RNA therapeutics came to global attention when mRNA-based vaccines provided an answer to the SARS-CoV-2 pandemic. The immense significance of this development notwithstanding, it is important to note that almost a decade prior to the pandemic, RNA drugs had made important inroads toward the amelioration of disease. The first class of RNA therapies to be introduced into clinical use were the antisense oligomers and siRNA drugs which generally induce a therapeutic effect by acting to brake or to modulate mRNA expression. RNA therapeutics is quickly becoming the fourth pillar of pharmacotherapy, and will have broad applications, including for the treatment of cardiovascular disease.

The United States (US) Food and Drug Administration (FDA) has approved several antisense oligomers (ASOs) and siRNA-based drugs to treat disorders associated with cardiovascular disease. In addition, multiple RNA-based drugs are in clinical trials to assess their safety and efficacy in patients with cardiovascular disorders, such as Zodasiran, a siRNA therapy that targets angiopoietin-like protein 3 (ANGPTL3) to reduce LDL cholesterol.

Because of limitless sequence choice; speed of design; and relative ease of synthesis, RNA drugs will be rapidly developed, will have broad applications, and will be generated at lower cost than other drug types. This review aims to highlight RNA therapies for cardiovascular diseases that are approved, and those that are under clinical evaluation.

Duchenne muscular dystrophy (DMD) is a severe disorder caused by mutations in the dystrophin gene, resulting in loss of functional dystrophin protein in muscle. While phosphorodiamidate morpholino oligomers (PMOs) are promising exon-skipping therapeutics aimed at restoring dystrophin expression, their effectiveness is often limited by poor muscle delivery. We developed AOC 1044, an antibody-oligonucleotide conjugate (AOC) that combines a PMO-targeting exon 44 with an antibody against the transferrin receptor (TfR1), enhancing delivery to muscle tissues for patients with DMD amenable to exon 44 skipping (DMD44). AOC 1044 induces dose-dependent exon 44 skipping and its mouse-active variant elicited dose-dependent dystrophin restoration in skeletal and cardiac muscle in a DMD mouse model. This treatment also reduced muscle damage, as evidenced by decreases in serum creatine kinase and key liver enzymes, suggesting that restored dystrophin is functionally active. In nonhuman primates, single or repeated AOC 1044 doses resulted in dose-dependent increases in PMO concentration and exon 44 skipping across a range of muscle tissues, including the heart. Collectively, these findings highlight AOC 1044 as a promising therapeutic candidate for patients with DMD44, offering improved muscle targeting and meaningful dystrophin restoration, with potential clinical benefits in reducing muscle degeneration.

Radiotherapy remains a cornerstone of cancer treatment, using high-energy radiation to induce DNA damage in tumor cells, leading to cell death. However, its efficacy is often hindered by challenges such as radiation resistance and side effects. As a powerful class of functional molecules, nucleic acid drugs (NADs) present a promising solution to these limitations. Engineered to target key pathways like DNA repair and tumor hypoxia, NADs can enhance radiotherapy sensitivity. NADs can also serve as delivery vehicles for radiotherapy agents such as radionuclides, improving targeting accuracy and minimizing side effects. This review explores the role of NADs in optimizing radiotherapy, highlighting their mechanisms, clinical applications, and synergies with radiotherapy, ultimately offering a promising strategy for improving patient outcomes in cancer therapy.

Nucleic-acid-based therapies have emerged as a pivotal domain within contemporary biomedical science, marked by significant advancements in recent years. These innovative treatments primarily operate through the precise binding of DNA or RNA molecules to discrete target genes, subsequently suppressing the expression of the target proteins. The spectrum of nucleic-acid-based therapies encompasses antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs), etc. Compared to more traditional medicinal approaches, nucleic-acid-based therapies stand out for their highly targeted action on specific genes, as well as their potential for chemical modification to improve resistance to nucleases, ensuring sustained therapeutic activity and mitigating immunogenicity concerns. Nevertheless, these molecules' limited cellular permeability necessitates the deployment of delivery vectors to enhance their intracellular uptake and stability. As nucleic-acid-based therapies progressively display promising pharmacodynamic profiles, there has been a burgeoning interest in these treatments for applications in clinical research. This review aims to summarize the variety of nucleic acid drugs and their mechanisms, evaluate the present status in research and application, discourse on prospective trends, and potential challenges ahead. These innovative therapeutics are anticipated to assume a pivotal role in the management of a wide array of diseases.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.

Tetrahedral framework nucleic acids (tFNAs) represent a promising advancement in nucleic acid nanotechnology due to their unique structural properties, high biocompatibility, and multifaceted biomedical applications. Constructed through a one-pot annealing method, four single-stranded DNAs self-assemble into stable, three-dimensional tetrahedral nanostructures with enhanced mechanical robustness and physiological stability, resisting enzymatic degradation. Their ability to permeate mammalian cells without transfection agents, coupled with modifiable surfaces, positions tFNAs as versatile carriers for drug and gene delivery systems. The tFNA-based platforms exhibit superior therapeutic efficacy, including antioxidative and anti-inflammatory effects, alongside efficient cellular uptake and tissue penetration. These features underpin their role in precision medicine, enabling targeted delivery of diverse therapeutic agents such as synthetic compounds, peptides, and nucleic acids. Additionally, tFNAs demonstrate significant potential in regenerative medicine, immune modulation, antibacterial strategies, and oncology. By addressing challenges in translational integration, tFNAs stand poised to accelerate the development of biomedical research and clinical applications, fostering novel therapies and enhancing therapeutic outcomes across a wide spectrum of diseases. This Perspective thoroughly details the unique attributes and diverse applications of tFNAs and critically evaluates tFNAs' clinical translational potential, outlining inherent implementation challenges and exploring potential solutions to these obstacles.

Nucleic acids have emerged as a powerful class of therapeutics. Through simple base pair complementarity, nucleic acids allow the targeting of a variety of pathologically relevant proteins and RNA molecules. However, despite the preliminary successes of nucleic acids as drugs in the clinic, limited biodistribution, inadequate delivery mechanisms, and target engagement remain key challenges in the field. A key area of research has been the chemical optimization of nucleic acid backbones to significantly enhance their "drug-like" properties. Alternatively, this review focuses on the next generation of nucleic acid chemical modifications: covalent biochemical conjugates. These conjugates are being applied to improve the delivery, functionality, and targeting. Exploiting research on heterobifunctionals, such as PROTACs, RIBOTACs, molecular glues, etc., has the potential to dramatically expand nucleic acid drug functionality and target engagement capabilities. Such next-generation chemistry-based enhancements have the potential to unlock nucleic acids as effective and versatile therapeutic agents.

Thirty years ago, in 1995, I proposed a fundamental treatment for Duchenne Muscular Dystrophy (DMD) using antisense oligonucleotides (ASOs) to induce exon skipping and restore dystrophin expression. DMD is a progressive and fatal muscular dystrophy, and the establishment of an effective therapy has been a pressing demand among patients worldwide. Exon-skipping therapy utilizing ASOs has garnered significant attention as one of the most promising treatments for DMD, stimulating global research and development efforts in ASO technology. Two decades later, in 2016, one ASO was conditionally approved by the U.S. FDA as the first DMD treatment. This review summarizes the current status and challenges of ASO-based exon-skipping therapies for DMD and explores the prospects of pseudoexon skipping using ASOs, which holds the potential for achieving a complete cure for DMD.

Personalized antisense oligonucleotides (ASOs) have achieved positive results in the treatment of rare genetic disease1. As clinical sequencing technologies continue to advance, the ability to identify patients with rare disease harbouring pathogenic genetic variants amenable to this therapeutic strategy will probably improve. Here we describe a scalable platform for generating patient-derived cellular models and demonstrate that these personalized models can be used for preclinical evaluation of patient-specific ASOs. We describe protocols for delivery of ASOs to patient-derived organoid models and confirm reversal of disease-associated phenotypes in cardiac organoids derived from a patient with Duchenne muscular dystrophy (DMD) with a structural deletion in the gene encoding dystrophin (DMD) that is amenable to treatment with existing ASO therapeutics. Furthermore, we designed novel patient-specific ASOs for two additional patients with DMD (siblings) with a deep intronic variant in the DMD gene that gives rise to a novel splice acceptor site, incorporation of a cryptic exon and premature transcript termination. We showed that treatment of patient-derived cardiac organoids with patient-specific ASOs results in restoration of DMD expression and reversal of disease-associated phenotypes. The approach outlined here provides the foundation for an expedited path towards the design and preclinical evaluation of personalized ASO therapeutics for a broad range of rare diseases.

Dysregulation of RNA processing has in recent years emerged as a significant contributor to neurodegeneration. The diverse mechanisms and molecular functions underlying RNA processing underscore the essential role of RNA regulation in maintaining neuronal health and function. RNA molecules are bound by RNA-binding proteins (RBPs), and interactions between RNAs and RBPs are commonly affected in neurodegeneration. In this review, we highlight recent progress in understanding dysregulated RNA-processing pathways and the causes of RBP dysfunction across various neurodegenerative diseases. We discuss both established and emerging mechanisms of RNA-mediated neuropathogenesis in this rapidly evolving field. Furthermore, we explore the development of potential RNA-targeting therapeutic approaches for the treatment of neurodegenerative diseases.

Recent years have seen enormous progress in the field of advanced therapeutics for the progressive muscle wasting disease Duchenne muscular dystrophy (DMD). In particular, four antisense oligonucleotide (ASO) therapies targeting various DMD-causing mutations have achieved FDA approval, marking major milestones in the treatment of this disease. These compounds are designed to induce alternative splicing events that restore the translation reading frame of the dystrophin gene, leading to the generation of internally-deleted, but mostly functional, pseudodystrophin proteins with the potential to compensate for the genetic loss of dystrophin. However, the efficacy of these compounds is very limited, with delivery remaining a key obstacle to effective therapy. There is therefore an urgent need for improved ASO technologies with better efficacy, and with applicability to a wider range of patient mutations. Here we discuss recent developments in ASO therapies for DMD, and future prospects with a focus on ASO chemical modification and bioconjugation strategies.

Amyotrophic lateral sclerosis (ALS) and Huntington's disease (HD) are diverse in clinical presentation and are caused by complex and multiple factors, including genetic mutations and environmental factors. Numerous therapeutic approaches have been developed based on the genetic causes and potential mechanisms of ALS and HD. Currently, available treatments for various neurodegenerative diseases can alleviate symptoms but do not provide a definitive cure. Gene therapy, which aims to modify or express specific proteins for neuroprotection or correction, is considered a powerful tool in managing neurodegenerative conditions. To date, antisense oligonucleotide (ASO) drugs targeting the pathological genes associated with ALS and HD have shown promising results in numerous animal studies and several clinical trials. This review provides a comprehensive overview of the development, mechanisms of action, limitations, and clinical applications of ASO drugs in neurodegenerative diseases, with a specific focus on ALS and HD therapeutic strategies.

Alternative splicing (AS) is a mechanism that generates translational diversity within a genome. Equally important is the dynamic adaptability of the splicing machinery, which can give preference to one isoform over others encoded by a single gene. These isoform preferences change in response to the cell's state and function. Particularly significant is the impact of physiological alternative splicing in T lymphocytes, where specific isoforms can enhance or reduce the cells' reactivity to stimuli. This process makes splicing isoforms defining features of cell states, exemplified by CD45 splice isoforms, which characterize the transition from naïve to memory states. Two developments have accelerated the use of AS dynamics for therapeutic interventions: advancements in long-read RNA sequencing and progress in nucleic acid chemical modifications. Improved oligonucleotide stability has enabled their use in directing splicing to specific sites or modifying sequences to enhance or silence particular splicing events. This review highlights immune regulatory splicing patterns with potential significance for enhancing anticancer immunotherapy.

Vascular smooth muscle cell (vSMC) dysfunction is a critical contributor to cardiovascular diseases, including atherosclerosis, restenosis and vein graft failure. Recent advances have unveiled a fascinating range of non-coding RNAs (ncRNAs) that play a pivotal role in regulating vSMC function. This review aims to provide an in-depth analysis of the mechanisms underlying vSMC dysfunction and the therapeutic potential of various ncRNAs in mitigating this dysfunction, either preventing or reversing it. We explore the intricate interplay of microRNAs, long-non-coding RNAs and circular RNAs, shedding light on their roles in regulating key signalling pathways associated with vSMC dysfunction. We also discuss the prospects and challenges associated with developing ncRNA-based therapies for this prevalent type of cardiovascular pathology. LINKED ARTICLES: This article is part of a themed issue Non-coding RNA Therapeutics. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v182.2/issuetoc.

The evolution of novel Severe Acute Respiratory Syndrome-related Coronavirus 2 (SARS-CoV-2) strains with greater degrees of infectivity, resistance to vaccine-induced acquired immunity, and more severe morbidity have contributed to the recent spread of COVID-19. In light of this, novel therapeutic alternatives with improved effectiveness and fewer side effects have become a necessity. Despite many new or repurposed antiviral agents recommended for Coronavirus disease (COVID-19) therapy, this objective remains unfulfilled. Under these circumstances, the scientific community holds the significant responsibility to develop classes of novel therapeutic modalities to combat SARS-CoV-2 with the least harmful side effects.

Antisense Oligonucleotides (ASOs) are short single-stranded oligonucleotides that allow the specific targeting of RNA, leading to its degradation. They may also prevent cellular factors or machinery from binding to the target RNA. It is possible to improve the pharmacokinetics and pharmacodynamics of ASOs by chemical modification or bioconjugation, which may provide conditions for customization of a particular clinical target. This study aimed to outline the potential use of ASOs in the treatment of COVID-19 disease, along with the use of antisense stabilization and transfer methods, as well as future challenges and limitations.

We have reviewed the structure and properties of ASOs containing nucleobase, sugar, or backbone modifications, and provided an overview of the therapeutic potential, delivery challenges, and strategies of ASOs in the treatment of COVID-19.

The first-line therapy for COVID-19-infected individuals, as well as the development of oligonucleotide- based drugs, warrants further investigation. Chemical changes in the oligonucleotide structure can affect the biological processes. These chemical alterations may lead to enhanced potency, while changing the pharmacokinetics and pharmacodynamics.

ASOs can be designed to target both coding and non-coding regions of the viral genome to disrupt or completely degrade the genomic RNA and thereby eliminate SARS-CoV-2. They may be very effective in areas, where vaccine distribution is challenging, and they may be helpful for future coronavirus pandemics.

Nowadays, nucleic acid derivatives capable of modulating gene expression at the RNA level have gained widespread recognition as promising therapeutic agents. A suitable degree of biological stability of oligonucleotide therapeutics is required for in vivo application; this can be most expeditiously achieved by the chemical modification of the internucleotidic phosphate group, which may also affect their cellular uptake, tissue distribution and pharmacokinetics. Our group has previously developed a strategy for the chemical modification of the phosphate group via the Staudinger reaction on a solid phase of the intermediate dinucleoside phosphite triester and a range of, preferably, electron deficient organic azides such as sulfonyl azides during automated solid-phase DNA synthesis according to the conventional β-cyanoethyl phosphoramidite scheme. Polyfluoro compounds are characterized by unique properties that have prompted their extensive application both in industry and in scientific research. We report herein the synthesis and isolation of novel oligodeoxyribonucleotides incorporating internucleotidic perfluoro-1-octanesulfonyl phosphoramidate or 2,2,2-trifluoroethanesulfonyl phosphoramidate groups. In addition, novel oligonucleotide derivatives with fluorinated zwitterionic phosphate mimics were synthesized by a tandem methodology, which involved (a) the introduction of a carboxylic ester group at the internucleotidic position via the Staudinger reaction with methyl 2,2-difluoro-3-azidosulfonylacetate; and (b) treatment with an aliphatic diamine, e.g., 1,1-dimethylethylenediamine or 1,3-diaminopropane. It was further shown that the polyfluoro oligonucleotides obtained were able to form complementary duplexes with either DNA or RNA, which were not significantly differing in stability from the natural counterparts. Long-chain perfluoroalkyl oligonucleotides were taken up into cultured human cells in the absence of a transfection agent. It may be concluded that the polyfluoro oligonucleotides described here can represent a useful platform for designing oligonucleotide therapeutics.

Nucleic acids are a class of drugs that can modulate gene and protein expression by various mechanisms, namely, RNAi, mRNA degradation by RNase H cleavage, splice modulation, and steric blocking of protein binding or mRNA translation, thus exhibiting immense potential to treat various genetic and rare diseases. Unlike protein-targeted therapeutics, the clinical use of nucleic acids relies on Watson-Crick sequence recognition to regulate aberrant gene expression and impede protein translation. Though promising, targeted delivery remains a bottleneck for the clinical adoption of nucleic acid-based therapeutics. To overcome the delivery challenges associated with nucleic acids, various chemical modifications and bioconjugation-based delivery strategies have been explored. Currently, liver targeting by N-acetyl galactosamine (GalNAc) conjugation has been at the forefront for the treatment of rare and various metabolic diseases, which has led to FDA approval of four nucleic acid drugs. In addition, various other bioconjugation strategies have been explored to facilitate active organ and cell-enriched targeting. This review briefly covers the different classes of nucleic acids, their mechanisms of action, and their challenges. We also elaborate on recent advances in bioconjugation strategies in developing a diverse set of ligands for targeted delivery of nucleic acid drugs.

Owing to the increased public interest and advances in chemical modifications, the approval of antisense therapeutics, a class of mRNA-targeting DNA-based oligonucleotide therapeutics, has accelerated in recent years. It was previously reported that siRNAs with several 4'-C-α-aminoethoxy-2'-O-methyl-uridine (4AEoU) analogs could maintain moderate thermal stability similar to the native ones while showing robust nuclease stability. In this study, we further expanded the application of 4AEo modification to antisense therapeutics and achieved superior thermal stability by adding the uracil 5-propynyl modification. Antisense oligonucleotides containing 4'-C-α-aminoethoxy-2'-O-methyl-5-propynyl-uridine (4AEopU) could efficiently activate RNase H-mediated antisense in vitro in the presence of native DNA gaps. These results encourage future studies of 4AEopU-containing antisense therapeutics.

Mutations that impact splicing play a significant role in disease etiology but are not fully understood. To characterize the impact of exonic variants on splicing in 71 clinically-actionable disease genes in asymptomatic people, we analyzed 32,112 exonic mutations from ClinVar and Geisinger MyCode using a minigene reporter assay. We identify 1,733 splice-disrupting mutations, of which the most extreme 1-2% of variants are likely to be deleterious. We report that these variants are not distributed evenly across exons but are mostly concentrated in the ∼8% of exons that are most susceptible to splicing mutations (i.e. hotspot exons). We demonstrate that splicing defects in these exons can be reverted by ASOs targeting the splice sites of either their upstream or downstream flanking exons. This finding supports the feasibility of developing single therapeutic ASOs that could revert all splice-altering variants localized to a particular exon.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematological disorders and are more commonly found in people over the age of 60. MDS patients exhibit peripheral blood cytopenias and carry an increased risk of disease progression to acute myeloid leukemia (AML). Splicing factor mutations (including genes SF3B1, SRSF2, U2AF1, and ZRSR2) are early events identified in more than 50% of MDS cases. These mutations cause aberrant pre-mRNA splicing and impact MDS pathophysiology. Emerging evidence shows that splicing factor-mutant cells are more sensitive to perturbations targeting the spliceosome, aberrantly spliced genes and/or their regulated molecular pathways. This review summarizes current therapeutic strategies and ongoing efforts targeting splicing factor mutations for the treatment of MDS.

Alternative splicing (AS) plays a pivotal role in protein diversity and mRNA maturation. Programmable control of targeted AS events is of longstanding interest in RNA biology, promising correction of dysregulated splicing in disease and discovery of AS events. This review explores four main strategies for programmable splicing manipulation: (1) inhibiting splicing signals with antisense oligonucleotides (ASOs), exemplified by therapies approved by the U.S. Food and Drug Administration, (2) applying DNA-targeting clustered regularly interspaced short palindromic repeats systems to edit splicing signals, (3) using synthetic splicing factors, including synthetic proteins and ribonucleoproteins, inspired by natural RNA-binding proteins, and (4) guiding endogenous splicing machinery with bifunctional ASOs and engineered small nuclear RNAs. While ASOs remain clinically prominent, emerging technologies aim for broad, scalable, durable, and precise splicing modulation, holding promise for transformative advancements in RNA biology and therapeutic interventions.

The study aimed to provide quantitative information on the utilization of MRI transverse relaxation time constant (MRI-T2) of leg muscles in DMD clinical trials by developing multivariate disease progression models of Duchenne muscular dystrophy (DMD) using 6-min walk distance (6MWD) and MRI-T2. Clinical data were collected from the prospective and longitudinal ImagingNMD study. Disease progression models were developed by a nonlinear mixed-effect modeling approach. Univariate models of 6MWD and MRI-T2 of five muscles were developed separately. Age at assessment was the time metric. Multivariate models were developed by estimating the correlation of 6MWD and MRI-T2 model variables. Full model estimation approach for covariate analysis and five-fold cross validation were conducted. Simulations were performed to compare the models and predict the covariate effects on the trajectories of 6MWD and MRI-T2. Sigmoid Imax and Emax models best captured the profiles of 6MWD and MRI-T2 over age. Steroid use, baseline 6MWD, and baseline MRI-T2 were significant covariates. The median age at which 6MWD is half of its maximum decrease in the five models was similar, while the median age at which MRI-T2 is half of its maximum increase varied depending on the type of muscle. The models connecting 6MWD and MRI-T2 successfully quantified how individual characteristics alter disease trajectories. The models demonstrate a plausible correlation between 6MWD and MRI-T2, supporting the use of MRI-T2. The developed models will guide drug developers in using the MRI-T2 to most efficient use in DMD clinical trials.

Nucleic acid drugs are one of the hot spots in the field of biomedicine in recent years, and play a crucial role in the treatment of many diseases. However, its low stability and difficulty in target drug delivery are the bottlenecks restricting its application. Hydrogels are proven to be promising for improving the stability of nucleic acid drugs, reducing the adverse effects of rapid degradation, sudden release, and unnecessary diffusion of nucleic acid drugs. In this review, the strategies of loading nucleic acid drugs in hydrogels are summarized for various biomedical research, and classify the mechanism principles of these strategies, including electrostatic binding, hydrogen bond based binding, hydrophobic binding, covalent bond based binding and indirect binding using various carriers. In addition, this review also describes the release strategies of nucleic acid drugs, including photostimulation-based release, enzyme-responsive release, pH-responsive release, and temperature-responsive release. Finally, the applications and future research directions of hydrogels for delivering nucleic acid drugs in the field of medicine are discussed.

High efficacy, selectivity and cellular targeting of therapeutic agents has been an active area of investigation for decades. Currently, most clinically approved therapeutics are small molecules or protein/antibody biologics. Targeted action of small molecule drugs remains a challenge in medicine. In addition, many diseases are considered 'undruggable' using standard biomacromolecules. Many of these challenges however, can be addressed using nucleic therapeutics. Nucleic acid drugs (NADs) are a new generation of gene-editing modalities characterized by their high efficiency and rapid development, which have become an active research topic in new drug development field. However, many factors, including their low stability, short half-life, high immunogenicity, tissue targeting, cellular uptake, and endosomal escape, hamper the delivery and clinical application of NADs. Scientists have used chemical modification techniques to improve the physicochemical properties of NADs. In contrast, modified NADs typically require carriers to enter target cells and reach specific intracellular locations. Multiple delivery approaches have been developed to effectively improve intracellular delivery and the in vivo bioavailability of NADs. Several NADs have entered the clinical trial recently, and some have been approved for therapeutic use in different fields. This review summarizes NADs development and evolution and introduces NADs classifications and general delivery strategies, highlighting their success in clinical applications. Additionally, this review discusses the limitations and potential future applications of NADs as gene therapy candidates.

Recent advances in genetics and nucleic acid chemistry have created fundamentally new tools, both for practical applications in therapy and diagnostics and for fundamental genome editing tasks. Nucleic acid-based therapeutic agents offer a distinct advantage of selectively targeting the underlying cause of the disease. Nevertheless, despite the success achieved thus far, there remain unresolved issues regarding the improvement of the pharmacokinetic properties of therapeutic nucleic acids while preserving their biological activity. In order to address these challenges, there is a growing focus on the study of safe and effective delivery methods utilising modified nucleic acid analogues and their lipid bioconjugates. The present review article provides an overview of the current state of the art in the use of chemically modified nucleic acid derivatives for therapeutic applications, with a particular focus on oligonucleotides conjugated to lipid moieties. A systematic analysis has been conducted to investigate the ability of amphiphilic oligonucleotides to self-assemble into micelle-like structures, as well as the influence of non-covalent interactions of such derivatives with serum albumin on their biodistribution and therapeutic effects.

Rare diseases affect almost 500 million people globally, predominantly impacting children and often leading to significantly impaired quality of life and high treatment costs. While significant contributions have been made to develop effective treatments for those with rare diseases, more rapid drug discovery strategies are needed. Therapeutic antisense oligonucleotides can modulate target gene expression with high specificity through various mechanisms determined by base sequences and chemical modifications; and have shown efficacy in clinical trials for a few rare neurological conditions. Therefore, this review will focus on the applications of antisense oligonucleotides, in particular splice-switching antisense oligomers as promising therapeutics for rare neurological diseases, with key examples of Duchenne muscular dystrophy and spinal muscular atrophy. Challenges and future perspectives in developing antisense therapeutics for rare conditions including target discovery, antisense chemical modifications, animal models for therapeutic validations, and clinical trial designs will also be briefly discussed.

Duchenne Muscular dystrophy (DMD), a yet-incurable X-linked recessive disorder that results in muscle wasting and loss of ambulation is due to mutations in the dystrophin gene. Exonic duplications of dystrophin gene are a common type of mutations found in DMD patients. In this study, we utilized a single guide RNA CRISPR strategy targeting intronic regions to delete the extra duplicated regions in patient myogenic cells carrying duplication of exon 2, exons 2-9, and exons 8-9 in the DMD gene. Immunostaining on CRISPR-corrected derived myotubes demonstrated the rescue of dystrophin protein. Subsequent RNA sequencing of the DMD cells indicated rescue of genes of dystrophin related pathways. Examination of predicted close-match off-targets evidenced no aberrant gene editing at these loci. Here, we further demonstrate the efficiency of a single guide CRISPR strategy capable of deleting multi-exon duplications in the DMD gene without significant off target effect. Our study contributes valuable insights into the safety and efficacy of using single guide CRISPR strategy as a potential therapeutic approach for DMD patients with duplications of variable size.

Small nucleic acid drugs, composed of nucleotides, represent a novel class of pharmaceuticals that differ significantly from conventional small molecule and antibody-based therapeutics. These agents function by selectively targeting specific genes or their corresponding messenger RNAs (mRNAs), further modulating gene expression and regulating translation-related processes. Prominent examples within this category include antisense oligonucleotides (ASO), small interfering RNAs (siRNAs), microRNAs (miRNAs), and aptamers. The emergence of small nucleic acid drugs as a focal point in contemporary biopharmaceutical research is attributed to their remarkable specificity, facile design, abbreviated development cycles, expansive target spectrum, and prolonged activity. Overcoming challenges such as poor stability, immunogenicity, and permeability issues have been addressed through the integration of chemical modifications and the development of drug delivery systems. This review provides an overview of the current status and prospective trends in small nucleic acid drug development. Commencing with a historical context, we introduce the primary classifications and mechanisms of small nucleic acid drugs. Subsequently, we delve into the advantages of the U.S. Food and Drug Administration (FDA) approved drugs and mainly discuss the challenges encountered during their development. Apart from researching chemical modification and delivery system that efficiently deliver and enrich small nucleic acid drugs to target tissues, promoting endosomal escape is a critical scientific question and important research direction in siRNA drug development. Future directions in this field will prioritize addressing these challenges to facilitate the clinical transformation of small nucleic acid drugs.

A synthetic protocol for the preparation of a new class of morpholino homonucleosides in enantiopure form starting from readily available 1,2-aminoalcohols or glycidol has been developed. Key intermediates of the synthetic sequence are 2-bromomethyl morpholines, diastereoselectively achieved from the corresponding alkenols by palladaelectro-catalyzed alkoxybromination of unactivated alkenes. The so obtained bromo derivatives are in turn susceptible to functionalization with nucleic bases for easy access to morpholino homonucleosides.

Standards of care exist to optimize outcomes in Duchenne and Becker muscular dystrophy (DBMD), caused by alterations in the DMD gene; however, there are limited data regarding health care access in these patients. This study aims to characterize outpatient subspecialty care utilization in pediatric patients with DBMD.

This retrospective cohort study used administrative claims data from IBM MarketScan Medicaid and Commercial Claims and Encounters Research Databases (2013-2018). Male patients 1-18 years with an ICD-9/10 diagnosis code for hereditary progressive muscular dystrophy between January 1, 2013, and December 31, 2017, were included. Participants were stratified into 3 age cohorts: 1-6 years, 7-12 years, and 13-18 years. The primary outcome was rate of annual neurology visits. Secondary outcomes included annual follow-up rates in other subspecialties and proportion of days covered (PDC) by corticosteroids.

A total of 1,386 patients met inclusion-347 (25.0%) age 1-6 years, 502 (36.2%) age 7-12 years, and 537 (38.7%) age 13-18 years. Heart failure, respiratory failure, and technology dependence increased with age (p for all<0.05). The rate of neurology visits per person-year was 0.36 and did not differ by age. Corticosteroid use was low; 30% of person-years (1452/4829) had a PDC ≥20%. Medicaid insurance was independently associated with a lower likelihood of annual neurology follow-up (OR 0.23; 95% CI 0.18-0.28).

The rate of annual neurology follow-up and corticosteroid use in patients with DBMD is low. Medicaid insurance status was independently associated with a decreased likelihood of neurology follow-up, while age was not, suggesting that factors other than disease severity influence neurology care access. Identifying barriers to regular follow-up is critical in improving outcomes for patients with DBMD.

Alternative splicing is a highly intricate process that plays a crucial role in post-transcriptional regulation and significantly expands the functional proteome of a limited number of coding genes in eukaryotes. Its regulation is multifactorial, with RNA structure exerting a significant impact. Aberrant RNA conformations lead to dysregulation of splicing patterns, which directly affects the manifestation of disease symptoms. In this review, the molecular mechanisms of RNA secondary structure-mediated splicing regulation are summarized, with a focus on the complex interplay between aberrant RNA conformations and disease phenotypes resulted from splicing defects. This study also explores additional factors that reshape structural conformations, enriching our understanding of the mechanistic network underlying structure-mediated splicing regulation. In addition, an emphasis has been placed on the clinical role of targeting aberrant splicing corrections in human diseases. The principal mechanisms of action behind this phenomenon are described, followed by a discussion of prospective development strategies and pertinent challenges.

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.].

Rapid advances in biological knowledge and technological innovation have greatly advanced the fields of stem cell and gene therapies to combat a broad spectrum of neurologic disorders. Researchers are currently exploring a variety of stem cell types (e.g., embryonic, progenitor, induced pluripotent) and various transplantation strategies, each with its own advantages and drawbacks. Similarly, various gene modification techniques (zinc finger, TALENs, CRISPR-Cas9) are employed with various delivery vectors to modify underlying genetic contributors to neurologic disorders. While these two individual fields continue to blaze new trails, it is the combination of these technologies which enables genetically engineered stem cells and vastly increases investigational and therapeutic opportunities. The capability to culture and expand stem cells outside the body, along with their potential to correct genetic abnormalities in patient-derived cells or enhance cells with extra gene products, unleashes the full biological potential for innovative, multifaceted approaches to treat complex neurological disorders. In this review, we provide an overview of stem cell and gene therapies in the context of neurologic disorders, highlighting recent advances and current shortcomings, and discuss prospects for future therapies in clinical settings.

Nonalcoholic steatohepatitis (NASH) represents a severe disease subtype of nonalcoholic fatty liver disease (NAFLD) that is thought to be highly associated with systemic metabolic abnormalities. It is characterized by a series of substantial liver damage, including hepatocellular steatosis, inflammation, and fibrosis. The end stage of NASH, in some cases, may result in cirrhosis and hepatocellular carcinoma (HCC). Nowadays a large number of investigations are actively under way to test various therapeutic strategies, including emerging oligonucleotide drugs (e.g., antisense oligonucleotide, small interfering RNA, microRNA, mimic/inhibitor RNA, and small activating RNA) that have shown high potential in treating this fatal liver disease. This article systematically reviews the pathogenesis of NASH/NAFLD, the promising druggable targets proven by current studies in chemical compounds or biological drug development, and the feasibility and limitations of oligonucleotide-based therapeutic approaches under clinical or pre-clinical studies.

RNA molecules have emerged as promising clinical therapeutics due to their ability to target "undruggable" proteins or molecules with high precision and minimal side effects. Nevertheless, the primary challenge in RNA therapeutics lies in rapid degradation and clearance from systemic circulation, the inability to traverse cell membranes, and the efficient intracellular delivery of bioactive RNA molecules. In this review, we explore the implications of RNAs in diseases and provide a chronological overview of the development of RNA therapeutics. Additionally, we summarize the technological advances in RNA-screening design, encompassing various RNA databases and design platforms. The paper then presents an update on FDA-approved RNA therapeutics and those currently undergoing clinical trials for various diseases, with a specific emphasis on RNA medicine and RNA vaccines.

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.

Accurate pre-mRNA splicing is essential for proper protein translation; however, aberrant splicing is commonly observed in the context of cancer and genetic disorders. Notably, in genetic diseases, these splicing abnormalities often play a pivotal role. Substantial challenges persist in accurately identifying and classifying disease-induced aberrant splicing, as well as in development of targeted therapeutic strategies. In this review, we examine prevalent forms of aberrant splicing and explore potential therapeutic approaches aimed at addressing these splicing-related diseases. This summary contributes to a deeper understanding of the complexities about aberrant splicing and provide a foundation for the development of effective therapeutic interventions in the field of genetic disorders and cancer.

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle-wasting disorder that leads to rapid loss of mobility and premature death. The absence of functional dystrophin in DMD patients reduces sarcolemma stiffness and increases contraction damage, triggering a cascade of events leading to muscle cell degeneration, chronic inflammation, and deposition of fibrotic and adipose tissue. Efforts in the last decade have led to the clinical approval of novel drugs for DMD that aim to restore dystrophin function. However, combination therapies able to restore dystrophin expression and target the myriad of cellular events found impaired in dystrophic muscle are desirable. Muscles are higher energy consumers susceptible to mitochondrial defects. Mitochondria generate a significant source of reactive oxygen species (ROS), and they are, in turn, sensitive to proper redox balance. In both DMD patients and animal models there is compelling evidence that mitochondrial impairments have a key role in the failure of energy homeostasis. Here, we highlighted the main aspects of mitochondrial dysfunction and oxidative stress in DMD and discussed the recent findings linked to mitochondria/ROS-targeted molecules as a therapeutic approach. In this respect, dual targeting of both mitochondria and redox homeostasis emerges as a potential clinical option in DMD.

The rapid evolution of gene editing technology has markedly improved the outlook for treating genetic diseases. Base editing, recognized as an exceptionally precise genetic modification tool, is emerging as a focus in the realm of genetic disease therapy. We provide a comprehensive overview of the fundamental principles and delivery methods of cytosine base editors (CBE), adenine base editors (ABE), and RNA base editors, with a particular focus on their applications and recent research advances in the treatment of genetic diseases. We have also explored the potential challenges faced by base editing technology in treatment, including aspects such as targeting specificity, safety, and efficacy, and have enumerated a series of possible solutions to propel the clinical translation of base editing technology. In conclusion, this article not only underscores the present state of base editing technology but also envisions its tremendous potential in the future, providing a novel perspective on the treatment of genetic diseases. It underscores the vast potential of base editing technology in the realm of genetic medicine, providing support for the progression of gene medicine and the development of innovative approaches to genetic disease therapy.

The last decade (2013-2023) has seen unprecedented successes in the clinical translation of therapeutic antisense oligonucleotides (ASOs). Eight such molecules have been granted marketing approval by the United States Food and Drug Administration (US FDA) during the decade, after the first ASO drug, fomivirsen, was approved much earlier, in 1998. Splice-modulating ASOs have also been developed for the therapy of inborn errors of metabolism (IEMs), due to their ability to redirect aberrant splicing caused by mutations, thus recovering the expression of normal transcripts, and correcting the deficiency of functional proteins. The feasibility of treating IEM patients with splice-switching ASOs has been supported by FDA permission (2018) of the first "N-of-1" study of milasen, an investigational ASO drug for Batten disease. Although for IEM, owing to the rarity of individual disease and/or pathogenic mutation, only a low number of patients may be treated by ASOs that specifically suppress the aberrant splicing pattern of mutant precursor mRNA (pre-mRNA), splice-switching ASOs represent superior individualized molecular therapeutics for IEM. In this work, we first summarize the ASO technology with respect to its mechanisms of action, chemical modifications of nucleotides, and rational design of modified oligonucleotides; following that, we precisely provide a review of the current understanding of developing splice-modulating ASO-based therapeutics for IEM. In the concluding section, we suggest potential ways to improve and/or optimize the development of ASOs targeting IEM.

Current therapies are unable to cure Duchenne muscular dystrophy (DMD), a severe and common form of muscular dystrophy, and instead aim to delay disease progression. Several treatments currently in phase I trials could increase the number of therapeutic options available to patients.

This review aims to provide an overview of current treatments undergoing or having recently undergone early-stage trials. Several exon-skipping and gene therapy approaches are currently being investigated at the clinical stage to address an unmet need for DMD treatments. This article also covers Phase I trials from the last 5 years that involve inhibitors, small molecules, a purified synthetic flavanol, a cell-based therapy, and repurposed cardiac or tumor medications.

With antisense oligonucleotide (AON) treatments making up the majority of conditionally approved DMD therapies, most of the clinical trials occurring within the last 5 years have also evaluated exon-skipping AONs. The approval of Elevidys, a micro-dystrophin therapy, is reflected in a recent trend toward gene transfer therapies in phase I DMD clinical trials, but their safety and efficacy are being established in this phase of development. Other Phase I clinical-stage approaches are diverse, but have a range in efficacy, safety, and endpoint measures.

Five small interfering RNA (siRNA)-based therapeutics have been approved by the Food and Drug Administration (FDA), namely patisiran, givosiran, lumasiran, inclisiran, and vutrisiran. Besides, siRNA delivery to the target site without toxicity is a big challenge for researchers, and naked-siRNA delivery possesses several challenges, including membrane impermeability, enzymatic degradation, mononuclear phagocyte system (MPS) entrapment, fast renal excretion, endosomal escape, and off-target effects. The siRNA therapeutics can silence any disease-specific gene, but their intracellular and extracellular barriers limit their clinical applications. For this purpose, several modifications have been employed to siRNA for better transfection efficiency. Still, there is a quest for better delivery systems for siRNA delivery to the target site. In recent years, nanoparticles have shown promising results in siRNA delivery with minimum toxicity and off-target effects. Patisiran is a lipid nanoparticle (LNP)-based siRNA formulation for treating hereditary transthyretin-mediated amyloidosis that ultimately warrants the use of nanoparticles from different classes, especially lipid-based nanoparticles. These nanoparticles may belong to different categories, including lipid-based, polymer-based, and inorganic nanoparticles. This review briefly discusses the lipid, polymer, and inorganic nanoparticles and their sub-types for siRNA delivery. Finally, several clinical trials related to siRNA therapeutics are addressed, followed by the future prospects and conclusions.

Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5'-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide.

The antisense RNA molecule is a unique DNA transcript consisting of 19-23 nucleotides, characterized by its complementary nature to mRNA. These antisense RNAs play a crucial role in regulating gene expression at various stages, including replication, transcription, and translation. Additionally, artificial antisense RNAs have demonstrated their ability to effectively modulate gene expression in host cells. Consequently, there has been a substantial increase in research dedicated to investigating the roles of antisense RNAs. These molecules have been found to be influential in various cellular processes, such as X-chromosome inactivation and imprinted silencing in healthy cells. However, it is important to recognize that in cancer cells; aberrantly expressed antisense RNAs can trigger the epigenetic silencing of tumor suppressor genes. Moreover, the presence of deletion-induced aberrant antisense RNAs can lead to the development of diseases through epigenetic silencing. One area of drug development worth mentioning is antisense oligonucleotides (ASOs), and a prime example of an oncogenic trans-acting long noncoding RNA (lncRNA) is HOTAIR (HOX transcript antisense RNA). NATs (noncoding antisense transcripts) are dysregulated in many cancers, and researchers are just beginning to unravel their roles as crucial regulators of cancer's hallmarks, as well as their potential for cancer therapy. In this review, we summarize the emerging roles and mechanisms of antisense RNA and explore their application in cancer therapy.