RNAi Trigger Research Articles

692. A Novel RNAi Trigger Design Retains Potent, Target Specific Activity Despite Emerging Mutations in the Target Site

Conventional si/shRNA designs are restricted to Watson/Crick pairing between guide strand and target RNA as this has been deemed crucial for potent Argonaute 2 based slicer activity. Despite individual publications partially challenging the requirement of sole Watson/Crick pairing these results have not yet found entry into RNAi designs.By rational incorporation of results from RNA crystallography structures and non-canonical and non-Watson/Crick pairings (nWCp) at certain positions we have developed a novel RNAi trigger design. Our approach allowed us to create shRNAs where a single guide strand suppresses a spectrum of target sequences through Argonaute 2 activity with target specificity, and similar or higher potency than a conventional design. Genomic off target effects were not observed in NGS analysis. In target/guide variant analysis several guide strands demonstrated potent RNAi activity even with G/U pairings at numerous positions or G/G pairings at position 10 or 11. Furthermore we demonstrate that this approach allows the application of RNAi against mutating targets as verified against HIV-1.Thus, our results demonstrate that an RNAi trigger can be applied to any therapeutic target (with an accessible RNA) prone to mutation-based resistance/escape offering significant improvement to the therapeutics of both infectious diseases and cancer. Additionally, we provide evidence that target and off-target prediction rules for microRNAs and shRNA/siRNAs may benefit from revisions to include a significantly wider set of pairings.

Abstract PR07: RNAi mouse models: Revolutionizing drug discovery in vivo

Abstract RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi tools including RNAi transgenic mice remains a significant limitation. One main hurdle is the identification of potent RNAi triggers, or short hairpin RNAs (shRNAs), that will induce stable and regulated gene silencing. Due to the lack of understanding of the requirements for shRNA biogenesis and target suppression, many predicted shRNAs fail to efficiently induce gene suppression. We have developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale and show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. In addition, we have engineered a new miRNA scaffold, miR-E, that is more efficiently processed and thus produces more potent knockdown of target genes than our previous miR30 system. By combining our sensored miR-E based shRNAs with high efficiency ES cell targeting, we have developed a fast, scalable pipeline for the production of shRNA transgenic mice with reversible gene silencing. We show that RNAi can cause sufficient knockdown to recapitulate the phenotypes of knockout mice, particularly in cancer models. More importantly, unlike traditional knockout models, RNAi has the powerful advantage of reversibility, since the endogenous gene remains intact. Using this system, we generated a number of inducible RNAi transgenic lines and demonstrate how this approach can identify predicted phenotypes and also unknown functions for well-studied genes. In addition, through regulated gene silencing we are able to mimic drug therapy in mice without the actual drug molecule, allowing us to determine the therapeutic value and/or toxic effects associated with systemic gene suppression. Using this approach, we have been able to pinpoint potential toxicities associated with gene inhibition, results that will guide drug development to avoid target failures which will likely cause harmful and intolerable effects in patients. In a model of hepatocellular carcinoma, we demonstrate that short-term inhibition of a ribosomal protein is sufficient to induce stable cell cycle arrest of liver tumor cells. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene – mice with enormous predictive power that will shape our development of better tolerated therapies. This abstract is also presented as Poster B30. Citation Format: Prem Premsrirut, Lukas Dow, Gregory Hannon, Johannes Zuber, Scott Lowe, Lars Zender, Christof Fellmann. RNAi mouse models: Revolutionizing drug discovery in vivo. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR07.

Abstract LB-318: A direct in vivo RNAi screen unveils a myosin as potent tumor suppressor in head & neck squamous cell carcinomas

Abstract Modern genomics is revealing hundreds of alterations associated with cancer. Mining this information for cancer therapies is now predicated on weeding out ‘bystander’ alterations, identifying the ‘driver’ mutations responsible for tumor initiation and/or metastasis, and elucidating how these mutations alter the fundamental molecular pathways governing tissue growth. We, therefore, devise and employ a direct in vivo RNAi methodology in mice that allows us to simultaneously test hundreds of candidate human cancer genes. We focus on head and neck squamous cell carcinomas (HNSCCs), which arise from the epithelial lining of the upper aerodigestive tract, including the oral cavity, tongue, lip, pharynx and larynx. Despite their frequency, aggressive recurrence and poor prognosis (6th most common cancer; 50% mortality) HNSCCs are relatively poorly studied. Using non-invasive, ultrasound-guided in utero lentiviral-mediated delivery of RNAi, we selectively transduce the single-layered surface ectoderm of living E9.5 mouse embryos where stem cells stably incorporate and propagate the desired genetic alterations into adulthood in a mosaic fashion. To screen for putative driver mutations and to dissect the physiological relevance of epigenetic changes in gene expression that occur in the development of SCC tumor-initiating (stem) cells, we employed various tumor-susceptible mouse backgrounds and identified nine tumor suppressors, seven of which have not been hitherto linked to tumor development. Our top hit, Myh9, encodes the non-muscle myosin-IIa heavy chain (NMHCIIa). On tumor-susceptible backgrounds, epithelial-specific Myh9 RNAi or Myh9 knockout trigger formation of multiple invasive SCCs and even distant lung metastasis. Surprisingly, myosin-IIa’s function is manifested not only in conventional actin-related processes, but also in regulating DNA damage-induced, post-transcriptional p53 activation and p53 nuclear accumulation. Clinically, we show that ∼20% of human HNSCCs have lost myosin-IIa protein expression, ∼5% harbor evolutionarily conserved domain-specific MYH9 mutations, and that low MYH9 expression in HNSCCs correlates with poor survival (uni-variant Kaplan-Meier analysis). These findings establish novel SCC suppressors with potential prognostic and therapeutic relevance highlighting the utility of direct in vivo RNAi to integrate cancer genomics and mouse modeling to rapidly discover and validate potent but low penetrance cancer driver mutations. Importantly, our methodology tests function within the native environment and immune system prior to the gross perturbations in tissue architecture that invariably accompanies cancer progression. Citation Format: Daniel Schramek, Ataman Sondoel, Jeremy P. Segal, Evan Heller, Slobodan Beronja, Daniel Oristian, Boris Reva, Elaine Fuchs. A direct in vivo RNAi screen unveils a myosin as potent tumor suppressor in head & neck squamous cell carcinomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-318. doi:10.1158/1538-7445.AM2014-LB-318

Abstract 2974: RNAi mouse models: Revolutionizing drug discovery in vivo

Abstract RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi tools including RNAi transgenic mice remains a significant limitation. One main hurdle is the identification of potent RNAi triggers, or short hairpin RNAs (shRNAs), that will induce stable and regulated gene silencing. Due to the lack of understanding of the requirements for shRNA biogenesis and target suppression, many predicted shRNAs fail to efficiently induce gene suppression. We have developed a “Sensor assay” that enables the biological identification of effective shRNAs at large scale and show that our assay reliably identifies potent shRNAs that are surprisingly rare and predominantly missed by existing algorithms. In addition, we have engineered a new miRNA scaffold, miR-E, that is more efficiently processed and thus produces more potent knockdown of target genes than our previous miR30 system. By combining our sensored miR-E based shRNAs with high efficiency ES cell targeting, we have developed a fast, scalable pipeline for the production of shRNA transgenic mice with reversible gene silencing. We show that RNAi can cause sufficient knockdown to recapitulate the phenotypes of knockout mice, particularly in cancer models. More importantly, unlike traditional knockout models, RNAi has the powerful advantage of reversibility, since the endogenous gene remains intact. Using this system, we generated a number of inducible RNAi transgenic lines and demonstrate how this approach can identify predicted phenotypes and also unknown functions for well-studied genes. In addition, through regulated gene silencing we are able to mimic drug therapy in mice without the actual drug molecule, allowing us to determine the therapeutic value and/or toxic effects associated with systemic gene suppression. Using this approach, we have been able to pinpoint potential toxicities associated with gene inhibition, results that will guide drug development to avoid target failures which will likely cause harmful and intolerable effects in patients. In a model of hepatocellular carcinoma, we demonstrate that short-term inhibition of a ribosomal protein is sufficient to induce stable cell cycle arrest of liver tumor cells. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene - mice with enormous predictive power that will shape our development of better tolerated therapies. Citation Format: Prem K. Premsrirut, BoYoung Yoon, Marina Pesic, Lukas E. Dow, Johannes Zuber, Scott W. Lowe, Gregory J. Hannon, Lars Zender, Christof Fellmann. RNAi mouse models: Revolutionizing drug discovery in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2974. doi:10.1158/1538-7445.AM2014-2974

Current issues of RNAi therapeutics delivery and development

12years following the discovery of the RNAi mechanism in Man, a number of RNAi therapeutics development candidates have emerged with profiles suggesting that they could become drugs of significant medical importance for diseases like TTR amyloidosis, HBV, solid cancers, and hemophilia. Despite this robust progress, the perception of RNAi therapeutics has been on a roller-coaster ride driven not only by science, but also regulatory trends, the stock markets, and Big Pharma business development decisions [1]. This presentation provides an update on the current state of RNAi therapeutics development with a particular focus on what RNAi delivery can achieve today and key challenges to be overcome to expand therapeutic opportunities.The delivery of RNAi triggers to disease-relevant cell types clearly represents the rate-limiting factor in broadly expanding the applicability of RNAi therapeutics. Today, with at least 3 delivery options (lipid nanoparticles/LNPs, GalNAc–siRNA conjugates, Dynamic PolyConjugates/DPCs) for which profound gene knockdowns have been demonstrated in non-human primates and in the clinic, RNAi therapeutics should in principle be able to address most diseases related to gene expression in the liver. Given the central importance of the liver in systemic physiology, this already represents a significant therapeutic and commercial opportunity rivaling that of e.g. monoclonal antibodies. Beyond the liver, there is a reason to believe that current RNAi therapeutics technologies can address a number of solid tumors (e.g. LNPs), diseases of the eye (e.g. self-delivering RNAi triggers) as well as diseases involving the respiratory epithelium (e.g. aerosolized LNPs), certain phagocytic cells (LNPs), hematopoietic stem cells and their progeny (lentiviral DNA–directed RNAi), vascular endothelial cells (cationic lipoplexes), and certain cell types in the kidney (self-delivering RNAi triggers, DPCs; Table 1).Despite this success, there has been a sense that the applications of RNAi therapeutics are rather limited. This is largely based on the observation that the biodistribution of RNAi formulations is typically more limited compared to small molecules and oral administration is not possible with current technologies. Similarly, the utility of a given RNAi formulation is limited to a few cell types and tissues at most and a universal delivery strategy should remain elusive for the foreseeable future. Therefore, to further expand on the therapeutic utility and patient convenience of RNAi, it is important to overcome a number of delivery-related technical and scientific challenges which will be discussed in this presentation. For systemic applications, these include the necessity for extended blood circulation times, vascular escape (probably the most rewarding inquiry currently), tissue penetration, cellular uptake, and escape into the cytoplasm. In terms of safety, it is important that these formulations do not accumulate in the body, do not cause excessive off-targeting due to ‘chemical stickiness’ (often useful for purposes of biodistribution), and overcome the physical/biological barriers in a controlled manner.The time for realizing the therapeutic potential of RNAi has come. At the same time, it is important to lay the foundations for the next leg of value creation by overcoming the challenges of delivering RNAi to new cell types. Based on results from exploratory research, the renewed interest in RNAi therapeutics and capital infusion, there is a reason to be optimistic that this can be achieved.

680. Multi-SubType Escape Reducing (MuSTER) RNAi Trigger Retains Potent Anti-Viral Activity Despite Emerging Mutations in the Target Site

680. Multi-SubType Escape Reducing (MuSTER) RNAi Trigger Retains Potent Anti-Viral Activity Despite Emerging Mutations in the Target Site

Simultaneous silencing of endo‐β‐1,4 xylanase genes reveals their roles in the virulence of Magnaporthe oryzae

Due to functional redundancy, it is often difficult to genetically analyse the biological function of fungal cell wall-degrading enzymes that belong to a gene family. To overcome this difficulty, we used RNAi to knock-down (KD) multiple xylanase genes to elucidate their roles in the pathogenicity of the blast fungus, Magnaporthe oryzae. To obtain the maximum average efficiency of gene silencing for the xylanase genes, we used the 'building blocks method', in which a 40 bp sequence was chosen from an endoxylanase gene, and 10 such sequences from 10 endoxylanases were combined to make an artificial RNAi trigger by synthetic DNA. Quantitative RT-PCR analysis revealed that the transcript levels of all the expressed xylanase genes were significantly reduced in KD mutants with the artificial RNAi trigger. Even though the KD mutants did not completely lose their pathogenicity to host plants, the number of lesions, rate of penetration and extent of infected cells were all reduced in KD mutant-infected leaves. The degree of pathogenicity reduction was associated with the silencing levels of xylanase mRNA and enzymatic activity in the KD mutants. Cytological analysis indicated that xylanases play significant roles in both vertical penetration and horizontal expansion of M. oryzae in infected plants.

Pharmacological Characterization of Chemically Synthesized Monomeric phi29 pRNA Nanoparticles for Systemic Delivery

Previous studies have shown that the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor folds into a compact structure, constituting a RNA nanoparticle that can be modularized with functional groups as a nanodelivery system. pRNA nanoparticles can also be self-assembled by the bipartite approach without altering folding property. The present study demonstrated that 2'-F-modified pRNA nanoparticles were readily manufactured through this scalable bipartite strategy, featuring total chemical synthesis and permitting diverse functional modularizations. The RNA nanoparticles were chemically and metabolically stable and demonstrated a favorable pharmacokinetic (PK) profile in mice (half-life (T(1/2)): 5-10 hours, clearance (Cl): <0.13 l/kg/hour, volume of distribution (V(d)): 1.2 l/kg). It did not induce an interferon (IFN) response nor did it induce cytokine production in mice. Repeat intravenous administrations in mice up to 30 mg/kg did not result in any toxicity. Fluorescent folate-pRNA nanoparticles efficiently and specifically bound and internalized to folate receptor (FR)-bearing cancer cells in vitro. It also specifically and dose-dependently targeted to FR(+) xenograft tumor in mice with minimal accumulation in normal tissues. This first comprehensive pharmacological study suggests that the pRNA nanoparticle had all the preferred pharmacological features to serve as an efficient nanodelivery platform for broad medical applications.

RNAi trigger fragment truncation attenuates soybean FAD2‐1 transcript suppression and yields intermediate oil phenotypes

Suppression of the microsomal ω6 oleate desaturase during the seed development of soybean (Glycine max) with the 420-bp soybean FAD2-1A intron as RNAi trigger shifts the conventional fatty acid composition of soybean oil from 20% oleic and 60% polyunsaturates to one containing greater than 80% oleic acid and less than 10% polyunsaturates. To determine whether RNAi could be attenuated by reducing the trigger fragment length, transgenic plants were generated to express successively shorter 5' or 3' deletion derivatives of the FAD2-1A intron. We observed a gradual reduction in transcript suppression with shorter trigger fragments. Fatty acid composition was less affected with shorter triggers, and triggers less than 60 bp had no phenotypic effect. No trigger sequences conferring significantly higher or lower suppression efficiencies were found, and the primary determinant of suppression effect was sequence length. The observed relationship of transcript suppression with the induced fatty acid phenotype indicates that RNAi is a saturation process and not a step change between suppressed and nonsuppressed states and intermediate suppression states can be achieved.

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.