Search Results (25)

Mucopolysaccharidosis type IIIC is a neurodegenerative lysosomal storage disorder (LSD) characterized by the accumulation of undegraded heparan sulfate (HS) due to the lack of an enzyme responsible for its degradation: acetyl-CoA:α-glucosaminide N-acetyltransferase (HGSNAT). Classical treatments are ineffective. Here, we attempt a new approach in genetic medicine, genetic substrate reduction therapy (gSRT), to counteract this neurological disorder. Briefly, we used synthetic oligonucleotides, particularly gapmer antisense oligonucleotides (ASOs), to target the synthesis of the accumulated compounds at the molecular level, downregulating a specific gene involved in the first step of HS biosynthesis, XYLT1. Our goal was to reduce HS production and, consequently, its accumulation. Initially, five gapmer ASOs were designed and their potential to decrease XYLT1 mRNA levels were tested in patient-derived fibroblasts. Subsequent analyses focused on the two best performing molecules alone. The results showed a high inhibition of the XYLT1 gene mRNA (around 90%), a decrease in xylosyltransferase I (XT-I) protein levels and a reduction in HS storage 6 and 10 days after transfection (up to 21% and 32%, respectively). Overall, our results are highly promising and may represent the initial step towards the development of a potential therapeutic option not only for MPS IIIC, but virtually for every other MPS III form. Ultimately, the same principle may also apply to other neuropathic MPS. Full article

Gapmer-type antisense oligonucleotides (ASOs) are an emerging class of therapeutic agents that directly inhibit pathogenic mRNA. In this study, three new 4′-C-substituted thymidine analogs were generated using a synthetic strategy recently established by our group, namely, 4′-C-(N-ethyl) aminoethyl (4′-EAE-T), 4′-C-(N-butyl) aminoethyl (4′-BAE-T), and 4′-C-(N-octyl) aminoethyl (4′-OAE-T). Their properties were evaluated and compared with those of previously reported analogs, including 4′-C-aminoethyl (4′-AE-T) and 4′-C-(N-methyl) aminoethyl (4′-MAE-T). The novel nucleoside analogs were subsequently incorporated into gapmer-type ASOs featuring phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in the wing regions. The incorporation of 4′-EAE-T and 4′-BAE-T analogs resulted in RNA binding affinities similar to that of the previously reported 4′-MAE-T analog, whereas a marked decrease in RNA affinity was noted for 4′-OAE-T, however, this reduction was mitigated when combined with other chemical modifications. Furthermore, the structural modifications conferred enhanced nuclease resistance under bovine serum conditions, with 4′-EAE-T resulting in the highest stability, followed by 4′-BAE-T and 4′-OAE-T. Additionally, oligonucleotides modified with the developed analogs preserved their RNase H cleavage susceptibility, albeit inducing minor alterations in the cleavage pattern. Finally, the oligonucleotides were applied in a gene silencing experiment targeting the KRAS gene, conducted without the use of transfection agents, displaying gene silencing activities comparable to that of the control, with the exception of the 4′-OAE-modified nucleotide, which exhibited low activity. Full article

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. Full article

Ribosomal frameshifting (RFS) at the slippery site of SARS-CoV-2 RNA is essential for the biosynthesis of the viral replication machinery. It requires the formation of a pseudoknot (PK) structure near the slippery site and can be inhibited by PK-disrupting oligonucleotide-based antivirals. We obtained and compared three types of such antiviral candidates, namely locked nucleic acids (LNA), LNA–DNA gapmers, and G-clamp-containing phosphorothioates (CPSs) complementary to PK stems. Using optical and electrophoretic methods, we showed that stem 2-targeting oligonucleotide analogs induced PK unfolding at nanomolar concentrations, and this effect was particularly pronounced in the case of LNA. For the leading PK-unfolding LNA and CPS oligonucleotide analogs, we also demonstrated dose-dependent RSF inhibition in dual luciferase assays (DLAs). Finally, we showed that the leading oligonucleotide analogs reduced SARS-CoV-2 replication at subtoxic concentrations in the nanomolar range in two human cell lines. Our findings highlight the promise of PK targeting, illustrate the advantages and limitations of various types of DNA modifications and may promote the future development of oligonucleotide-based antivirals. Full article

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. Full article

Facioscapulohumeral muscular dystrophy (FSHD), one of the most common muscular dystrophies, is caused by an abnormal expression of the DUX4 gene in skeletal muscles, resulting in muscle weakness. In this study, we investigated MT-DUX4-ASO, a novel gapmer antisense oligonucleotide (ASO). MT-DUX4-ASO decreased the expression of DUX4 and its target genes in FSHD patient-derived myoblasts. For the first time, we demonstrated that a systemically administered ASO, even without a ligand for drug delivery, could significantly improve muscle injury and motor function in the ACTA1-MCM/FLExDUX4 (DUX4-TG) mouse model of FSHD. Tamoxifen (TMX) injection transiently induces skeletal-muscle-specific DUX4 expression in DUX4-TG mice, while the skeletal muscles of TMX-untreated DUX4-TG mice have leaky DUX4 expression in a small subset of myofibers similar to those of FSHD patients. Subcutaneous 10 mg/kg of MT-DUX4-ASO at two-week intervals significantly suppressed muscular DUX4 target gene expression, histological muscle injury, and blood muscle injury marker elevation in TMX-untreated DUX4-TG mice. Notably, MT-DUX4-ASO at 10 mg/kg every other week significantly prevented the TMX-induced declines in treadmill test running speed and muscle force in DUX4-TG mice. Thus, the systemically administered unconjugated MT-DUX4-ASO suppressed disease progression in DUX4-TG mice, extending the potential of unconjugated ASOs as a promising FSHD treatment strategy. Full article

Previously reported (S)-5′-C-aminopropyl-2′-arabinofluoro-thymidine (5ara-T) and newly synthesized (S)-5′-C-aminopropyl-2′-arabinofluoro-5-methyl-cytidine (5ara-^MeC) analogs were incorporated into a series of antisense gapmers containing multiple phosphorothioate (PS) linkages and locked nucleic acids (LNAs) in their wing regions. The functional properties of the gapmers were further evaluated in vitro. Compared with the positive control, for the LNA-wing full PS gapmer without 5ara modification, it was revealed that each gapmer could have a high affinity and be thermally stable under biological conditions. Although the cleavage pattern was obviously changed; gapmers with 5ara modification could still efficiently activate E. coli RNase H1. In addition, incorporating one 5ara modification into the two phosphodiester linkages could reverse the destabilization in enzymatic hydrolysis caused by fewer PS linkages. In vitro cellular experiments were also performed, and the Lipofectamine^® 2000 (LFA)+ group showed relatively higher antisense activity than the LFA-free group. KN5ara-10, which contains fewer PS linkages, showed similar or slightly better antisense activity than the corresponding full PS-modified KN5ara-3. Hence, KN5ara-10 may be the most promising candidate for KNTC2-targeted cancer therapy. Full article

The effect of glycemic stress on de novo lipogenesis (DNL) in podocytes and tubular epithelial cells is understudied. This study is aimed (A) to show the effect of glycemic stress on DNL, and (B) to assess the effect of acetyl-Co A (ACC) inhibition on halting upregulation of DNL, on the expression of other lipid regulatory genes in the DNL pathway, and on markers of fibrosis and apoptosis in podocytes and tubular epithelial cells. We used cultured mouse primary tubular epithelial cells, mouse proximal tubular (BUMPT) cells, and immortal mouse podocytes and measured their percentage of labeled ¹³C₂-palmitate as a marker of DNL after incubation with ¹³C₂ acetate in response to high glucose concentration (25 mM). We then tested the effect of ACC inhibition by complimentary strategies utilizing CRISPR/cas9 deletion or incubation with Acaca and Acacb GapmeRs or using a small molecule inhibitor on DNL under hyperglycemic concentration. Exposure to high glucose concentration (25 mM) compared to osmotic controlled low glucose concentration (5.5 mM) significantly increased labeled palmitate after 24 h up to 72 h in podocytes and primary tubular cells. Knocking out of the ACC coding Acaca and Acacb genes by CRISPR/cas9, downregulation of Acaca and Acacb by specific antisense LNA GapmeRs and inhibition of ACC by firsocostat similarly halted/mitigated upregulation of DNL and decreased markers of fibrosis and programmed cell death in podocytes and various tubular cells. ACC inhibition is a potential therapeutic target to mitigate or halt hyperglycemia-induced upregulation of DNL in podocytes and tubular cells. Full article

The development of oligonucleotide conjugates for in vivo targeting is one of the most exciting areas for oligonucleotide therapeutics. A major breakthrough in this field was the development of multifunctional GalNAc-oligonucleotides with high affinity to asialoglycoprotein receptors (ASGPR) that directed therapeutic oligonucleotides to hepatocytes. In the present study, we explore the use of G-rich sequences functionalized with one unit of GalNAc at the 3′-end for the formation of tetrameric GalNAc nanostructures upon formation of a parallel G-quadruplex. These compounds are expected to facilitate the synthetic protocols by providing the multifunctionality needed for the binding to ASGPR. To this end, several G-rich oligonucleotides carrying a TGGGGGGT sequence at the 3′-end functionalized with one molecule of N-acetylgalactosamine (GalNAc) were synthesized together with appropriate control sequences. The formation of a self-assembled parallel G-quadruplex was confirmed through various biophysical techniques such as circular dichroism, nuclear magnetic resonance, polyacrylamide electrophoresis and denaturation curves. Binding experiments to ASGPR show that the size and the relative position of the therapeutic cargo are critical for the binding of these nanostructures. The biological properties of the resulting parallel G-quadruplex were evaluated demonstrating the absence of the toxicity in cell lines. The internalization preferences of GalNAc-quadruplexes to hepatic cells were also demonstrated as well as the enhancement of the luciferase inhibition using the luciferase assay in HepG2 cell lines versus HeLa cells. All together, we demonstrate that tetramerization of G-rich oligonucleotide is a novel and simple route to obtain the beneficial effects of multivalent N-acetylgalactosamine functionalization. Full article

The capability of the adult central nervous system to self-repair/regenerate was demonstrated repeatedly throughout the last decades but remains in debate. Reduced neurogenic niche activity paralleled by a profound neuronal loss represents fundamental hallmarks in the disease course of neurodegenerative disorders. We and others have demonstrated the endogenous TGFβ system to represent a potential pathogenic participant in disease progression, of amyotrophic lateral sclerosis (ALS) in particular, by generating and promoting a disequilibrium of neurodegenerative and neuroregenerative processes. The novel human/primate specific LNA Gapmer Antisense Oligonucleotide “NVP-13”, targeting TGFBR2, effectively reduced its expression and lowered TGFβ signal transduction in vitro and in vivo, paralleled by boosting neurogenic niche activity in human neuronal progenitor cells and nonhuman primate central nervous system. Here, we investigated NVP-13 in vivo pharmacology, safety, and tolerability following repeated intrathecal injections in nonhuman primate cynomolgus monkeys for 13 weeks in a GLP-toxicology study approach. NVP-13 was administered intrathecally with 1, 2, or 4 mg NVP-13/animal within 3 months on days 1, 15, 29, 43, 57, 71, and 85 in the initial 13 weeks. We were able to demonstrate an excellent local and systemic tolerability, and no adverse events in physiological, hematological, clinical chemistry, and microscopic findings in female and male Cynomolgus Monkeys. Under the conditions of this study, the no observed adverse effect level (NOAEL) is at least 4 mg/animal NVP-13. Full article

Mutant p53 proteins result from missense mutations in the TP53 gene, the most mutated in human cancer, and have been described to contribute to cancer initiation and progression. Therapeutic strategies for targeting mutant p53 proteins in cancer cells are limited and have proved unsuitable for clinical application due to problems related to drug delivery and toxicity to healthy tissues. Therefore, the discovery of efficient and safe therapeutic strategies that specifically target mutant p53 remains challenging. In this study, we generated gold nanoparticles (AuNPs) chemically modified with low molecular branched polyethylenimine (bPEI) for the efficient delivery of gapmers targeting p53 mutant protein. The AuNPs formulation consists of a combination of polymeric mixed layer of polyethylene glycol (PEG) and PEI, and layer-by-layer assembly of bPEI through a sensitive linker. These nanoparticles can bind oligonucleotides through electrostatic interactions and release them in the presence of a reducing agent as glutathione. The nanostructures generated here provide a non-toxic and powerful system for the delivery of gapmers in cancer cells, which significantly downregulated mutant p53 proteins and altered molecular markers related to cell growth and apoptosis, thus overcoming chemoresistance to gemcitabine. Full article

Long non-coding RNAs (lncRNAs) play key roles in Angiotensin II (AngII) signaling but their role in chondrogenic transformation of vascular smooth muscle cells (VSMCs) is unknown. We describe a novel AngII-induced lncRNA Alivec (Angiotensin II-induced lncRNA in VSMCs eliciting chondrogenic phenotype) implicated in VSMC chondrogenesis. In rat VSMCs, Alivec and the nearby gene Acan, a chondrogenic marker, were induced by growth factors AngII and PDGF and the inflammatory cytokine TNF-α. AngII co-regulated Alivec and Acan through the activation of AngII type1 receptor signaling and Sox9, a master transcriptional regulator of chondrogenesis. Alivec knockdown with GapmeR antisense-oligonucleotides attenuated the expression of AngII-induced chondrogenic marker genes, including Acan, and inhibited the chondrogenic phenotype of VSMCs. Conversely, Alivec overexpression upregulated these genes and promoted chondrogenic transformation. RNA-pulldown coupled to mass-spectrometry identified Tropomyosin-3-alpha and hnRNPA2B1 proteins as Alivec-binding proteins in VSMCs. Furthermore, male rats with AngII-driven hypertension showed increased aortic expression of Alivec and Acan. A putative human ortholog ALIVEC, was induced by AngII in human VSMCs, and this locus was found to harbor the quantitative trait loci affecting blood pressure. Together, these findings suggest that AngII-regulated lncRNA Alivec functions, at least in part, to mediate the AngII-induced chondrogenic transformation of VSMCs implicated in vascular dysfunction and hypertension. Full article

Mesenchymal stem cell (MSC) transplantation has emerged as a promising approach for bone regeneration. Importantly, the beneficial effects of MSCs can be improved by modulating the expression levels of specific genes to stimulate MSC osteogenic differentiation. We have previously shown that Smurf1 silencing by using Locked Nucleic Acid-Antisense Oligonucleotides, in combination with a scaffold that sustainably releases low doses of BMP-2, was able to increase the osteogenic potential of MSCs in the presence of BMP-2 doses significantly smaller than those currently used in the clinic. This would potentially allow an important reduction in this protein in MSs-based treatments, and thus of the side effects linked to its administration. We have further improved this system by specifically targeting the Wnt pathway modulator Sfrp1. This approach not only increases MSC bone regeneration efficiency, but is also able to induce osteogenic differentiation in osteoporotic human MSCs, bypassing the need for BMP-2 induction, underscoring the regenerative potential of this system. Achieving successful osteogenesis with the sole use of LNA-ASOs, without the need of administering pro-osteogenic factors such as BMP-2, would not only reduce the cost of treatments, but would also open the possibility of targeting these LNA-ASOs specifically to MSCs in the bone marrow, allowing us to treat systemic bone loss such as that associated with osteoporosis. Full article

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides. Full article

Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2′-O-methylation. Full article