differed across tissues. The results presented here reveal that the uptake of RNAi triggers is distinct and specific to the cell type in vivo.

RNA INTERFERENCE IN THRIPS VECTORS: A STEP FORWARD TOWARD SUSTAINABLE MANAGEMENT

Thrips are major pests of agricultural and horticultural crops worldwide. In addition to causing direct damage, thrips are the most common vector of tospoviruses. Currently, thrips control is mostly based on chemical insecticides, but effective control can be difficult to achieve because it has developed resistance to various insecticides. Therefore, there is an urgent need for novel alternative management options. A viable strategy for the management of the virus-vector complex involves understanding the molecular links between thrips and viruses and disrupting their association. RNA interference, a sequence-specific method to suppress target gene expression, is a conserved process in all eukaryotic organisms and has emerged as a new tool for reverse genetics and potential pest control. Advancements in RNA interference-based gene silencing techniques in the past few years could potentially be utilized in the management of the thrips-tospovirus complexes. RNA interference-mediated plant protection strategies have the potential to revolutionize pest management practices in a sustainable and effective way if development is carried out profoundly by discerning bioinformatics identification, in-silico designing of RNAi trigger molecules, laboratory and field-based toxicity and biosafety investigations.

Abstract 13054: The Novel Galnac-Conjugated Rnai Trigger Downregulates Apoc3 and Triglyceride Levels in Humanized Liver Mouse Model

Background: Hypertriglyceridemia is a critical risk factor for coronary artery disease. Apolipoprotein C-III (APOC3), produced by liver hepatocytes, regulates triglyceride (TG) levels. We studied effect of novel GalNAc-conjugated APOC3 targeting RNAi trigger (STP125G) on APOC3 and TG levels in humanized liver mouse model. Methods: In dose response study, female uPA-SCID mice (n=4 mice/group), group 1 received PBS control, group 2 received 10mg/kg and group 3 received 30mg/kg STP125G, subcutaneously on day 0. Liver biopsies, plasma and serum samples were collected at week 2. In duration response study, male uPA-SCID mice (n=4 mice/group/timepoint), group 1 received PBS control and group 2 received 10mg/kg STP125G, subcutaneously on day 0. Liver biopsies, plasma and serum samples were collected for each group at weeks 2, 4, 8 and 12. Human APOC3 mRNA (qPCR) and protein (ELISA) levels were measured in liver biopsies and plasma samples, respectively. TGs and total cholesterol (TC) levels were measured in serum samples. Results: In dose response study, animals reported significant suppression of mRNA and reduction of plasma protein levels in 10mg/kg (88% and 91%, respectively) and 30mg/kg (92% and 98%, respectively) dose groups as compared to control group ( p <0.001). Animals also reported significant reductions in TGs and TC levels at 10mg/kg (25% and 33%, respectively) and 30mg/kg (43% and 44%, respectively) dose groups as compared to control group ( p <0.05). In duration response study, treated animals reported significant suppression of mRNA levels at week 2 (83%), week 4 (85%) and week 8 (48%) as compared to control group ( p <0.0001). Treated animals reported significant reduction in plasma protein levels at week 2 (82%; p <0.0001), week 4 (89%; p <0.0001) and week 8 (70%; p =0.0005) as compared to control group. At week 4, treated animals reported significant reduction in TGs (53%; p=0.0079) and TC (36%; p=0.0072). Conclusions: The studies demonstrated that a single administration of STP125G, in a humanized liver mouse model, results in significant suppression of human APOC3 mRNA and plasma protein levels that lasts for up to 8 weeks. It also suggests treatment with STP125G results in lowering of triglycerides and total cholesterol in the treated animals.

Initial results from the phase 1 study of ARO-HIF2 to silence HIF2-alpha in patients with advanced ccRCC (AROHIF21001).

339 Background: Hypoxia inducible factor-2 alpha (HIF2a) is a transcription factor and key tumorigenic driver of clear cell renal cell carcinoma (ccRCC). Normally, HIF2a is expressed at low levels and targeted for degradation by the von Hippel-Lindau ( VHL) tumor suppressor protein. VHL inactivation, the causative event of ccRCC, induces tumorigenesis through uncontrolled accumulation of HIF2a and constitutive expression of downstream target genes implicated in cell survival, cell proliferation and angiogenesis. ARO-HIF2 is a synthetic double-stranded RNAi trigger with an alpha-v beta3 targeting ligand, designed to silence HIF2a expression. Herein, we present initial results from a phase 1 clinical trial of ARO-HIF2 in advanced ccRCC (NCT04169711). Methods: Advanced ccRCC patients (pts) (ECOG ≤ 1), with progressive disease on prior anti-VEGF and checkpoint inhibitor therapy, were enrolled into three escalating dose cohorts (up to 10 per cohort) to receive 225, 525 or 1,050 mg i.v. weekly of ARO-HIF2 until progression or unacceptable toxicity. The primary endpoint was incidence and severity of AEs. Secondary endpoints included tumor response based on RECIST. All pts underwent tumor biopsy at baseline and post-dose for analysis of changes in HIF2a mRNA (qPCR) and protein expression (IHC). Results: At the time of data cut, twenty-three pts (median age 66.5) were enrolled across 3 cohorts. Seventy four percent of pts received ≥ 3 prior lines of therapy. The most common treatment emergent AE was fatigue (39%). Three SAEs in 3 pts were reported by investigators as possibly drug related including, myocarditis (in a pt with a history of TKI induced cardiomyopathy and NSTEMI requiring stent placement), demyelinating neuropathy (in a pt with autoimmune sequelae secondary to checkpoint inhibitors), and hypoxia (in a pt with a pulmonary infiltrate treated with antibiotics). No cases of drug related anemia were reported. Among pts with adequate biopsy for qPCR (n=7), mean (max) reductions in HIF2a mRNA were 38% (47%), 25% (30%) and 28% (28%) in cohorts 1, 2, and 3, respectively. Among pts with adequate biopsy for IHC (n = 12), 8 showed reductions in HIF2a protein with mean (max) reductions of 45% (82%), 57% (90%) and 63% (63%) in cohorts 1, 2, 3, respectively. Across all 3 cohorts, disease control (CR+PR+SD) was seen in 7 (30%) of pts. One pt (cohort 2, with prior sunitinib, nivolumab, axitinib) experienced a partial response with 66% reduction in sum of longest diameters. Six pts had a best response of stable disease. Three pts remain on drug with stable disease and treatment durations between 20 and 32 weeks. Conclusions: HIF2a is a clinically validated driver of ccRCC which can be targeted with a RNAi therapeutic. This ongoing phase 1 study provides initial proof of target engagement based on reductions in HIF2a expression as well as a favorable safety profile in response to escalating doses of ARO-HIF2. Clinical trial information: NCT04169711.

Exploiting somatic piRNAs in Bemisia tabaci enables novel gene silencing through RNA feeding.

RNAi promises to reshape pest control by being nontoxic, biodegradable, and species specific. However, due to the plastic nature of RNAi, there is a significant variability in responses. In this study, we investigate small RNA pathways and processing of ingested RNAi trigger molecules in a hemipteran plant pest, the whitefly Bemisia tabaci Unlike Drosophila, where the paradigm for insect RNAi technology was established, whitefly has abundant somatic piwi-associated RNAs (piRNAs). Long regarded as germline restricted, piRNAs are common in the soma of many invertebrates. We sought to exploit this for a novel gene silencing approach. The main principle of piRNA biogenesis is the recruitment of target RNA fragments into the pathway. As such, we designed synthetic RNAs to possess complementarity to the loci we annotated. Following feeding of these exogenous piRNA triggers knockdown as effective as conventional siRNA-only approaches was observed. These results demonstrate a new approach for RNAi technology that could be applicable to dsRNA-recalcitrant pest species and could be fundamental to realizing insecticidal RNAi against pests.

Abstract 4775: Optimizing the potency and dosing design for ARO-HIF2: An RNAi therapeutic for clear cell renal cell carcinoma

Abstract Background: Clear cell renal cell carcinoma (ccRCC) frequently involves the inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Loss of VHL functions lead to the accumulation of hypoxia-inducible factors (HIFs). HIF2α has been regarded as a key tumorigenic driver of ccRCC and an attractive therapeutic target. Arrowhead has developed a RNA interference therapeutic (HIF2 RNAi) to selectively target and silence HIF2α expression, using a proprietary targeted-RNAi molecule (TRiM™) delivery platform for the treatment of ccRCC. The TRiM™ based Hif2 construct comprises a highly potent RNAi trigger using stabilization chemistries, targeting ligands to facilitate delivery, and structures to enhance pharmacokinetics (PK). The optimization of HIF2 RNAi to enhance the potency and safety profile to maximize the potential clinical success is described. Methods: Functional optimization of HIF2 RNAi was evaluated in an orthotopic ccRCC tumor xenograft model established with A498 ccRCC cells that stably expresses the reporter gene SEAP (secreted embryonic alkaline phosphatase) as a serum biomarker for monitoring tumor growth. HIF2 RNAi was delivered by intravenous injections. HIF2α gene silencing was evaluated by isolating tumor RNA and measuring relative gene expression by qRT-PCR. Results: We demonstrate that to achieve deep HIF2α mRNA knockdown (KD), functionalizing HIF2 RNAi with PK enhancement and tumor targeting ligand (TTL) is required. Optimization of the HIF2 RNAi construct enabled a 10-fold improvement in potency. Evaluation of a loading dose regimen improved overall HIF2α mRNA KD compared to a single administration of equal total dosage. Utilizing this strategy, we demonstrated that silencing of HIF2α mRNA (85% KD) resulted in tumor growth inhibition in the A498 xenograft model. Significant improvement in overall survival was also seen in a patient derived xenograft model. Histology evaluation of tumor samples revealed extensive tumor destruction with clusters of apoptotic cells and necrosis. Follow-up studies suggest that loading doses can be administered four hours apart without loss in potency. This allows dosing to be completed in one day and may be more acceptable in clinical settings. The maximum HIF2α mRNA KD after a single dose of HIF2 RNAi was achieved about 7 days after dosing and sustained for about one week in the xenograft model. This suggests that dosing can likely be less frequent in clinical settings. An exploratory toxicity study in rats predicts a wide safety margin. Conclusions: We demonstrate that the TRiM™ delivery platform can be utilized to deliver a RNAi therapeutic selectively targeting HIF2α for the treatment of ccRCC. This represents a novel therapeutic approach either as a monotherapy or in combination with other therapies in seeking better tolerated and/or more effective treatment for ccRCC. Citation Format: So C. Wong, Anthony Nicholas, Jeff Carlson, Dongxu Shu, Che Liu, Rui Chu, Amanda Frankiewicz, Holly Hamilton, Casi Schienebeck, Aaron Andersen, Matthew Fowler-Watters, Stephanie Bertin, Xiaokai Li, Bo Chen, Josh Schumacher, Julia Hegge, Bruce Given, Zhen Li. Optimizing the potency and dosing design for ARO-HIF2: An RNAi therapeutic for clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4775.

Ribosome clearance during RNA interference.

In the course of identifying and cleaving RNA, the RNAi machinery must encounter and contend with the megadalton-sized ribosomes that carry out translation. We investigated this interface by examining the fate of actively translated mRNAs subjected to RNAi in C. elegans. Quantifying RNA levels (RNA-seq) and ongoing translation (Ribo-seq), we found there is a greater fold repression of ongoing translation than expected from loss of RNA alone, observing stronger translation repression relative to RNA repression for multiple, independent double-stranded RNA triggers, and for multiple genes. In animals that lack the RNA helicase SKI complex and the ribosome rescue factor PELOTA, ribosomes stall on the 3′ edges of mRNAs at and upstream of the RNAi trigger. One model to explain these observations is that ribosomes are actively cleared from mRNAs by SKI and PELO during or following mRNA cleavage. Our results expand prior studies that show a role for the SKI RNA helicase complex in removing RNA targets following RNAi in flies and plants, illuminating the widespread role of the nonstop translation surveillance in RNA silencing during RNAi. Our results are also consistent with proposals that RNAi can attack messages during active translation.

RNAi-Mediated β-Catenin Inhibition Promotes T Cell Infiltration and Antitumor Activity in Combination with Immune Checkpoint Blockade.

Wnt/β-catenin signaling mediates cancer immune evasion and resistance to immune checkpoint therapy, in part by blocking cytokines that trigger immune cell recruitment. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is a nanoparticle drug product containing a chemically optimized RNAi trigger targeting CTNNB1, the gene that encodes β-catenin. In syngeneic mouse tumor models, β-catenin inhibition with DCR-BCAT significantly increased T cell infiltration and potentiated the sensitivity of the tumors to checkpoint inhibition. The combination of DCR-BCAT and immunotherapy yielded significantly greater tumor growth inhibition (TGI) compared to monotherapy in B16F10 melanoma, 4T1 mammary carcinoma, Neuro2A neuroblastoma, and Renca renal adenocarcinoma. Response to the RNAi-containing combination therapy was not dependent on Wnt activation status of the tumor. Importantly, this drug combination was associated with elevated levels of biomarkers of T cell-mediated cytotoxicity. Finally, when CTLA-4 and PD-1 antibodies were combined with DCR-BCAT in MMTV-Wnt1 transgenic mice, a genetic model of spontaneous Wnt-driven tumors, complete regressions were achieved in the majority of treated subjects. These data support RNAi-mediated β-catenin inhibition as an effective strategy to increase response rates to cancer immunotherapy.

WS09.4 Targeting αENaC with an epithelial RNAi trigger delivery platform for the treatment of cystic fibrosis

WS09.4 Targeting αENaC with an epithelial RNAi trigger delivery platform for the treatment of cystic fibrosis

SiRNAs and piRNAs collaborate for transposon control in the two-spotted spider mite.

RNAi has revolutionized genetic research, and is being commercialized as an insect pest control technology. Mechanisms exploited for this purpose are antiviral and therefore rapidly evolving. Ideally, RNAi will also be used for noninsect pests; however, differences in RNAi biology make this uncertain. Tetranychus urticae (two-spotted spider mite) is a destructive noninsect pest, which has a proclivity to develop pesticide resistance. Here we provide a comprehensive study of the endogenous RNAi pathways of spider mites to inform design of exogenous RNAi triggers. This effort revealed unexpected roles for small RNAs and novel genome surveillance pathways. Spider mites have an expanded RNAi machinery relative to insects, encoding RNA dependent RNA polymerase (Rdrp) and extra Piwi-class effectors. Through analyzing T. urticae transcriptome data we explored small RNA biogenesis, and discovered five siRNA loci that appear central to genome surveillance. These RNAs are expressed in the gonad, which we hypothesize to trigger production of piRNAs for control of transposable elements (TEs). This work highlights the need to investigate endogenous RNAi biology as lessons from model organisms may not hold in other species, impacting development of an RNAi strategy.

β-Catenin mRNA Silencing and MEK Inhibition Display Synergistic Efficacy in Preclinical Tumor Models.

Colorectal carcinomas harbor well-defined genetic abnormalities, including aberrant activation of Wnt/β-catenin and MAPK pathways, often simultaneously. Although the MAPK pathway can be targeted using potent small-molecule drugs, including BRAF and MEK inhibitors, β-catenin inhibition has been historically challenging. RNAi approaches have advanced to the stage of clinical viability and are especially well suited for transcriptional modulators, such as β-catenin. In this study, we report therapeutic effects of combined targeting of these pathways with pharmacologic agents. Using a recently described tumor-selective nanoparticle containing a β-catenin-targeting RNAi trigger, in combination with the FDA-approved MEK inhibitor (MEKi) trametinib, we demonstrate synergistic tumor growth inhibition in in vivo models of colorectal cancer, melanoma, and hepatocellular carcinoma. At dose levels that were insufficient to significantly impact tumor growth as monotherapies, combination regimens resulted in synergistic efficacy and complete tumor growth inhibition. Importantly, dual MEKi/RNAi therapy dramatically improved survival of mice bearing colorectal cancer liver metastases. In addition, pharmacologic silencing of β-catenin mRNA was effective against tumors that are inherently resistant or that acquire drug-induced resistance to trametinib. These results provide a strong rationale for clinical evaluation of this dual-targeting approach for cancers harboring Wnt/β-catenin and MAPK pathway mutations. Mol Cancer Ther; 17(2); 544-53. ©2017 AACR.

Structural properties and gene-silencing activity of chemically modified DNA-RNA hybrids with parallel orientation.

We report, herein, a new class of RNAi trigger molecules based on the unconventional parallel hybridization of two oligonucleotide chains. We have prepared and studied several parallel stranded (ps) duplexes, in which the parallel orientation is achieved through incorporation of isoguanine and isocytosine to form reverse Watson-Crick base pairs in ps-DNA:DNA, ps-DNA:RNA, ps-(DNA-2′F-ANA):RNA, and ps-DNA:2′F-RNA duplexes. The formation of these duplexes was confirmed by UV melting experiments, FRET and CD studies. In addition, NMR structural studies were conducted on a ps-DNA:RNA hybrid for the first time. Finally, we provide evidence for the unprecedented finding that ps-DNA:RNA and ps-DNA:2′F-RNA hybrids can engage the RNAi pathway to silence gene expression in vitro.

HIF2α-Targeted RNAi Therapeutic Inhibits Clear Cell Renal Cell Carcinoma.

Targeted therapy against VEGF and mTOR pathways has been established as the standard-of-care for metastatic clear cell renal cell carcinoma (ccRCC); however, these treatments frequently fail and most patients become refractory requiring subsequent alternative therapeutic options. Therefore, development of innovative and effective treatments is imperative. About 80%-90% of ccRCC tumors express an inactive mutant form of the von Hippel-Lindau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong genetic and experimental evidence supports the correlate that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF2α) and that an overabundance of HIF2α functions as a tumorigenic driver of ccRCC. In this report, we describe an RNAi therapeutic for HIF2α that utilizes a targeting ligand that selectively binds to integrins αvβ3 and αvβ5 frequently overexpressed in ccRCC. We demonstrate that functional delivery of a HIF2α-specific RNAi trigger resulted in HIF2α gene silencing and subsequent tumor growth inhibition and degeneration in an established orthotopic ccRCC xenograft model. Mol Cancer Ther; 17(1); 140-9. ©2017 AACR.

Induction of RNAi Responses by Short Left-Handed Hairpin RNAi Triggers.

Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses. However, further guide strand deletion inhibited LHP activity, thereby defining the minimal length guide targeting length to 13 nucleotides. To dissect LHP processing, we examined LHP species that coimmunoprecipitated with Argonaute 2 (Ago2), the catalytic core of RNA-induced silencing complex, and found that the Ago2-associated processed LHP species was of a length that correlated with Ago2 cleavage of the passenger strand. Placement of a blocking 2'-OMe blocking modification at the LHP predicted Ago2 cleavage site resulted in an intact LHP loaded into Ago2 and no RNAi response. Taken together, these data argue that in the absence of a substantial loop, this novel class of small LHP RNAs enters the RNAi pathway by a Dicer-independent mechanism that involves Ago2 cleavage and results in an extended guide strand. This work establishes LHPs as an alternative RNAi trigger that can be produced from a single synthesis for potential use as an RNAi therapeutic.

Core-shell hybrid nanostructured delivery platforms for advanced RNAi therapeutics.

Study was aimed at combining the advantages of nonclassical RNAi-triggering oligonucleotides with nanoparticle-based advanced delivery platforms for developing efficient therapeutic systems. We utilized a core-shell hybrid nanostructured platform for effectively delivering nonclassical RNAi triggers, namely long double stranded interfering RNA and tripodal interfering RNA. Core-shell structure was prepared by stably anchoring thiol-modified cationic polymer on the surface of growing crystal gold (Au) seeds, and the resulting particles were further complexed with nonclassical RNAi candidates via electrostatic interactions. Our studies clearly demonstrated that the unique combination of nonclassical RNAi structures with an advanced core-shell hybrid nanostructured platform is an effective module for advanced RNAi-based therapeutic development.

Abstract 2064: Novel HIF-2α targeted RNAi therapeutic for renal cell carcinoma

Abstract Background: Targeted therapy including VEGF and mTOR pathway inhibitors has dramatically transformed treatment options and outcomes for patients with metastatic clear cell renal cell carcinoma (ccRCC). However, alternate treatments are needed as resistance to these initially promising agents occurs frequently. RNAi interference (RNAi), an innate gene silencing mechanism, has been explored as a new class of therapeutics where conventional treatments are lacking or have failed. The challenge in leveraging this promising approach has been efficient delivery of an RNAi trigger (siRNA) to target tissue. Over 90% of ccRCC tumors express a mutant inactive form of the von Hippel-Landau protein (pVHL), an E3 ubiquitin ligase that promotes target protein degradation. Strong evidence supports the observation that pVHL functional loss leads to the accumulation of the transcription factor hypoxia-inducible factor 2α (HIF-2α), a tumorigenic driver of ccRCC. Methods: We have developed a targeted delivery platform called Dynamic Polyconjugte™ (DPC) as an RNAi-based therapeutic targeting HIF-2α for advanced ccRCC. The ccRCC-specific DPC (ITG-DPC) comprises a membrane active polymer to promote RNAi trigger endosomal release, a ligand that binds to αV-containing integrin receptors expressed on tumor cells, reversible masking to prevent polymer activity before reaching the endosomal compartment, and a potent and specific RNAi trigger to HIF-2α. The modular nature of this delivery platform allows for flexibility to optimize each functional component independently. The ligand-dependent delivery of ITG-DPC was first evaluated in cultured tumor cells and then confirmed in ccRCC tumors established in nude mice using fluorescently-labeled ITG-DPC and confocal microscopy. To validate silencing of HIF-2α as an effective therapeutic approach, an inducible shRNA to HIF-2α was expressed in ccRCC tumors established in mice that significantly silenced HIF-2α gene expression and induced tumor regression. Results: Bi-weekly injection of ITG-DPC into nude mice with established orthotopic A498 ccRCC tumors resulted in >80% knockdown of HIF-2α mRNA and significant tumor growth inhibition. Histological examination of tumor sections showed substantial cell killing and destruction of tumor architecture. Down-regulation of HIF-2α regulated pathways genes including VEGF-A, and the corresponding reduction in tumor-associated CD31 positive neovascularization, corroborated on-target effects of HIF-2α gene silencing. Conclusion: Targeted delivery of a HIF-2α specific RNAi-based-therapeutic has the potential to radically impact the late-stage ccRCC treatment paradigm. Citation Format: So Wong, Weijun Cheng, Darren Wakefield, Aaron Almeida, Andrei Blokhin, Holly Hamilton, Vladimir Subbotin, Julia Hegge, Zane Neal, Guofeng Zhang, David Rozema, David Lewis, Steven Kanner. Novel HIF-2α targeted RNAi therapeutic for renal cell carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2064.

Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers

Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers

Broad distribution of ataxin 1 silencing in rhesus cerebella for spinocerebellar ataxia type 1 therapy.

Spinocerebellar ataxia type 1 is one of nine polyglutamine expansion diseases and is characterized by cerebellar ataxia and neuronal degeneration in the cerebellum and brainstem. Currently, there are no effective therapies for this disease. Previously, we have shown that RNA interference mediated silencing of ATXN1 mRNA provides therapeutic benefit in mouse models of the disease. Adeno-associated viral delivery of an engineered microRNA targeting ATXN1 to the cerebella of well-established mouse models improved motor phenotypes, neuropathy, and transcriptional changes. Here, we test the translatability of this approach in adult rhesus cerebella. Nine adult male and three adult female rhesus macaque were unilaterally injected with our therapeutic vector, a recombinant adeno-associated virus type 1 (rAAV1) expressing our RNAi trigger (miS1) and co-expressing enhanced green fluorescent protein (rAAV1.miS1eGFP) into the deep cerebellar nuclei using magnetic resonance imaging guided techniques combined with a Stealth Navigation system (Medtronics Inc.). Transduction was evident in the deep cerebellar nuclei, cerebellar Purkinje cells, the brainstem and the ventral lateral thalamus. Reduction of endogenous ATXN1 messenger RNA levels were ≥30% in the deep cerebellar nuclei, the cerebellar cortex, inferior olive, and thalamus relative to the uninjected hemisphere. There were no clinical complications, and quantitative and qualitative analyses suggest that this therapeutic intervention strategy and subsequent reduction of ATXN1 is well tolerated. Collectively the data illustrate the biodistribution and tolerability of rAAV1.miS1eGFP administration to the adult rhesus cerebellum and are supportive of clinical application for spinocerebellar ataxia type 1.

Corrigendum

Insect Molecular BiologyVolume 24, Issue 4 p. 502-502 CorrigendumFree Access Corrigendum This article corrects the following: Inducing RNA interference in the arbovirus vector, Culicoides sonorensis M. K. Mills, D. Nayduch, K. Michel, Volume 24Issue 1Insect Molecular Biology pages: 105-114 First Published online: October 7, 2014 First published: 25 May 2015 https://doi.org/10.1111/imb.12178AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat In Mills et al., 2015, Insect Molecular Biology Volume 24 Issue 1, pp. 105–114, the authors discovered that an error was introduced inadvertently in the analysis of the quantitative RT-PCR data using the method established by Pfaffl et al. (2001). The changes had the following consequences for the results of the qRT-PCR analyses: (i) transcript levels of dsGFP treatments are closer to untreated controls (UT); (ii) CsIAP1 transcripts were reduced on average by 33% instead of the initially reported 40%; (iii) variation of both dsGFP and dsCsIAP1 transcript levels increased slightly; (iv) the combination of increased variation and lowered reduction in transcript levels resulted in a loss of the originally reported statistical significance (Student's t-test, P=0.07). Figure 4 and its legend (Page 109) were updated: Figure 4. Relative expression of CsIAP1 transcripts after dsRNA injection (changes italicized): Graph depicts CsIAP1 mean transcript levels at 5 dpi for dsGFP and dsCsIAP1-injected midges relative to untreated controls. QRT-PCR results were analyzed using EF1b as reference gene and untreated midges as calibrator condition. On average, mRNA levels of CsIAP1 were unaffected by dsGFP injection, and reduced by 33% after dsCsIAP1 treatment compared to untreated controls (UT). However, expression levels between dsGFP and dsCsIAP1-treated midges were not statistically significantly different (Student's t-test, P = 0.07). Data are presented as mean ± one S.E.M., from four biological replicates (Figure S2). The last paragraph of the Experimental Procedures (Page 112) should read (changes italicized): Fold change for each treatment (dsGFP or dsCsIAP1) was assessed using a modified ΔΔCt method (Pfaffl, 2001), which takes potentially different primer efficiencies (Figure S10) into account. All qRT-PCRs were performed with 3 technical and four biological replicates. Untreated controls were used as the calibrator in each biological replicate. Expression data between dsGFP and dsCsIAP1 treatments were compared statistically with Student's t-test. Statistical analyses were performed using GraphPad Prism software version 5.01 (GraphPad Software Inc., La Jolla, CA, USA). Sentences 5 and 6 of paragraph 5 (Discussion - Page 110) should read (changes italicized): The 33% transcript reduction of CsIAP1 induced by a comparatively high dose of dsRNA (Miller et al., 2012) suggests that the initial RNAi trigger is not amplified in C. sonorensis, and is likely not taken up by all tissues. Similar results have been obtained from closely related Culicidae (Blandin et al., 2002, Zhu et al., 2003). In addition, we observed considerable variation in overall transcript reduction levels between biological replicates. References Pfaffl, M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45. CrossrefCASPubMedWeb of Science®Google Scholar Mills, M.K., Nayduch, D. and Michel, K. (2015) Inducing RNA interference in the arbovirus vector, Culicoides sonorensis. Insect Mol Biol 24: 105– 114. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume24, Issue4August 2015Pages 502-502 ReferencesRelatedInformation

699. shRNA Sense Strand Neutralization Reduces Off-Targeting, Ameliorates Toxicity and Enhances Efficacy of RNAi-Based HBV Gene Therapy

A decade after first reports of successful inhibition of hepatitis B virus (HBV) replication in vivo using shRNAs, only a single RNAi therapeutic is under phase II efficacy investigation in Hepatitis B patients. This slow clinical translation is puzzling, as current standard-of-care requires life-long and daily drug treatment which is often accompanied by side effects and occurrence of resistant HBV mutants. Also, while preventing the generation of infectious HBV virions, reverse-transcriptase inhibitors fail to suppress HBV surface and e-antigen, which enable viral persistence by modulating the host immune system and contribute to HBV-associated hepatocellular carcinoma. These problems may be solved using RNAi as anti-HBV therapeutic since (i) long-term suppression can be achieved by a single dose of an shRNA-encoding vector, and (ii) all four HBV transcripts share a common 3 end, allowing concurrent inhibition of HBV pre-genomic RNA and all viral proteins with a single RNAi molecule, and potentially restoring antiviral immunity and preventing carcinogenesis. Still, clinical translation remains hampered by safety concerns as shRNA over-expression in the mouse liver causes elevated liver transaminases, jaundice and weight loss. One explanation is that ectopic RNAi triggers overload the endogenous miRNA pathway, perturbing miRNA biogenesis and/or activity, and causing cytotoxicity. Evidence for this saturation model is that (i) shRNAs circumvent Drosha processing, a gatekeeper for miRNA biogenesis, (ii) embedding shRNAs in a recombinant miRNA context yields lower and safer levels of mature RNAi triggers, and (iii) overexpression of Argonaute-2 (Ago2) ameliorates toxicity and enhances RNAi efficacy. Moreover, RNAi triggers can perturb cell physiology through unwanted inhibition of off-target genes with partial complementarities to one of the two strands of the RNAi molecule. To alleviate such unintended gene silencing, we have developed a novel bi-cistronic AAV vector that expresses, in addition to the shRNA, a second RNA hairpin called tough decoy or TuD. In cell culture, the TuD effectively sequestered and inactivated shRNA sense strands, and thereby improved RNAi specificity. These remarkable features translated well into an HBV-transgenic mouse model, where sense strand off-target activity was likewise prevented, as validated by transcriptome analysis of liver RNA, coinciding with ameliorated toxicity. Enhanced in vivo safety was also noted for two alternative AAV vectors, either co-expressing the shRNA and Ago2, or embedding the shRNA in a miR-122 backbone. Notably, the new shRNA/TuD vector outperformed the other expression strategies regarding efficacy with stable HBV reduction of up to 98% over 3 months. We speculate that its enhanced antiviral efficacy results from increased loading of the desired antisense strand into RISC in absence of the sense strand, possibly also further amending toxicity by attenuating RISC saturation. The simple TuD design and the versatility of our new AAV vector pave the way for adaptation of our strategy to other applications and should facilitate clinical translation.