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.

Gapmer antisense oligonucleotides (ASOs) hold therapeutic promise for allele-specific silencing, but face challenges in distinguishing between mutant and wild-type transcripts. This study explores new design strategies to enhance ASO specificity, focusing on a common dominant mutation in COL6A3 gene associated with Ullrich congenital muscular dystrophy. Initial gapmer ASO design exhibited high efficiency but poor specificity for the mutant allele. We then adopted a mixmer design, incorporating additional RNA bases based on computational predictions of secondary structures for both mutant and wild-type alleles, aiming to enhance ASO accessibility to mutant transcripts. The mixmer ASO design demonstrated up to a 3-fold increase in specificity compared with the classical gapmer design. Further refinement involved introducing a nucleotide mismatch as a structural modification, resulting in a 10-fold enhancement in specificity compared with the gapmer design and a 3-fold over the mixmer design. Additionally, we identified for the first time a potential role of the RNA-induced silencing complex (RISC), alongside RNase H1, in gapmer-mediated silencing, in contrast with what was observed with mixmer ASOs, where only RNase H1 was involved. In conclusion, this study presents a novel design concept for allele-specific ASOs leveraging mRNA secondary structures and nucleotide mismatching and suggests a potential involvement of RISC in gapmer-mediated silencing.

An increasing number of antisense oligonucleotides (ASOs) have been approved for clinical use. However, improvements of both efficacy and safety in the central nervous system (CNS) are crucial for the treatment with CNS diseases. We aimed to overcome the crucial issues by our development of various gapmer ASOs with a novel nucleoside derivative including a 2\',4\'-BNA/LNA with 9-(aminoethoxy)phenoxazine (BNAP-AEO). The various gapmer ASOs with BNAP-AEO were evaluated for thermal stability, in vitro and in vivo efficacy, and acute CNS toxicity. Thermal stability analysis of the duplexes with their complementary RNAs showed that ASOs with BNAP-AEO had a higher binding affinity than those without BNAP-AEO. In vitro assays, when transfected into neuroblastoma cell lines, demonstrated that ASOs with BNAP-AEO, had a more efficient gene silencing effect than those without BNAP-AEO. In vivo assays, involving intracerebroventricular injections into mice, revealed ASOs with BNAP-AEO potently suppressed gene expression in the brain. Surprisingly, the acute CNS toxicity in mice, as assessed through open field tests and scoring systems, was significantly lower for ASOs with BNAP-AEO than for those without BNAP-AEO. This study underscores the efficient gene-silencing effect and low acute CNS toxicity of ASOs incorporating BNAP-AEO, indicating the potential for future therapeutic applications.

The development of messenger RNA (mRNA) vaccines and therapeutics necessitates the production of high-quality in vitro-transcribed mRNA drug substance with specific critical quality attributes (CQAs), which are closely tied to the uniformity of linear DNA template. The supercoiled plasmid DNA is the precursor to the linear DNA template, and the supercoiled DNA percentage is commonly regarded as a key in-process control (IPC) during the manufacturing of linear DNA template. In this study, we investigate the influence of supercoiled DNA percentage on key mRNA CQAs, including purity, capping efficiency, double-stranded RNA (dsRNA), and distribution of poly(A) tail. Our findings reveal a significant impact of supercoiled DNA percentage on mRNA purity and in vitro transcription yield. Notably, we observe that the impact on mRNA purity can be mitigated through oligo-dT chromatography, alleviating the tight range of DNA supercoiled percentage to some extent. Overall, this study provides valuable insights into IPC strategies for DNA template chemistry, manufacturing, and controls (CMC) and process development for mRNA drug substance.

Effective therapeutics are necessary for managing severe COVID-19 disease despite the availability of vaccines. Small interfering RNA (siRNA) can silence viral genes and restrict SARS-CoV-2 replication. Cell-penetrating peptides is a robust method for siRNA delivery, enhancing siRNA stability and targeting specific receptors. We developed a peptide HE25 that blocks SARS-CoV-2 replication by various mechanisms, including the binding of multiple receptors involved in the virus\'s internalization, such as ACE2, integrins and NRP1. HE25 not only acts as a vehicle to deliver the SARS-CoV-2 RNA-dependent RNA polymerase siRNA into cells but also facilitates their internalization through endocytosis. Once inside endosomes, the siRNA is released into the cytoplasm through the Histidine-proton sponge effect and the selective cleavage of HE25 by cathepsin B. These mechanisms effectively inhibited the replication of the ancestral SARS-CoV-2 and the Omicron variant BA.5 in vitro. When HE25 was administered in vivo, either by intravenous injection or inhalation, it accumulated in lungs, veins and arteries, endothelium, or bronchial structure depending on the route. Furthermore, the siRNA/HE25 complex caused gene silencing in lung cells in vitro. The SARS-CoV-2 siRNA/HE25 complex is a promising therapeutic for COVID-19, and a similar strategy can be employed to combat future emerging viral diseases.

Small interfering RNAs (siRNAs) are revolutionizing the treatment of liver-associated indications. Yet, robust delivery to extrahepatic tissues remains a challenge. Conjugating lipids (e.g., docosanoic acid [DCA]) to siRNA supports extrahepatic delivery, but tissue accumulation remains lower than that achieved in liver by approved siRNA therapeutics. Early evidence suggests that functionalizing DCA with a head group (e.g., phosphatidylcholine [PC]) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC head group chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC head group enhances siRNA accumulation in heart, muscle, lung, pancreas, duodenum, urinary bladder, and fat. Varying the size of the linker between the phosphate and choline moiety of the PC head group altered the extrahepatic accumulation of siRNA, with the optimal linker length being different for different tissues. Increasing PC head group valency also improved extrahepatic accumulation in a tissue-specific manner. This study demonstrates the structural impact of the PC moiety on the biodistribution of lipid-conjugated siRNA and introduces multiple novel PC variants for the chemical optimization of DCA-conjugated siRNA. These chemical variants can be used in the context of other lipids to increase the repertoire of conjugates for the extrahepatic distribution of siRNAs.

Locked nucleic acids (LNAs) are a subtype of antisense oligonucleotides (ASOs) that are characterized by a bridge within the sugar moiety. LNAs owe their robustness to this chemical modification, which as the name suggests, locks it in one conformation. This perspective includes two components: a general overview on ASOs from one side and on delivery issues focusing on lipid nanoparticles (LNPs) on the other side. Throughout, a screening of the ongoing clinical trials involving ASOs is given, as well as a take on the versatility and challenges of using LNAs. Finally, we highlight the potential of LNPs as carriers for the successful delivery of LNAs.