Glis2 is an early effector of polycystin signaling and a target for therapy in polycystic kidney disease.

Mouse models of autosomal dominant polycystic kidney disease (ADPKD) show that intact primary cilia are required for cyst growth following the inactivation of polycystin-1. The signaling pathways underlying this process, termed cilia-dependent cyst activation (CDCA), remain unknown. Using translating ribosome affinity purification RNASeq on mouse kidneys with polycystin-1 and cilia inactivation before cyst formation, we identify the differential 'CDCA pattern' translatome specifically dysregulated in kidney tubule cells destined to form cysts. From this, Glis2 emerges as a candidate functional effector of polycystin signaling and CDCA. In vitro changes in Glis2 expression mirror the polycystin- and cilia-dependent changes observed in kidney tissue, validating Glis2 as a cell culture-based indicator of polycystin function related to cyst formation. Inactivation of Glis2 suppresses polycystic kidney disease in mouse models of ADPKD, and pharmacological targeting of Glis2 with antisense oligonucleotides slows disease progression. Glis2 transcript and protein is a functional target of CDCA and a potential therapeutic target for treating ADPKD.

Therapeutic targeting of telomerase ameliorates experimental choroidal neovascularization.

Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.

A novel transient receptor potential C3/C6 selective activator induces the cellular uptake of antisense oligonucleotides.

Antisense oligonucleotide (ASO) therapy is a novel therapeutic approach in which ASO specifically binds target mRNA, resulting in mRNA degradation; however, cellular uptake of ASOs remains critically low, warranting improvement. Transient receptor potential canonical (TRPC) channels regulate Ca2+ influx and are activated upon stimulation by phospholipase C-generated diacylglycerol. Herein, we report that a novel TRPC3/C6/C7 activator, L687, can induce cellular ASO uptake. L687-induced ASO uptake was enhanced in a dose- and incubation-time-dependent manner. L687 enhanced the knockdown activity of various ASOs both in vitro and in vivo. Notably, suppression of TRPC3/C6 by specific siRNAs reduced ASO uptake in A549 cells. Application of BAPTA-AM, a Ca2+ chelator, and SKF96365, a TRPC3/C6 inhibitor, suppressed Ca2+ influx via TRPC3/C6, resulting in reduced ASO uptake, thereby suggesting that Ca2+ influx via TRPC3/C6 is critical for L687-mediated increased ASO uptake. L687 also induced dextran uptake, indicating that L687 increased endocytosis. Adding ASO to L687 resulted in endosome accumulation; however, the endosomal membrane disruptor UNC7938 facilitated endosomal escape and enhanced knockdown activity. We discovered a new function for TRPC activators regarding ASO trafficking in target cells. Our findings provide an opportunity to formulate an innovative drug delivery system for the therapeutic development of ASO.

Antisense oligonucleotide therapeutic approach for Timothy syndrome.

Timothy syndrome (TS) is a severe, multisystem disorder characterized by autism, epilepsy, long-QT syndrome and other neuropsychiatric conditions1. TS type 1 (TS1) is caused by a gain-of-function variant in the alternatively spliced and developmentally enriched CACNA1C exon 8A, as opposed to its counterpart exon 8. We previously uncovered several phenotypes in neurons derived from patients with TS1, including delayed channel inactivation, prolonged depolarization-induced calcium rise, impaired interneuron migration, activity-dependent dendrite retraction and an unanticipated persistent expression of exon 8A2-6. We reasoned that switching CACNA1C exon utilization from 8A to 8 would represent a potential therapeutic strategy. Here we developed antisense oligonucleotides (ASOs) to effectively decrease the inclusion of exon 8A in human cells both in vitro and, following transplantation, in vivo. We discovered that the ASO-mediated switch from exon 8A to 8 robustly rescued defects in patient-derived cortical organoids and migration in forebrain assembloids. Leveraging a transplantation platform previously developed7, we found that a single intrathecal ASO administration rescued calcium changes and in vivo dendrite retraction of patient neurons, suggesting that suppression of CACNA1C exon 8A expression is a potential treatment for TS1. Broadly, these experiments illustrate how a multilevel, in vivo and in vitro stem cell model-based approach can identify strategies to reverse disease-relevant neural pathophysiology.

Peptide nanocarriers co-delivering an antisense oligonucleotide and photosensitizer elicit synergistic cytotoxicity.

Combination therapies demand co-delivery platforms with efficient entrapment of distinct payloads and specific delivery to cells and possibly organelles. Herein, we introduce the combination of two therapeutic modalities, gene and photodynamic therapy, in a purely peptidic platform. The simultaneous formation and cargo loading of the multi-micellar platform is governed by self-assembly at the nanoscale. The multi-micellar architecture of the nanocarrier and the positive charge of its constituent micelles offer controlled dual loading capacity with distinct locations for a hydrophobic photosensitizer (PS) and negatively charged antisense oligonucleotides (ASOs). Moreover, the nuclear localization signal (NLS) sequence built-in the peptide targets PS + ASO-loaded nanocarriers to the nucleus. Breast cancer cells treated with nanocarriers demonstrated photo-triggered enhancement of radical oxygen species (ROS) associated with increased cell death. Besides, delivery of ASO payloads resulted in up to 90 % knockdown of Bcl-2, an inhibitor of apoptosis that is overexpressed in more than half of all human cancers. Simultaneous delivery of PS and ASO elicited synergistic apoptosis to an extent that could not be reached by singly loaded nanocarriers or the free form of the drugs. Both, the distinct location of loaded compounds that prevents them from interfering with each other, and the highly efficient cellular delivery support the great potential of this versatile peptide platform in combination therapy.

Inhibition of MER proto-oncogene tyrosine kinase by an antisense oligonucleotide enhances treatment efficacy of immunoradiotherapy.

BACKGROUND: The combination of radiotherapy and immunotherapy (immunoradiotherapy) has been increasingly used for treating a wide range of cancers. However, some tumors are resistant to immunoradiotherapy. We have previously shown that MER proto-oncogene tyrosine kinase (MerTK) expressed on macrophages mediates resistance to immunoradiotherapy. We therefore sought to develop therapeutics that can mitigate the negative impact of MerTK. We designed and developed a MerTK specific antisense oligonucleotide (ASO) and characterized its effects on eliciting an anti-tumor immune response in mice. METHODS: 344SQR cells were injected into the right legs on day 0 and the left legs on day 4 of 8-12 weeks old female 129sv/ev mice to establish primary and secondary tumors, respectively. Radiation at a dose of 12 Gy was given to the primary tumors on days 8, 9, and 10. Mice received either anti-PD-1, anti-CTLA-4 or/and MerTK ASO starting from day 1 post tumor implantation. The composition of the tumor microenvironment and the level of MerTK on macrophages in the tumor were evaluted by flow cytometry. The expression of immune-related genes was investigated with NanoString. Lastly, the impact of MerTK ASO on the structure of the eye was histologically evaluated. RESULTS: Remarkably, the addition of MerTK ASO to XRT+anti-PD1 and XRT+anti-CTLA4 profoundly slowed the growth of both primary and secondary tumors and significantly extended survival. The ASO significantly reduced the expression of MerTK in tumor-associated macrophages (TAMs), reprograming their phenotype from M2 to M1. In addition, MerTK ASO increased the percentage of Granzyme B+ CD8+ T cells in the secondary tumors when combined with XRT+anti-CTLA4. NanoString results demonstrated that the MerTK ASO favorably modulated immune-related genes for promoting antitumor immune response in secondary tumors. Importantly, histological analysis of eye tissues demonstrated that unlike small molecules, the MerTK ASO did not produce any detectable pathology in the eyes. CONCLUSIONS: The MerTK ASO can significantly downregulate the expression of MerTK on TAMs, thereby promoting antitumor immune response. The combination of MerTK ASO with immunoradiotherapy can safely and significantly slow tumor growth and improve survival.

Anisamide-conjugated hairpin antisense oligonucleotides prodrug co-delivering doxorubicin exhibited enhanced anticancer efficacy.

Antisense oligonucleotides (ASONs)-based therapeutics offers tremendous promise for the treatment of diverse diseases. However, there is still a need to develop ASONs with enhanced stability against enzymes, improved drug delivery, and enhanced biological potency. In this study, we propose a novel anisamide (AA)-conjugated hairpin oligonucleotide prodrug loading with chemotherapeutic agent (doxorubicin, DOX) (AA-loop-ASON/DOX) for oncotherapy. Results indicated that the introduction of a hairpin conformation and AA ligand in prodrug significantly improved the stability against enzymatic hydrolysis, as well as the cellar uptake of ASONs and DOX. The incorporation of disulfide bonds could trigger mechanical opening, resulting in the release of ASON and DOX in response to the intracellular glutathione (GSH) in tumors. Moreover, the composite of DOX-loading ASONs prodrug exhibited a robust and selective inhibition of tumor cell proliferation. This paper introduces a novel design concept for nucleic acid-based therapeutics, aiming to enhance the delivery of drug and improve biological effectiveness.

A comparative analysis of peptide-delivered antisense antibiotics using diverse nucleotide mimics.

Antisense oligomer (ASO)-based antibiotics that target mRNAs of essential bacterial genes have great potential for counteracting antimicrobial resistance and for precision microbiome editing. To date, the development of such antisense antibiotics has primarily focused on using phosphorodiamidate morpholino (PMO) and peptide nucleic acid (PNA) backbones, largely ignoring the growing number of chemical modalities that have spurred the success of ASO-based human therapy. Here, we directly compare the activities of seven chemically distinct 10mer ASOs, all designed to target the essential gene acpP upon delivery with a KFF-peptide carrier into Salmonella. Our systematic analysis of PNA, PMO, phosphorothioate (PTO)-modified DNA, 2'-methylated RNA (RNA-OMe), 2'-methoxyethylated RNA (RNA-MOE), 2'-fluorinated RNA (RNA-F), and 2'-4'-locked RNA (LNA) is based on a variety of in vitro and in vivo methods to evaluate ASO uptake, target pairing and inhibition of bacterial growth. Our data show that only PNA and PMO are efficiently delivered by the KFF peptide into Salmonella to inhibit bacterial growth. Nevertheless, the strong target binding affinity and in vitro translational repression activity of LNA and RNA-MOE make them promising modalities for antisense antibiotics that will require the identification of an effective carrier.

Eplontersen: First Approval.

Eplontersen (Wainua™) is a ligand-conjugated antisense oligonucleotide directed to TTR, which is being developed by Ionis Pharmaceuticals and AstraZeneca for the treatment of TTR-mediated amyloidosis (ATTR). Eplontersen, which is targeted to the liver by a ligand containing three N-acetyl galactosamine residues, binds to wild-type and variant TTR mRNA, thus reducing the levels of circulating TTR protein and amyloid deposition. Subcutaneous eplontersen reduced serum TTR levels, inhibited neuropathy progression and improved health-related quality of life in patients with polyneuropathy of hereditary ATTR (ATTRv-PN; v for variant) in a phase III trial. Based on these results, eplontersen was approved in the USA for the treatment of ATTRv-PN on 21 December 2023 and is currently undergoing regulatory review for a similar indication in the EU, the UK, Switzerland and Canada. Eplontersen is also undergoing phase III development for ATTR cardiomyopathy. This article summarizes the milestones in the development of eplontersen leading to this first approval for ATTRv-PN.

Theranostic DNA nanostructure based on phenotype-specific activation of antisense oligonucleotides.

Antisense oligonucleotide (ASO) is a powerful agent for gene therapy, designed to form complementary pairs with specific mRNA to inhibit gene expression. However, low specificity limits its potential. To overcome this challenge, we developed a Y-shape DNA nanostructure that enhances the specificity in ASO-based treatment by introducing a detection trigger. The design incorporates the phenotype-specific miR21 activation and the sequential release of Bcl2 ASO. As a result, our Y-shape DNA nanostructure downregulates >50 % Bcl2 mRNA expression and induces >60 % cell death in breast cancer cells. Meanwhile, this approach shows no obvious damage to the non-cancerous cells, indicating the therapeutic potential as a theranostics agent in precision medicine with the combination of biomarker sensing and treatment. Overall, our Y-shape DNA nanostructure serves as a promising strategy providing potential in customized conformation design with specific target sequences in gene therapy.

Impact of the Inhibition of Organic Anion Transporter on Tricyclo-DNA-Mediated Exon Skipping in the mdx Mouse Model.

Antisense-mediated exon skipping is one of the most promising therapeutic strategies for Duchenne muscular dystrophy (DMD) and some antisense oligonucleotide (ASO) drugs have already been approved by the U.S. FDA for DMD. The potential of this therapy is still limited by several challenges including the poor distribution of ASOs to target tissues. Indeed, most of them accumulate in the kidney and tend to be rapidly eliminated after systemic delivery. We hypothesized here that preventing renal clearance of ASO using organic anion transporter (OAT) inhibitor could increase the bioavailability of ASOs and thus their distribution to target tissues and ultimately their efficacy in muscles. Mdx mice were, therefore, treated with ASO with or without the OAT inhibitor named probenecid. Our findings indicate that OAT inhibition, or at least using probenecid, does not improve the therapeutic potential of ASO-mediated exon-skipping approaches for the treatment of DMD.

Systemic administration of NIS-lncRNA antisense oligonucleotide alleviates neuropathic pain.

OBJECTIVE: The antisense oligonucleotide (ASO) is an FDA-approved strategy in the treatment of neurological diseases. We have shown the viability of using intrathecal ASO to suppress nerve injury-specific long noncoding RNA (NIS-lncRNA) in dorsal root ganglion (DRG), resulting in a stable and long-lasting antinociceptive effect on NP. This study examined whether systemic administration of NIS-lncRNA ASO relieved the chronic constriction injury (CCI)-induced nociceptive hypersensitivity. METHODS: A single subcutaneous injection of NIS-lncRNA ASO at a dose of 1,000 µg was carried out 7 days after CCI or sham surgery in male mice. Behavioral tests were performed one day before surgery and at different days after surgery. DRG and spinal cord were finally collected for quantitative real-time RT-PCR and Western blot assays. RESULTS: NIS-lncRNA ASO significantly alleviated CCI-induced mechanical allodynia, heat hyperalgesia, and cold hyperalgesia starting on day 14 or 21 post-ASO injection and lasting for at least 7 days on the ipsilateral side. Additionally, CCI-induced spontaneous pain and ipsilateral dorsal horn neuronal and astrocyte hyperactivation were blocked on day 28 after NIS-lncRNA ASO injection. As predicted, the CCI-induced increases in the levels of NIS-lncRNA and its downstream target C-C motif chemokine ligand 2 in the ipsilateral lumbar 3 and 4 DRGs were attenuated on day 28 following NIS-lncRNA ASO injection. CONCLUSION: Our findings indicate that systemic administration of NIS-lncRNA ASO also produces a stable and long-lasting antinociceptive effect on neuropathic pain. NIS-lncRNA ASO may have potential clinical application in the treatment of this disorder.

Antiviral Efficacy of RNase H-Dependent Gapmer Antisense Oligonucleotides against Japanese Encephalitis Virus.

RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.

High-order framework nucleic acid for targeted-delivery of antisense peptide nucleic acids to overcome drug resistance.

A sophisticated high-order framework nucleic acid (FNA) was engineered for the targeted delivery and responsive release of environment tolerant antisense peptide nucleic acids (asPNAs). The dendritic FNA-asPNAs system was constructed via simple one-pot modular assembly and demonstrated a good synergistic effect with chemotherapy on drug resistant cancer cells.

TGF-ß1 signaling and Smad7 control T-cell responses in health and immune-mediated disorders.

Transforming growth factor (TGF)-ß1, a member of the TGF-ß superfamily, is produced by many immune and nonimmune cells and has pleiotropic effects on both innate and adaptive immunity, especially in the control of T-cell differentiation and function. Consistently, loss of TGF-ß1 function is associated with exacerbated T-cell-dependent inflammatory responses that culminate in pathological processes in allergic and immune-mediated diseases. In this review, we highlight the roles of TGF-ß1 in immunity, focusing mainly on its ability to promote differentiation of regulatory T cells, T helper (Th)-17, and Th9 cells, thus contributing to amplifying or restricting T-cell responses in health and human diseases (e.g., inflammatory bowel diseases, type 1 diabetes, asthma, and MS). In addition, we discuss the involvement of Smad7, an inhibitor of TGF-ß1 signaling, in immune-mediated disorders (e.g., psoriasis, rheumatoid arthritis, MS, and inflammatory bowel diseases), as well as the discordant results of clinical trials with mongersen, an oral pharmaceutical compound containing a Smad7 antisense oligonucleotide, in patients with Crohn's disease. Further work is needed to ascertain the reasons for such a discrepancy as well as to identify better candidates for treatment with Smad7 inhibitors.

Delivery Characterization of SPL84 Inhaled Antisense Oligonucleotide Drug for 3849 + 10 kb C- > T Cystic Fibrosis Patients.

Recent advances in the therapeutic potential of RNA-related treatments, specifically for antisense oligonucleotide (ASO)-based drugs, have led to increased numbers of ASO regulatory approvals. In this study, we focus on SPL84, an inhaled ASO-based drug, developed for the treatment of the pulmonary disease cystic fibrosis (CF). Pulmonary drug delivery is challenging, due to a variety of biological, physical, chemical, and structural barriers, especially when targeting the cell nucleus. The distribution of SPL84 throughout the lungs, penetration into the epithelial cells and nucleus, and structural stability are critical parameters that will impact drug efficacy in a clinical setting. In this study, we demonstrate broad distribution, as well as cell and nucleus penetration of SPL84 in mouse and monkey lungs. In vivo and in vitro studies confirmed the stability of our inhaled drug in CF patient-derived mucus and in lung lysosomal extracts. The mobility of SPL84 through hyperconcentrated mucus was also demonstrated. Our results, supported by a promising preclinical pharmacological effect of full restoration of cystic fibrosis transmembrane conductance regulator channel activity, emphasize the high potential of SPL84 as an effective drug for the treatment of CF patients. In addition, successfully tackling the lung distribution of SPL84 offers immense opportunities for further development of SpliSense's inhaled ASO-based drugs for unmet needs in pulmonary diseases.

Novel strategy of liver cancer treatment with modified antisense oligonucleotides targeting human vasohibin-2.

Vasohihibin-2 (VASH2) is a homolog of vasohibin-1 (VASH1) and is overexpressed in various cancers. Vasohihibin-2 acts on both cancer cells and cancer microenvironmental cells. Previous analyses have shown that VASH2 promotes cancer progression and abrogation of VASH2 results in significant anticancer effects. We therefore propose VASH2 to be a practical molecular target for cancer treatment. Modifications of antisense oligonucleotide (ASO) such as bridged nucleic acids (BNA)-based modification increases the specificity and stability of ASO, and are now applied to the development of a number of oligonucleotide-based drugs. Here we designed human VASH2-ASOs, selected an optimal one, and developed 2',4'-BNA-based VASH2-ASO. When systemically administered, naked 2',4'-BNA-based VASH2-ASO accumulated in the liver and showed its gene-silencing activity. We then examined the effect of 2',4'-BNA-based VASH2-ASO in liver cancers. Intraperitoneal injection of naked 2',4'-BNA-based VASH2-ASO exerted a potent antitumor effect on orthotopically inoculated human hepatocellular carcinoma cells. The same manipulation also showed potent antitumor activity on the splenic inoculation of human colon cancer cells for liver metastasis. These results provide a novel strategy for the treatment of primary as well as metastatic liver cancers by using modified ASOs targeting VASH2.

Targeting LncRNA LLNLR-299G3.1 with antisense oligonucleotide inhibits malignancy of esophageal squamous cell carcinoma cells in vitro and in vivo.

Accumulating evidence has indicated that long non-coding RNAs (lncRNAs) play critical roles in the development and progression of cancers, including esophageal squamous cell carcinoma (ESCC). However, the mechanisms of lncRNAs in ESCC are still incompletely understood and therapeutic attempts for in vivo targeting cancer-associated lncRNA remain a challenge. By RNA-sequencing analysis, we identified that LLNLR-299G3.1 was a novel ESCC-associated lncRNA. LLNLR-299G3.1 was up-regulated in ESCC tissues and cells and promoted ESCC cell proliferation and invasion. Silencing of LLNLR-299G3.1 with ASO (antisense oligonucleotide) resulted in opposite effects. Mechanistically, LLNLR-299G3.1 bound to cancer-associated RNA binding proteins and regulated the expression of cancer-related genes, including OSM, TNFRSF4, HRH3, and SSTR3. ChIRP-seq (chromatin isolation by RNA purification and sequencing) revealed that these genes contained enriched chromatin binding sites for LLNLR-299G3.1. Rescue experiments confirmed that the effects of LLNLR-299G3.1 on ESCC cell proliferation were dependent on interaction with HRH3 and TNFRSF4. Therapeutically, intravenous delivery of placental chondroitin sulfate A binding peptide-coated nanoparticles containing antisense oligonucleotide (pICSA-BP-ANPs) strongly inhibited ESCC tumor growth and significantly improved animal survival in vivo. Overall, our results suggest that LLNLR-299G3.1 promotes ESCC malignancy through regulating gene-chromatin interactions and targeting ESCC by pICSA-BP-ANPs may be an effective strategy for the treatment of lncRNA-associated ESCC.

mTOR Inhibition Enhances Delivery and Activity of Antisense Oligonucleotides in Uveal Melanoma Cells.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Owing to a lack of effective treatments, patients with metastatic disease have a median survival time of 6-12 months. We recently demonstrated that the Survival Associated Mitochondrial Melanoma Specific Oncogenic Non-coding RNA (SAMMSON) is essential for UM cell survival and that antisense oligonucleotide (ASO)-mediated silencing of SAMMSON impaired cell viability and tumor growth in vitro and in vivo. By screening a library of 2911 clinical stage compounds, we identified the mammalian target of rapamycin (mTOR) inhibitor GDC-0349 to synergize with SAMMSON inhibition in UM. Mechanistic studies revealed that mTOR inhibition enhanced uptake and reduced lysosomal accumulation of lipid complexed SAMMSON ASOs, improving SAMMSON knockdown and further decreasing UM cell viability. We found mTOR inhibition to also enhance target knockdown in other cancer cell lines as well as normal cells when combined with lipid nanoparticle complexed or encapsulated ASOs or small interfering RNAs (siRNAs). Our results are relevant to nucleic acid treatment in general and highlight the potential of mTOR inhibition to enhance ASO and siRNA-mediated target knockdown.

FXYD2 antisense oligonucleotide provides an efficient approach for long-lasting relief of chronic peripheral pain.

Chronic pain, whether of inflammatory or neuropathic origin, affects about 18% of the population of developed countries, and most current treatments are only moderately effective and/or cause serious side effects. Therefore, the development of novel therapeutic approaches still represents a major challenge. The Na,K-ATPase modulator FXYD2 is critically required for the maintenance of neuropathic pain in rodents. Here, we set up a therapeutic protocol based on the use of chemically modified antisense oligonucleotides (ASOs) to inhibit FXYD2 expression and treat chronic pain. We identified an ASO targeting a 20-nucleotide stretch in the FXYD2 mRNA that is evolutionarily conserved between rats and humans and is a potent inhibitor of FXYD2 expression. We used this sequence to synthesize lipid-modified forms of ASO (FXYD2-LASO) to facilitate their entry into dorsal root ganglia neurons. We established that intrathecal or intravenous injections of FXYD2-LASO in rat models of neuropathic or inflammatory pain led to a virtually complete alleviation of their pain symptoms, without causing obvious side effects. Remarkably, by using 2'-O-2-methoxyethyl chemical stabilization of the ASO (FXYD2-LASO-Gapmer), we could significantly prolong the therapeutic action of a single treatment up to 10 days. This study establishes FXYD2-LASO-Gapmer administration as a promising and efficient therapeutic strategy for long-lasting relief of chronic pain conditions in human patients.