Oligonucleotide Therapy Research Articles

Current and Possible Future Therapeutic Options for Huntington's Disease.

Huntington’s disease (HD) is an autosomal neurodegenerative disease that is characterized by an excessive number of CAG trinucleotide repeats within the huntingtin gene (HTT). HD patients can present with a variety of symptoms including chorea, behavioural and psychiatric abnormalities and cognitive decline. Each patient has a unique combination of symptoms, and although these can be managed using a range of medications and non-drug treatments there is currently no cure for the disease. Current therapies prescribed for HD can be categorized by the symptom they treat. These categories include chorea medication, antipsychotic medication, antidepressants, mood stabilizing medication as well as non-drug therapies. Fortunately, there are also many new HD therapeutics currently undergoing clinical trials that target the disease at its origin; lowering the levels of mutant huntingtin protein (mHTT). Currently, much attention is being directed to antisense oligonucleotide (ASO) therapies, which bind to pre-RNA or mRNA and can alter protein expression via RNA degradation, blocking translation or splice modulation. Other potential therapies in clinical development include RNA interference (RNAi) therapies, RNA targeting small molecule therapies, stem cell therapies, antibody therapies, non-RNA targeting small molecule therapies and neuroinflammation targeted therapies. Potential therapies in pre-clinical development include Zinc-Finger Protein (ZFP) therapies, transcription activator-like effector nuclease (TALEN) therapies and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) therapies. This comprehensive review aims to discuss the efficacy of current HD treatments and explore the clinical trial progress of emerging potential HD therapeutics.

RNA Drugs

RNA Drugs

Suppression of Estrogen Receptor in Breast Cancer Cells via Transferrin Coated Gold Nanoparticles Carrying Antisens Oligonucleotides

Breast cancer is the most common cancer of women. In breast cancer treatment the estrogen receptor is an established target in receptor positive tumors. A novel approach to therapy is gene regulation by oligonucleotide drugs. Antisens oligonucleotide therapy is facing with some difficulties such as targeting a polianionic nucleotide into the tumor cell. In order to form an efficient carrier system to protect and target antisens oligonucleotides against estrogen receptor α mRNA, a gold nanoparticle based construct is formed. Uptake of amino and thiol modified gold nanoparticles was analysed by confocal microscopy. Gold nanaparticles were functionalysed by transferrin coating and estrogen receptor α antisens oligonucleotide and were applied to MCF7 breast cancer cells. It was found that funtionalized gold nanoparticles decreased the estrogen receptor expression by 51% as well as cell proliferation upto 41.00 ± 5.6 % of untreated cells. Therefore, this targeting construct using the transferrin receptor and receptor mediated endocytosis process delivers the antisens oligonucleotide efficiently. Since estrogen receptor α is an important regulator of cell proliferation, this system is able to inhibit proliferation by means of decreasing the expression level of this major regulatory molecule. It is concluded that, this functionalysed gold nanoparticle system may be designed for other targets in tumors and may also be used as a teranostic agent.

Antisense Oligonucleotide Therapy: From Design to the Huntington Disease Clinic.

Huntington disease (HD) is a fatal progressive neurodegenerative disorder caused by an inherited mutation in the huntingtin (HTT) gene, which encodes mutant HTT protein. Though HD remains incurable, various preclinical studies have reported a favorable response to HTT suppression, emphasizing HTT lowering strategies as prospective disease-modifying treatments. Antisense oligonucleotides (ASOs) lower HTT by targeting transcripts and are well suited for treating neurodegenerative disorders as they distribute broadly throughout the central nervous system (CNS) and are freely taken up by neurons, glia, and ependymal cells. With the FDA approval of an ASO therapy for another disease of the CNS, spinal muscular atrophy, ASOs have become a particularly attractive therapeutic option for HD. However, two types of ASOs were recently assessed in human clinical trials for the treatment of HD, and both were halted early. In this review, we will explore the differences in chemistry, targeting, and specificity of these HTT ASOs as well as preliminary clinical findings and potential reasons for and implications of these halted trials.

Human \u03b1-synuclein overexpression in mouse serotonin neurons triggers a depressive-like phenotype. Rescue by oligonucleotide therapy

Anxiety and depression affect 35–50% of patients with Parkinson’s disease (PD), often precede the onset of motor symptoms, and have a negative impact on their quality of life. Dysfunction of the serotonergic (5-HT) system, which regulates mood and emotional pathways, occurs during the premotor phase of PD and contributes to a variety of non-motor symptoms. Furthermore, α-synuclein (α-Syn) aggregates were identified in raphe nuclei in the early stages of the disease. However, there are very few animal models of PD-related neuropsychiatric disorders. Here, we develop a new mouse model of α-synucleinopathy in the 5-HT system that mimics prominent histopathological and neuropsychiatric features of human PD. We showed that adeno-associated virus (AAV5)-induced overexpression of wild-type human α-Syn (h-α-Syn) in raphe 5-HT neurons triggers progressive accumulation, phosphorylation, and aggregation of h-α-Syn protein in the 5-HT system. Specifically, AAV5-injected mice displayed axonal impairment in the output brain regions of raphe neurons, and deficits in brain-derived neurotrophic factor (BDNF) expression and 5-HT neurotransmission, resulting in a depressive-like phenotype. Intracerebroventricular treatment with an indatraline-conjugated antisense oligonucleotide (IND-ASO) for four weeks induced an effective and safe reduction of h-α-Syn synthesis in 5-HT neurons and its accumulation in the forebrain, alleviating early deficits of 5-HT function and improving the behavioural phenotype. Altogether, our findings show that α-synucleinopathy in 5-HT neurons negatively affects brain circuits that control mood and emotions, resembling the expression of neuropsychiatric symptoms occurring at the onset of PD. Early preservation of 5-HT function by reducing α-Syn synthesis/accumulation may alleviate PD-related depressive symptoms.

Therapeutic RNA-silencing oligonucleotides in metabolic diseases.

Recent years have seen unprecedented activity in the development of RNA-silencing oligonucleotide therapeutics for metabolic diseases. Improved oligonucleotide design and optimization of synthetic nucleic acid chemistry, in combination with the development of highly selective and efficient conjugate delivery technology platforms, have established and validated oligonucleotides as a new class of drugs. To date, there are five marketed oligonucleotide therapies, with many more in clinical studies, for both rare and common liver-driven metabolic diseases. Here, we provide an overview of recent developments in the field of oligonucleotide therapeutics in metabolism, review past and current clinical trials, and discuss ongoing challenges and possible future developments.

Antisense Oligonucleotide Technologies to Combat Obesity and Fatty Liver Disease.

Synthetic oligonucleotide technologies are DNA or RNA based molecular compounds that are utilized to disrupt gene transcription or translation in target tissues or cells. Optimally, oligonucleotides are 10–30 base pairs in length, and mediate target gene suppression through directed sequence homology with messenger RNA (mRNA), leading to mRNA degradation. Examples of specific oligonucleotide technologies include antisense oligonucleotides (ASO), short hairpin RNAs (shRNA), and small interfering RNAs (siRNA). In vitro and in vivo studies that model obesity related disorders have demonstrated that oligonucleotide technologies can be implemented to improve the metabolism of cells and tissues, exemplified by improvements in fat utilization and hepatic insulin signaling, respectively. Oligonucleotide therapy has also been associated with reductions in lipid accumulation in both the liver and adipose tissue in models of diet-induced obesity. Recent advances in oligonucleotide technologies include the addition of chemical modifications such as N-acetylgalactosamine (GalNAc) conjugates that have been successful at achieving affinity for the liver, in turn improving specificity, and thus reducing off target effects. However, some challenges are still yet to be overcome relating to hepatic injury and off-target effects that have been reported with some compounds, including ASOs. In summary, oligonucleotide-based therapies are an effective tool to elucidate mechanistic insights into metabolic pathways and provide an attractive avenue for translational research into the clinic.

Supportive Oligonucleotide Therapy (SOT) as an Alternative Treatment Option in Cancer: A Preliminary Study.

An early evaluation concerning the effectiveness of supportive oligonucleotide therapy (SOT) in cancer as a monotherapy and in combination with other types of treatment. This study evaluated the clinical condition and performance status (Karnofsky-Index) of 95 patients, post-SOT administration. Furthermore, circulating tumor cells (CTCs) from 47 patients' pre- and post-SOT administration were measured and analyzed by repeated-measures ANOVA. Improvement of the clinical condition was observed in all patients who used SOT (77.89%), SOT in combination with other therapy (69.77%) and SOT as a monotherapy or no information was given concerning another therapy (84.31%). Positive results for Karnofsky-Index were also observed in 71.58%, 61.36%, and 80.39%, respectively. Finally, statistically significant reductions in CTCs were observed for both SOT as a monotherapy and SOT as an adjunctive therapy. The preliminary results indicate that SOT therapy can be used both as monotherapy as well as in combination with other therapies for cancer.

New antisense oligonucleotide therapies reach first base in ALS.

New antisense oligonucleotide therapies reach first base in ALS.

Potential of antisense oligonucleotide therapies for amyotrophic lateral sclerosis

Potential of antisense oligonucleotide therapies for amyotrophic lateral sclerosis

Long Noncoding RNAs as Therapeutic Targets.

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of cellular functions including maintenance of cellular homeostasis as well as the onset and progression of disease. LncRNAs often exhibit cell-, tissue-, and disease-specific expression patterns, making them desirable therapeutic targets. LncRNAs are commonly targeted using oligonucleotide therapeutics, and advances in oligonucleotide chemistry including C2 ribose sugar modifications such as 2'-fluoro, 2'-O-methyl, and 2-O-methoxyethyl modifications; 2'4'-constrained nucleotides such as locked nucleic acids and constrained 2'-O-ethyl (cEt) nucleotides; and phosphorothioate bonds have dramatically improved efficacy of oligonucleotide therapies. Novel delivery platforms such as viral vectors and nanoparticles have also improved pharmacokinetic properties of oligonucleotides targeting lncRNAs. Accumulating pre-clinical studies have utilized these strategies to therapeutically target lncRNAs and alter progression of many different disease states including Snhg12 and Chast in cardiovascular disease, Mirt2 and HOTTIP in sepsis and autoimmune disease, and Malat1 and HOXB-AS3 in cancer. Emerging oligonucleotide conjugation methods including the use of peptide nucleic acids hold promise to facilitate targeting to specific tissue types. Here, we review recent advances in lncRNA therapeutics and provide examples of how lncRNAs have been successfully targeted in pre-clinical models of disease. Finally, we detail remaining challenges facing the lncRNA field and how advances in delivery platforms and oligonucleotide chemistry might help overcome these barriers to catalyze the translation of pre-clinical studies to successful pharmaceutical development.

A New Modality in Dyslipidemia Treatment: Antisense Oligonucleotide Therapy.

There are unmet needs for pharmacologic agents beyond current medications, such as statins, to effectively lower low-density lipoprotein cholesterol levels to target goals, especially in patients with very high or extremely high risk. Pharmacological targeting of mRNA represents an emerging, innovative approach with the potential to expand upon current therapies. In RNA-targeted therapeutics, a novel approach is the use of chemically modified oligonucleotides to inhibit the production of target proteins at their sites of gene coding. There are two main classes of RNA-targeted therapeutics: single-stranded antisense oligonucleotides (ASOs) and double-stranded small inhibiting RNAs. ASOs are synthetic molecules with a length of 15–30 nucleotides that are designed specifically to bind to a target mRNA in a sequence-specific manner. Using these agents to inhibit the translation of key regulatory proteins, such as apolipoprotein CIII, apolipoprotein(a), and angiopoietin-like protein 3, has demonstrated treatment efficacy for dyslipidemia. Many cardiovascular outcome trials with ASOs are ongoing. As clinicians, we must carefully monitor the long-term safety and efficacy of this new modality through large clinical trials in the future.

Individualized Antisense Oligonucleotide Therapies: How to Approach the Challenge of Manufacturing These Oligos from a Chemistry, Manufacturing, and Control-Regulatory Standpoint.

With the development of antisense oligonucleotides over more than 30 years and the increasing number of identified unique severely debilitating or life-threatening diseases affecting only 1 person in the world-now referred to as N-of-1 diseases-it is more and more appealing to use antisense technology to treat N-of-1 diseases when they are caused by well-identified mutations in single genes. N-of-1 patients present unique challenges to the health care system because the patient may be, and often is, the single patient in the world with the specific mutation in question, thus requiring an approach particular to that patient. Yet, we now know that there are millions of such patients, requiring scalable solutions. This article offers suggestions on how a specific and very regulated area of the new drug development process, chemistry, manufacturing, and control, could be addressed for N-of-1 oligonucleotides from a regulatory standpoint.

Advances in Therapeutic L-Nucleosides and L-Nucleic Acids with Unusual Handedness.

Nucleic-acid-based small molecule and oligonucleotide therapies are attractive topics due to their potential for effective target of disease-related modules and specific control of disease gene expression. As the non-naturally occurring biomolecules, modified DNA/RNA nucleoside and oligonucleotide analogues composed of L-(deoxy)riboses, have been designed and applied as innovative therapeutics with superior plasma stability, weakened cytotoxicity, and inexistent immunogenicity. Although all the chiral centers in the backbone are mirror converted from the natural D-nucleic acids, L-nucleic acids are equipped with the same nucleobases (A, G, C and U or T), which are critical to maintain the programmability and form adaptable tertiary structures for target binding. The types of L-nucleic acid drugs are increasingly varied, from chemically modified nucleoside analogues that interact with pathogenic polymerases to nanoparticles containing hundreds of repeating L-nucleotides that circulate durably in vivo. This article mainly reviews three different aspects of L-nucleic acid therapies, including pharmacological L-nucleosides, Spiegelmers as specific target-binding aptamers, and L-nanostructures as effective drug-delivery devices.

Assessment of cerebral spinal fluid biomarkers and microRNA-mediated disease mechanisms in spinal muscular atrophy patient samples.

Cerebral spinal fluid (CSF) is a promising biospecimen for the detection of central nervous system biomarkers to monitor therapeutic efficacy at the cellular level in neurological diseases. Spinal muscular atrophy (SMA) patients receiving intrathecal antisense oligonucleotide (nusinersen) therapy tend to show improved motor function, but the treatment effect on cellular health remains unknown. The objective of this study was to assess the potential of extracellular RNAs and microRNAs in SMA patient CSF as indicators of neuron and glial health following nusinersen treatment. Extracellular RNA analysis of CSF samples revealed ongoing cellular stress related to inflammation and glial differentiation, even after treatment administration. Downregulated microRNA expression associated with SMA-specific or general motor neuron dysfunction in animal and cellular models, tended to increase in nusinersen-treated patient CSF samples and correlated with SMA Type 1 and 2 motor functioning improvements. However, miR-146a, known to be upregulated in SMA-induced pluripotent stem cell (iPSC)-derived astrocytes, showed increased expression in nusinersen-treated CSF samples. We then used mRNA sequencing and multi-electrode arrays to assess the transcriptional and functional effects of miR-146a on healthy and SMA iPSC-derived motor neurons. miR-146a treatment on iPSC-derived motor neurons led to a downregulation of extracellular matrix genes associated with synaptic perineuronal net and alterations in spontaneous electrophysiological activity. Altogether, this study suggests that extracellular RNAs and microRNAs may serve as useful biomarkers to monitor cellular health during nusinersen treatment. Moreover, these data highlight the importance of addressing astrocyte health and response to nusinersen in SMA pathogenesis and treatment strategies.

A Brief Journey through Protein Misfolding in Transthyretin Amyloidosis (ATTR Amyloidosis).

Transthyretin (TTR) amyloidogenesis involves the formation, aggregation, and deposition of amyloid fibrils from tetrameric TTR in different organs and tissues. While the result of amyloidoses is the accumulation of amyloid fibrils resulting in end-organ damage, the nature, and sequence of the molecular causes leading to amyloidosis may differ between the different variants. In addition, fibril accumulation and toxicity vary between different mutations. Structural changes in amyloidogenic TTR have been difficult to identify through X-ray crystallography; but nuclear magnetic resonance spectroscopy has revealed different chemical shifts in the backbone structure of mutated and wild-type TTR, resulting in diverse responses to the cellular conditions or proteolytic stress. Toxic mechanisms of TTR amyloidosis have different effects on different tissues. Therapeutic approaches have evolved from orthotopic liver transplants to novel disease-modifying therapies that stabilize TTR tetramers and gene-silencing agents like small interfering RNA and antisense oligonucleotide therapies. The underlying molecular mechanisms of the different TTR variants could be responsible for the tropisms to specific organs, the age at onset, treatment responses, or disparities in the prognosis.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

Antisense oligonucleotide therapy reduces seizures and extends life span in an SCN2A gain-of-function epilepsy model.

De novo variation in SCN2A can give rise to severe childhood disorders. Biophysical gain of function in SCN2A is seen in some patients with early seizure onset developmental and epileptic encephalopathy (DEE). In these cases, targeted reduction in SCN2A expression could substantially improve clinical outcomes. We tested this theory by central administration of a gapmer antisense oligonucleotide (ASO) targeting Scn2a mRNA in a mouse model of Scn2a early seizure onset DEE (Q/+ mice). Untreated Q/+ mice presented with spontaneous seizures at P1 and did not survive beyond P30. Administration of the ASO to Q/+ mice reduced spontaneous seizures and significantly extended life span. Across a range of behavioral tests, Scn2a ASO-treated Q/+ mice were largely indistinguishable from WT mice, suggesting treatment is well tolerated. A human SCN2A gapmer ASO could likewise impact the lives of patients with SCN2A gain-of-function DEE.

Promise of Nucleic Acid Therapeutics for Amyotrophic Lateral Sclerosis.

Nucleic acid therapeutics have been attracting attention as novel drug discovery modalities for intractable diseases, including amyotrophic lateral sclerosis. This review provides an overview of the current status and prospects of antisense oligonucleotide treatment for amyotrophic lateral sclerosis. Recently, the results of a phase I/II study using the antisense oligonucleotides Tofersen to treat familial amyotrophic lateral sclerosis with superoxide dismutase 1 mutation have been reported. Intrathecal Tofersen administration resulted in a 36% reduction in superoxide dismutase 1 level in the cerebrospinal fluid. Another report described 2 patients with mutant superoxide dismutase 1 treated with an adeno-associated virus encoding a microRNA targeting superoxide dismutase 1. The first patient, who possessed the fast progressive mutant A5V, received a single intrathecal infusion. Although the patient died of respiratory arrest 16months after treatment, autopsy findings showed a reduction of >90% in superoxide dismutase 1 level in the spinal cord. Clinical trials on antisense oligonucleotide therapies targeting other major amyotrophic lateral sclerosis-causative genes, fused in sarcoma and chromosome 9 open reading frame 72, are ongoing. To attenuate the pathology of TDP-43, strategies targeting regulators of TDP-43 (ataxin 2) and proteins downstream of TDP-43 (stathmin 2) by antisense oligonucleotides are being developed. The advent of nucleic acid therapeutics has enabled to specifically attack the molecules in the amyotrophic lateral sclerosis pathological cascade, expanding the options for therapeutic targets. ANN NEUROL 2022;91:13-20.

Future Perspectives of Oral Delivery of Next Generation Therapies for Treatment of Skin Diseases.

Gene therapies have conspicuously bloomed in recent years as evidenced by the increasing number of cell-, gene-, and oligo-based approved therapies. These therapies hold great promise for dermatological disorders with high unmet need, for example, epidermolysis bullosa or pachyonychia congenita. Furthermore, the recent clinical success of clustered regularly interspaced short palindromic repeats (CRISPR) for genome editing in humans will undoubtedly contribute to defining a new wave of therapies. Like biologics, naked nucleic acids are denatured inside the gastrointestinal tract and need to be administered via injections. For a treatment to be effective, a sufficient amount of a given regimen needs to reach systemic circulation. Multiple companies are racing to develop novel oral drug delivery approaches to circumvent the proteolytic and acidic milieu of the gastrointestinal tract. In this review, we provide an overview of the evolution of the gene therapy landscape, with a deep focus on gene and oligonucleotide therapies in clinical trials aimed at treating skin diseases. We then examine the progress made in drug delivery, with particular attention on the peptide field and drug-device combinations that deliver macromolecules into the gastrointestinal tract. Such novel devices could potentially be applied to administer other therapeutics including genes and CRISPR-based systems.