Antisense oligonucleotides (ASOs) are a therapeutic modality for incurable diseases. However, systemic injection of gapmer-type ASOs causes class-related toxicities, including prolongation of activated partial thromboplastin time (aPTT) and thrombocytopenia. We previously reported that cholesterol-conjugated DNA/RNA heteroduplex oligonucleotides (Chol-HDOs) exhibit significantly enhanced gene-silencing effects compared to ASOs, even in the central nervous system, by crossing the blood-brain barrier. In the present study, we initially evaluated the effect of the HDO structure on class-related toxicities. The HDO structure ameliorated the class-related toxicities associated with ASOs, but they remained to some extent. As a further antidote, we have developed artificial cationic oligopeptides, L-2,4-diaminobutanoic acid oligomers (DabOs), which bind to the phosphates in the major groove of the A-type double-helical structure of HDOs. The DabO/Chol-HDO complex showed significantly improved aPTT prolongation and thrombocytopenia in mice while maintaining gene-silencing efficacy. Moreover, the conjugation with DabOs effectively prevented cerebral infarction, a condition frequently observed in mice intravenously injected with high-dose Chol-HDO. These approaches, combining HDO technology with DabOs, offer distinct advantages over conventional strategies in reducing toxicities. Consequently, the DabO/HDO complex represents a promising platform for overcoming the class-related toxicities associated with therapeutic ASOs.

A hexanucleotide (G4C2) repeat expansion (HRE) within intron one of C9ORF72 is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). C9ORF72 haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for C9ORF72 driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different C9ORF72 transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing C9ORF72 mRNA variants are found to preferentially localize to the nucleus, and thus C9ORF72 mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting C9ORF72 mRNA variants specifically or non-selectively reduce the expression of C9ORF72 mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all C9ORF72 mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing C9ORF72 mRNA transcripts. Combined, these data support RNAi-based degradation of C9ORF72 as a potential therapeutic paradigm.

The role of CD4+ T cells in the induction of protective CD8+ T cells by mRNA lipid nanoparticle (LNP) vaccines is unknown. We used B6 or Tlr9 -/- mice depleted or not of CD4+ T cells and LNP vaccines loaded with mRNAs encoding the ectromelia virus (ECTV) MHC class I H-2 Kb-restricted immunodominant CD8+ T cell epitope TSYKFESV (TSYKFESV mRNA-LNPs) or the ECTV EVM158 protein, which contains TSYKFESV (EVM-158 mRNA-LNPs). Following prime and boost with 10 μg of either vaccine, Kb-TSYKFESV-specific CD8+ T cells fully protected male and female mice from ECTV at 29 (both mRNA-LNPs) or 90 days (EVM158 mRNA-LNPs) post boost (dpb) independently of CD4+ T cells. However, at 29 dpb with 1 μg mRNA-LNPs, males had lower frequencies of Kb-TSYKFESV-specific CD8+ T cells and were much less well protected than females from ECTV, also independently of CD4+ T cells. At 90 dpb with 1 μg EVM158 mRNA-LNPs, the frequencies of Kb-TSYKFESV-specific CD8+ T cells in males and females were similar, and both were similarly partially protected from ECTV, independently of CD4+ T cells. Therefore, at optimal or suboptimal doses of mRNA-LNP vaccines, CD4+ T cell help is unnecessary to induce protective anti-poxvirus CD8+ T cells specific to a dominant epitope. At suboptimal doses, protection of males requires more time to develop.

Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease, presents considerable challenges in both diagnosis and treatment. It is categorized into sporadic and familial amyotrophic lateral sclerosis (fALS); the latter accounts for approximately 10% of cases and is primarily inherited in an autosomal dominant manner. This review summarizes the molecular genetics of fALS, highlighting key mutations that contribute to its pathogenesis, such as mutations in SOD1, FUS, and C9orf72. Central to this discourse is exploring antisense oligonucleotides (ASOs) that target these genetic aberrations, providing a promising therapeutic strategy. This review provides a detailed overview of the molecular mechanisms underlying fALS and the potential therapeutic value of ASOs, offering new insights into treating neurodegenerative diseases.

Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation in vivo. At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation (Irf8, Cebpa, Itgam) and antigen-presentation (Ciita, Il12a, B2m)-related genes with concomitant reduction of leukemia-promoting Runx1. Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing in vivo, IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.

The implementation of targeted molecular therapies and immunotherapy in melanoma vastly improved the therapeutic outcome in patients with limited efficacy of surgical intervention. Nevertheless, a large fraction of patients with melanoma still remain refractory or acquire resistance to these new forms of treatment, illustrating a need for improvement. Here, we report that the clinically relevant combination of mitogen-activated protein (MAP) kinase pathway inhibitors dabrafenib and trametinib synergize with RIG-I agonist-induced immunotherapy to kill BRAF-mutated human and mouse melanoma cells. Kinase inhibition did not compromise the agonist-induced innate immune response of the RIG-I pathway in host immune cells. In a melanoma transplantation mouse model, the triple therapy outperformed individual therapies. Our study suggests that agonist-induced activation of RIG-I with its synthetic ligand 3pRNA could vastly improve tumor control in a substantial fraction of patients with melanoma receiving MAP kinase inhibitors.

Allergic contact dermatitis is a prevalent occupational disease with limited therapeutic options. The chemokine CCL22, a ligand of the chemokine receptor CCR4, directs the migration of immune cells. Here, it is shown that genetic deficiency of CCL22 effectively ameliorated allergic reactions in contact hypersensitivity (CHS), a commonly used mouse model of allergic contact dermatitis. For the pharmacological inhibition of CCL22, DNA aptamers specific for murine CCL22 were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Nine CCL22-binding aptamers were initially selected and functionally tested in vitro. The 29-nt DNA aptamer AJ102.29m profoundly inhibited CCL22-dependent T cell migration and did not elicit undesired Toll-like receptor-dependent immune activation. AJ102.29m efficiently ameliorated CHS in vivo after systemic application. Moreover, CHS-associated allergic symptoms were also reduced following topical application of the aptamer on the skin. Microscopic analysis of skin treated with AJ102.29m ex vivo demonstrated that the aptamer could penetrate into the epidermis and dermis. The finding that epicutaneous application of the aptamer AJ102.29m in a cream was as effective in suppressing the allergic reaction as intraperitoneal injection paves the way for therapeutic use of aptamers beyond the current routes of systemic administration.

Ocular neurodegenerative diseases like glaucoma lead to progressive retinal ganglion cell (RGC) loss, causing irreversible vision impairment. Neuroprotection is needed to preserve RGCs across debilitating conditions. Nerve growth factor (NGF) protein therapy shows efficacy, but struggles with limited bioavailability and a short half-life. Here we explore a novel approach to address this deficiency by utilizing circular RNA (circRNA)-based therapy. We show that circRNAs exhibit an exceptional capacity for prolonged protein expression and circRNA-expressed NGF protects cells from glucose deprivation. In a mouse optic nerve crush model, lipid nanoparticle (LNP)-formulated circNGF administered intravitreally protects RGCs and axons from injury-induced degeneration. It also significantly outperforms NGF protein therapy without detectable retinal toxicity. Furthermore, single-cell transcriptomics revealed LNP-circNGF\'s multifaceted therapeutic effects, enhancing genes related to visual perception while reducing trauma-associated changes. This study signifies the promise of circRNA-based therapies for treating ocular neurodegenerative diseases and provides an innovative intervention platform for other ocular diseases.

Although recent advancements in cancer immunology have resulted in the approval of numerous immunotherapies, minimal progress has been observed in addressing hard-to-treat cancers. In this context, therapeutic oligonucleotides, including interfering RNAs, antisense oligonucleotides, aptamers, and DNAzymes, have gained a central role in cancer therapeutic approaches due to their capacity to regulate gene expression and protein function with reduced toxicity compared with conventional chemotherapeutics. Nevertheless, systemic administration of naked oligonucleotides faces many extra- and intracellular challenges that can be overcome by using effective delivery systems. Thus, viral and non-viral carriers can improve oligonucleotide stability and intracellular uptake, enhance tumor accumulation, and increase the probability of endosomal escape while minimizing other adverse effects. Therefore, gaining more insight into fundamental mechanisms of actions of various oligonucleotides and the challenges posed by naked oligonucleotide administration, this article provides a comprehensive review of the recent progress on oligonucleotide delivery systems and an overview of completed and ongoing cancer clinical trials that can shape future oncological treatments.

Huntington\'s disease (HD) is an autosomal dominant disease caused by the expansion of cytosine-adenine-guanine (CAG) repeats in one copy of the HTT gene (mutant HTT, mHTT). The unaffected HTT gene encodes wild-type HTT (wtHTT) protein, which supports processes important for the health and function of the central nervous system. Selective lowering of mHTT for the treatment of HD may provide a benefit over nonselective HTT-lowering approaches, as it aims to preserve the beneficial activities of wtHTT. Targeting a heterozygous single-nucleotide polymorphism (SNP) where the targeted variant is on the mHTT gene is one strategy for achieving allele-selective activity. Herein, we investigated whether stereopure phosphorothioate (PS)- and phosphoryl guanidine (PN)-containing oligonucleotides can direct allele-selective mHTT lowering by targeting rs362273 (SNP3). We demonstrate that our SNP3-targeting molecules are potent, durable, and selective for mHTT in vitro and in vivo in mouse models. Through comparisons with a surrogate for the nonselective investigational compound tominersen, we also demonstrate that allele-selective molecules display equivalent potency toward mHTT with improved durability while sparing wtHTT. Our preclinical findings support the advancement of WVE-003, an investigational allele-selective compound currently in clinical testing (NCT05032196) for the treatment of patients with HD.