Non-coding RNA expression has shown to have cell type-specificity. The regulatory characteristics of these molecules are impacted by changes in their expression levels. We performed next-generation sequencing and examined small RNA-seq data obtained from 6 different types of blood cells separated by fluorescence-activated cell sorting of severe COVID-19 patients and healthy control donors. In addition to examining the behavior of piRNA in the blood cells of severe SARS-CoV-2 infected patients, our aim was to present a distinct piRNA differential expression portrait for each separate cell type. We observed that depending on the type of cell, different sorted control cells (erythrocytes, monocytes, lymphocytes, eosinophils, basophils, and neutrophils) have altering piRNA expression patterns. After analyzing the expression of piRNAs in each set of sorted cells from patients with severe COVID-19, we observed 3 significantly elevated piRNAs - piR-33,123, piR-34,765, piR-43,768 and 9 downregulated piRNAs in erythrocytes. In lymphocytes, all 19 piRNAs were upregulated. Monocytes were presented with a larger amount of statistically significant piRNA, 5 upregulated (piR-49039 piR-31623, piR-37213, piR-44721, piR-44720) and 35 downregulated. It has been previously shown that piR-31,623 has been associated with respiratory syncytial virus infection, and taking in account the major role of piRNA in transposon silencing, we presume that the differential expression patterns which we observed could be a signal of indirect antiviral activity or a specific antiviral cell state. Additionally, in lymphocytes, all 19 piRNAs were upregulated.

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.

piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.

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.

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, while there is a lack of pharmaceutical interventions to halt AAA progression presently. To address the multifaceted pathology of AAA, this work develops a novel multifunctional gene delivery system to simultaneously deliver two siRNAs targeting MMP-2 and MMP-9. The system (TPNs-siRNA), formed through the oxidative polymerization and self-assembly of epigallocatechin gallate (EGCG), efficiently encapsulates siRNAs during self-assembly. TPNs-siRNA safeguards siRNAs from biological degradation, facilitates intracellular siRNA transfection, promotes lysosomal escape, and releases siRNAs to silence MMP-2 and MMP-9. Additionally, TPNs, serving as a multi-bioactive material, mitigates oxidative stress and inflammation, fosters M1-to-M2 repolarization of macrophages, and inhibits cell calcification and apoptosis. In experiments with AAA mice, TPNs-siRNA accumulated and persisted in aneurysmal tissue after intravenous delivery, demonstrating that TPNs-siRNA can be significantly distributed in macrophages and VSMCs relevant to AAA pathogenesis. Leveraging the carrier\'s intrinsic multi-bioactive properties, the targeted siRNA delivery by TPNs exhibits a synergistic effect for enhanced AAA therapy. Furthermore, TPNs-siRNA is gradually metabolized and excreted from the body, resulting in excellent biocompatibility. Consequently, TPNs emerges as a promising multi-bioactive nanotherapy and a targeted delivery nanocarrier for effective AAA therapy.

Macrophages show high plasticity and play a vital role in the progression of metabolic dysfunction-associated steatohepatitis (MASH). X-box binding protein 1 (XBP1), a key sensor of the unfolded protein response, can modulate macrophage-mediated pro-inflammatory responses in the pathogenesis of MASH. However, how XBP1 influences macrophage plasticity and promotes MASH progression remains unclear. Herein, we formulated an Xbp1 siRNA delivery system based on folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles (FT@XBP1) to explore the precise role of macrophage-specific Xbp1 deficiency in the progression of MASH. FT@XBP1 was specifically internalized into hepatic macrophages and subsequently inhibited the expression of spliced XBP1 both in vitro and in vivo. It promoted M1-phenotype macrophage repolarization to M2 macrophages, reduced the release of pro-inflammatory factors, and alleviated hepatic steatosis, liver injury, and fibrosis in mice with fat-, fructose- and cholesterol-rich diet-induced MASH. Mechanistically, FT@XBP1 promoted macrophage polarization toward the M2 phenotype and enhanced the release of exosomes that could inhibit the activation of hepatic stellate cells. A promising macrophage-targeted siRNA delivery system was revealed to pave a promising strategy in the treatment of MASH.

Targeting non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), has recently emerged as a promising strategy for treating malignancies and other diseases. In recent years, the development of ncRNA-based therapeutics for targeting protein-coding and non-coding genes has also gained momentum. This review systematically examines ongoing and completed clinical trials to provide a comprehensive overview of the emerging landscape of ncRNA-based therapeutics. Significant efforts have been made to advance ncRNA therapeutics to early clinical studies. The most advanced trials have been conducted with small interfering RNAs (siRNAs), miRNA replacement using nanovector-entrapped miRNA mimics, or miRNA silencing by antisense oligonucleotides. While siRNA-based therapeutics have already received FDA approval, miRNA mimics, inhibitors, and lncRNA-based therapeutics are still under evaluation in preclinical and early clinical studies. We critically discuss the rationale and methodologies of ncRNA targeting strategies to illustrate this rapidly evolving field.

The prognosis after heart transplantation continues to improve. Therefore, the prevention of chronic post-transplant sequelae, such as chronic kidney disease, allograft vasculopathy, and malignancies is becoming increasingly important. Everolimus, an inhibitor of the mammalian target of rapamycin (mTOR), is increasingly used for immunosuppression after heart transplantation. However, everolimus may cause a characteristic complex of adverse effects, including dyslipidemia. Currently there are no guidelines for the long-term screening and treatment of dyslipidemia in heart transplant recipients treated with everolimus. This article presents a clinical case of hypercholesterolemia that developed after the start of the everolimus treatment in a heart recipient. The patient was a 39-year-old man who underwent orthotopic heart transplantation for ischemic cardiomyopathy in 2012 (at the age of 27). In 2019, the patient\'s immunosuppressive therapy was converted from mycophenolate mofetil to everolimus due to the development of cardiac allograft vasculopathy. The change in the immunosuppressive therapy was associated with increases in total cholesterol and low-density lipoprotein cholesterol, which were not reversed with a combined lipid-lowering therapy (maximum doses of rosuvastatin, ezetimibe, fenofibrate). A decrease in lipid levels was achieved with a blocker of hepatic proprotein convertase subtilisin/kexin type 9 synthesis at the level of microribonucleic acid (inclisiran). This case demonstrates the difficulties in correcting dyslipidemia in patients with cardiac allograft, since the treatment with the immunosuppressant everolimus worsens existing dyslipidemia. However, the combination lipid-lowering therapy, that affects various elements of the pathogenesis (specifically, the combined inhibition of hydroxymethylglutaryl-CoA reductase with a statin, cholesterol absorption from the small intestine with ezetimibe, and PCSK9 messenger RNA with inclisiran), provides an effective control of blood lipids and minimizing the adverse effects of immunosuppressive therapy, such as cardiac allograft vasculopathy.

UNASSIGNED: Dysregulated calcium homeostasis and consequentially aberrant Ca2+ signalling could enhance survival, proliferation and metastasis in various cancers. Despite rapid development in exploring the ion channel functions in relation to cancer, most of the mechanisms accounting for the impact of ion channel modulators have yet to be fully clarified. Although harnessing small interfering RNA (siRNA) to specifically silence gene expression has the potential to be a pivotal approach, its success in therapeutic intervention is dependent on an efficient delivery system. Nanoparticles have the capacity to strongly bind siRNAs. They remain in the circulation and eventually deliver the siRNA payload to the target organ. Afterward, they interact with the cell surface and enter the cell via endocytosis. Finally, they help escape the endo-lysosomal degradation system prior to unload the siRNAs into cytosol. Carbonate apatite (CA) nanocrystals primarily is composed of Ca2+, carbonate and phosphate. CA possesses both anion and cation binding domains to target negatively charged siRNA molecules.
UNASSIGNED: Hybrid CA was synthesized by complexing CA NPs with a hydrophilic polysaccharide - hyaluronic acid (HA). The average diameter of the composite particles was determined using Zetasizer and FE-SEM and their zeta potential values were also measured.
UNASSIGNED: The stronger binding affinity and cellular uptake of a fluorescent siRNA were observed for HA-CA NPs as compared to plain CA NPs. Hybrid CA was electrostatically bound individually and combined with three different siRNAs to silence expression of calcium ion channel and transporter genes, TRPC6, TRPM8 and SLC41A1 in a human breast cancer cell line (MCF-7) and evaluate their potential for treating breast cancer. Hybrid NPs carrying TRPC6, TRPM8 and SLC41A1 siRNAs could significantly enhance cytotoxicity both in vitro and in vivo. The resultant composite CA influenced biodistribution of the delivered siRNA, facilitating reduced off target distribution and enhanced breast tumor targetability.

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