Design Of Antisense Oligonucleotides Research Articles

Mechanistic insights into ASO-RNA complexation: Advancing antisense oligonucleotide design strategies

Mechanistic insights into ASO-RNA complexation: Advancing antisense oligonucleotide design strategies

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

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. 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. 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. Overall, by harnessing the specificity and efficacy of ASOs, we present an effective and adaptable therapeutic platform for NSCLC treatment. 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.

Down syndrome and DYRK1A overexpression: relationships and future therapeutic directions.

Down syndrome is a genetic-based disorder that results from the triplication of chromosome 21, leading to an overexpression of many triplicated genes, including the gene encoding Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A). This protein has been observed to regulate numerous cellular processes, including cell proliferation, cell functioning, differentiation, and apoptosis. Consequently, an overexpression of DYRK1A has been reported to result in cognitive impairment, a key phenotype of individuals with Down syndrome. Therefore, downregulating DYRK1A has been explored as a potential therapeutic strategy for Down syndrome, with promising results observed from in vivo mouse models and human clinical trials that administered epigallocatechin gallate. Current DYRK1A inhibitors target the protein function directly, which tends to exhibit low specificity and selectivity, making them unfeasible for clinical or research purposes. On the other hand, antisense oligonucleotides (ASOs) offer a more selective therapeutic strategy to downregulate DYRK1A expression at the gene transcript level. Advances in ASO research have led to the discovery of numerous chemical modifications that increase ASO potency, specificity, and stability. Recently, several ASOs have been approved by the U.S. Food and Drug Administration to address neuromuscular and neurological conditions, laying the foundation for future ASO therapeutics. The limitations of ASOs, including their high production cost and difficulty delivering to target tissues can be overcome by further advances in ASO design. DYRK1A targeted ASOs could be a viable therapeutic approach to improve the quality of life for individuals with Down syndrome and their families.

Mechanisms of Action of the US Food and Drug Administration-Approved Antisense Oligonucleotide Drugs.

Antisense oligonucleotides (ASOs) are single stranded nucleic acids that target RNA. The US Food and Drug Administration has approved ASOs for several diseases. ASOs utilize three principal modes of action (MOA). The first MOA is initiated by base-pairing between the ASO and its target mRNA, followed by RNase H-dependent mRNA degradation. The second MOA is triggered by ASOs that occlude splice acceptor sites in pre-mRNAs leading to skipping of a mutation-bearing exon. The third MOA involves ASOs that sterically hinder mRNA function, often inhibiting translation. ASOs contain a variety of modifications to the sugar-phosphate backbone and bases that stabilize the ASO or render them resistant to RNase activity. RNase H-dependent ASOs include inotersen and eplontersen (for hereditary transthyretin amyloidosis), fomiversen (for opportunistic cytomegalovirus infection), mipomersen (for familial hypercholesterolemia), and tofersen [for amyotrophic lateral sclerosis (ALS)]. Splice modulating ASOs include nursinersen (for spinal muscular atrophy) and eteplirsen, golodirsen, viltolarsen, and casimersen (all for the treatment of Duchenne muscular dystrophy). In addition, a designer ASO, milasen, was used to treat a single individual afflicted with Batten disease. Since ASO design relies principally upon knowledge of mRNA sequence, the bench to bedside pipeline for ASOs is expedient compared with protein-directed drugs.[Graphical abstract available.].

Strategies to improve the design of gapmer antisense oligonucleotide on allele-specific silencing

Gapmer antisense oligonucleotides (ASOs) hold therapeutic promise for allele-specific silencing, but face challenges in distinguishing between mutant and wild-type transcripts. This study explores new design strategies to enhance ASO specificity, focusing on a common dominant mutation in COL6A3 gene associated with Ullrich congenital muscular dystrophy. Initial gapmer ASO design exhibited high efficiency but poor specificity for the mutant allele. We then adopted a mixmer design, incorporating additional RNA bases based on computational predictions of secondary structures for both mutant and wild-type alleles, aiming to enhance ASO accessibility to mutant transcripts. The mixmer ASO design demonstrated up to a 3-fold increase in specificity compared with the classical gapmer design. Further refinement involved introducing a nucleotide mismatch as a structural modification, resulting in a 10-fold enhancement in specificity compared with the gapmer design and a 3-fold over the mixmer design. Additionally, we identified for the first time a potential role of the RNA-induced silencing complex (RISC), alongside RNase H1, in gapmer-mediated silencing, in contrast with what was observed with mixmer ASOs, where only RNase H1 was involved. In conclusion, this study presents a novel design concept for allele-specific ASOs leveraging mRNA secondary structures and nucleotide mismatching and suggests a potential involvement of RISC in gapmer-mediated silencing.

Exon 44 skipping in Duchenne muscular dystrophy: NS-089/NCNP-02, a dual-targeting antisense oligonucleotide

Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated antisense oligonucleotides for exon 44 skipping (applicable to approximately 6% of all Duchenne muscular dystrophy patients) to improve activity by using a novel antisense oligonucleotide design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to target two splicing regulators in exon 44 simultaneously, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a Duchenne muscular dystrophy patient to whom exon 44 skipping is applicable. We also assessed the invivo activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an antisense oligonucleotide with a novel connected-sequence design, showed highly efficient exon skipping both invitro and invivo.

Ensemble-Learning and Feature Selection Techniques for Enhanced Antisense Oligonucleotide Efficacy Prediction in Exon Skipping.

Antisense oligonucleotide (ASO)-mediated exon skipping has become a valuable tool for investigating gene function and developing gene therapy. Machine-learning-based computational methods, such as eSkip-Finder, have been developed to predict the efficacy of ASOs via exon skipping. However, these methods are computationally demanding, and the accuracy of predictions remains suboptimal. In this study, we propose a new approach to reduce the computational burden and improve the prediction performance by using feature selection within machine-learning algorithms and ensemble-learning techniques. We evaluated our approach using a dataset of experimentally validated exon-skipping events, dividing it into training and testing sets. Our results demonstrate that using a three-way-voting approach with random forest, gradient boosting, and XGBoost can significantly reduce the computation time to under ten seconds while improving prediction performance, as measured by R2 for both 2'-O-methyl nucleotides (2OMe) and phosphorodiamidate morpholino oligomers (PMOs). Additionally, the feature importance ranking derived from our approach is in good agreement with previously published results. Our findings suggest that our approach has the potential to enhance the accuracy and efficiency of predicting ASO efficacy via exon skipping. It could also facilitate the development of novel therapeutic strategies. This study could contribute to the ongoing efforts to improve ASO design and optimize gene therapy approaches.

Recent Progress and Challenges in the Development of Antisense Therapies for Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a dominant genetic disease in which the expansion of long CTG trinucleotides in the 3' UTR of the myotonic dystrophy protein kinase (DMPK) gene results in toxic RNA gain-of-function and gene mis-splicing affecting mainly the muscles, the heart, and the brain. The CUG-expanded transcripts are a suitable target for the development of antisense oligonucleotide (ASO) therapies. Various chemical modifications of the sugar-phosphate backbone have been reported to significantly enhance the affinity of ASOs for RNA and their resistance to nucleases, making it possible to reverse DM1-like symptoms following systemic administration in different transgenic mouse models. However, specific tissue delivery remains to be improved to achieve significant clinical outcomes in humans. Several strategies, including ASO conjugation to cell-penetrating peptides, fatty acids, or monoclonal antibodies, have recently been shown to improve potency in muscle and cardiac tissues in mice. Moreover, intrathecal administration of ASOs may be an advantageous complementary administration route to bypass the blood-brain barrier and correct defects of the central nervous system in DM1. This review describes the evolution of the chemical design of antisense oligonucleotides targeting CUG-expanded mRNAs and how recent advances in the field may be game-changing by forwarding laboratory findings into clinical research and treatments for DM1 and other microsatellite diseases.

High Concentration of an ISS-N1-Targeting Antisense Oligonucleotide Causes Massive Perturbation of the Transcriptome.

Intronic splicing silencer N1 (ISS-N1) located within Survival Motor Neuron 2 (SMN2) intron 7 is the target of a therapeutic antisense oligonucleotide (ASO), nusinersen (Spinraza), which is currently being used for the treatment of spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. The discovery of ISS-N1 as a promising therapeutic target was enabled in part by Anti-N1, a 20-mer ASO that restored SMN2 exon 7 inclusion by annealing to ISS-N1. Here, we analyzed the transcriptome of SMA patient cells treated with 100 nM of Anti-N1 for 30 h. Such concentrations are routinely used to demonstrate the efficacy of an ASO. While 100 nM of Anti-N1 substantially stimulated SMN2 exon 7 inclusion, it also caused massive perturbations in the transcriptome and triggered widespread aberrant splicing, affecting expression of essential genes associated with multiple cellular processes such as transcription, splicing, translation, cell signaling, cell cycle, macromolecular trafficking, cytoskeletal dynamics, and innate immunity. We validated our findings with quantitative and semiquantitative PCR of 39 candidate genes associated with diverse pathways. We also showed a substantial reduction in off-target effects with shorter ISS-N1-targeting ASOs. Our findings are significant for implementing better ASO design and dosing regimens of ASO-based drugs.

ANTISENSE OLIGONUCLEOTIDES AND RADIONUCLIDES – PERSPECTIVES

Over the past decade, antisense technology has successfully established itself as an entirely innovative platform for research and creation of new therapies. Significant advances in the design of antisense oligonucleotides, as well as a deeper understanding of their mechanisms of action, have led to their successful clinical application in many RNA-targeted therapies. In addition, their potential for in vivo imaging by radiolabeling has been identified. Here are discussed the prospects for the use of antisense oligonucleotides in nuclear medicine and highlighted some of the advantages and disadvantages of labelling them with radionuclides.

PFRED: A computational platform for siRNA and antisense oligonucleotides design.

PFRED a software application for the design, analysis, and visualization of antisense oligonucleotides and siRNA is described. The software provides an intuitive user-interface for scientists to design a library of siRNA or antisense oligonucleotides that target a specific gene of interest. Moreover, the tool facilitates the incorporation of various design criteria that have been shown to be important for stability and potency. PFRED has been made available as an open-source project so the code can be easily modified to address the future needs of the oligonucleotide research community. A compiled version is available for downloading at https://github.com/pfred/pfred-gui/releases/tag/v1.0 as a java Jar file. The source code and the links for downloading the precompiled version can be found at https://github.com/pfred.

Methods Used to Make Lipid Nanoparticles to Deliver LNA Gapmers Against lncRNAs into Acute Myeloid Leukemia (AML) Blasts.

Developing strategies to target lncRNAs are needed. In this chapter, we describe in detail a method to deliver antisense oligonucleotides into acute myeloid leukemia cells using lipid nanoparticles tagged with the transferrin receptor. While this chapter is focused on the delivery method, we also discuss important considerations about the design of antisense oligonucleotides (ASOs). The strategy described here has been used successfully to deliver ASOs into leukemic blasts and stem cells.

Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists

Oligonucleotide-based therapeutics have become a reality, and are set to transform management of many diseases. Nevertheless, the modulatory activities of these molecules on immune responses remain incompletely defined. Here, we show that gene targeting 2′-O-methyl (2′OMe) gapmer antisense oligonucleotides (ASOs) can have opposing activities on Toll-Like Receptors 7 and 8 (TLR7/8), leading to divergent suppression of TLR7 and activation of TLR8, in a sequence-dependent manner. Surprisingly, TLR8 potentiation by the gapmer ASOs was blunted by locked nucleic acid (LNA) and 2′-methoxyethyl (2′MOE) modifications. Through a screen of 192 2′OMe ASOs and sequence mutants, we characterized the structural and sequence determinants of these activities. Importantly, we identified core motifs preventing the immunosuppressive activities of 2′OMe ASOs on TLR7. Based on these observations, we designed oligonucleotides strongly potentiating TLR8 sensing of Resiquimod, which preserve TLR7 function, and promote strong activation of phagocytes and immune cells. We also provide proof-of-principle data that gene-targeting ASOs can be selected to synergize with TLR8 agonists currently under investigation as immunotherapies, and show that rational ASO selection can be used to prevent unintended immune suppression of TLR7. Taken together, our work characterizes the immumodulatory effects of ASOs to advance their therapeutic development.

Mind the Gapmer: Implications of Co-transcriptional Cleavage by Antisense Oligonucleotides.

While gapmers efficiently knock down as well as terminate transcription of nascent lncRNAs and mRNAs, Lee and Mendell (2020) and Lai etal. (2020) also demonstrate that Pol II termination is not observed with gapmers targeting the 3' terminal portions of the transcript.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Albumin-Binding Fatty Acid-Modified Gapmer Antisense Oligonucleotides for Modulation of Pharmacokinetics.

Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASOs. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.

The assembly and evaluation of antisense oligonucleotides applied in exon skipping for titin-based mutations in dilated cardiomyopathy

The leading cause of genetic dilated cardiomyopathy (DCM) is due to mutations in the TTN gene, impacting approximately 15–20% of familial and 18% of sporadic DCM cases. Currently, there is potential for a personalized RNA-based therapeutic approach in titin-based DCM, utilizing antisense oligonucleotide (AON) mediated exon-skipping, which attempts to reframe mutated titin transcripts, resulting in shortened, functional protein. However, the TTN gene is massive with 363 exons; each newly identified TTN exon mutation provides a challenge to address when considering the potential application of AON mediated exon skipping. In the initial phase of this strategy, the mutated TTN exon requires specific AON design and evaluation to assess the exon skipping effectiveness for subsequent experiments.Here, we present a detailed protocol to effectively assemble and evaluate AONs for efficient exon-skipping in targeted TTN exons. We chose a previously identified TTN 1-bp deletion mutation in exon 335 as an exemplary target exon, which causes a frameshift mutation leading to truncated A-band titin in DCM. We designed two specific AONs to mask the Ttn exon 335 and confirmed successfully mediated exon skipping without disrupting the Ttn reading frame. In addition, we evaluated and confirmed AON-treated HL-1 cells show maintained store-operated calcium entry, fractional shortening as well as preserved sarcomeric formation in comparison to control samples, indicating the treated cardiomyocytes retain adequate, essential cell function and structure, proving the treated cells can compensate for the loss of exon 335. These results indicate our method offers the first systematic protocol in designing and evaluating AONs specifically for mutated TTN target exons, expanding the framework of future advancements in the therapeutic potential of antisense-mediated exon skipping in titin-based DCM.

Thorough in silico and in vitro cDNA analysis of 21 putative BRCA1 and BRCA2 splice variants and a complex tandem duplication in BRCA2 allowing the identification of activated cryptic splice donor sites in BRCA2 exon 11.

For 21 putative BRCA1 and BRCA2 splice site variants, the concordance between mRNA analysis and predictions by in silico programs was evaluated. Aberrant splicing was confirmed for 12 alterations. In silico prediction tools were helpful to determine for which variants cDNA analysis is warranted, however, predictions for variants in the Cartegni consensus region but outside the canonical sites, were less reliable. Learning algorithms like Adaboost and Random Forest outperformed the classical tools. Further validations are warranted prior to implementation of these novel tools in clinical settings. Additionally, we report here for the first time activated cryptic donor sites in the large exon 11 of BRCA2 by evaluating the effect at the cDNA level of a novel tandem duplication (5' breakpoint in intron 4; 3' breakpoint in exon 11) and of a variant disrupting the splice donor site of exon 11 (c.6841+1G>C). Additional sites were predicted, but not activated. These sites warrant further research to increase our knowledge on cis and trans acting factors involved in the conservation of correct transcription of this large exon. This may contribute to adequate design of ASOs (antisense oligonucleotides), an emerging therapy to render cancer cells sensitive to PARP inhibitor and platinum therapies.

Antisense Oligonucleotide Design and Evaluation of Splice-Modulating Properties Using Cell-Based Assays.

Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as a therapeutic strategy for a number of hereditary conditions. AONs are small modified single-stranded RNA or DNA molecules that are complementary to splice enhancer or silencer target sites. Upon pre-mRNA binding, AONs will prevent or stimulate binding of the spliceosome thereby modulating splicing events. AONs can be designed and applied for different genes and genetic disorders as the specificity depends on their nucleotide sequence. Here we provide a guideline for setting up AON-based splice-modulation experiments by describing a detailed protocol to design and evaluate AONs using a combination of in silico and in vitro analyses.

Electronic Structures of LNA Phosphorothioate Oligonucleotides.

Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules.