Recent investigations have defined the pathophysiological basis of many hereditary ataxias (HAs), including loss-of-function as well as gain-of-function mechanisms at either the RNA or protein level. Preclinical studies have assessed gene editing, gene and protein replacement, gene enhancement, and gene knockdown strategies. Methodologies include viral vector delivery of genes, oligonucleotide therapies, cell-penetrating peptides, synthetic transcription factors, and technologies to deliver therapies to defined targets. In this review, we focus on Friedreich ataxia (FRDA) and the polyglutamine ataxias in which translational research is active. However, much remains to be done to identify safe and effective molecules, create ideal delivery methods, and perform innovative clinical trials to prove the safety and efficacy of treatments for these rare but devastating diseases.

Global change is exacerbating the prevalence of plant diseases caused by pathogenic fungi in forests worldwide. The conventional use of chemical fungicides, which is commonplace in agricultural settings, is not sanctioned for application in forest ecosystems, so novel control strategies are imperative. SIGS (Spray-Induced Gene Silencing) is a promising approach that can modulate the expression of target genes in eukaryotes in response to double-stranded RNA (dsRNA) present in the environment that triggers the RNA interference (RNAi) mechanism. SIGS exhibited notable success in reducing virulence when deployed against some crop fungal pathogens, such as Fusarium graminearum, Botrytis cinerea and Sclerotinia sclerotiorum, among others. However, there is a conspicuous dearth of studies evaluating the applicability of SIGS for managing forest pathogens. This research aimed to determine whether SIGS could be used to control Fusarium circinatum, a widely impactful forest pathogen that causes Pine Pitch Canker disease. Through a bacterial synthesis, we produced dsRNA molecules to target fungal essential genes involved to vesicle trafficking (Vps51, DCTN1, and SAC1), signal transduction (Pp2a, Sit4, Ppg1, and Tap42), and cell wall biogenesis (Chs1, Chs2, Chs3b, Gls1) metabolic pathways. We confirmed that F. circinatum is able to uptake externally applied dsRNA, triggering an inhibition of the pathogen\'s virulence. Furthermore, this study pioneers the demonstration that recurrent applications of dsRNAs in SIGS are more effective in protecting plants than single applications. Therefore, SIGS emerges as an effective and sustainable approach for managing plant pathogens, showcasing its efficacy in controlling a globally significant forest pathogen subject to quarantine measures.

Many studies have demonstrated that long double-stranded RNAs (dsRNAs) targeting essential genes of white spot syndrome virus (WSSV) can induce a sequence-specific antiviral RNA interference (RNAi) response in shrimp, thereby offering protection against WSSV infection. However, further experimental data on the required dose of dsRNAs and the duration of protection from a single administration are necessary to establish RNAi-mediated methods as effective and practical antiviral measures. In this study, we evaluated the protective efficacy and the duration of protection provided by a single administration of various doses of long dsRNA targeting WSSV ribonucleotide reductase 2 (rr2) in white-leg shrimp Litopenaeus vannamei. The protective efficacy of long dsRNA targeting WSSV rr2 was not diminished by the reduction of the dose to 100 ng g-1 of body weight, suggesting that a relatively low dose can effectively induce an RNAi response in shrimp. Furthermore, shrimp were well-protected against WSSV challenges for up to 4 wk post-administration of the rr2-targeting long dsRNA, although the protective effect almost disappeared at 6 wk post-administration. These results suggest that long dsRNAs can provide protection against WSSV for at least 1 mo, and monthly administration of long dsRNAs could serve as a long-term protective strategy for shrimp against WSSV.

RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl-/H+ antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.

Nosema ceranae, a parasite that parasitizes and reproduces in the gut of honeybees, has become a serious threat to the global apiculture industry. RNA interference (RNAi) technology can be used to inhibit N. ceranae growth by targeting silencing the thioredoxin reductase (TrxR) in N. ceranae. However, suitable carriers are one of the reasons limiting the application of RNAi due to the easy degradation of dsRNA in honeybees. As a vesicle composed of a lipid bilayer, liposomes are a good carrier for nucleic acid delivery, but studies in honeybees are lacking. In this study, liposomes were used for double-stranded RNA (dsRNA) dsTrxR delivery triggering RNAi to inhibit the N. ceranae growth in honeybees. Compared to naked dsTrxR, liposome-dsTrxR reduced N. ceranae numbers in the midgut and partially restored midgut morphology without affecting bee survival and gut microbial composition. The results of this study confirmed that liposomes could effectively protect dsRNA from entering the honeybee gut and provide a reference for using RNAi technology to suppress honeybee pests and diseases.

Insect hemocytes eliminate foreign substances from the hemocoel through various immune reactions. Integrins, receptor proteins present on the cell membrane, are formed as a heterodimer from α and β subunits and are known to be involved in various immune reactions. To elucidate the role of integrins in the immunity of the lepidoptera Mythimna separata, genes encoding integrins were screened from the genome, resulting in the identification of eight α and four β integrin genes. The expression levels of the integrin genes did not change in response to the injection of small abiotic beads undergoing phagocytosis in M. separata larvae. However, significant inductions of some integrin gene expressions were observed in hemocytes that formed capsules around large abiotic beads during encapsulation, especially in MysIntα2. Under biotic stimulation, induction of the MysIntα2 was evident after exposures to Gram-negative bacteria (Escherichia coli) and entomopathogenic nematodes (Steinernema carpocapsae), but not to Gram-positive bacteria (Micrococcus luteus). Immunostaining analysis revealed that MysIntα2 was specifically localized to hemocytes surrounding the beads during the encapsulation reaction. Furthermore, the spreading and encapsulation abilities of hemocytes were significantly inhibited by incubation with MysIntα2 antibodies. Suppression of MysIntα2 expression in M. separata larvae by injecting double-stranded RNA also resulted in a decrease in encapsulation activity. Collectively, these results indicate that MysIntα2 plays pivotal roles in the cellular immune response of M. separata, particularly during encapsulation. This likely occurs through the regulation of hemocyte spreading activity, thereby facilitating the formation of multilayered capsules around large invaders.

BACKGROUND: Numerous insect species undertake long-distance migrations on an enormous scale, with great implications for ecosystems. Given that take-off is the point where it all starts, whether and how the external light and internal circadian rhythm are involved in regulating the take-off behaviour remains largely unknown. Herein, we explore this issue in a migratory pest, Cnaphalocrocis medinalis, via behavioural observations and RNAi experiments.
RESULTS: The results showed that C. medinalis moths took off under conditions where the light intensity gradually weakened to 0.1 lx during the afternoon or evening, and the take-off proportions under full spectrum or blue light were significantly higher than that under red and green light. The ultraviolet-A/blue light-sensitive type 1 cryptochrome gene (Cmedcry1) was significantly higher in take-off moths than that of non-take-off moths. In contrast, the expression of the light-insensitive CRY2 (Cmedcry2) and circadian genes (Cmedtim and Cmedper) showed no significant differences. After silencing Cmedcry1, the take-off proportion significantly decreased. Thus, Cmedcry1 is involved in the decrease in light intensity induced take-off behaviour in C. medinalis.
CONCLUSIONS: This study can help further explain the molecular mechanisms behind insect migration, especially light perception and signal transmission during take-off phases.

Anxiety and depression are central nervous system illnesses that are among the most prevalent medical concerns of the twenty-first century. Patients with this condition and their families bear psychological, financial, and societal hardship. There are currently restrictions when utilizing the conventional course of treatment. RNA interference is expected to become an essential approach in anxiety and depression due to its potent and targeted gene silencing. Silencing of genes by post-transcriptional modification is the mechanism of action of small interfering RNA (siRNA). The suppression of genes linked to disease is typically accomplished by siRNA molecules in an efficient and targeted manner. Unfavourable immune responses, off-target effects, naked siRNA instability, nuclease vulnerability, and the requirement to create an appropriate delivery method are some of the challenges facing the clinical application of siRNA. This review focuses on the use of siRNA in the treatment of anxiety and depression.

RNA interference (RNAi) is a powerful tool for sequence-specific gene knockdown in therapeutic and research applications. However, spatiotemporal control of RNAi is required to decrease nonspecific targeting, potential toxicity, and allow targeting of essential genes. Herein we describe a class of de-novo-designed RNA switches that enable sequence-specific regulation of RNAi in mammalian cells. Using cis-repressing RNA elements, we engineer RNA devices that only initiate microRNA biogenesis when binding with cognate trigger RNAs. We demonstrate that this conditional RNAi system, termed Orthogonal RNA Interference induced by Trigger RNA (ORIENTR), provides up to 14-fold increases in artificial miRNA biogenesis upon activation in orthogonal libraries. We show that integration of ORIENTR triggers with dCas13d enhances dynamic range to up to 31-fold. We further demonstrate that ORIENTR can be applied to detect endogenous RNA signals and to conditionally knockdown endogenous genes, thus enabling regulatory possibilities including cell-type-specific RNAi and rewiring of transcriptional networks via RNA profile.

BACKGROUND: Genetic-driven deregulation of the amyloid pathway and overproduction of downstream amyloid-β are known to cause early-onset Alzheimer\'s disease (EOAD)1. ALN-APP is an investigational intrathecally (IT) administered RNAi therapeutic designed to reduce upstream intracellular and extracellular amyloid precursor protein (APP) levels by lowering APP mRNA. As a result, we hypothesize that ALN-APP may alter the cascade of events that result in neurodegeneration, potentially slowing, halting, or reversing Alzheimer\'s disease progression.
METHODS: ALN-APP-001 (NCT05231785) Part A is an ongoing randomized, double-blind, placebo-controlled, Phase 1 single-ascending dose study in patients with EOAD. Patients are required to have disease onset at age <65 years, Clinical Dementia Rating® global score of 0.5 or 1.0, and Mini Mental State Examination score >20. Patients are being evaluated over 6 months, with further follow-up of up to 6 months as needed. The primary endpoint is the safety and tolerability of ALN-APP. Secondary objectives include the evaluation of pharmacokinetics and pharmacodynamic effects of ALN-APP.
RESULTS: 12 patients were enrolled and randomized 2:1 to receive ALN-APP or placebo in 25mg and 75mg dose cohorts. Baseline characteristics are shown in Table 1. Mean (SD) duration on study was 6.7 (1.7) months for cohort 1 (25mg) and 2.0 (1.0) months for cohort 2 (75mg). Dose-dependent reductions of soluble APPα and APPβ (sAPPα and sAPPβ) levels in cerebrospinal fluid (CSF) at day 15 were observed following a single dose of ALN-APP, with mean reductions from baseline of 55% (sAPPα) and 69% (sAPPβ), and maximum reductions of 71% (sAPPα) and 83% (sAPPβ) in the 75mg cohort (n = 4) (Table 2). All adverse events (AEs) by data cut-off on 01/17/2023 were mild or moderate (Table 3), with no AEs deemed related to study drug by the investigators. Additional cohort data will be presented at the meeting.
CONCLUSIONS: This first clinical study of a CNS-administered RNAi therapeutic demonstrates target engagement of APP, with reductions in CSF sAPPα and sAPPβ. To date, ALN-APP remains generally well tolerated with all reported AEs mild or moderate. These interim results support further evaluation of ALN-APP in patients with EOAD. Reference: 1. Hampel H et al. Molecular Psychiatry (2021). 26:5481-5503.