Antisense Oligonucleotides Research Articles

Recent Progress in Gene-Targeting Therapies for Spinal Muscular Atrophy: Promises and Challenges.

Spinal muscular atrophy (SMA) is a severe genetic disorder characterized by the loss of motor neurons, leading to progressive muscle weakness, loss of mobility, and respiratory complications. In its most severe forms, SMA can result in death within the first two years of life if untreated. The condition arises from mutations in the SMN1 (survival of motor neuron 1) gene, causing a deficiency in the survival motor neuron (SMN) protein. Humans possess a near-identical gene, SMN2, which modifies disease severity and is a primary target for therapies. Recent therapeutic advancements include antisense oligonucleotides (ASOs), small molecules targeting SMN2, and virus-mediated gene replacement therapy delivering a functional copy of SMN1. Additionally, recognizing SMA's broader phenotype involving multiple organs has led to the development of SMN-independent therapies. Evidence now indicates that SMA affects multiple organ systems, suggesting the need for SMN-independent treatments along with SMN-targeting therapies. No single therapy can cure SMA; thus, combination therapies may be essential for comprehensive treatment. This review addresses the SMA etiology, the role of SMN, and provides an overview of the rapidly evolving therapeutic landscape, highlighting current achievements and future directions.

Factor XI inhibitors – Rising stars in anti-thrombotic therapy?

The “holy grail” of preventing and treating thrombosis and thromboembolism would be a drug that was highly effective (preventing clots) and at the same time had a low risk of bleeding. From a hemostasiological perspective, the inhibition of factor XI represents a promising target because a reduced level of factor XI protects against thrombosis without significantly increasing the risk of spontaneous bleeding.Currently, three different classes of drugs of factor XI-inhibition are tested. These are (1) monoclonal antibodies (mAbs), (2) so-called synthetic, small molecules and (3) antisense oligonucleotides (ASOs). This article provides a narrative overview of the current status of studies on all three classes of drugs.Tests with mAbs have been conducted primarily in DVT prevention after knee replacement surgery. One large phase 3 study is testing the mAbs Abelacimab in patients with atrial fibrillation. The synthetic, small molecules Asundexian and Milvexian are tested in several phase 3 trials, mainly in patients with non-cardioembolic ischemic stroke. Results can be expected in the coming years. Clinical testing of ASOs to inhibit factor XI are still in their infancies.

Matrix stiffness-dependent regulation of immunomodulatory genes in human MSCs is associated with the lncRNA CYTOR

Cell-matrix interactions in 3D environments significantly differ from those in 2D cultures. As such, mechanisms of mechanotransduction in 2D cultures are not necessarily applicable to cell-encapsulating hydrogels that resemble features of tissue architecture. Accordingly, the characterization of molecular pathways in 3D matrices is expected to uncover insights into how cells respond to their mechanical environment in physiological contexts, and potentially also inform hydrogel-based strategies in cell therapies. In this study, a bone marrow-mimetic hydrogel was employed to systematically investigate the stiffness-responsive transcriptome of mesenchymal stromal cells. High matrix rigidity impeded integrin-collagen adhesion, resulting in changes in cell morphology characterized by a contractile network of actin proximal to the cell membrane. This resulted in a suppression of extracellular matrix-regulatory genes involved in the remodeling of collagen fibrils, as well as the upregulation of secreted immunomodulatory factors. Moreover, an investigation of long noncoding RNAs revealed that the cytoskeleton regulator RNA (CYTOR) contributes to these 3D stiffness-driven changes in gene expression. Knockdown of CYTOR using antisense oligonucleotides enhanced the expression of numerous mechanoresponsive cytokines and chemokines to levels exceeding those achievable by modulating matrix stiffness alone. Taken together, our findings further our understanding of mechanisms of mechanotransduction that are distinct from canonical mechanotransductive pathways observed in 2D cultures.

Therapeutic potential of APP antisense oligonucleotides for Alzheimer’s disease and down syndrome-related Alzheimer’s disease

Therapeutic potential of APP antisense oligonucleotides for Alzheimer’s disease and down syndrome-related Alzheimer’s disease

Safety analysis of laboratory parameters in paediatric patients with spinal muscular atrophy treated with nusinersen

BackgroundSpinal muscular atrophy (SMA) is a progressive neurodegenerative disorder that can be treated with intrathecal nusinersen, an antisense oligonucleotide. In addition to efficacy, safety is a determining factor in the success of any therapy. Here, we aim to assess the safety of nusinersen therapy in paediatric patients with SMA.MethodsLaboratory data of paediatric patients with SMA who received nusinersen between October 2019 and May 2022 were retrospectively analysed.ResultsDuring the observation period, 46 infants and children aged 2.9 months to 13.6 years received a total of 213 nusinersen doses without safety concerns. Inflammatory markers were stable throughout the study. International normalized ratio was increased by 0.09 per injection. Urea levels were increased by 0.108 mmol/L, and cystatin C decreased by 0.029 mg/L per injection. There were no significant changes in platelet count, activated partial thrombin time, creatinine levels or liver enzyme levels during treatment. The cerebrospinal fluid (CSF) leukocyte count remained stable, and total protein increased by 24.038 mg/L per injection.ConclusionOur data showed that nusinersen therapy is generally safe in children with SMA. Laboratory monitoring did not identify any persistent or significantly abnormal findings. CSF protein should be monitored to gain more insights.

Engineering of RNase P Ribozymes for Therapy against Human Cytomegalovirus Infection.

Nucleic acid-based gene interference and editing strategies, such as antisense oligonucleotides, ribozymes, RNA interference (RNAi), and CRISPR/Cas9 coupled with guide RNAs, are exciting research tools and show great promise for clinical applications in treating various illnesses. RNase P ribozymes have been engineered for therapeutic applications against human viruses such as human cytomegalovirus (HCMV). M1 ribozyme, the catalytic RNA subunit of RNase P from Escherichia coli, can be converted into a sequence-specific endonuclease, M1GS ribozyme, which is capable of hydrolyzing an mRNA target base-pairing with the guide sequence. M1GS RNAs have been shown to hydrolyze essential HCMV mRNAs and block viral progeny production in virus-infected cell cultures. Furthermore, RNase P ribozyme variants with enhanced hydrolyzing activity can be generated by employing in vitro selection procedures and exhibit better ability in suppressing HCMV gene expression and replication in cultured cells. Additional studies have also examined the antiviral activity of RNase P ribozymes in mice in vivo. Using cytomegalovirus infection as an example, this review summarizes the principles underlying RNase P ribozyme-mediated gene inactivation, presents recent progress in engineering RNase P ribozymes for applications in vitro and in mice, and discusses the prospects of using M1GS technology for therapeutic applications against HCMV as well as other pathogenic viruses.

Get Spliced: Uniting Alternative Splicing and Arthritis.

Immune responses demand the rapid and precise regulation of gene protein expression. Splicing is a crucial step in this process; ~95% of protein-coding gene transcripts are spliced during mRNA maturation. Alternative splicing allows for distinct functional regulation, as it can affect transcript degradation and can lead to alternative functional protein isoforms. There is increasing evidence that splicing can directly regulate immune responses. For several genes, immune cells display dramatic changes in isoform-level transcript expression patterns upon activation. Recent advances in long-read RNA sequencing assays have enabled an unbiased and complete description of transcript isoform expression patterns. With an increasing amount of cell types and conditions that have been analyzed with such assays, thousands of novel transcript isoforms have been identified. Alternative splicing has been associated with autoimmune diseases, including arthritis. Here, GWASs revealed that SNPs associated with arthritis are enriched in splice sites. In this review, we will discuss how alternative splicing is involved in immune responses and how the dysregulation of alternative splicing can contribute to arthritis pathogenesis. In addition, we will discuss the therapeutic potential of modulating alternative splicing, which includes examples of spliceform-based biomarkers for disease severity or disease subtype, splicing manipulation using antisense oligonucleotides, and the targeting of specific immune-related spliceforms using antibodies.

Tofersen decreases neurofilament levels supporting the pathogenesis of the SOD1 p.D91A variant in amyotrophic lateral sclerosis patients

BackgroundSince the antisense oligonucleotide tofersen has recently become available for the treatment of amyotrophic lateral sclerosis (ALS) caused by mutations in SOD1, determining the causality of the over 230 SOD1 variants has become even more important. The most common SOD1 variant worldwide is p.D91A (c.272A > C), whose causality for ALS is contested when in a heterozygous state. The reason is the high allele frequency of SOD1D91A in Europe, exceeding 1% in Finno-Scandinavia.MethodsWe present the clinical disease course and serum neurofilament light chain (NfL) results of treating 11 patients either homo- or heterozygous for the SOD1D91A allele for up to 16 months with tofersen.ResultsTofersen decreases serum neurofilament levels (sNFL), which are associated with the ALS progression rate, in the 6 ALS patients homozygous for SOD1D91A. We observe significantly lower sNfL levels in the 5 patients heterozygous for SOD1D91A. The results indicate that both mono- and bi-allelic SOD1D91A are causally relevant targets, with a possibly reduced effect size of SOD1D91Ahet.ConclusionsThe finding is relevant for decision making regarding tofersen treatment, patient counseling and inclusion of SOD1D91A patients in drug trials. As far as we are aware, the approach is conceptually new since it provides evidence for the causality of an ALS variant based on a biomarker response to gene-specific treatment.

Contemporary directions in the therapy of sensory hearing loss

<b>Introduction:</b> More than 5% of the world’s population experience hearing impairment. The most common form is presbycusis (age-related hearing loss; ARHL). It affects almost one in three people over the age of 65. The hair cells of the cochlea play an important role in the process of sound registration. Genetic mutations, aging and environmental factors can cause damage that contributes to the hearing loss.<b>Methods and results:</b> The currently explored research directions include drug treatments, gene therapies, and stem cell therapies. To date, no significant differences in the therapeutic effect depending on the route of corticosteroid administration have been demonstrated in patients with moderate to severe hearing loss. New dexamethasone-containing hydrogel formulations, as well as lipid formulations, thermosensitive polymers, and nanoparticles, have been developed to achieve high drug concentrations in the inner ear structures. Otoprotective effects of antioxidants or substances that modify the toxic effects of e.g. cisplatin, are also being studied. Attempts at auditory cells’ regeneration seem promising in hearing loss research. Substances that regulate the central mechanisms of the Notch and Wnt pathways are being explored to this end. The genetic determinants of presbycusis suggest that interference at the level of specific genes may be a promising option for the treatment of this condition. With the CRISPR/Cas9 technology, the functions of inner ear genes can be effectively studied by disrupting normal gene alleles. The CRISPR/Cas9 complexes developed to target specific genes are delivered using cationic lipids, proteins, and viral vectors. They are then transported through the round window membrane by diffusion, without the need to surgically disrupt the inner ear. The potential of using antisense oligonucleotides to treat hereditary deafness caused by hair cell degeneration has also been established. Another research direction is related to stem cells being used for the development of in vitro 3D models of the human inner ear. Studies are also pursued to identify the mechanisms underlying the formation of cochlear organoids from pluripotent cells as well as determine the critical time points and events for cochlear sensory epithelial development and targeted hair cell differentiation.<b>Conclusions:</b> In summary, significant progress has been made over the past decade in the search for novel therapies for sensory hearing loss. This line of research remains an ambitious and important area for further exploration.

Structural Unfolding of G-Quadruplexes: From Small Molecules to Antisense Strategies.

G-quadruplexes (G4s) are non-canonical nucleic acid secondary structures that have gathered significant interest in medicinal chemistry over the past two decades due to their unique structural features and potential roles in a variety of biological processes and disorders. Traditionally, research efforts have focused on stabilizing G4s, while in recent years, the attention has progressively shifted to G4 destabilization, unveiling new therapeutic perspectives. This review provides an in-depth overview of recent advances in the development of small molecules, starting with the controversial role of TMPyP4. Moreover, we described effective metal complexes in addition to G4-disrupting small molecules as well as good G4 stabilizing ligands that can destabilize G4s in response to external stimuli. Finally, we presented antisense strategies as a promising approach for destabilizing G4s, with a particular focus on 2'-OMe antisense oligonucleotide, peptide nucleic acid, and locked nucleic acid. Overall, this review emphasizes the importance of understanding G4 dynamics as well as ongoing efforts to develop selective G4-unfolding strategies that can modulate their biological function and therapeutic potential.

Down syndrome and DYRK1A overexpression: relationships and future therapeutic directions.

Down syndrome is a genetic-based disorder that results from the triplication of chromosome 21, leading to an overexpression of many triplicated genes, including the gene encoding Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A). This protein has been observed to regulate numerous cellular processes, including cell proliferation, cell functioning, differentiation, and apoptosis. Consequently, an overexpression of DYRK1A has been reported to result in cognitive impairment, a key phenotype of individuals with Down syndrome. Therefore, downregulating DYRK1A has been explored as a potential therapeutic strategy for Down syndrome, with promising results observed from in vivo mouse models and human clinical trials that administered epigallocatechin gallate. Current DYRK1A inhibitors target the protein function directly, which tends to exhibit low specificity and selectivity, making them unfeasible for clinical or research purposes. On the other hand, antisense oligonucleotides (ASOs) offer a more selective therapeutic strategy to downregulate DYRK1A expression at the gene transcript level. Advances in ASO research have led to the discovery of numerous chemical modifications that increase ASO potency, specificity, and stability. Recently, several ASOs have been approved by the U.S. Food and Drug Administration to address neuromuscular and neurological conditions, laying the foundation for future ASO therapeutics. The limitations of ASOs, including their high production cost and difficulty delivering to target tissues can be overcome by further advances in ASO design. DYRK1A targeted ASOs could be a viable therapeutic approach to improve the quality of life for individuals with Down syndrome and their families.

Levels of Exon-Skipping Are Not Artificially Overestimated Because of the Increased Affinity of Tricyclo-DNA-Modified Antisense Oligonucleotides to the Target DMD Exon.

Antisense oligonucleotides (ASO) are very promising drugs for numerous diseases including neuromuscular disorders such as Duchenne muscular dystrophy (DMD). Several ASO drugs have already been approved by the US Food and Drug Administration for DMD and global efforts are still ongoing to improve further their potency, notably by developing new delivery systems or alternative chemistries. In this context, a recent study investigated the potential of different chemically modified ASO to induce exon-skipping in mouse models of DMD. Importantly, the authors reported a strong discrepancy between exon-skipping and protein restoration levels, which was mainly owing to the high affinity of locked nucleic acid (LNA) modifications to the target RNA, thereby interfering with the amplification of the unskipped product and resulting in artificial overamplification of the exon-skipped product. These findings urged us to verify whether a similar phenomenon could occur with tricyclo-DNA (tcDNA)-ASO that also display high-affinity properties to the target RNA. We thus ran a series of control experiments and demonstrate here that exon-skipping levels are not overestimated owing to an interference of tcDNA-ASO with the unskipped product in contrast to what was observed with LNA-containing ASO.

Safety, tolerability, and pharmacokinetics of antisense oligonucleotide BIIB078 in adults with C9orf72-associated amyotrophic lateral sclerosis: a phase 1, randomised, double blinded, placebo-controlled, multiple ascending dose study

Hexanucleotide repeat expansion of C9orf72 is a common genetic cause of amyotrophic lateral sclerosis (ALS). No C9orf72-targeted treatments are available. BIIB078 is an investigational antisense oligonucleotide targeting C9orf72 sense RNA. We aimed to assess the safety, tolerability, and pharmacokinetics of BIIB078 in participants with C9orf72-associated ALS. This phase 1, randomised controlled trial was done at 22 sites in six countries (Canada, Ireland, Netherlands, Switzerland, UK, and USA). Adults with ALS and a pathogenic repeat expansion in C9orf72 were randomly assigned within six cohorts, via Interactive Response Technology in a 3:1 ratio per cohort, to receive BIIB078 (5 mg, 10 mg, 20 mg, 35 mg, 60 mg, or 90 mg in cohorts 1-6, respectively) or placebo, via an intrathecal bolus injection. The treatment period consisted of three loading doses of study treatment, administered approximately once every 2 weeks, followed by monthly maintenance doses during a treatment period of about 3 months for cohorts 1-3 and about 6 months for cohorts 4-6. Patients and investigators were masked to treatment assignment. The primary endpoint was the incidence of adverse events and serious adverse events. This trial was registered with ClinicalTrials.gov (NCT03626012) and is completed. Between Sept 10, 2018, and Nov 17, 2021, 124 patients were screened for inclusion in the study. 18 patients were excluded and 106 participants were enrolled and randomly assigned to receive 5 mg (n=6), 10 mg (n=9), 20 mg (n=9), 35 mg (n=19), 60 mg (n=18), or 90 mg (n=18) of BIIB078, or placebo (n=27). 58 (55%) of 106 patients were female. All patients received at least one dose of study treatment and were included in all analyses. All participants had at least one adverse event; most adverse events were mild or moderate in severity and did not lead to treatment discontinuation. The most common adverse events in BIIB078-treated participants were falls, procedural pain, headache, and post lumbar puncture syndrome. 14 (18%) of 79 patients who received any dose of BIIB078 reported serious adverse events, compared with nine (33%) of 27 patients who received placebo. Five participants who received BIIB078 and three participants who received placebo had fatal adverse events: respiratory failure in a participant who received 10 mg BIIB078, ALS worsening in two participants who received 35 mg BIIB078, traumatic intracerebral haemorrhage in one participant who received 35 mg BIIB078, pulmonary embolism in one participant who received 60 mg BIIB078, and respiratory failure in three participants who received placebo. All deaths were assessed as not related to the study treatment by the reporting investigator. On the basis of these phase 1 study results, including secondary and exploratory findings showing no reduction in neurofilament levels and no benefit on clinical outcomes relative to the placebo cohort, BIIB078 clinical development has been discontinued. However, these results will be informative in furthering our understanding of the complex pathobiology of C9orf72-associated ALS. Biogen.

ASO-mediated knockdown of GPNMB in mutant- GRN and Grn -deficient peripheral myeloid cells disrupts lysosomal function and immune responses.

Increases in GPNMB are detectable in FTD- GRN cerebrospinal fluid (CSF) and post-mortem brain, and brains of aged Grn -deficient mice. Although no upregulation of GPNMB is observed in the brains of young Grn -deficient mice, peripheral immune cells of these mice do exhibit this increase in GPNMB. Importantly, the functional significance of GPNMB upregulation in progranulin-deficient states is currently unknown. Given that GPNMB has been discussed as a potential therapeutic target in GRN -mediated neurodegeneration, it is vital for the field to determine what the normal function of GPNMB is in the immune system, and whether targeting GPNMB will elicit beneficial or deleterious effects. The effects of GPNMB knock-down via antisense oligonucleotide (ASO) were assessed in peripheral blood mononuclear cells (PBMCs) from 25 neurologically healthy controls (NHCs) and age- and sex-matched FTD- GRN patients, as well as peritoneal macrophages (pMacs) from progranulin-deficient ( Grn -/- ) and B6 mice. Lysosomal function, antigen presentation and MHC-II processing and recycling were assessed, as well as cytokine release and transcription. We demonstrate here that ASO-mediated knockdown of GPNMB increases lysosomal burden and cytokine secretion in FTD-GRN carrier and neurologically healthy controls (NHCs) monocytes. ASO-mediated knockdown of GPNMB in Grn -deficient macrophages decreased lysosomal pan-cathepsin activity and protein degradation. In addition, ASO-mediated knockdown of GPNMB increased MHC-II surface expression, which was driven by decreased MHC-II uptake and recycling, in macrophages from Grn -deficient females. Finally, ASO-mediated knockdown of GPNMB dysregulated IFNγ-stimulated cytokine transcription and secretion by mouse macrophages due to the absence of regulatory actions of the GPNMB extracellular fragment (ECF). Our data herein reveals that GPNMB has a regulatory effect on multiple immune effector functions, including capping inflammation and immune responses in myeloid cells via secretion of its ECF. Therefore, in progranulin-deficient states, the drastic upregulation in GPNMB transcript and protein may represent a compensatory mechanism to preserve lysosomal function in myeloid cells. These novel findings indicate that targeted depletion in FTD- GRN would not be a rational therapeutic strategy because it is likely to dysregulate important immune cell effector functions.

Navigating Lipodystrophy: Insights from Laminopathies and Beyond.

Recent research into laminopathic lipodystrophies-rare genetic disorders caused by mutations in the LMNA gene-has greatly expanded our knowledge of their complex pathology and metabolic implications. These disorders, including Hutchinson-Gilford progeria syndrome (HGPS), Mandibuloacral Dysplasia (MAD), and Familial Partial Lipodystrophy (FPLD), serve as crucial models for studying accelerated aging and metabolic dysfunction, enhancing our understanding of the cellular and molecular mechanisms involved. Research on laminopathies has highlighted how LMNA mutations disrupt adipose tissue function and metabolic regulation, leading to altered fat distribution and metabolic pathway dysfunctions. Such insights improve our understanding of the pathophysiological interactions between genetic anomalies and metabolic processes. This review merges current knowledge on the phenotypic classifications of these diseases and their associated metabolic complications, such as insulin resistance, hypertriglyceridemia, hepatic steatosis, and metabolic syndrome, all of which elevate the risk of cardiovascular disease, stroke, and diabetes. Additionally, a range of published therapeutic strategies, including gene editing, antisense oligonucleotides, and novel pharmacological interventions aimed at addressing defective adipocyte differentiation and lipid metabolism, will be explored. These therapies target the core dysfunctional lamin A protein, aiming to mitigate symptoms and provide a foundation for addressing similar metabolic and genetic disorders.

Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO-PS Backbone for Splice-Switching Applications.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson-Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO's pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO-PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs.

Cytokines in cerebrospinal fluid as a prognostic predictor after treatment of nusinersen in SMA patients

ObjectivesRecent studies have suggested that neuroinflammation may play a role in the progression of spinal muscular atrophy (SMA), and this may influence the efficacy of antisense oligonucleotide treatment. This study explored the biomarkers associated with SMA and the efficacy of nusinersen therapy. Methods: Fifteen patients with SMA were enrolled and their motor function (World Health Organization motor milestone, Hammersmith Functional Motor Scale Expanded (HFMSE), and Revised Upper Limb Module [RULM] scores, and 6-minute walking test) was evaluated before, during (63 days), and after (6 months) nusinersen treatment. The concentrations of monocyte chemoactive protein 1 (MCP1), tumour necrosis factor-alpha (TNF-α), and interleukin (IL)-10 in the cerebrospinal fluid were measured at the indicated time points, and their correlations with motor function were analysed. Results: A significant increase in MCP1 was observed after 6 month’s treatment compared with that before treatment, while TNF-α gradually decreased over the course of treatment. IL-10 levels were negatively correlated with HFMSE scores before treatment, and reductions in IL-10 levels were correlated with improvements in RULM scores. Conclusions: This study suggests that neuroinflammation may be associated with the severity of SMA and with the therapeutic effects of nusinersen, which could have clinical implications in the treatment of SMA.

Natural products and long noncoding RNA signatures in gallbladder cancer: a review focuses on pathogenesis, diagnosis, and drug resistance.

Gallbladder cancer (GBC) is an aggressive and lethal malignancy with a poor prognosis. Long noncoding RNAs (lncRNAs) and natural products have emerged as key orchestrators of cancer pathogenesis through widespread dysregulation across GBC transcriptomes. Functional studies have revealed that lncRNAs interact with oncoproteins and tumor suppressors to control proliferation, invasion, metastasis, angiogenesis, stemness, and drug resistance. Curcumin, baicalein, oleanolic acid, shikonin, oxymatrine, arctigenin, liensinine, fangchinoline, and dioscin are a few examples of natural compounds that have demonstrated promising anticancer activities against GBC through the regulation of important signaling pathways. The lncRNAs, i.e., SNHG6, Linc00261, GALM, OIP5-AS1, FOXD2-AS1, MINCR, DGCR5, MEG3, GATA6-AS, TUG1, and DILC, are key players in regulating the aforementioned processes. For example, the lncRNAs FOXD2-AS1, DILC, and HOTAIR activate oncogenes such as DNMT1, Wnt/β-catenin, BMI1, and c-Myc, whereas MEG3 and GATA6-AS suppress the tumor proteins NF-κB, EZH2, and miR-421. Clinically, specific lncRNAs can serve as diagnostic or prognostic biomarkers based on overexpression correlating with advanced TNM stage, metastasis, chemoresistance, and poor survival. Therapeutically, targeting aberrant lncRNAs with siRNA or antisense oligos disrupts their oncogenic signaling and inhibits GBC progression. Overall, dysfunctional lncRNA regulatory circuits offer multiple avenues for precision medicine approaches to improve early GBC detection and overcome this deadly cancer. They have the potential to serve as novel biomarkers as they are detectable in bodily fluids and tissues. These findings enhance gallbladder treatments, mitigating resistance to chemo- and radiotherapy.

Changes in Cerebrospinal Fluid Concentrations of Selenium Species Induced by Tofersen Administration in Subjects with Amyotrophic Lateral Sclerosis Carrying SOD1 Gene Mutations.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting the brain and spinal cord motor neurons. On 25 April 2023, the drug tofersen, an antisense oligonucleotide, received the US Food and Drug Administration approval for treating ALS in adults carrying mutations of the SOD1 gene. We aimed at assessing whether cerebrospinal fluid concentrations of selenium, an element of both toxicological and nutritional interest possibly involved in disease etiology and progression, are modified by tofersen administration. We determined concentrations of selenium species by anion exchange chromatography hyphenated to inductively coupled plasma-dynamic reaction cell-mass spectrometry and overall selenium by using inductively coupled plasma sector-field mass spectrometry, at baseline and 6months after active tofersen treatment in ten Italian ALS patients carrying the SOD1 gene mutation. Concentrations of total selenium and many selenium species substantially increased after the intervention, particularly of inorganic (tetravalent and hexavalent) selenium and of the organic species selenomethionine and a compound co-eluting with the selenocystine standard. Overall, these findings suggest that tofersen treatment markedly alters selenium status and probably the redox status within the central nervous system, possibly due to a direct effect on neurons and/or the blood-brain barrier. Further studies are required to investigate the biological and clinical relevance of these findings and how they might relate to the pharmacological effects of the drug and to disease progression.

Characterization of the TLR9-Activating Potential of LNA-Modified Antisense Oligonucleotides.

Early characterization of the immunostimulatory potential of therapeutic antisense oligonucleotides (ASOs) is crucial. At present, little is known about the toll-like receptor 9 (TLR9)-mediated immunostimulatory potential of third-generation locked nucleic acid (LNA)-modified ASOs. In this study, we have systematically investigated the TLR9-activating potential of LNA-modified oligonucleotides using different mouse and human cell culture systems. Although it has been reported that LNA modifications as well as cytosine methylation of 5'-cytosine-phosphate-guanine-3' (CpG) motifs can reduce TLR9 stimulation by phosphorothioate (PTO)-modified oligonucleotides, we identified CpG-containing LNA gapmers with substantial TLR9-stimulatory activity. We further identified immunostimulatory LNA gapmers without CpG motifs. Unexpectedly, methylation of cytosines only within the CpG motif did not necessarily reduce but could even increase TLR9 activation. In contrast, systematic methylation of all cytosines reduced or even abrogated TLR9 activation in most cases. Context dependently, the introduction of LNA-modifications into the flanks could either increase or decrease TLR9 stimulation. Overall, our results indicate that TLR9-dependent immunostimulatory potential is an individual feature of an oligonucleotide and needs to be investigated on a case-by-case basis.