[Angelman syndrome: current approach and the future of therapies]. / Síndrome de Angelman: abordaje actual y el futuro de las terapias.

Angelman syndrome is a severe neurodevelopmental disorder secondary to disruption of the UBE3A gene in the maternal allele of chromosome 15. Its manifestations are mainly neurological, but a multidisciplinary management is required for its treatment. There are consensus guidelines available for best clinical management. Current clinical trials with antisense oligonucleotides promise, for the first time, to treat the cause by activating the UBE3A gene in the paternal allele, showing encouraging preliminary clinical effects. Inoculation of UBE3A gene through a viral vector has been tested in animal models and is underway for future clinical trials.

El síndrome de Angelman es un grave desorden del neurodesarrollo secundario a disrupción del gen UBE3A en el alelo materno del cromosoma 15. Sus manifestaciones son principalmente neurológicas, pero se requiere de un manejo multidisciplinario para su tratamiento. Existen guías por consenso para el manejo clínico adecuado. Actuales ensayos clínicos con oligonucleótidos antisentido prometen, por primera vez, tratar la causa por medio de activación del gen UBE3A en el alelo paterno, demostrando efectos clínicos preliminares alentadores. La inoculación del gen UBE3A a través de un vector viral ha sido probada en modelos animales y está en vías para futuros ensayos clínicos.

Customised design of antisense oligonucleotides targeting EGFR driver mutants for personalised treatment of non-small cell lung cancer.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are currently the standard therapy for patients with non-small cell lung cancer (NSCLC) bearing mutations in epidermal growth factor receptor (EGFR). Unfortunately, drug-acquired resistance is inevitable due to the emergence of new mutations in EGFR. Moreover, the TKI treatment is associated with severe toxicities due to the unspecific inhibition of wild-type (WT) EGFR. Thus, treatment that is customised to an individual's genetic alterations in EGFR may offer greater therapeutic benefits for patients with NSCLC. METHODS: In this study, we demonstrate a new therapeutic strategy utilising customised antisense oligonucleotides (ASOs) to selectively target activating mutations in the EGFR gene in an individualised manner that can overcome drug-resistant mutations. We use extracellular vesicles (EVs) as a vehicle to deliver ASOs to NSCLC cells. FINDINGS: Specifically guided by the mutational profile identified in NSCLC patients, we have successfully developed ASOs that selectively inhibit point mutations in the EGFR gene, including L858R and T790M, while sparing the WT EGFR. Delivery of the EGFR-targeting ASOs by EVs significantly reduced tumour growth in xenograft models of EGFR-L858R/T790M-driven NSCLC. Importantly, we have also shown that EGFR-targeting ASOs exhibit more potent anti-cancer effect than TKIs in NSCLC with EGFR mutations, effectively suppressing a patient-derived TKI-resistant NSCLC tumour. INTERPRETATION: Overall, by harnessing the specificity and efficacy of ASOs, we present an effective and adaptable therapeutic platform for NSCLC treatment. FUNDING: This study was funded by Singapore's Ministry of Health (NMRC/OFIRG/MOH-000643-00, OFIRG21nov-0068, NMRC/OFLCG/002-2018, OFYIRG22jul-0034), National Research Foundation (NRF-NRFI08-2022, NRF-CRP22-2019-0003, NRF-CRP23-2019-0004), A∗STAR, and Ministry of Education.

TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition.

Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b-/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.

TMPRSS2-specific antisense oligonucleotides inhibit host cell entry of emerging viruses.

Emerging viruses, such as novel influenza A viruses (IAV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose a constant threat to animal and human health. Identification of host cell factors necessary for viral replication but dispensable for cellular survival might reveal novel, attractive targets for therapeutic intervention. Proteolytic activation of IAV hemagglutinin (HA) and SARS-CoV-2 spike protein (S) by the type II transmembrane serine protease (TTSPs), e.g. TMPRSS2 is sought to be critical for viral spread and pathogenesis. Here, we investigated the secondary structure of TMPRSS2 mRNA coding sequence and designed TMPRSS2-specific antisense oligonucleotides (ASOs). Several of these ASOs markedly reduced the TMPRSS2 expression and decreased IAV infection and SARS-CoV-2 entry into cells.

Ceramide synthesis inhibitors prevent lipid-induced insulin resistance through the DAG-PKCε-insulin receptorT1150 phosphorylation pathway.

Inhibition of the ceramide synthetic pathway with myriocin or an antisense oligonucleotide (ASO) targeting dihydroceramide desaturase (DES1) both improved hepatic insulin sensitivity in rats fed either a saturated or unsaturated fat diet and was associated with reductions in both hepatic ceramide and plasma membrane (PM)-sn-1,2-diacylglycerol (DAG) content. The insulin sensitizing effects of myriocin and Des1 ASO were abrogated by acute treatment with an ASO against DGAT2, which increased hepatic PM-sn-1,2-DAG but not hepatic C16 ceramide content. Increased PM-sn-1,2-DAG content was associated with protein kinase C (PKC)ε activation, increased insulin receptor (INSR)T1150 phosphorylation leading to reduced insulin-stimulated INSRY1152/AktS473 phosphorylation, and impaired insulin-mediated suppression of endogenous glucose production. These results demonstrate that inhibition of de novo ceramide synthesis by either myriocin treatment or DES1 knockdown protects against lipid-induced hepatic insulin resistance through a C16 ceramide-independent mechanism and that they mediate their effects to protect from lipid-induced hepatic insulin resistance via the PM-sn-1,2-DAG-PKCε-INSRT1150 phosphorylation pathway.

Targeting Apolipoprotein C-III for the Management of Severe Hypertriglyceridemia: Current Research and Future Directions.

Hypertriglyceridemia is characterized by elevated triglyceride levels in the blood, which increases the risk of cardiovascular disease and pancreatitis. This condition stems from multiple factors including lifestyle choices, genetics, and conditions such as diabetes and metabolic syndrome. Apolipoprotein C-III (APOC3), a protein for lipid metabolism, hinders enzymes necessary for breaking down triglycerides and thus plays a key role in hypertriglyceridemia. Variations in the APOC3 gene are associated with varying triglyceride levels among individuals. Recent genetic studies and clinical trials have shed light on the potential of targeting APOC3 as a potentially promising therapeutic modality of hypertriglyceridemia. Antisense oligonucleotides like volanesorsen have displayed effectiveness in lowering triglyceride levels in individuals with severe hypertriglyceridemia. This review article delves into how APOC3 influences triglyceride control and its potential use in targeting APOC3 to manage severe hypertriglyceridemia.

Therapeutic Antisense Oligonucleotides in Oncology: From Bench to Bedside.

Advancements in our comprehension of tumor biology and chemoresistance have spurred the development of treatments that precisely target specific molecules within the body. Despite the expanding landscape of therapeutic options, there persists a demand for innovative approaches to address unmet clinical needs. RNA therapeutics have emerged as a promising frontier in this realm, offering novel avenues for intervention such as RNA interference and the utilization of antisense oligonucleotides (ASOs). ASOs represent a versatile class of therapeutics capable of selectively targeting messenger RNAs (mRNAs) and silencing disease-associated proteins, thereby disrupting pathogenic processes at the molecular level. Recent advancements in chemical modification and carrier molecule design have significantly enhanced the stability, biodistribution, and intracellular uptake of ASOs, thereby bolstering their therapeutic potential. While ASO therapy holds promise across various disease domains, including oncology, coronary angioplasty, neurological disorders, viral, and parasitic diseases, our review manuscript focuses specifically on the application of ASOs in targeted cancer therapies. Through a comprehensive examination of the latest research findings and clinical developments, we delve into the intricacies of ASO-based approaches to cancer treatment, shedding light on their mechanisms of action, therapeutic efficacy, and prospects.

Liver-targeted Angptl4 silencing by antisense oligonucleotide treatment attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice.

BACKGROUND AND AIMS: Angiopoietin-like 3 (ANGPTL3) and 4 (ANGPTL4) inhibit lipoprotein lipase to regulate tissue fatty acid uptake from triglyceride-rich lipoproteins such as VLDL. While pharmacological inhibition of ANGPTL3 is being evaluated as lipid-lowering strategy, systemic ANGPTL4 inhibition is not pursued due to adverse effects. This study aimed to compare the therapeutic potential of liver-specific Angptl3 and Angptl4 silencing to attenuate hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, a well-established humanized model for lipoprotein metabolism. METHODS AND RESULTS: Mice were subcutaneously injected twice-weekly with saline or liver-targeted antisense oligonucleotides against Angptl3, Angptl4, both, or a scrambled oligonucleotide. Plasma lipid levels, VLDL clearance and hepatic VLDL production were determined, and atherosclerosis development was assessed. For toxicological evaluation, cynomolgus monkeys were treated with three dosages of liver-targeted ANGPTL4-silencing oligonucleotides.Liver-targeted Angptl4 silencing reduced plasma triglycerides (-48%) and total cholesterol (-56%), explained by higher VLDL-derived fatty acid uptake by brown adipose tissue and lower VLDL production by the liver. Accordingly, Angptl4 silencing reduced atherosclerotic lesion size (-86%) and improved lesion stability. Hepatic Angptl3 silencing similarly attenuated hyperlipidemia and atherosclerosis development. While Angptl3 and Angptl4 silencing lowered plasma triglycerides in the refed and fasted state, respectively, combined Angptl3/4 silencing lowered plasma triglycerides independent of nutritional state. In cynomolgus monkeys, anti-ANGPTL4 ASO treatment was well tolerated without adverse effects. CONCLUSIONS: Liver-targeted Angptl4 silencing potently attenuates hyperlipidemia and atherosclerosis development in APOE*3-Leiden.CETP mice, and liver-targeted ANGPTL4 silencing is well-tolerated in non-human primates. These data warrant further clinical development of liver-targeted ANGPTL4 silencing.

Increasing MicroRNA Abundance by Targeting Biogenesis from the Primary Transcript with Steric-Blocking Antisense Oligonucleotides.

MicroRNAs (miRNAs) are regulators of gene expression, and their dysregulation is linked to cancer and other diseases, making them important therapeutic targets. Several strategies for targeting and modulating miRNA activity are being explored. For example, steric blocking antisense oligonucleotides (ASOs) can reduce miRNA activity by either blocking binding sites on specific mRNAs or base-pairing to the miRNA itself to prevent its interaction with the target mRNAs. ASOs have been less explored as a tool to elevate miRNA levels, which could also be beneficial for treating disease. In this study, using the PKD1/miR-1225 gene locus as an example, where miR-1225 is located within a PKD1 intron, we demonstrate an ASO-based strategy that increases miRNA abundance by enhancing biogenesis from the primary miRNA transcript. Disruptions in PKD1 and miR-1225 are associated with autosomal dominant polycystic kidney disease (ADPKD) and various cancers, respectively, making them important therapeutic targets. We investigated PKD1 sequence variants reported in ADPKD that are located within the sequence shared by miR-1225 and PKD1, and identified one that causes a reduction in miR-1225 without affecting PKD1. We show that this reduction in miR-1225 can be recovered by treatment with a steric-blocking ASO. The ASO-induced increase in miR-1225 correlates with a decrease in the abundance of predicted miR-1225 cellular mRNA targets. This study demonstrates that miRNA abundance can be elevated using ASOs targeted to the primary transcript. This steric-blocking ASO-based approach has broad potential application as a therapeutic strategy for diseases that could be treated by modulating miRNA biogenesis.

Antisense targeting of FOXP3+ Tregs to boost anti-tumor immunity.

Our goal is to improve the outcomes of cancer immunotherapy by targeting FOXP3+ T-regulatory (Treg) cells with a next generation of antisense oligonucleotides (ASO), termed FOXP3 AUMsilence ASO. We performed in vitro experiments with human healthy donor PBMC and clinical samples from patients with lung cancer, mesothelioma and melanoma, and tested our approach in vivo using ASO FOXP3 in syngeneic murine cancer models and in humanized mice. ASO FOXP3 had no effects on cell viability or cell division, did not affect expression of other FOXP members, but decreased expression of FOXP3 mRNA in PBMC by 54.9% and in cancer samples by 64.7%, with corresponding 41.0% (PBMC) and 60.0% (cancer) decreases of Treg numbers (all p<0.0001). Hence, intratumoral Treg were more sensitive to the effects of ASO FOXP3 than peripheral blood Tregs. Isolated human Treg, incubated with ASO FOXP3 for 3.5 hours, had significantly impaired suppressive function (66.4%) versus Scramble control. In murine studies, we observed a significant inhibition of tumor growth, while 13.6% (MC38) to 22% (TC1) of tumors were completely resorbed, in conjunction with ~50% decrease of Foxp3 mRNA by qPCR and decreased numbers of intratumoral Tregs. In addition, there were no changes in FOXP3 mRNA expression or in the numbers of Tregs in draining lymph nodes and in spleens of tumor bearing mice, confirming that intratumoral Treg had enhanced sensitivity to ASO FOXP3 in vivo compared to other Treg populations. ASO FOXP3 Treg targeting in vivo and in vitro was accompanied by significant downregulation of multiple exhaustion markers, and by increased expression of perforin and granzyme-B by intratumoral T cells. To conclude, we report that targeting the key Treg transcription factor FOXP3, with ASO FOXP3, has a powerful anti-tumoral effect and enhances T cell response in vitro and in vivo.

Addressing residual risk beyond statin therapy: New targets in the management of dyslipidaemias-A report from the European Society of Cardiology Cardiovascular Round Table.

Cardiovascular (CV) disease is the most common cause of death in Europe. Despite proven benefits, use of lipid-lowering therapy remains suboptimal. Treatment goals are often not achieved, even in patients at high risk with atherosclerotic CV disease (ASCVD). The occurrence of CV events in patients on lipid-lowering drugs is defined as "residual risk", and can result from inadequate control of plasma lipids or blood pressure, inflammation, diabetes, and environmental hazards. Assessment of CV risk factors and vascular imaging can aid in the evaluation and management decisions for individual patients. Lifestyle measures remain the primary intervention for lowering CV risk. Where drug therapies are required to reach lipid treatment targets, their effectiveness increases when they are combined with lifestyle measures delivered through formal programs. However, lipid drug dosage and poor adherence to treatment remain major obstacles to event-free survival. This article discusses guideline-supported treatment algorithms beyond statin therapy that can help reduce residual risk in specific patient profiles while also likely resulting in substantial healthcare savings through better patient management and treatment adherence.

Androgen receptor modulatory miR-1271-5p can promote hormone sensitive prostate cancer cell growth.

In most patients with advanced prostate cancer treated with hormonal therapy, androgen independence eventually emerges, leading to death. Androgen receptor signalling remains an important prostate cancer driver, even in the advanced disease stage. MicroRNAs (miRs), non-coding RNAs that regulate gene expression by inhibiting translation and/or promoting degradation of target mRNAs, can act as tumour suppressors or "oncomiRs" and modulate tumour growth. Because of their stability in tissues and in circulation, and their specificity, microRNAs have emerged as potential biomarkers, as well as therapeutic targets in cancer. We identified miR-1271-5p as an androgen receptor modulatory microRNA and we show it can promote hormone sensitive prostate cancer cell growth. Inhibition or overexpression of miR-1271-5p levels affects prostate cancer cell growth, apoptosis and expression of both androgen receptor target genes and other genes that are likely direct targets, dependent on androgen receptor status, and tumour stage. We conclude that miR-1271-5p has the potential to drive progression of hormone-dependent disease and that the use of specific inhibitors of miR-1271-5p may have therapeutic potential in prostate cancer.

Current status of nucleic acid therapy and its new progress in cancer treatment.

Nucleic acid is an essential biopolymer in all living cells, performing the functions of storing and transmitting genetic information and synthesizing protein. In recent decades, with the progress of science and biotechnology and the continuous exploration of the functions performed by nucleic acid, more and more studies have confirmed that nucleic acid therapy for living organisms has great medical therapeutic potential. Nucleic acid drugs began to become independent therapeutic agents. As a new therapeutic method, nucleic acid therapy plays an important role in the treatment of genetic diseases, viral infections and cancers. There are currently 19 nucleic acid drugs approved by the Food and Drug Administration (FDA). In the following review, we start from principles and advantages of nucleic acid therapy, and briefly describe development history of nucleic acid drugs. And then we give examples of various RNA therapeutic drugs, including antisense oligonucleotides (ASO), mRNA vaccines, small interfering RNA (siRNA) and microRNA (miRNA), aptamers, and small activating RNA (saRNA). In addition, we also focused on the current status of nucleic acid drugs used in cancer therapy and the breakthrough in recent years. Clinical trials of nucleic acid drugs for cancer treatment are under way, conventional radiotherapy and chemotherapy combined with the immunotherapies such as checkpoint inhibitors and nucleic acid drugs may be the main prospects for successful cancer treatment.

Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis.

TGFß1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFß. Our work investigates the potential of blocking the 'splicing in' of EDA with antisense oligonucleotides to inhibit TGFß1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFß1 in human renal proximal tubule cells (PTEC). Human primary PTEC were treated with TGFß1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFß, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed. Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFß1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA. Exogenous TGFß1 induced endogenous TGFß, αSMA, MMP2, MMP9 and Col I mRNA. TGFß1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFß1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFß, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFß1 was confirmed by the use of a TGFß receptor inhibitor. Our results demonstrate a critical role of FN EDA+ in a cycle of TGFß driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.

FLI1 in PBMCs contributes to elevated inflammation in combat-related posttraumatic stress disorder.

Post-traumatic stress disorder (PTSD) is a debilitating psychiatric condition with significant public health implications that arise following exposure to traumatic events. Recent studies highlight the involvement of immune dysregulation in PTSD, characterized by elevated inflammatory markers. However, the precise mechanisms underlying this immune imbalance remain unclear. Previous research has implicated friend leukemia virus integration 1 (FLI1), an erythroblast transformation-specific (ETS) transcription factor, in inflammatory responses in sepsis and Alzheimer's disease. Elevated FLI1 levels in peripheral blood mononuclear cells (PBMCs) have been linked to lupus severity. Yet, FLI1's role in PTSD-related inflammation remains unexplored. In our study, PBMCs were collected from Veterans with and without PTSD. We found significantly increased FLI1 expression in PBMCs from PTSD-afflicted Veterans, particularly in CD4+ T cells, with no notable changes in CD8+ T cells. Stimulation with LPS led to heightened FLI1 expression and elevated levels of inflammatory cytokines IL-6 and IFNγ in PTSD PBMCs compared to controls. Knockdown of FLI1 using Gapmers in PTSD PBMCs resulted in a marked reduction in inflammatory cytokine levels, restoring them to control group levels. Additionally, co-culturing PBMCs from both control and PTSD Veterans with the human brain microglia cell line HMC3 revealed increased inflammatory mediator levels in HMC3. Remarkably, HMC3 cells co-cultured with PTSD PBMCs treated with FLI1 Gapmers exhibited significantly lower inflammatory mediator levels compared to control Gapmer-treated PTSD PBMCs. These findings suggest that suppressing FLI1 may rebalance immune activity in PBMCs and mitigate microglial activation in the brain. Such insights could provide novel therapeutic strategies for PTSD.

Construction of charge-reversible coordination-crosslinked spherical nucleic acids to deliver dual anti-cancer genes and ferroptosis payloads.

Spherical nucleic acids (SNAs) are nanostructures with the DNA arranged radially on the surface, thus allowing specific binding with cancer cells expressing high levels of scavenger receptor-A to enhance cellular uptake. However, conventional carriers for SNAs are cytotoxic, not degradable and difficult to deliver multiple payloads. In this study, we developed charge-reversible coordination-crosslinked SNAs to deliver dual anti-cancer genes and ferroptosis payload for anti-cancer purposes. To this end, we modified poly(lactic acid) (PLA) with functionalized side chains to allow its binding with antisense oligonucleotides (ASOs) and siRNA, annealed two single-stranded RNAs to obtain double-stranded RNA, and introduced a polyethylene glycol (PEG) shell to enhance the circulation time. Additionally, the ferroptosis payload imidazole was coordinated with iron ions as a core-crosslinked group to enhance the stability of SNAs and efficiency to kill cancer cells. We demonstrated that this novel nanocomplex efficiently internalized and killed CT-26 cells in vitro. In vivo data confirmed that the dual gene delivery system successfully targeted CT-26 tumors in tumor-bearing BALB/c mice, and exhibited strong tumor suppression ability, without inducing adverse toxic effects. Taken together, our dual gene therapy system offered an enhanced anti-tumor solution by simultaneously delivering dual anti-cancer genes and ferroptosis payload in tumor microenvironment.

Splice modulation strategy applied to deep intronic variants in COL7A1 causing recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and most often severe genetic disease characterized by recurrent blistering and erosions of the skin and mucous membranes after minor trauma, leading to major local and systemic complications. The disease is caused by loss-of-function variants in COL7A1 encoding type VII collagen (C7), the main component of anchoring fibrils, which form attachment structures stabilizing the cutaneous basement membrane zone. Alterations in C7 protein structure and/or expression lead to abnormal, rare or absent anchoring fibrils resulting in loss of dermal-epidermal adherence and skin blistering. To date, more than 1,200 distinct COL7A1 deleterious variants have been reported and 19% are splice variants. Here, we describe two RDEB patients for whom we identified two pathogenic deep intronic pathogenic variants in COL7A1. One of these variants (c.7795-97C > G) promotes the inclusion of a pseudoexon between exons 104 and 105 in the COL7A1 transcript, while the other causes partial or complete retention of intron 51. We used antisense oligonucleotide (ASO) mediated exon skipping to correct these aberrant splicing events in vitro. This led to increased normal mRNA splicing above 94% and restoration of C7 protein expression at a level (up to 56%) that should be sufficient to reverse the phenotype. This first report of exon skipping applied to counteract deep intronic variants in COL7A1 represents a promising therapeutic strategy for personalized medicine directed at patients with intronic variants at a distance of consensus splice sites.

Allele-Selective Thiomorpholino Antisense Oligonucleotides as a Therapeutic Approach for Fused-in-Sarcoma Amyotrophic Lateral Sclerosis.

Pathogenic variations in the fused in sarcoma (FUS) gene are associated with rare and aggressive forms of amyotrophic lateral sclerosis (ALS). As FUS-ALS is a dominant disease, a targeted, allele-selective approach to FUS knockdown is most suitable. Antisense oligonucleotides (AOs) are a promising therapeutic platform for treating such diseases. In this study, we have explored the potential for allele-selective knockdown of FUS. Gapmer-type AOs targeted to two common neutral polymorphisms in FUS were designed and evaluated in human fibroblasts. AOs had either methoxyethyl (MOE) or thiomorpholino (TMO) modifications. We found that the TMO modification improved allele selectivity and efficacy for the lead sequences when compared to the MOE counterparts. After TMO-modified gapmer knockdown of the target allele, up to 93% of FUS transcripts detected were from the non-target allele. Compared to MOE-modified AOs, the TMO-modified AOs also demonstrated reduced formation of structured nuclear inclusions and SFPQ aggregation that can be triggered by phosphorothioate-containing AOs. How overall length and gap length of the TMO-modified AOs affected allele selectivity, efficiency and off-target gene knockdown was also evaluated. We have shown that allele-selective knockdown of FUS may be a viable therapeutic strategy for treating FUS-ALS and demonstrated the benefits of the TMO modification for allele-selective applications.

KCTD17-mediated Ras stabilization promotes hepatocellular carcinoma progression.

Background/Aims: Potassium channel tetramerization domain containing 17 (KCTD17) protein, an adaptor for the cullin3 (Cul3) ubiquitin ligase complex, has been implicated in various human diseases; however, its role in hepatocellular carcinoma (HCC) remains elusive. Here, we aimed to elucidate the clinical features of KCTD17, and investigate the mechanisms by which KCTD17 affects HCC progression. Methods: We analyzed transcriptomic data from patients with HCC. Hepatocyte-specific KCTD17 deficient mice were treated with diethylnitrosamine (DEN) to assess its effect on HCC progression. Additionally, we tested KCTD17-directed antisense oligonucleotides for their therapeutic potential in vivo. Results: Our investigation revealed the upregulation of KCTD17 expression in both tumors from patients with HCC and mouse models of HCC, in comparison to non-tumor controls. We identified the leucine zipper-like transcriptional regulator 1 (Lztr1) protein, a previously identified Ras destabilizer, as a substrate for KCTD17-Cul3 complex. KCTD17-mediated Lztr1 degradation led to Ras stabilization, resulting in increased proliferation, migration, and wound healing in liver cancer cells. Hepatocyte-specific KCTD17 deficient mice or liver cancer xenograft models were less susceptible to carcinogenesis or tumor growth. Similarly, treatment with KCTD17-directed antisense oligonucleotides (ASO) in a mouse model of HCC markedly lowered tumor volume as well as Ras protein levels, compared to those in control ASO-treated mice. Conclusions: KCTD17 induces the stabilization of Ras and downstream signaling pathways and HCC progression and may represent a novel therapeutic target for HCC.