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RNA (New York, N.Y.) Oct 2023Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient...
Current methods for detecting unlabeled antisense oligonucleotide (ASO) drugs rely on immunohistochemistry (IHC) and/or conjugated molecules, which lack sufficient sensitivity, specificity, and resolution to fully investigate their biodistribution. Our aim was to demonstrate the qualitative and quantitative distribution of unlabeled bepirovirsen, a clinical stage ASO, in livers and kidneys of dosed mice using novel staining and imaging technologies at subcellular resolution. ASOs were detected in formalin-fixed paraffin-embedded (FFPE) and frozen tissues using an automated chromogenic in situ hybridization (ISH) assay: miRNAscope. This was then combined with immunohistochemical detection of cell lineage markers. ASO distribution in hepatocytes versus nonparenchymal cell lineages was quantified using HALO AI image analysis. To complement this, hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) imaging microscopy was used to specifically detect the unique cellular Raman spectral signatures following ASO treatment. Bepirovirsen was localized primarily in nonparenchymal liver cells and proximal renal tubules. Codetection of ASO with distinct cell lineage markers of liver and kidney populations aided target cell identity facilitating quantification. Positive liver signal was quantified using HALO AI, with 12.9% of the ASO localized to the hepatocytes and 87.1% in nonparenchymal cells. HS-CARS imaging specifically detected ASO fingerprints based on the unique vibrational signatures following unlabeled ASO treatment in a totally nonperturbative manner at subcellular resolution. Together, these novel detection and imaging modalities represent a significant increase in our ability to detect unlabeled ASOs in tissues, demonstrating improved levels of specificity and resolution. These methods help us understand their underlying mechanisms of action and ultimately improve the therapeutic potential of these important drugs for treating globally significant human diseases.
Topics: Mice; Humans; Animals; Oligonucleotides, Antisense; Tissue Distribution; Liver; In Situ Hybridization; Staining and Labeling
PubMed: 37460153
DOI: 10.1261/rna.079699.123 -
Methods in Molecular Biology (Clifton,... 2020Although technological advances in molecular genetics over the last few decades have greatly expedited the identification of mutations in many genetic diseases, the... (Review)
Review
Although technological advances in molecular genetics over the last few decades have greatly expedited the identification of mutations in many genetic diseases, the translation of the genetic mechanisms into a clinical setting has been quite challenging, with a minimum number of effective treatments available. The advancements in antisense therapy have revolutionized the field of neuromuscular disorders as well as lipid-mediated diseases. With the approval of splice-switching antisense oligonucleotide (AO) therapy for nusinersen and eteplirsen for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD), several modified AOs are now being evaluated in clinical trials for the treatment of a number of disorders. In order to activate RNase H-mediated cleavage of the target mRNA, as well as to increase the binding affinity and specificity, gapmer AOs are designed that have a PS backbone flanked with the modified AOs on both sides. Mipomersen (trade name Kynamro), a 2'-O-methoxyethyl (MOE) gapmer, was approved by the Food and Drug Administration (FDA) for the treatment of homozygous familial hypercholesterolemia (HoFH) in 2013. Volanesorsen, another 20-mer MOE gapmer has shown to be successful in lowering the levels of triglycerides (TGs) in several lipid disorders and has received conditional approval in the European Union for the treatment of Familial chylomicronemia syndrome (FCS) in May 2019 following successful results from phase II/III clinical trials. This chapter focuses on the clinical applications of gapmer AOs for genetic dyslipidemia and lipodystrophy.
Topics: Animals; Drug Development; Dyslipidemias; Genetic Therapy; History, 20th Century; History, 21st Century; Humans; Lipodystrophy; Morpholinos; Oligonucleotides; Oligonucleotides, Antisense
PubMed: 32865783
DOI: 10.1007/978-1-0716-0771-8_5 -
Cancer Science Feb 2022There has been accumulating evidence that RNA splicing is frequently dysregulated in a variety of cancers and that hotspot mutations affecting key splicing factors,... (Review)
Review
There has been accumulating evidence that RNA splicing is frequently dysregulated in a variety of cancers and that hotspot mutations affecting key splicing factors, SF3B1, SRSF2 and U2AF1, are commonly enriched across cancers, strongly suggesting that aberrant RNA splicing is a new class of hallmark that contributes to the initiation and/or maintenance of cancers. In parallel, some studies have demonstrated that cancer cells with global splicing alterations are dependent on the transcriptional products derived from wild-type spliceosome for their survival, which potentially creates a therapeutic vulnerability in cancers with a mutant spliceosome. It has been c. 10 y since the frequent mutations affecting splicing factors were reported in cancers. Based on these surprising findings, there has been a growing interest in targeting altered splicing in the treatment of cancers, which has promoted a wide variety of investigations including genetic, molecular and biological studies addressing how altered splicing promotes oncogenesis and how cancers bearing alterations in splicing can be targeted therapeutically. In this mini-review we present a concise trajectory of what has been elucidated regarding the pathogenesis of cancers with aberrant splicing, as well as the development of therapeutic strategies to target global splicing alterations in cancers.
Topics: Antineoplastic Agents; Humans; Mutation; Neoplasms; Oligonucleotides, Antisense; RNA Splicing; RNA Splicing Factors; RNA-Binding Proteins; Spliceosomes
PubMed: 34812550
DOI: 10.1111/cas.15213 -
F1000Research 2019Recent approvals of oligonucleotide analogue drugs to alter gene expression have been welcomed by patient communities but not universally supported. These compounds... (Review)
Review
Recent approvals of oligonucleotide analogue drugs to alter gene expression have been welcomed by patient communities but not universally supported. These compounds represent a class of drugs that are designed to target a specific gene transcript, and they include a number of chemical entities to evoke different antisense mechanisms, depending upon the disease aetiology. To date, oligonucleotide therapeutics that are in the clinic or at advanced stages of translation target rare diseases, posing challenges to clinical trial design, recruitment and evaluation and requiring new evaluation paradigms. This review discusses the currently available and emerging therapeutics that alter exon selection through an effect on pre-mRNA splicing and explores emerging concerns over safety and efficacy. Although modification of synthetic nucleic acids destined for therapeutic application is common practice to protect against nuclease degradation and to influence drug function, such modifications may also confer unexpected physicochemical and biological properties. Negatively charged oligonucleotides have a strong propensity to bind extra- and intra-cellular proteins, whereas those analogues with a neutral backbone show inefficient cellular uptake but excellent safety profiles. In addition, the potential for incorporation of chemically modified nucleic acid monomers, yielded by nuclease degradation of exogenous oligonucleotides, into biomolecules has been raised and the possibility not entirely discounted. We conclude with a commentary on the ongoing efforts to develop novel antisense compounds and enhance oligonucleotide delivery in order to further improve efficacy and accelerate implementation of antisense therapeutics for human disease.
Topics: Exons; Gene Expression; Humans; Oligonucleotides, Antisense; RNA Splicing
PubMed: 31164976
DOI: 10.12688/f1000research.18466.1 -
Nucleic Acid Therapeutics Jun 2023In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin...
In the last two decades, antisense oligonucleotides (AONs) that induce corrective exon skipping have matured as promising therapies aimed at tackling the dystrophin deficiency that underlies the severe and progressive muscle fiber degeneration in Duchenne muscular dystrophy (DMD) patients. Pioneering first generation exon 51 skipping AONs like drisapersen and eteplirsen have more recently been followed up by AONs for exons 53 and 45, with, to date, a total of four exon skipping AON drugs having reached (conditional) regulatory US Food and Drug Administration (FDA) approval for DMD. Nonetheless, considering the limited efficacy of these drugs, there is room for improvement. The aim of this study was to develop more efficient [2'--methyl-modified phosphorothioate (2'OMePS) RNA] AONs for exon 51 skipping by implementing precision chemistry as well as identifying a more potent target binding site. More than a hundred AONs were screened in muscle cell cultures, followed by a selective comparison in the hDMD and hDMDdel52/ mouse models. Incorporation of 5-methylcytosine and position-specific locked nucleic acids in AONs targeting the drisapersen/eteplirsen binding site resulted in 15-fold higher exon 51 skipping levels compared to drisapersen in hDMDdel52/ mice. However, with similarly modified AONs targeting an alternative site in exon 51, 65-fold higher skipping levels were obtained, restoring dystrophin up to 30% of healthy control. Targeting both sites in exon 51 with a single AON further increased exon skipping (100-fold over drisapersen) and dystrophin (up to 40%) levels. These dystrophin levels allowed for normalization of creatine kinase (CK) and lactate dehydrogenase (LDH) levels, and improved motor function in hDMDdel52/ mice. As no major safety observation was obtained, the improved therapeutic index of these next generation AONs is encouraging for further (pre)clinical development.
Topics: Mice; Animals; Muscular Dystrophy, Duchenne; Dystrophin; Oligonucleotides, Antisense; Mice, Inbred mdx; Genetic Therapy; Exons
PubMed: 37036788
DOI: 10.1089/nat.2022.0063 -
Trends in Molecular Medicine Apr 2022The question of a loss or toxic gain of function in FUS-related amyotrophic lateral sclerosis is still debated. Recently, Korobeynikov et al. argued that FUS mutations...
The question of a loss or toxic gain of function in FUS-related amyotrophic lateral sclerosis is still debated. Recently, Korobeynikov et al. argued that FUS mutations lead to a gain of function and showed that lowering wild-type and mutant FUS levels could be a promising therapeutic strategy.
Topics: Amyotrophic Lateral Sclerosis; DNA-Binding Proteins; Humans; Mutation; Oligonucleotides, Antisense; RNA-Binding Protein FUS
PubMed: 35246398
DOI: 10.1016/j.molmed.2022.02.006 -
Expert Opinion on Investigational Drugs Mar 2024Current therapies are unable to cure Duchenne muscular dystrophy (DMD), a severe and common form of muscular dystrophy, and instead aim to delay disease progression.... (Review)
Review
INTRODUCTION
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.
AREAS COVERED
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.
EXPERT OPINION
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.
Topics: Humans; Genetic Therapy; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; RNA Splicing; Clinical Trials as Topic
PubMed: 38291016
DOI: 10.1080/13543784.2024.2313105 -
Hearing Research Dec 2022Hearing loss affects more than 430 million people, worldwide, and is the third most common chronic physical condition in the United States and Europe (GBD Hearing Loss... (Review)
Review
Hearing loss affects more than 430 million people, worldwide, and is the third most common chronic physical condition in the United States and Europe (GBD Hearing Loss Collaborators, 2021; NIOSH, 2021; WHO, 2021). The loss of hearing significantly impacts motor and cognitive development, communication, education, employment, and overall quality of life. The inner ear houses the sensory organs for both hearing and balance and provides an accessible target for therapeutic delivery. Antisense oligonucleotides (ASOs) use various mechanisms to manipulate gene expression and can be tailor-made to treat disorders with defined genetic targets. In this review, we discuss the preclinical advancements within the field of the highly promising ASO-based therapies for hereditary hearing loss disorders. Particular focus is on ASO mechanisms of action, preclinical studies on ASO treatments of hearing loss, timing of therapeutic intervention, and delivery routes to the inner ear.
Topics: Humans; Quality of Life; Hearing Loss; Deafness; Oligonucleotides, Antisense; Gene Expression
PubMed: 35649738
DOI: 10.1016/j.heares.2022.108523 -
PeerJ 2023The rapid emergence of antibiotic-resistant bacteria directly contributes to a wave of untreatable infections. The lack of new drug development is an important driver of...
BACKGROUND
The rapid emergence of antibiotic-resistant bacteria directly contributes to a wave of untreatable infections. The lack of new drug development is an important driver of this crisis. Most antibiotics today are small molecules that block vital processes in bacteria. To optimize such effects, the three-dimensional structure of targeted bacterial proteins is imperative, although such a task is time-consuming and tedious, impeding the development of antibiotics. The development of RNA-based therapeutics has catalyzed a new platform of antibiotics-antisense oligonucleotides (ASOs). These molecules hybridize with their target mRNAs with high specificity, knocking down or interfering with protein translation. This study aims to develop a bioinformatics pipeline to identify potent ASO targets in essential bacterial genes.
METHODS
Three bacterial species (, , and ) were used to demonstrate the utility of the pipeline. Open reading frames of bacterial essential genes were downloaded from the Database of Essential Genes (DEG). After filtering for specificity and accessibility, ASO candidates were ranked based on their self-hybridization score, predicted melting temperature, and the position on the gene in an operon. Enrichment analysis was conducted on genes associated with putative potent ASOs.
RESULTS
A total of 45,628 ASOs were generated from 348 unique essential genes in . A total of 1,117 of them were considered putative. A total of 27,273 ASOs were generated from 191 unique essential genes in . A total of 847 of them were considered putative. A total of 175,606 ASOs were generated from 346 essential genes in . A total of 7,061 of them were considered putative. Critical biological processes associated with these genes include translation, regulation of cell shape, cell division, and peptidoglycan biosynthetic process. Putative ASO targets generated for each bacterial species are publicly available here: https://github.com/EricSHo/AOA. The results demonstrate that our bioinformatics pipeline is useful in identifying unique and accessible ASO targets in bacterial species that post major public health issues.
Topics: Oligonucleotides, Antisense; Anti-Bacterial Agents; Staphylococcus aureus; Oligonucleotides
PubMed: 38025700
DOI: 10.7717/peerj.16343 -
Zhejiang Da Xue Xue Bao. Yi Xue Ban =... Aug 2023RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes or encode proteins for the synthesis...
RNA therapeutics inhibit the expression of specific proteins/RNAs by targeting complementary sequences of corresponding genes or encode proteins for the synthesis desired genes to treat genetic diseases. RNA-based therapeutics are categorized as oligonucleotide drugs (antisense oligonucleotides, small interfering RNA, RNA aptamers), and mRNA drugs. The antisense oligonucleotides and small interfering RNA for treatment of genetic diseases have been approved by the FDA in the United States, while RNA aptamers and mRNA drugs are still in clinical trials. Chemical modifications can be applied to RNA drugs, such as pseudouridine modification of mRNA, to reduce immunogenicity and improve the efficacy. The secure and effective delivery systems such as lipid-based nanoparticles, extracellular vesicles, and virus-like particles are under development to address stability, specificity, and safety issues of RNA drugs. This article provides an overview of the specific molecular mechanisms of eleven RNA drugs currently used for treating genetic diseases, and discusses the research progress of chemical modifications and delivery systems of RNA drugs.
Topics: Aptamers, Nucleotide; RNA, Small Interfering; RNA, Messenger; Oligonucleotides, Antisense
PubMed: 37643975
DOI: 10.3724/zdxbyxb-2023-0190