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.

The role of voltage-gated calcium channel α2δ-1 in the occurrence and development in myofascial orofacial pain.

Patients who suffer from myofascial orofacial pain could affect their quality of life deeply. The pathogenesis of pain is still unclear. Our objective was to assess Whether Voltage-gated calcium channel α2δ-1(Cavα2δ-1) is related to myofascial orofacial pain. Rats were divided into the masseter tendon ligation group and the sham group. Compared with the sham group, the mechanical pain threshold of the masseter tendon ligation group was reduced on the 4th, 7th, 10th and 14th day after operation(P < 0.05). On the 14th day after operation, Cavα2δ-1 mRNA expression levels in trigeminal ganglion (TG) and the trigeminal spinal subnucleus caudalis and C1-C2 spinal cervical dorsal horn (Vc/C2) of the masseter tendon ligation group were increased (PTG=0.021, PVc/C2=0.012). Rats were divided into three groups. On the 4th day after ligating the superficial tendon of the left masseter muscle of the rats, 10 ul Cavα2δ-1 antisense oligonucleotide, 10 ul Cavα2δ-1 mismatched oligonucleotides and 10 ul normal saline was separately injected into the left masseter muscle of rats in Cavα2δ-1 antisense oligonucleotide group, Cavα2δ-1 mismatched oligonucleotides group and normal saline control group twice a day for 4 days. The mechanical pain threshold of the Cavα2δ-1 antisense oligonucleotides group was higher than Cavα2δ-1 mismatched oligonucleotides group on the 7th and 10th day after operation (P < 0.01). After PC12 cells were treated with lipopolysaccharide, Cavα2δ-1 mRNA expression level increased (P < 0.001). Cavα2δ-1 may be involved in the occurrence and development in myofascial orofacial pain.

Introduction of sugar-modified nucleotides into CpG-containing antisense oligonucleotides inhibits TLR9 activation.

Antisense oligonucleotides (ASOs) are synthetic single-stranded oligonucleotides that bind to RNAs through Watson-Crick base pairings. They are actively being developed as therapeutics for various human diseases. ASOs containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are known to trigger innate immune responses via interaction with toll-like receptor 9 (TLR9). However, the TLR9-stimulatory properties of ASOs, specifically those with lengths equal to or less than 20 nucleotides, phosphorothioate linkages, and the presence and arrangement of sugar-modified nucleotides-crucial elements for ASO therapeutics under development-have not been thoroughly investigated. In this study, we first established SY-ODN18, an 18-nucleotide phosphorothioate oligodeoxynucleotide with sufficient TLR9-stimulatory activity. We demonstrated that an unmethylated CpG motif near its 5'-end was indispensable for TLR9 activation. Moreover, by utilizing various sugar-modified nucleotides, we systematically generated model ASOs, including gapmer, mixmer, and fully modified designs, in accordance with the structures of ASO therapeutics. Our results illustrated that introducing sugar-modified nucleotides in such designs significantly reduces TLR9-stimulatory activity, even without methylation of CpG motifs. These findings would be useful for drug designs on several types of ASOs.

Effects of local reduction of endogenous α-synuclein using antisense oligonucleotides on the fibril-induced propagation of pathology through the neural network in wild-type mice.

In Parkinson's disease and other synucleinopathies, fibrillar forms of α-synuclein (aSyn) are hypothesized to structurally convert and pathologize endogenous aSyn, which then propagates through the neural connections, forming Lewy pathologies and ultimately causing neurodegeneration. Inoculation of mouse-derived aSyn preformed fibrils (PFFs) into the unilateral striatum of wild-type mice causes widespread aSyn pathologies in the brain through the neural network. Here, we used the local injection of antisense oligonucleotides (ASOs) against Snca mRNA to confine the area of endogenous aSyn protein reduction and not to affect the PFFs properties in this model. We then varied the timing and location of ASOs injection to examine their impact on the initiation and propagation of aSyn pathologies in the whole brain and the therapeutic effect using abnormally-phosphorylated aSyn (pSyn) as an indicator. By injecting ASOs before or 0-14 days after the PFFs were inoculated into the same site in the left striatum, the reduction in endogenous aSyn in the striatum leads to the prevention and inhibition of the regional spread of pSyn pathologies to the whole brain including the contralateral right hemisphere. ASO post-injection inhibited extension from neuritic pathologies to somatic ones. Moreover, injection of ASOs into the right striatum prevented the remote regional spread of pSyn pathologies from the left striatum where PFFs were inoculated and no ASO treatment was conducted. This indicated that the reduction in endogenous aSyn protein levels at the propagation destination site can attenuate pSyn pathologies, even if those at the propagation initiation site are not inhibited, which is consistent with the original concept of prion-like propagation that endogenous aSyn is indispensable for this regional spread. Our results demonstrate the importance of recruiting endogenous aSyn in this neural network propagation model and indicate a possible potential for ASO treatment in synucleinopathies.

Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease.

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.

Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer's disease.

BACKGROUND: Microglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer's disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs). METHODS: In this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo. RESULTS: We proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-ß plaques in vivo in a model of AD. CONCLUSIONS: This study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

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.

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-mediated terminal intron retention of endoglin: A potential strategy to inhibit renal interstitial fibrosis.

TGF-ß is considered an important cytokine in the development of interstitial fibrosis in chronic kidney disease. The TGF-ß co-receptor endoglin (ENG) tends to be upregulated in kidney fibrosis. ENG has two membrane bound isoforms generated via alternative splicing. Long-ENG was shown to enhance the extent of renal fibrosis in an unilateral ureteral obstruction mouse model, while short-ENG inhibited renal fibrosis. Here we aimed to achieve terminal intron retention of endoglin using antisense-oligo nucleotides (ASOs), thereby shifting the ratio towards short-ENG to inhibit the TGF-ß1-mediated pro-fibrotic response. We isolated mRNA from kidney biopsies of patients with chronic allograft disease (CAD) (n = 12) and measured total ENG and short-ENG mRNA levels. ENG mRNA was upregulated 2.3 fold (p < 0.05) in kidneys of CAD patients compared to controls, while the percentage short-ENG of the total ENG mRNA was significantly lower (1.8 fold; p < 0.05). Transfection of ASOs that target splicing regulatory sites of ENG into TK173 fibroblasts led to higher levels of short-ENG (2 fold; p < 0.05). In addition, we stimulated these cells with TGF-ß1 and measured a decrease in upregulation of ACTA2, COL1A1 and FN1 mRNA levels, and protein expression of αSMA, collagen type I, and fibronectin. These results show a potential for ENG ASOs as a therapy to reduce interstitial fibrosis in CKD.

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.

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.

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration.

Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon-intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

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.

Interferon-α receptor antisense oligonucleotides reduce neuroinflammation and neuropathology in a mouse model of cerebral interferonopathy.

Chronic and elevated levels of the antiviral cytokine IFN-α in the brain are neurotoxic. This is best observed in patients with genetic cerebral interferonopathies such as Aicardi-Goutières syndrome. Cerebral interferonopathies typically manifest in early childhood and lead to debilitating disease and premature death. There is no cure for these diseases with existing treatments largely aimed at managing symptoms. Thus, an effective therapeutic strategy is urgently needed. Here, we investigated the effect of antisense oligonucleotides targeting the murine IFN-α receptor (Ifnar1 ASOs) in a transgenic mouse model of cerebral interferonopathy. Intracerebroventricular injection of Ifnar1 ASOs into transgenic mice with brain-targeted chronic IFN-α production resulted in a blunted cerebral interferon signature, reduced neuroinflammation, restoration of blood-brain barrier integrity, absence of tissue destruction, and lessened neuronal damage. Remarkably, Ifnar1 ASO treatment was also effective when given after the onset of neuropathological changes, as it reversed such disease-related features. We conclude that ASOs targeting the IFN-α receptor halt and reverse progression of IFN-α-mediated neuroinflammation and neurotoxicity, opening what we believe to be a new and promising approach for the treatment of patients with cerebral interferonopathies.

Biodistribution of Radioactively Labeled Splice Modulating Antisense Oligonucleotides After Intracerebroventricular and Intrathecal Injection in Mice.

Antisense oligonucleotides (AONs) are promising therapeutic candidates, especially for neurological diseases. Intracerebroventricular (ICV) injection is the predominant route of administration in mouse studies, while in clinical trials, intrathecal (IT) administration is mostly used. There is little knowledge on the differences in distribution of these injection methods within the same species over time. In this study, we compared the distribution of splice-switching AONs targeting exon 15 of amyloid precursor protein pre-mRNA injected via the ICV and IT route in mice. The AON was labeled with radioactive indium-111 and mice were imaged using single-photon emission computed tomography (SPECT) 0, 4, 24, 48, 72, and 96 h after injection. In vivo SPECT imaging showed 111In-AON activity diffused throughout the central nervous system (CNS) in the first hours after injection. The 111In-AON activity in the CNS persisted over the course of 4 days, while signal in the kidneys rapidly decreased. Postmortem counting in different organs and tissues showed very similar distribution of 111In-AON activity throughout the body, while the signal in the different brain regions was higher with ICV injection. Overall, IT and ICV injection have very similar distribution patterns in the mouse, but ICV injection is much more effective in reaching the brain.

Types of RNA therapeutics.

RNA therapy is one of the new treatments using small RNA molecules to target and regulate gene expression. It involves the application of synthetic or modified RNA molecules to inhibit the expression of disease-causing genes specifically. In other words, it silences genes and suppresses the transcription process. The main theory behind RNA therapy is that RNA molecules can prevent the translation into proteins by binding to specific messenger RNA (mRNA) molecules. By targeting disease-related mRNA molecules, RNA therapy can effectively silence or reduce the development of harmful proteins. There are different types of RNA molecules used in therapy, including small interfering RNAs (siRNAs), microRNAs (miRNAs), aptamer, ribozyme, and antisense oligonucleotides (ASOs). These molecules are designed to complement specific mRNA sequences, allowing them to bind and degrade the targeted mRNA or prevent its translation into protein. Nanotechnology is also highlighted to increase the efficacy of RNA-based drugs. In this chapter, while examining various methods of RNA therapy, we discuss the advantages and challenges of each.

Evaluation of antisense oligonucleotide therapy targeting Hsd17b13 in a fibrosis mice model.

Human genetic evidence suggests a protective role of loss-of-function variants in 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13) for liver fibrotic diseases. Although there is limited preclinical experimental data on Hsd17b13 antisense oligonucleotide (ASO) or siRNA in a fibrosis model, several ASO and siRNA approaches are being tested clinically as potential therapies for nonalcoholic steatohepatitis (NASH). The aim of this study was to assess the therapeutic potential of Hsd17b13 ASO in a preclinical advanced NASH-like hepatic fibrosis in vivo model. In vitro testing on primary hepatocytes demonstrated that Hsd17b13 ASO exhibited strong efficacy and specificity for knockdown of the Hsd17b13 gene. In choline-deficient, L-amino acid-defined, HFD (CDAHFD)-induced steatotic and fibrotic mice, therapeutic administration of Hsd17b13 ASO resulted in a significant and dose-dependent reduction of hepatic Hsd17b13 gene expression. The CDAHFD group exhibited considerably elevated liver enzyme levels, hepatic steatosis score, hepatic fibrosis, and increased fibrotic and inflammatory gene expression, indicating an advanced NASH-like hepatic fibrosis phenotype. Although Hsd17b13 ASO therapy significantly affected hepatic steatosis, it had no effect on hepatic fibrosis. Our findings demonstrate, for the first time, that Hsd17b13 ASO effectively suppressed Hsd17b13 gene expression both in vitro and in vivo, and had a modulatory effect on hepatic steatosis in mice, but did not affect fibrosis in the CDAHFD mouse model of NASH.

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.