Nanoparticle-Based RNAi Therapeutics Targeting Cancer Stem Cells: Update and Prospective

Cancer stem cells (CSCs) are characterized by intrinsic self-renewal and tumorigenic properties, and play important roles in tumor initiation, progression, and resistance to diverse forms of anticancer therapy. Accordingly, targeting signaling pathways that are critical for CSC maintenance and biofunctions, including the Wnt, Notch, Hippo, and Hedgehog signaling cascades, remains a promising therapeutic strategy in multiple cancer types. Furthermore, advances in various cancer omics approaches have largely increased our knowledge of the molecular basis of CSCs, and provided numerous novel targets for anticancer therapy. However, the majority of recently identified targets remain ‘undruggable’ through small-molecule agents, whereas the implications of exogenous RNA interference (RNAi, including siRNA and miRNA) may make it possible to translate our knowledge into therapeutics in a timely manner. With the recent advances of nanomedicine, in vivo delivery of RNAi using elaborate nanoparticles can potently overcome the intrinsic limitations of RNAi alone, as it is rapidly degraded and has unpredictable off-target side effects. Herein, we present an update on the development of RNAi-delivering nanoplatforms in CSC-targeted anticancer therapy and discuss their potential implications in clinical trials.

MDA5 disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoinflammatory disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more stably to synthetic Alu:Alu dsRNA. CryoEM structures of MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 sidechain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle- RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via an allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides insights on how MDA5 recognizes dsRNA in health and disease.

Lab-to-Field Transition of RNA Spray Applications – How Far Are We?

The drastic loss of biodiversity has alarmed the public and raised sociopolitical demand for chemical pesticide-free plant production, which is now treated by governments worldwide as a top priority. Given this global challenge, RNAi-based technologies are rapidly evolving as a promising substitute to conventional chemical pesticides. Primarily, genetically modified (GM) crops expressing double-stranded (ds)RNA-mediating gene silencing of foreign transcripts have been developed. However, since the cultivation of GM RNAi crops is viewed negatively in numerous countries, GM-free exogenous RNA spray applications attract tremendous scientific and political interest. The sudden rise in demand for pesticide alternatives has boosted research on sprayable RNA biopesticides, generating significant technological developments and advancing the potential for field applications in the near future. Here we review the latest advances that could pave the way for a quick lab-to-field transition for RNA sprays, which, as safe, selective, broadly applicable, and cost-effective biopesticides, represent an innovation in sustainable crop production. Given these latest advances, we further discuss technological limitations, knowledge gaps in the research, safety concerns and regulatory requirements that need to be considered and addressed before RNA sprays can become a reliable and realistic agricultural approach.

Exogenous miRNAs induce post-transcriptional gene silencing in plants

Plants seem to take up exogenous RNA that was artificially designed to target specific genes, followed by activation of the RNA interference (RNAi) machinery. It is, however, not known whether plants use RNAs themselves as signalling molecules in plant-to-plant communication, other than evidence that an exchange of small RNAs occurs between parasitic plants and their hosts. Exogenous RNAs from the environment, if taken up by some living organisms, can indeed induce RNAi. This phenomenon has been observed in nematodes and insects, and host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver plant small RNAs into Botrytis cinerea. Here we show that micro-RNAs (miRNAs) produced by plants act as signalling molecules affecting gene expression in other, nearby plants. Exogenous miRNAs, such as miR156 and miR399, trigger RNAi via a mechanism requiring both AGO1 and RDR6. This emphasizes that the production of secondary small interfering RNAs is required. This evidence highlights the existence of a mechanism in which miRNAs represent signalling molecules that enable communication between plants.

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

Pseudouridine (ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes ~7% of the total uridine level. However, ψ constitutes only ~0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based ψ mapping technique called photo-crosslinking assisted ψ sequencing (PA-ψ-seq) and use it to map ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add ψ residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of ψ addition on cellular mRNAs to each of these three PUS enzymes, the ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of ψ residues detected on total human mRNA below the ~0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of ψ residues to human mRNAs remains to be defined.

Decoupling expression and editing preferences of ADAR1 p150 and p110 isoforms

Human adenosine deaminase acting on RNA 1 (ADAR1) catalyzes adenosine-to-inosine deamination reactions on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Defective ADAR1 editing leads to disorders such as Aicardi-Goutières syndrome, an autoinflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. Two ADAR1 protein isoforms, p150 (150 kDa) and p110 (110 kDa), are expressed and can edit RNA, but the contribution of each isoform to the editing landscape remains unclear, largely because of the challenges in expressing p150 without p110. In this study, we demonstrate that p110 is coexpressed with p150 from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. The presence of a strong Kozak consensus context surrounding the p110 start codon suggests the p150 mRNA is optimized to leak p110 alongside expression of p150. To reduce leaky scanning and translation initiation at the p110 start codon, we introduced synonymous mutations in the coding region between the p150 and p110 start codons. Cells expressing p150 constructs with these mutations produced significantly reduced levels of p110. Editing analysis of total RNA from ADAR1 knockout cells reconstituted separately with modified p150 and p110 revealed that more than half of the A-to-I edit sites are selectively edited by p150, and the other half are edited by either p150 or p110. This method of isoform-selective editing analysis, making use of the modified p150, has the potential to be adapted for other cellular contexts.

Exogenous RNA as a Regulatory Signal during a Plant’s Interaction with the Biotic Environment: An Evolutionary Perspective and Future Applications in Agriculture

Environmental RNAi (eRNAi) is a sequence-specific regulation of endogenous gene expression in a responsive organism by exogenous RNA. While exogenous RNA transfer between organisms of different kingdoms of life have been unambiguously identified in nature, our understanding of the biological significance of this phenomenon remains obscure, particularly within an evolutionary context. During the last decade multiple reports utilizing various mechanisms of natural eRNAi phenomena have been attempted to develop new agricultural traits and products including weed, disease and insect control. Although these attempts yielded mixed results, this concept remains extremely attractive for many agricultural applications. To better utilize eRNAi for practical applications, we would like to emphasize the necessity of understanding the biological significance of this phenomenon within an evolutionary context and learn from nature by developing advanced tools to identify and study new cases of exogeneous RNA transfer and eRNAi. In this opinion article we would like to look at the exogeneous RNA transfer from an evolutionary perspective, propose that new cases of exogeneous RNA transfer still remain to be identified in nature, and address a knowledge gap in understanding the biological function and significance of RNA transfer. We believe such approach may eventually result in a more successful use of this phenomenon for practical applications in agriculture.

MDA5 autoimmune disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoimmune disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more tightly to synthetic Alu:Alu dsRNA. CryoEM structures MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 side-chain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle-RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via a novel allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides new insights on how MDA5 recognizes dsRNA in health and disease.

Predicting Broad-Spectrum Antiviral Drugs against RNA Viruses Using Transcriptional Responses to Exogenous RNA

All RNA viruses deliver their genomes into target host cells through processes distinct from normal trafficking of cellular RNA transcripts. The delivery of viral RNA into most cells hence triggers innate antiviral defenses that recognize viral RNA as foreign. In turn, viruses have evolved mechanisms to subvert these defenses, allowing them to thrive in target cells. Therefore, drugs activating defense to exogenous RNA could serve as broad-spectrum antiviral drugs. Here we show that transcriptional signatures associated with cellular responses to the delivery of a non-viral exogenous RNA sequence into human cells predict small molecules with broad-spectrum antiviral activity. In particular, transcriptional responses to the delivery of Cas9 mRNA into human hematopoietic stem and progenitor cells (HSPCs) highly matches those triggered by small molecules with broad-spectrum antiviral activity such as emetine, homoharringtonine, pyrvinium pamoate and anisomycin, indicating that these drugs are potentially active against other RNA viruses. Furthermore, these drugs have been approved for other indications and could thereby be repurposed to novel viruses. We propose that the antiviral activity of these drugs to SARS-CoV-2 should therefore be determined as they have been shown as active against other coronaviruses including SARS-CoV-1 and MERS-CoV. Indeed, two of these drugs- emetine and homoharringtonine- were independently shown to inhibit SARS-CoV-2 as this article was in preparation. These drugs could also be explored as potential adjuvants to COVID-19 vaccines in development due to their potential effect on the innate antiviral defenses that could bolster adaptive immunity when delivered alongside vaccine antigens.