Molecular mechanisms and antisense oligonucleotide therapies of familial amyotrophic lateral sclerosis.

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.

Advancing cancer treatments: The role of oligonucleotide-based therapies in driving progress.

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.

Circular RNA-based therapy provides sustained and robust neuroprotection for retinal ganglion cells.

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.

RNA therapeutics in targeting G protein-coupled receptors: Recent advances and challenges.

G protein-coupled receptors (GPCRs) are the major targets of existing drugs for a plethora of human diseases and dominate the pharmaceutical market. However, over 50% of the GPCRs remain undruggable. To pursue a breakthrough and overcome this situation, there is significant clinical research for developing RNA-based drugs specifically targeting GPCRs, but none has been approved so far. RNA therapeutics represent a unique and promising approach to selectively targeting previously undruggable targets, including undruggable GPCRs. However, the development of RNA therapeutics faces significant challenges in areas of RNA stability and efficient in vivo delivery. This review presents an overview of the advances in RNA therapeutics and the diverse types of nanoparticle RNA delivery systems. It also describes the potential applications of GPCR-targeted RNA drugs for various human diseases.

Unlocking the therapeutic potential of locked nucleic acids through lipid nanoparticle delivery.

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.

Phosphatidylcholine head group chemistry alters the extrahepatic accumulation of lipid-conjugated siRNA.

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.

Synthetic RIG-I agonist-mediated cancer immunotherapy synergizes with MAP kinase inhibition against BRAF-mutated melanoma.

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.

Engineering lentivirus envelope VSV-G for liver targeted delivery of IDOL-shRNA to ameliorate hypercholesterolemia and atherosclerosis.

Lentiviral vectors (LVs) have been widely used as a tool for gene therapies. However, tissue-selective transduction after systemic delivery remains a challenge. Inducible degrader of low-density lipoprotein receptor is an attractive target for treating hypercholesterolemia. Here, a liver-targeted LV, CS8-LV-shIDOL, is developed by incorporating a hepatocyte-targeted peptide derived from circumsporozoite protein (CSP) into the lentivirus envelope for liver-targeted delivery of IDOL-shRNA (short hairpin RNA) to alleviate hypercholesterolemia. Tail-vein injection of CS8-LV-shIDOL results in extremely high accumulation in liver and nearly undetectable levels in other organs in mice. In addition, it shows superior therapeutic efficacy in lowering serum low-density lipoprotein cholesterol (LDL-C) and reducing atherosclerotic lesions over unmodified LV-shIDOL in hyperlipidemic mice. Mechanically, the envelope-engineered CS8-LV-shIDOL can enter liver cells via low-density lipoprotein receptor-related protein (LRP). Thus, this study provides a novel approach for liver-targeted delivery of IDOL-shRNA to treat hypercholesterolemia by using an envelope-engineered LV, and this delivery system has great potential for liver-targeted transgene therapy.

Single intravitreal administration of a tetravalent siRNA exhibits robust and efficient gene silencing in mouse and pig photoreceptors.

Inherited retinal dystrophies caused by dominant mutations in photoreceptor (PR) cell expressed genes are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets PR cell expressed genes. Here, we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best PR accumulation. A single intravitreal administration induces multimonths efficacy in rodent and porcine retinas while demonstrating a good safety profile. The data suggest that this configuration can treat retinal diseases caused by PR cell expressed genes with 1-2 intravitreal injections per year.

CD3 aptamers promote expansion and persistence of tumor-reactive T cells for adoptive T cell therapy in cancer.

The CD3/T cell receptor (TCR) complex is responsible for antigen-specific pathogen recognition by T cells, and initiates the signaling cascade necessary for activation of effector functions. CD3 agonistic antibodies are commonly used to expand T lymphocytes in a wide range of clinical applications, including in adoptive T cell therapy for cancer patients. A major drawback of expanding T cell populations ex vivo using CD3 agonistic antibodies is that they expand and activate T cells independent of their TCR antigen specificity. Therapeutic agents that facilitate expansion of T cells in an antigen-specific manner and reduce their threshold of T cell activation are therefore of great interest for adoptive T cell therapy protocols. To identify CD3-specific T cell agonists, several RNA aptamers were selected against CD3 using Systematic Evolution of Ligands by EXponential enrichment combined with high-throughput sequencing. The extent and specificity of aptamer binding to target CD3 were assessed through surface plasma resonance, P32 double-filter assays, and flow cytometry. Aptamer-mediated modulation of the threshold of T cell activation was observed in vitro and in preclinical transgenic TCR mouse models. The aptamers improved efficacy and persistence of adoptive T cell therapy by low-affinity TCR-reactive T lymphocytes in melanoma-bearing mice. Thus, CD3-specific aptamers can be applied as therapeutic agents which facilitate the expansion of tumor-reactive T lymphocytes while conserving their tumor specificity. Furthermore, selected CD3 aptamers also exhibit cross-reactivity to human CD3, expanding their potential for clinical translation and application in the future.

Oligonucleotide therapies for nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) represents a severe disease subtype of nonalcoholic fatty liver disease (NAFLD) that is thought to be highly associated with systemic metabolic abnormalities. It is characterized by a series of substantial liver damage, including hepatocellular steatosis, inflammation, and fibrosis. The end stage of NASH, in some cases, may result in cirrhosis and hepatocellular carcinoma (HCC). Nowadays a large number of investigations are actively under way to test various therapeutic strategies, including emerging oligonucleotide drugs (e.g., antisense oligonucleotide, small interfering RNA, microRNA, mimic/inhibitor RNA, and small activating RNA) that have shown high potential in treating this fatal liver disease. This article systematically reviews the pathogenesis of NASH/NAFLD, the promising druggable targets proven by current studies in chemical compounds or biological drug development, and the feasibility and limitations of oligonucleotide-based therapeutic approaches under clinical or pre-clinical studies.

Tissue pharmacokinetics of antisense oligonucleotides.

Pharmacokinetics (PK) of antisense oligonucleotides (ASOs) is characterized by rapid distribution from plasma to tissue and slow terminal plasma elimination driven by re-distribution from tissue. Quantitative understanding of tissue PK and RNA knockdown for various ASO chemistries, conjugations, and administration routes is critical for successful drug discovery. Here, we report concentration-time and RNA knockdown profiles for a gapmer ASO with locked nucleic acid ribose chemistry in mouse liver, kidney, heart, and lung after subcutaneous and intratracheal administration. Additionally, the same ASO with liver targeting conjugation (galactosamine-N-acetyl) is evaluated for subcutaneous administration. Data indicate that exposure and knockdown differ between tissues and strongly depend on administration route and conjugation. In a second study, we show that tissue PK is similar between the three different ribose chemistries locked nucleic acid, constrained ethyl and 2'-O-methoxyethyl, both after subcutaneous and intratracheal administration. Further, we show that the half-life in mouse liver may vary with ASO sequence. Finally, we report less than dose-proportional increase in liver concentration in the dose range of 3-30 µmol/kg. Overall, our studies contribute pivotal data to support design and interpretation of ASO in vivo studies, thereby increasing the probability of delivering novel ASO therapies to patients.

Unique quinoline orientations shape the modified aptamer to sclerostin for enhanced binding affinity and bone anabolic potential.

Osteogenesis imperfecta (OI) is a rare genetic disease characterized by bone fragility and bone formation. Sclerostin could negatively regulate bone formation by antagonizing the Wnt signal pathway, whereas it imposes severe cardiac ischemic events in clinic. Our team has screened an aptamer that could promote bone anabolic potential without cardiovascular risk. However, the affinity of the aptamer is lower and needs to be improved. In the study, hydrophobic quinoline molecule with unique orientations (seven subtypes) were incorporated into key sites of a bone anabolic aptamer against sclerostin to form a modified aptamer library. Among all the quinoline modifications, 5-quinoline modification could shape the molecular recognition of modified aptamers to sclerostin to facilitate enhancing its binding to sclerostin toward the highest affinity by interacting with newly participated binding sites in sclerostin. Further, 5-quinoline modification could facilitate the modified aptamer attenuating the suppressed effect of the transfected sclerostin on both Wnt signaling and bone formation marker expression levels in vitro, promoting bone anabolism in OI mice (Col1a2+/G610C). The proposed quinoline-oriented modification strategy could shape the molecular recognition of modified aptamers to proteins to facilitate enhancing its binding affinity and therapeutic potency.

Oligo-PROTAC strategy for cell-selective and targeted degradation of activated STAT3.

Decoy oligodeoxynucleotides (ODNs) allow targeting undruggable transcription factors, such as STAT3, but their limited potency and lack of delivery methods hampered translation. To overcome these challenges, we conjugated a STAT3-specific decoy to thalidomide, a ligand to cereblon in E3 ubiquitin ligase complex, to generate a proteolysis-targeting chimera (STAT3DPROTAC). STAT3DPROTAC downregulated STAT3 in target cells, but not STAT1 or STAT5. Computational modeling of the STAT3DPROTAC ternary complex predicted two surface lysines, K601 and K626, in STAT3 as potential ubiquitination sites. Accordingly, K601/K626 point mutations in STAT3, as well as proteasome inhibition or cereblon deletion, alleviated STAT3DPROTAC effect. Next, we conjugated STAT3DPROTAC to a CpG oligonucleotide targeting Toll-like receptor 9 (TLR9) to generate myeloid/B cell-selective C-STAT3DPROTAC. Naked C-STAT3DPROTAC was spontaneously internalized by TLR9+ myeloid cells, B cells, and human and mouse lymphoma cells but not by T cells. C-STAT3DPROTAC effectively decreased STAT3 protein levels and also STAT3-regulated target genes critical for lymphoma cell proliferation and/or survival (BCL2L1, CCND2, and MYC). Finally, local C-STAT3DPROTAC administration to human Ly3 lymphoma-bearing mice triggered tumor regression, while control C-STAT3D and C-SCR treatments had limited effects. Our results underscore the feasibility of using a PROTAC strategy for cell-selective, decoy oligonucleotide-based STAT3 targeting of and potentially other tumorigenic transcription factors for cancer therapy.

Anti-miR-141-3p maintains homeostasis between autophagy and apoptosis by targeting Yy1 in the fetal lumbosacral defecation center of rats.

Anorectal malformations (ARMs) are congenital diseases that lead to postoperative fecal incontinence, constipation, and soiling, despite improvements in surgery; however, their pathological mechanisms remain unclear. Here, we report the role of microRNA-141-3p in maintaining homeostasis between apoptosis and autophagy in the lumbosacral defecation center of fetal rats with ARMs. Elevated microRNA-141-3p expression inhibited YIN-YANG-1 expression by binding its 3' UTR, and repressed autophagy and triggered apoptosis simultaneously. Then, adenylate cyclase 3 was screened to be the downstream target gene of YIN-YANG-1 by chromatin immunoprecipitation sequencing experiments, and Yin Yang 1 could positively activate the transcription of adenylate cyclase 3 by directly interacting with the motif GAGATGG and ATGG in its promoter. Intraamniotic microinjection of adeno-rno-microRNA-141-3p-sponge-GFP in fetal rats with ARMs on embryonic day 15 restored apoptosis-autophagy homeostasis. These findings reveal that microRNA-141-3p upregulation impaired homeostasis between apoptosis and autophagy by inhibiting the YIN-YANG-1/adenylate cyclase 3 axis, and that intraamniotic injection of anti-microRNA-141-3p helped maintain homeostasis in the lumbosacral defecation center of ARMs during embryogenesis.

Optimized allele-specific silencing of the dominant-negative COL6A1 G293R substitution causing collagen VI-related dystrophy.

Collagen VI-related dystrophies (COL6-RDs) are a group of severe, congenital-onset muscular dystrophies for which there is no effective causative treatment. Dominant-negative mutations are common in COL6A1, COL6A2, and COL6A3 genes, encoding the collagen α1, α2, and α3 (VI) chains. They act by incorporating into the hierarchical assembly of the three α (VI) chains and consequently produce a dysfunctional collagen VI extracellular matrix, while haploinsufficiency for any of the COL6 genes is not associated with disease. Hence, allele-specific transcript inactivation is a valid therapeutic strategy, although selectively targeting a pathogenic single nucleotide variant is challenging. Here, we develop a small interfering RNA (siRNA) that robustly, and in an allele-specific manner, silences a common glycine substitution (G293R) caused by a single nucleotide change in COL6A1 gene. By intentionally introducing an additional mismatch into the siRNA design, we achieved enhanced specificity toward the mutant allele. Treatment of patient-derived fibroblasts effectively reduced the levels of mutant transcripts while maintaining unaltered wild-type transcript levels, rescuing the secretion and assembly of collagen VI matrix by reducing the dominant-negative effect of mutant chains. Our findings establish a promising treatment approach for patients with the recurrent dominantly negative acting G293R glycine substitution.

A novel multitargeted self-assembling peptide-siRNA complex for simultaneous inhibition of SARS-CoV-2-host cell interaction and replication.

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.

Strategies to improve the design of gapmer antisense oligonucleotide on allele-specific silencing.

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.

Topical application of a CCL22-binding aptamer suppresses contact allergy.

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.

The roles of CD4+ T cell help, sex, and dose in the induction of protective CD8+ T cells against a lethal poxvirus by mRNA-LNP vaccines.

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.