RNA-targeted Therapy Research Articles

The Role of RNA-Targeted Therapeutics to Reduce ASCVD Risk: What Have We Learned Recently?

To discuss advances on the RNA-targeted therapies to treat dyslipidemia with the aim of reducing atherosclerotic cardiovascular disease (ASCVD). Genetic studies have paved the way for therapies that reduce translation of proteins that play causal roles in dyslipidemia and atherosclerosis like proprotein convertase subtilisin/kexin type 9 (PCSK9), apolipoprotein B-100 (apoB), apolipoprotein(a) [apo(a)], apolipoprotein C3 (apoC3), and angiopoietin-like 3 (ANGPTL3). Either antisense oligonucleotide (ASO) therapies and small interfering RNA (siRNA) molecules inhibit protein synthesis and consequently improve dyslipidemia. Most of these molecules contain N-acetylgalactosamine (GalNAc) moieties that have high specificity for hepatocytes and therefore reduce concentration in other tissues. Inclisiran, an siRNA for PCSK9, has shown robust LDL-C reductions, with good tolerability, in severe forms of hypercholesterolemia as well as in high cardiovascular disease patients with injections every 3 to 6 months. Pelacarsen is an ASO against apolipoprotein(a) that reduces Lp(a) up to 80% with good tolerability. Either inclisiran or pelacarsen is being tested to show it can prevent ASCVD. AMG 890, an siRNA compound aimed at reducing apo(a) synthesis, is also under investigation. Volanesorsen is an ASO against apoC3 that reduces triglyceride levels up to 70% and is being tested in severe hypertriglyceridemic patients. Vupanorsen is an ASO against ANGPTL3 that reduced triglyceride levels 36-53% among moderate hypertriglyceridemic individuals. Interestingly, it also reduces ApoC3 and non-HDL cholesterol and apoB; however, it lowers HDL cholesterol. RNA-targeted therapies are being extensively tested for dyslipidemia treatment with promising results.

Huntingtin and the Synapse.

Huntington disease (HD) is a monogenic disease that results in a combination of motor, psychiatric and cognitive symptoms. HD is caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, which results in the production of a pathogenic mutant HTT protein (mHTT). Although there is no cure at present for HD, a number of RNA-targeting therapies have recently entered clinical trials which aim to lower mHTT production through the use of antisense oligonucleotides (ASOs) and RNAi. However, many of these treatment strategies are non-selective in that they cannot differentiate between non-pathogenic wild type HTT (wtHTT) and the mHTT variant. As HD patients are already born with decreased levels of wtHTT, these genetic therapies may result in critically low levels of wtHTT. The consequence of wtHTT reduction in the adult brain is currently under debate, and here we argue that wtHTT loss is not well-tolerated at the synaptic level. Synaptic dysfunction is an extremely sensitive measure of subsequent cell death, and is known to precede neurodegeneration in numerous brain diseases including HD. The present review focuses on the prominent role of wtHTT at the synapse and considers the consequences of wtHTT loss on both pre- and postsynaptic function. We discuss how wtHTT is implicated in virtually all major facets of synaptic neurotransmission including anterograde and retrograde transport of proteins to/from terminal buttons and dendrites, neurotransmitter release, endocytic vesicle recycling, and postsynaptic receptor localization and recycling. We conclude that wtHTT presence is essential for proper synaptic function.

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.

Current Emerging Therapy for Amyloidosis Neuropathy

Peripheral neuropathy is a type of neurological disorder in which patients with complex inherited neurological defects present significant phenotype in the peripheral nervous system. Hereditary amyloidogenic transthyretin (hATTR) neuropathy is typical polyneuropathy caused by single-nucleotide variants in the gene encoding transthyretin (TTR) and leads to transthyretin misfolding and systemic deposition of amyloid. One of the clinical hallmarks of hATTR neuropathy is polyneuropathy of the destruction of the somatic and autonomic peripheral nervous system, leading to loss of autonomy. Progressive amyloid accumulation also causes multi-organ dysfunction and death. There are many therapeutics that have been proposed and developed in these years. These therapies aim to reduce or eliminate hATTR from the plasma, stabilize the hATTR to prevent deposition, and dissolute the amyloid misfolding matrix. Recently, gene therapy strategy is being deployed to treat recessive genetic disorders by eliminating the expression of the mutated genes. Thus, gene-silencing approaches have been used to manage this amyloidosis neuropathy in the broad stages and produce some degree of improvement of this disease. Food and Drug Administration (FDA) approved Inotersen (an antisense oligonucleotide (ASO)) and patisiran (a small interfering ribonucleic acid (siRNA) for the treatment of hATTR polyneuropathy to suppress hATTR expression. Inotersen, a 2’-O-methoxyethylmodified ASO, which acts by reducing the production of transthyretin, and has been demonstrated to improve the quality of life in early hereditary transthyretin amyloidosis polyneuropathy. I here focus on the RNA-targeted therapy with particular emphasis on the molecular mechanisms by which antisense oligonucleotide can be designed to modulate transthyretin RNA function for being a novel therapy for hereditary amyloidosis neuropathy.

Recent therapeutic prospects for Machado-Joseph disease.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a fatal, dominantly inherited, neurodegenerative disease caused by expansion of a CAG repeat in the coding region of the ATXN3 gene. No disease-modifying treatment is yet available for MJD/SCA3. This review discusses recently developed therapeutic strategies that hold promise as future effective treatments for this incurable disease. As a result of the exploration of multiple therapeutic approaches over the last decade, the MJD/SCA3 field is finally starting to see options for disease-modifying treatments for this disease come into view on the horizon. Recently developed strategies include DNA-targeted and RNA-targeted therapies, and approaches targeting protein quality control pathways and cellular homeostasis. While still in preclinical testing stages, antisense oligonucleotides, short hairpin RNAs and citalopram all show promise to reaching testing in clinical trials for MJD/SCA3. Two pharmacological approaches in early stages of development, the slipped-CAG DNA binding compound naphthyridine-azaquinolone and autophagosome-tethering compounds, also show potential therapeutic capacity for MJD/SCA3. Overall, a handful of therapeutic options are currently showing potential as future successful treatments for fatal MJD/SCA3.

From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases.

Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.

RNA-Targeted Therapies and High-Throughput Screening Methods.

RNA-binding proteins (RBPs) are involved in regulating all aspects of RNA metabolism, including processing, transport, translation, and degradation. Dysregulation of RNA metabolism is linked to a plethora of diseases, such as cancer, neurodegenerative diseases, and neuromuscular disorders. Recent years have seen a dramatic shift in the knowledge base, with RNA increasingly being recognised as an attractive target for precision medicine therapies. In this article, we are going to review current RNA-targeted therapies. Furthermore, we will scrutinise a range of drug discoveries targeting protein-RNA interactions. In particular, we will focus on the interplay between Lin28 and let-7, splicing regulatory proteins and survival motor neuron (SMN) pre-mRNA, as well as HuR, Musashi, proteins and their RNA targets. We will highlight the mechanisms RBPs utilise to modulate RNA metabolism and discuss current high-throughput screening strategies. This review provides evidence that we are entering a new era of RNA-targeted medicine.

Small Interfering RNAs and RNA Therapeutics in Cardiovascular Diseases.

Ribonucleic acid (RNA) is being exploited and understood in its many aspects of function and structure for development of valuable tools in the therapeutics of various diseases such as cardiovascular etc. The expanded knowledge regarding function of RNA in the genomics and inside the cell has dramatically changed the therapeutic strategies in the past few years. RNA has become a spotlight of attention for developing novel therapeutic schemes and hence variety of therapeutic strategies is being coming into the picture that includes RNA interference, use of aptamers, role of microRNA (miRNA) that can alter the complex gene expression patterns. It is due to the fact that RNA offers various advantages in disease management as it can be edited and modified in its various forms such as secondary and tertiary structures. Although scientists are in process of manufacturing RNA-targeting therapies using variety of endogenous gene silencing regulators, Small interfering RNAs (Si RNAs), aptamers and microRNA for cardiovascular diseases yet the development of a novel, risk free therapeutic strategy is a major challenge and need of the hour in cardiovascular medicine. In this regard these agents are required to overcome pleothra of barriers such as stability of drug targets, immunogenicity, adequate binding, targeted delivery etc. to become effective drugs. Recent years have witnessed the progress of RNA therapeutic strategies in cardiovascular diseases that are likely to significantly expand the cardiovascular therapeutic repertoire within the next decade. The present manuscript has been compiled to summarize various approaches of siRNA based therapies in cardiovascular diseases along with the advantages, outcomes and limitations if any in this regard. In addition, the future prospects of RNA therapeutic modalities in cardiovascular diseases are summarized.

Better living through peptide-conjugated chemistry: next-generation antisense oligonucleotides.

Two different antisense oligonucleotide-based (ASO-based) therapies are currently in clinical use to treat neuromuscular diseases. This success, for Duchenne muscular dystrophy and spinal muscular atrophy, offers hope not only for additional neuromuscular diseases, but also for other disorders that could benefit from RNA-targeted therapies. A major limitation for more widespread application of ASOs relates to relatively poor tissue penetration. In this issue of the JCI, Klein et al. showed that conjugating an ASO with an arginine-rich cell-penetrating peptide, Pip6a, enhanced delivery, resulting in corrective outcome for a mouse model of myotonic dystrophy. Linking ASOs to cell-penetrating peptides, or even other moieties, is an approach currently under development with treatment potential to expand to other disorders.

RNA Therapeutics in Cardiovascular Precision Medicine.

Since our knowledge on structure and function of messenger RNA (mRNA) has expanded from merely being an intermediate molecule between DNA and proteins to the notion that RNA is a dynamic gene regulator that can be modified and edited, RNA has become a focus of interest into developing novel therapeutic schemes. Therapeutic modulation of RNA molecules by DNA- and RNA-based therapies has broadened the scope of therapeutic targets in infectious diseases, cancer, neurodegenerative diseases and most recently in cardiovascular diseases as well. Currently, antisense oligonucleotides (ASO), small interfering RNAs (siRNAs), and microRNAs are the most widely applied therapeutic strategies to target RNA molecules and regulate gene expression and protein production. However, a number of barriers have to be overcome including instability, inadequate binding affinity and delivery to the tissues, immunogenicity, and off-target toxicity in order for these agents to evolve into efficient drugs. As cardiovascular diseases remain the leading cause of mortality worldwide, a large number of clinical trials are under development investigating the safety and efficacy of RNA therapeutics in clinical conditions such as familial hypercholesterolemia, diabetes mellitus, hypertriglyceridemia, cardiac amyloidosis, and atrial fibrillation. In this review, we summarize the clinical trials of RNA-targeting therapies in cardiovascular disease and critically discuss the advances, the outcomes, the limitations and the future directions of RNA therapeutics in precision transcriptomic medicine.

2342 Protein production as an early pharmacodynamics biomarker for RNA-targeting therapies

OBJECTIVES/SPECIFIC AIMS: We aimed to develop an assay to measure new protein synthesis after Antisense Oligonucleotide treatment, which we hypothesized to be the earliest biochemical identification of RNA-targeting therapy efficacy. METHODS/STUDY POPULATION: We treated 2 transgenic animal models expressing proteins implicated in neurodegenerative disease: human tau protein (hTau) and human superoxide dismutase 1 (hSOD1), with ASO against these mRNA transcripts. Animals received isotope-labeled 13C6-Leucine via drinking water to label newly synthesized proteins. We assayed target protein synthesis and concentration after ASO treatment to determine the earliest identification of ASO target engagement. RESULTS/ANTICIPATED RESULTS: hTau ASO treatment in transgenic mice lowered hTau protein concentration 23 days post-treatment in cortex (95% CI: 0.05%–64.0% reduction). In the same tissue, we observed lowering of hTau protein synthesis as early as 13 days (95% CI: 29.4%–123%). In hSOD1 transgenic rats, we observed lowering of 13C6-leucine-labeled hSOD1 in the cerebrospinal fluid 30 days after ASO treatment compared with inactive ASO control (95% CI: 12.0%–48.4%). DISCUSSION/SIGNIFICANCE OF IMPACT: In progressive neurodegenerative diseases, it is crucial to develop measurements that identify treatment efficacy early to improve patient outcomes. These data support the use of stable isotope labeling of amino acids to measure new protein synthesis as an early pharmacodynamics measurement for therapies that target RNA and inhibit the translation of proteins.

RNA-Targeted Therapies and Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motor disease in adults. Its pathophysiology remains mysterious, but tremendous advances have been made with the discovery of the most frequent mutations of its more common familial form linked to the C9ORF72 gene. Although most cases are still considered sporadic, these genetic mutations have revealed the role of RNA production, processing and transport in ALS, and may be important players in all ALS forms. There are no disease-modifying treatments for adult human neurodegenerative diseases, including ALS. As in spinal muscular atrophy, RNA-targeted therapies have been proposed as potential strategies for treating this neurodegenerative disorder. Successes achieved in various animal models of ALS have proven that RNA therapies are both safe and effective. With careful consideration of the applicability of such therapies in humans, it is possible to anticipate ongoing in vivo research and clinical trial development of RNA therapies for treating ALS.

Short (16-mer) locked nucleic acid splice-switching oligonucleotides restore dystrophin production in Duchenne Muscular Dystrophy myotubes.

Splice-switching antisense oligonucleotides (SSOs) offer great potential for RNA-targeting therapies, and two SSO drugs have been recently approved for treating Duchenne Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA). Despite promising results, new developments are still needed for more efficient chemistries and delivery systems. Locked nucleic acid (LNA) is a chemically modified nucleic acid that presents several attractive properties, such as high melting temperature when bound to RNA, potent biological activity, high stability and low toxicity in vivo. Here, we designed a series of LNA-based SSOs complementary to two sequences of the human dystrophin exon 51 that are most evolutionary conserved and evaluated their ability to induce exon skipping upon transfection into myoblasts derived from a DMD patient. We show that 16-mers with 60% of LNA modification efficiently induce exon skipping and restore synthesis of a truncated dystrophin isoform that localizes to the plasma membrane of patient-derived myotubes differentiated in culture. In sum, this study underscores the value of short LNA-modified SSOs for therapeutic applications.

Principles of Chemical Biology: From Antibiotic Resistance to RNA-Targeted Therapy via Autoxidation and Lipid Droplets

This month: Ribosome mutations and multi-antibiotic resistance, role of autoxidation in ferroptosis, ceramide metabolism, and finding RNA drug targets.

Downregulation of long non-coding RNA MEG3 in nasopharyngeal carcinoma.

In our previous whole-transcriptome sequencing analysis, downregulation of a long non-coding RNA, maternally expressed gene 3 (MEG3), was identified in NPC samples. This finding suggests the possible role of MEG3 as a tumor suppressor in this distinctive disease. In the present study, two MEG3 variants, AF119863 (MEG3-AF) and BX247998 (MEG3-BX), were found abundantly expressed in a normal nasopharyngeal epithelial cell line, NP69. Significant downregulation of MEG3-AF was further verified in a panel of NPC samples including xenografts and primary biopsies. MEG3 is an imprinted gene located within chromosome 14q32, a common deleted region in NPC. Both DNA copy number loss and aberrant promoter methylation contributed to MEG3 inactivation. Interestingly, MEG3 expression could successfully be rescued by the treatment of a demethylation agent. Besides, ectopic expression of MEG3 in NPC cell lines resulted in considerable repression of in vitro anchorage-independent growth and in vivo tumorigenicity, in addition to significant inhibition in cell proliferation, colony formation, and induction of cell cycle arrest. Finally, we revealed the association between MEG3 activity and the p53 signaling cascade. Our findings characterize MEG3 as a tumor suppressive long non-coding RNA in NPC and encourage the development of precise long non-coding RNA-targeted epigenetic therapy against this malignancy. © 2016 Wiley Periodicals, Inc.

SMALL NONCODING RNA AS PERSPECTIVE BIOMARKERS: BIOGENESIS AND THERAPEUTIC STRATIGIES

The review presents the opening story, biogenesis and functions of basic groups of human’s small noncoding RNA: microRNA and short interfering RNA. These RNA molecules inhibit gene expression during translation by RNA interference. It was found that microRNA and short interfering RNA circulate in bioliquids and can serve as biomarkers of different human diseases because of its conservative sequences, tissue specificity and resistance to environment factors. The paper considers techniques to study noncoding RNA (cloning, bioinformatics analysis and hybridization methods: northern-blotting, RT-PCR, in situ hybridization, microarray analysis, reporter analysis). Possible noncoding RNA-targeted therapy can suggest delivery microRNA, anti-microRNA, antagomirs, microRNAsponges to target tissue by virus molecules, liposomes or nanoparticles.

Red‐Light‐Controlled Protein–RNA Crosslinking with a Genetically Encoded Furan

Well red: A protein-RNA crosslinker has been genetically encoded that can be controlled with red light, thus offering high penetration depths in biological materials. This should enable the discovery and mapping of transient protein-RNA interactions and enable the design of peptide- and protein-based drugs for RNA-targeted photodynamic therapy.

Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

Antisense oligonucleotides and short interfering RNAs (siRNAs) are nucleic acids-targeting reagents for gene expression modulation that are being developed as drugs for many applications. A number of useful synthetic nucleic acids analogues have been introduced recently to greatly improve their properties for use as therapeutics. However, their full effectiveness in cells and in vivo has often only been realized through development of suitable nonviral delivery systems. Among these, a range of natural and synthetic peptides have been found useful for enhancing cellular uptake and/or cell targeting of oligonucleotide analogues and siRNA. Such peptides are synthetically conjugated, used as noncovalent complexes, or used in combination with polymer, liposomal or exosome formulation techniques. This review begins by describing the modes of action of antisense reagents and siRNA and goes on to focus on recent advances in their peptide-mediated cell and in vivo delivery and how peptide use has influenced drug development. The review discusses the challenges associated with understanding the physiological and toxicological aspects of peptide-mediated delivery. Developments towards clinical use are also highlighted, with particular emphasis on peptide conjugates of oligonucleotide analogues used for treatment of neuromuscular diseases.

Functional Rescue of Dystrophin-deficient mdx Mice by a Chimeric Peptide-PMO

Splice modulation using antisense oligonucleotides (AOs) has been shown to yield targeted exon exclusion to restore the open reading frame and generate truncated but partially functional dystrophin protein. This has been successfully demonstrated in dystrophin-deficient mdx mice and in Duchenne muscular dystrophy (DMD) patients. However, DMD is a systemic disease; successful therapeutic exploitation of this approach will therefore depend on effective systemic delivery of AOs to all affected tissues. We have previously shown the potential of a muscle-specific/arginine-rich chimeric peptide-phosphorodiamidate morpholino (PMO) conjugate, but its long-term activity, optimized dosing regimen, capacity for functional correction and safety profile remain to be established. Here, we report the results of this chimeric peptide-PMO conjugate in the mdx mouse using low doses (3 and 6 mg/kg) administered via a 6 biweekly systemic intravenous injection protocol. We show 100% dystrophin-positive fibers and near complete correction of the dystrophin transcript defect in all peripheral muscle groups, with restoration of 50% dystrophin protein over 12 weeks, leading to correction of the DMD pathological phenotype and restoration of muscle function in the absence of detectable toxicity or immune response. Chimeric muscle-specific/cell-penetrating peptides therefore represent highly promising agents for systemic delivery of splice-correcting PMO oligomers for DMD therapy.

RNA-targeted splice-correction therapy for neuromuscular disease

Splice-modulation therapy, whereby molecular manipulation of premessenger RNA splicing is engineered to yield genetic correction, is a promising novel therapy for genetic diseases of muscle and nerve-the prototypical example being Duchenne muscular dystrophy. Duchenne muscular dystrophy is the most common childhood genetic disease, affecting one in 3500 newborn boys, causing progressive muscle weakness, heart and respiratory failure and premature death. No cure exists for this disease and a number of promising new molecular therapies are being intensively studied. Duchenne muscular dystrophy arises due to mutations that disrupt the open-reading-frame in the DMD gene leading to the absence of the essential muscle protein dystrophin. Of all novel molecular interventions currently being investigated for Duchenne muscular dystrophy, perhaps the most promising method aiming to restore dystrophin expression to diseased cells is known as 'exon skipping' or splice-modulation, whereby antisense oligonucleotides eliminate the deleterious effects of DMD mutations by modulating dystrophin pre-messenger RNA splicing, such that functional dystrophin protein is produced. Recently this method was shown to be promising and safe in clinical trials both in The Netherlands and the UK. These trials studied direct antisense oligonucleotide injections into single peripheral lower limb muscles, whereas a viable therapy will need antisense oligonucleotides to be delivered systemically to all muscles, most critically to the heart, and ultimately to all other affected tissues including brain. There has also been considerable progress in understanding how such splice-correction methods could be applied to the treatment of related neuromuscular diseases, including spinal muscular atrophy and myotonic dystrophy, where defects of splicing or alternative splicing are closely related to the disease mechanism.