Making Sense of Antisense Oligonucleotide Therapeutics Targeting Bcl-2.
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ABSTRACT:
SUBMITTER: Gagliardi M
PROVIDER: S-EPMC8778715 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Antisense oligonucleotide (ASO) technology has become an attractive therapeutic modality for various diseases, including Mendelian disorders. ASOs can modulate the expression of a target gene by promoting mRNA degradation or changing pre-mRNA splicing, nonsense-mediated mRNA decay, or translation. Advances in medicinal chemistry and a deeper understanding of post-transcriptional mechanisms have led to the approval of several ASO drugs for diseases that had long lacked therapeutic options. For instance, an ASO drug called nusinersen became the first approved drug for spinal muscular atrophy, improving survival and the overall disease course. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Although Trikafta and other CFTR-modulation therapies benefit most CF patients, there is a significant unmet therapeutic need for a subset of CF patients. In this review, we introduce ASO therapies and their mechanisms of action, describe the opportunities and challenges for ASO therapeutics for CF, and discuss the current state and prospects of ASO therapies for CF.
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Project description:Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients' chondrocytes. This work introduces a versatile biomolecular modular-based "Plug-and-Play" platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics.
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Project description:To expand the antisense oligonucleotide (ASO) fluorescence labeling toolbox beyond covalent conjugation of external dyes (e.g. ATTO-, Alexa Fluor-, or cyanine dyes), we herein explore fluorescent base analogues (FBAs) as a novel approach to endow fluorescent properties to ASOs. Both cytosine and adenine analogues (tC, tCO, 2CNqA, and pA) were incorporated into a 16mer ASO sequence with a 3-10-3 cEt-DNA-cEt (cEt = constrained ethyl) gapmer design. In addition to a comprehensive photophysical characterization, we assess the label-induced effects on the gapmers' RNA affinities, RNA-hybridized secondary structures, and knockdown efficiencies. Importantly, we find practically no perturbing effects for gapmers with single FBA incorporations in the biologically critical gap region and, except for pA, the FBAs do not affect the knockdown efficiencies. Incorporating two cytosine FBAs in the gap is equally well tolerated, while two adenine analogues give rise to slightly reduced knockdown efficiencies and what could be perturbed secondary structures. We furthermore show that the FBAs can be used to visualize gapmers inside live cells using fluorescence microscopy and flow cytometry, enabling comparative assessment of their uptake. This altogether shows that FBAs are functional ASO probes that provide a minimally perturbing in-sequence labeling option for this highly relevant drug modality.
| S-EPMC8166847 | biostudies-literature
Project description:Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2'-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5' region of exon 51. These results indicated that locked nucleic acid/2'-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.
| S-EPMC6951410 | biostudies-literature
Project description:Cancer is one of the leading causes of death worldwide, and conventional cancer therapies such as surgery, chemotherapy, and radiotherapy do not address the underlying molecular pathologies, leading to inadequate treatment and tumor recurrence. Angiogenic factors, such as EGF, PDGF, bFGF, TGF-?, TGF-?, VEGF, endoglin, and angiopoietins, play important roles in regulating tumor development and metastasis, and they serve as potential targets for developing cancer therapeutics. Nucleic acid-based therapeutic strategies have received significant attention in the last two decades, and antisense oligonucleotide-mediated intervention is a prominent therapeutic approach for targeted manipulation of gene expression. Clinical benefits of antisense oligonucleotides have been recognized by the U.S. Food and Drug Administration, with full or conditional approval of Vitravene, Kynamro, Exondys51, and Spinraza. Herein we review the scope of antisense oligonucleotides that target angiogenic factors toward tackling solid cancers.
| S-EPMC6307321 | biostudies-other
Project description:To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)(7), also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.
| S-EPMC3164722 | biostudies-literature
Project description:BackgroundCancer cells are known to develop mechanisms to circumvent effective anti-tumor immunity. The two ectonucleotidases CD39 and CD73 are promising drug targets, as they act in concert to convert extracellular immune-stimulating ATP to adenosine. CD39 is expressed by different immune cell populations as well as cancer cells of different tumor types and supports the tumor in escaping immune recognition and destruction. Thus, increasing extracellular ATP and simultaneously reducing adenosine concentrations in the tumor can lead to effective anti-tumor immunity.MethodsWe designed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) with specificity for human or mouse CD39 that do not need a transfection reagent or delivery system for efficient target knockdown. Knockdown efficacy of ASOs on mRNA and protein level was investigated in cancer cell lines and in primary human T cells. The effect of CD39 knockdown on ATP-degrading activity was evaluated by measuring levels of ATP in tumor cell supernatants and analysis of T cell proliferation in the presence of extracellular ATP. The in vivo effects of CD39-specific ASOs on target expression, anti-tumor immune responses and on tumor growth were analyzed in syngeneic mouse tumor models using multi-color flow cytometry.ResultsCD39-specific ASOs suppressed expression of CD39 mRNA and protein in different murine and human cancer cell lines and in primary human T cells. Degradation of extracellular ATP was strongly reduced by CD39-specific ASOs. Strikingly, CD39 knockdown by ASOs was associated with improved CD8+ T cell proliferation. Treatment of tumor-bearing mice with CD39-specific ASOs led to dose-dependent reduction of CD39-protein expression in regulatory T cells (Tregs) and tumor-associated macrophages. Moreover, frequency of intratumoral Tregs was substantially reduced in CD39 ASO-treated mice. As a consequence, the ratio of CD8+ T cells to Tregs in tumors was improved, while PD-1 expression was induced in CD39 ASO-treated intratumoral CD8+ T cells. Consequently, CD39 ASO treatment demonstrated potent reduction in tumor growth in combination with anti-PD-1 treatment.ConclusionTargeting of CD39 by ASOs represents a promising state-of-the art therapeutic approach to improve immune responses against tumors.
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Project description: Not available
| S-EPMC10570754 | biostudies-literature
Project description:Sustained therapeutic responses from traditional and next-generation antiandrogen therapies remain elusive in clinical practice due to inherent and/or acquired resistance resulting in persistent androgen receptor (AR) activity. Antisense oligonucleotides (ASO) have the ability to block target gene expression and associated protein products and provide an alternate treatment strategy for castration-resistant prostate cancer (CRPC). We demonstrate the efficacy and therapeutic potential of this approach with a Generation-2.5 ASO targeting the mouse AR in genetically engineered models of prostate cancer. Furthermore, reciprocal feedback between AR and PI3K/AKT signaling was circumvented using a combination approach of AR-ASO therapy with the potent pan-AKT inhibitor, AZD5363. This treatment strategy effectively improved treatment responses and prolonged survival in a clinically relevant mouse model of advanced CRPC. Thus, our data provide preclinical evidence to support a combination strategy of next-generation ASOs targeting AR in combination with AKT inhibition as a potentially beneficial treatment approach for CRPC.
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Project description:High expression and phosphorylation of signal transducer and transcription activator 3 (STAT3) are correlated with progression and poor prognosis in various types of cancer. The constitutive activation of STAT3 in cancer affects processes such as cell proliferation, apoptosis, metastasis, angiogenesis, and drug resistance. The importance of STAT3 in cancer makes it a potential therapeutic target. Various methods of directly and indirectly blocking STAT3 activity at different steps of the STAT3 pathway have been investigated. However, the outcome has been limited, mainly by the number of upstream proteins that can reactivate STAT3 or the relatively low specificity of the inhibitors. A new branch of molecules with significant therapeutic potential has emerged thanks to recent developments in the regulatory function of non-coding nucleic acids. Oligonucleotide-based therapeutics can silence target transcripts or edit genes, leading to the modification of gene expression profiles, causing cell death or restoring cell function. Moreover, they can reach untreatable targets, such as transcription factors. This review briefly describes oligonucleotide-based therapeutics that found application to target STAT3 activity in cancer. Additionally, this review comprehensively summarizes how the inhibition of STAT3 activity by nucleic acid-based therapeutics such as siRNA, shRNA, ASO, and ODN-decoy affected the therapy of different types of cancer in preclinical and clinical studies. Moreover, due to some limitations of oligonucleotide-based therapeutics, the importance of carriers that can deliver nucleic acid molecules to affect the STAT3 in cancer cells and cells of the tumor microenvironment (TME) was pointed out. Combining a high specificity of oligonucleotide-based therapeutics toward their targets and functionalized nanoparticles toward cell type can generate very efficient formulations.
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