Next Article in Journal
18F-Fluorocholine PET and Multiphase CT Integrated in Dual Modality PET/4D-CT for Preoperative Evaluation of Primary Hyperparathyroidism
Previous Article in Journal
Pituitary Disease in AIP Mutation-Positive Familial Isolated Pituitary Adenoma (FIPA): A Kindred-Based Overview
 
 
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Review

Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development

1
Department of Pharmaceutical Science, University of Connecticut, Storrs, CT 06269, USA
2
Department of Genetics, Yale University, New Haven, CT 06520, USA
3
Department of Radiation Oncology, Vanderbilt University Medical Center, Nashville, TN 37232-5671, USA
4
Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA
5
Division of Cardiovascular Medicine, Department of Internal Medicine, The University of Iowa, Iowa City, IA 52242, USA
*
Author to whom correspondence should be addressed.
J. Clin. Med. 2020, 9(6), 2004; https://doi.org/10.3390/jcm9062004
Received: 24 April 2020 / Revised: 20 June 2020 / Accepted: 24 June 2020 / Published: 26 June 2020
(This article belongs to the Section Pharmacology)

Abstract

:
Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.

1. Introduction

Aberrant protein production or metabolism is associated with numerous devastating diseases and disorders [1,2,3,4]. As proteins are produced by decoding information stored in messenger RNA (mRNA), aberrant protein production can be regulated by targeting mRNA. Additionally, a greater understanding of RNA has unraveled its multifaceted roles. Until the advent of non-coding RNAs (ncRNAs), mRNA was only considered as the mediator between DNA and the ribosome for protein synthesis. Among ncRNAs, microRNA (miRNA) [5], transfer RNA-derived small RNA [6], pseudogenes [7], PIWI-interacting RNA [8], long ncRNAs (lncRNAs) [9], and circular RNAs [10] have been identified as critical regulators of biological functions through modulation of gene expression. Hence, the antisense strategy comprising of targeting pre-mRNA, mRNA, or ncRNAs can alter the production of disease-causing proteins for therapeutic interventions. Unlike small molecule-based protein targeting, antisense drugs exhibit their effect by Watson–Crick base pairing rules with target RNA sequence. This principle of Watson–Crick molecular recognition provides the antisense field more flexibility in RNA-based drug design and expedites its development, which is imperative for targeting a myriad of rare and genetic diseases [11]. The amalgamation of chemical structure modifications of oligonucleotides and diverse delivery platforms provides an additional boost to the antisense field. Recent United States Food and Drug Administration (FDA) approval of several nucleic acid-based drugs has further spurred interest in the antisense research. Presently, numerous antisense drug candidates are in clinical trials to treat cardiovascular, metabolic, endocrine, neurological, neuromuscular, inflammatory, and infectious diseases [12]. This review provides a brief overview of the structural modifications of new generation antisense oligonucleotides (ASOs), their mechanisms of action, delivery strategies, and comprehensive information about FDA-approved antisense therapies and current antisense-based drug candidates in clinical trials.

2. Oligonucleotide Modifications

In prior studies, ASOs based on phosphodiester backbone (also known as unmodified ASOs) were used to target RNA with moderate success. However, due to the presence of a phosphodiester bond, unmodified ASOs are susceptible to nuclease degradation [13]. In addition, the large size and charge of unmodified ASOs restrict their passive diffusion into the cell [14]. Hence, newer generation, chemically modified ASOs have been explored to increase their efficacy, enzymatic stability, and decrease immune response and off-target toxicity (Table 1).

2.1. Phosphorothioate (PS)

Phosphorothioate belongs to the first generation of ASOs that work by an mRNA cleavage-based mechanism [15]. In phosphorothioate (PS) ASOs, the non-bridging oxygen of the phosphate group is replaced by a sulfur group, resulting in the formation of a PS bond, which is resistant to nuclease-based degradation [16,17]. Compared to unmodified ASOs, the PS-ASOs strongly bind to serum proteins such as albumin, which further reduces their renal clearance and facilitates longer in vivo circulation [18]. Pharmacokinetic study in mice after intravenous (IV) administration of 30 mg kg−1 dose of PS-ASOs revealed 40% excretion in urine in 48 h [19]. Compared to unmodified ASOs, PS-ASOs show a predominant distribution in liver, kidney, and spleen when administered systemically, and demonstrate good cellular uptake. Following systemic administration in monkeys, PS-ASOs demonstrate biphasic plasma elimination with an initial half-life of 30–48 min, followed by a second-long half-life of 35–50 h [20]. However, repeated IV or intradermal administration of PS-ASOs in rodents elicits an immune reaction, highlighting the toxicity associated with them [21].

2.2. Phosphorodiamidate Morpholino Oligomer (PMO)

In phosphorodiamidate morpholino oligomers, the five-membered sugar moiety is substituted with a six-membered morpholine subunit, and each morpholine ring is inter-connected with phosphorodiamidate linkage [22]. PMOs are charge neutral and exert their antisense effect by steric hindrance or splice modulation. The absence of the carbonyl group in the PMO structure provides resistance against proteases and esterases [23]. The morpholine ring also increases its water solubility. Morpholinos have been extensively used in developmental biology for numerous antisense-based applications [24].

2.3. Peptide Nucleic Acids (PNA)

Peptide nucleic acids are synthetic nucleic acid mimics that contain neutral N-2-aminoethyl glycine units, with nucleobases connected by a flexible methyl carbonyl linker. Due to their neutral and unnatural backbone, PNAs are resistant to enzymatic degradation and possess a strong binding affinity with RNA sequences as compared to unmodified ASOs [25]. Significant challenges are associated with PNAs due to their low water solubility and decreased cellular uptake. Hence, various chemically modified PNAs, cationic PNAs [26], alpha and gamma guanidium PNAs [27], and lysine PNAs [28,29] have been developed to resolve these issues.

2.4. Locked Nucleic Acids (LNAs)

Locked nucleic acids contain a constrained methylene bridge between 2′ oxygen and 4′ carbon of the ribose ring and display a strong binding affinity to the target DNA or RNA sequences due to their preorganized structure. Each locked nucleic acid (LNA) modification increases the melting temperature of the duplex by 2–8°C [30]. LNAs express their antisense ability by steric hindrance mechanism. Diverse, comprehensive LNA designs, gapmer, mixmer, and other modifications have been evaluated for their antisense activity and compared with unmodified ASOs. Gapmers are LNA-DNA-LNA-based designs that contain continuous DNA nucleotides interspaced between two over hanged LNA nucleotides at the terminal regions (Figure 1). It has been well established that gapmer design stimulates RNase H1-based cleavage mechanism [31]. Mixmer designs contain interspaced DNA nucleotides between LNA nucleotides throughout the sequence and do not induce RNase H1 cleavage, but rather modulate mRNA expression through steric hindrance mechanism [32]. It has been noted that a minimum stretch of four LNA nucleotides at each terminal of the sequence (end block design) is adequate to provide a half-life of 15 h to LNAs, which is ~10-fold higher than that of unmodified ASOs [33].

Ribose Modifications

Chemical modification at the 2′ position of ribose sugars improves their binding affinity and provides resistance to enzymatic degradation. 2′ ribose modification includes 2′ fluoro (2′ F), 2′-O-methyl (2′-O-Me), and 2′-O-methoxyethyl (2′-O-MOE)-based ASOs [34]. Each change at the 2′ position increases the melting temperature of the duplex by 2 °C [35]. 2′-O-Me modification also decreases nonspecific protein binding during in vivo administration. As explained above, gapmer designs also apply to the 2′ ribose-modified class of ASOs. Here, in a gapmer design, a central region of unmodified antisense nucleotides is flanked by 2′ modified nucleotides at both ends. The unmodified nucleotides induce RNase H1 cleavage of the target RNA, and 2′ modified nucleotides improve the binding affinity of ASO to the region of interest and protect against endonucleases [36]. In a study by Shen et al. in mice, 2′ F-modified ASOs exhibits increase binding affinity to intracellular proteins like drosophila behavior/human splicing (DBHS) proteins, reducing DHBS proteins and resulting in hepatotoxicity [37]. Therefore, caution needs to be taken during in vivo studies as 2′ F modifications can lead to toxicity.

2.5. Nucleobase Modification

Nucleobase modifications are introduced to improve the properties of ASOs. Among nucleobase modifications, cytosine analogs have been used extensively. It has been noted that PS-ASOs that contain CpG dinucleotide stretches activate the toll-like receptor and cause immune stimulation [38]. Hence, 5-methyl cytosine-based analogs were used with PS-based chemistry to minimize the immune stimulation. Similarly, another cytosine analog, G-clamp has been used to increase the efficacy of ASOs. G-clamp modifications contain phenoxazine residues that form a total of five hydrogen bonds with complementary guanine nucleobase in the target sequence by Watson–Crick as well as Hoogsteen-base pairing [39]. It has been demonstrated that single G-clamp substitutions can increase the binding affinity by 23 °C [40,41].

3. Antisense Mechanism of Action

About four decades ago, Zamecnik and Stephenson first reported that 13-mer synthetic single-stranded ASOs could cause translational arrest by targeting Rous sarcoma virus mRNA [42]. After several studies, it has been well established that active ASOs are generally 15–20 nucleotides in length and can target complementary RNA by Watson–Crick base pairing without causing any significant off-target toxicity [43]. In addition, comprehensive studies established that the mechanism of synthetic ASOs can be of two types: (1) RNA cleavage and (2) RNA blockage (Figure 2) [44,45].

3.1. RNase H1 Mediated Degradation

ASOs target RNA, forming ASO-RNA heteroduplexes, which act as substrates for RNase enzymes present in the cytoplasm (Figure 2) [46]. RNases degrade RNA in the heteroduplex. Gapmer design contains a central region of unmodified nucleotides that aids in RNase H1 activity and flanking modified nucleotides increase its binding affinity and enzymatic resistance properties. Most of the drugs that have been FDA approved exert their antisense effect via RNases [47,48].

3.2. RNA Interference (RNAi)

Exogenous small interfering RNAs (siRNAs) are double-stranded 22 nucleotide RNA sequences with a 2-nucleotide overhang on the 3′ end of either strand [49]. This sequence associates with Argonaute 2 (Ago 2) enzyme to form the RNA induced silencing complex (RISC) where the passenger strand is degraded [50,51]. The remaining guide strand directs the RISC to the complementary mRNA region where Ago 2 enzyme cleaves the mRNA and exerts its gene silencing effect (Figure 2) [52]. RNAi works by a mechanism similar to the RNase H-dependent mechanism. The significant difference as compared to RNase-based degradation is that the siRNA is associated with the cleavage protein before interacting with the target site.

3.3. Steric Blockage

3.3.1. Translation Arrest Due to Steric Hindrance

These classes of ASOs bind to target RNA sequence and cause translational arrest by inhibiting their interaction with the 40S ribosomal subunit or preventing their assembly on the 40S or 60S ribosomal subunit (Figure 2) [53,54]. ASOs based on steric hindrance do not activate RNase H1-mediated cleavage; therefore, the pre-mRNA structure is retained. Steric hindrance is directly related to the binding affinity of ASOs. An increase in binding affinity results in superior hybridization with target RNA, resulting in a translational arrest. In addition, synthetic single-stranded oligonucleotides of 20–25 nucleotides in length are designed to bind the miRNA and prevent their interaction with mRNA by steric hindrance-based mechanism, further controlling the gene expression [55]. A few studies have also reported that ASOs can bind to pre-miRNA in the nucleus and exert steric hindrance [56].

3.3.2. Splice Modulation or Splice Switching-Based Mechanism

ASOs can also exert their effects by alternative splicing [57,58,59]. Splice modulation can be of two types: (1) exon skipping and (2) exon inclusions [60,61]. Frameshift mutations alter pre-mRNA splicing patterns that result in abnormal protein production or a translation arrest of full-length functional proteins [62,63]. In exon skipping, ASOs bind to the pre-mRNA transcripts, correct the disrupted reading frame and produce a short but functional protein [64]. Whereas in exon inclusion, ASOs bind to the pre-mRNA site and prevent the spliceosome and splicing factors from accessing the transcript sites (Figure 2) [65]. In 1993, for the first time, it was shown that 2′-O-Me precisely spliced the mutated beta-globin pre-mRNA in vitro to produce splice variant mRNA that restored hemoglobin production [66].

4. Delivery of ASOs.

While delivery of ASOs has always been a significant hurdle for their broad clinical applications, various strategies have been employed to deliver them [67,68,69].

4.1. Enhanced Stabilization Chemistry-Based Delivery

Alnylam extensively used enhanced stabilization chemistry (ESC) to deliver siRNA. In ESC, siRNAs are conjugated to N-Acetyl galactosamine (GalNAc) [70]. GalNac selectively targets the asialoglycoprotein receptor (ASGPR) that is highly expressed in hepatocytes [71]. GalNAc also stabilizes the siRNA conjugates in hepatocytes, plasma, as well as the lymphatic system, and reduces immune stimulation.

4.2. Nanoformulation-Based Delivery

Numerous polymeric nanoparticle-based delivery systems like PLGA, PBAE, and PEI have been used to deliver ASOs [72,73]. PBAE and PEI exert a ‘proton sponge’ effect that decreases the endosomal entrapment of ASOs and increases their delivery [74]. However, the advancement of these delivery systems to the clinic has been limited due to toxicity caused by excessive cationic charge as well as multiple non-specific interactions with serum and tissue proteins. On the contrary, PLGA being biocompatible and an FDA approved polymer, has been widely used to formulate nanoparticles and deliver ASOs [75,76].

4.3. Lipid-Based Delivery Systems

Several lipid-based delivery systems, lipoplexes, liposomes, and lipid nanoparticles (LNP) have been extensively used to deliver ASOs and siRNAs [77,78]. LNPs are typically coated with polyethylene glycol (PEG), which increases blood circulation time [79]. LNPs also show accumulation in the tumor microenvironment by the enhanced permeability and retention (EPR) effect. Patisiran contains LNP-based siRNA formulation.

5. FDA-Approved Formulations

5.1. Fomivirsen (VitraveneTM)

In 1998, Fomivirsen was the first ASO-based drug developed by Ionis Pharmaceuticals. Fomivirsen received approval by the United States FDA for the treatment of retinitis caused by opportunistic cytomegalovirus (CMV) infection in immunocompromised AIDS patients (Table 2). CMV infection results in loss of vision in AIDS patients. Fomivirsen is a PS-based 21-mer ASO that hybridizes with the coding sequence of CMV mRNA from the major immediate-early region-2, which is responsible for CMV replication [80]. Fomivirsen inhibited CMV replication in a dose-dependent manner in preclinical studies. It exhibited a half-life of 78 h after intravitreal administration of 115 µg dose in monkeys [81]. Fomivirsen demonstrated clinical efficacy in a randomized clinical trial and delayed the progression of CMV infection in AIDS patients by 71 days as compared to 13 days in the deferred patient cohort [82]. However, the same period witnessed the success of the highly active antiretroviral therapy (HAART), which reduced the incidences of opportunistic inf-ections in AIDS patients and thus reduced market demand for Fomivirsen. Eventually, Fomivirsen was voluntarily withdrawn from the market by its manufacturers.

5.2. Mipomersen (KynamroTM)

Homozygous familial hypercholesterolemia (HoFH) is an autosomal dominant genetic disorder that results in elevated low-density lipoprotein (LDL) levels. HoFH is caused by a mutation in the gene for LDL cholesterol receptor or pro-protein convertase subtilisin/kexin 9 (PCSK9) or apolipoprotein B (apo B) [83]. The elevated LDL levels result in high risk for developing coronary heart disease (CHD) or atherosclerosis at a young age. Mipomersen, an ASO developed by Genzyme, targets mRNA that encodes apo-B-100 mRNA and inhibits the synthesis of apolipoprotein B-100. It contains PS-based modifications throughout the sequence and five 2′-O-MOE-based nucleotides on both terminal ends. Mipomersen is administered subcutaneously at a dose of 200 mg once weekly and also used in combination with cholesterol-lowering drugs along with recommended lifestyle changes. In a placebo-controlled clinical trial study comprised of HoFH patients already on a lipid-lowering drug, Mipomersen reduced the LDL-cholesterol level by 25% compared to the placebo group. In addition, the patients treated with Mipomersen showed a reduction in serum VLDL cholesterol, non-HDL cholesterol, and lipoprotein(a) concentrations [84,85].

5.3. Nusinersen (Spinraza®)

Spinal muscular atrophy (SMA) is a genetic and autosomal recessive motor neuron disease caused by a homozygous deletion in the survival motor neuron 1 (SMN1) on chromosome 5q13 [86]. The SMN1 gene produces full-length mRNA that codes for functional SMN protein, which is required for normal functioning of motor neurons. Deficiency of SMN proteins results in the degeneration of lower muscle motor neurons, which leads to progressive paralysis due to muscle atrophy. In general, the SMN family contains two genes, SMN1 and SMN2. SMN2 differs from SMN1 by the presence of a thymine nucleotide instead of cytosine at the 840th position on the gene. This results in the exclusion of exon 7 from ~85% SMN2 pre-mRNA during splicing, resulting in the production of truncated SMN, which is unstable and rapidly degraded. However, the remaining 15% of SMN2 mRNA produces full-length functional SMN protein due to exon 7 inclusion [87]. Typically, everyone has the SMN1 and SMN2 gene; however, it becomes critical for SMA patients to present at least one copy of the SMN2 gene to allow for splice modulation to produce full-length SMN protein. Nusinersen is an ASO-based drug developed by Biogen for SMA treatment in pediatric and adult patients. It contains 2′-O-MOE and PS-based modifications [88]. Nusinersen hybridizes with SMN2 pre-mRNA and prevents the recruitment of splicing repressor, heterogeneous nuclear riboprotein A1 and A2, which favors the inclusion of exon 7 in the SMN2 mRNA and leads to increase in the production of functional SMN protein form [89,90]. Nusinersen treated SMA1 infants exhibited improved neuromuscular functions, possibly not requiring the use of permanent assisted ventilation as compared to the untreated SMA1 infants [91]. Clinically, a loading dose of 12 mg Nusinersen in a 5 mL volume is given intrathecally on the first, second, fourth, and ninth week, followed by maintenance therapy after every four-month interval [92]. One ongoing clinical trial is evaluating the efficacy of Nusinersen in improving the motor functions in adult SMN patients [93].

5.4. Patisiran (Onpattro®)

Transthyretin (TTR) is a tetrameric protein involved in the transport of thyroxin and retinol-binding protein vitamin A complex [94]. In healthy individuals, the TTR protein is present in cerebrospinal fluid and serum [95]. However, point mutations in the TTR gene produce aberrant TTR proteins that are more susceptible to misfolding and get deposited as TTR amyloid fibrils in extracellular spaces of the liver, heart, nerve, and gastrointestinal tract, eventually leading to organ dysfunction [96]. The aforementioned pathological condition is known as hereditary transthyretin-mediated amyloidosis (hATTR) and presents with severe symptoms of nausea, pain, and weakness. hATTR is estimated to affect around 50,000 patients worldwide. Patisiran is the first siRNA-based drug developed by Alnylam for hATTR treatment. Patisiran is a LNP-based formulation that is injected intravenously at a concentration of 2 mg/mL. In particular, D-Lin-MC3-DMA is an ionizable cationic lipid delivery vehicle used to encapsulate the siRNA. D-Lin-MC3-DMA has an acid dissociation constant (pKa) of 6.4 necessary to maintain a low surface charge to prevent early clearance of the siRNA from the body [97]. At a pKa of 6.4, the ionizable lipid group is positively charged, which aids in endosomal escape following endocytosis. Subsequently, siRNA is then released in the cell cytoplasm where it interacts with TTR mRNA, reducing TTR protein translation, thereby inhibiting the formation and deposition of amyloid plaques [98]. In a randomized, double-blinded placebo-controlled phase III study, patients treated with Patisiran showed an 80% reduction in serum TTR levels at doses between 0.15–0.5 mg kg−1 [99]. Three additional post-approval clinical trials are ongoing. The first trial is investigating the safety and efficacy of Patisiran in hATTR patients after liver transplant. The second study will assess the long-term safety of Patisiran treatment, and the third trial aims to evaluate and compare the efficacy of Vutrisiran to Patisiran for hATTR treatment [100,101,102].

5.5. Inotersen (Tegsedi®)

Inotersen was developed by Ionis Pharmaceuticals for hATTR treatment in 2018. Inotersen consists of gapmer design with five 2′-O-MOE nucleotides present at 5′ and 3′ ends with PS modifications present throughout the sequence. The 2′-O-MOE modification increases the ASO binding with the TTR mRNA without affecting its antisense activity. In a randomized, double-blinded placebo-controlled phase III clinical study, the safety and efficacy of subcutaneously administered Inotersen was evaluated in 112 patients and compared with 60 patients in a placebo group for 66 weeks. Composite scores based on the modified Neuropathy Impairment Score +7 (mNIS+7) and total score on the Norfolk Quality of Life-Diabetic Neuropathy (QOL-DN) questionnaire were the primary endpoints of the above-mentioned clinical study. In general, High mNIS+7 corresponds to inadequate response to therapy, and a high score on Norfolk QOL-DN is a measure of poor quality of life. The difference in the least-squares mean change from baseline to week 66 between the Inotersen and placebo-treated groups, which was −19.7 points for mNIS+7 and −11.7 points for the Norfolk QOL-DN, indicates the potential clinical efficacy of Inotersen. However, thrombocytopenia and glomerulonephritis were reported in a few patients during the study, warranting careful monitoring of hematological as well as a metabolic panel during the Inotersen-based treatment regimen [103].

5.6. Eteplirsen (Exondys 51®)

DMD is a fatal muscle degenerative disorder caused by mutations in the DMD gene, which encodes dystrophin. This partial gene deletion causes a reading frame shift resulting in an early stop codon that not only would prevent translation to functional dystrophin protein but, most importantly, induces mRNA destruction by nonsense-mediated RNA decay [104]. This results in loss of functional dystrophin, leading to muscle wasting and degeneration. Eteplirsen is a 30-mer oligonucleotide containing PMO-based chemical modifications developed by Sarepta Therapeutics for DMD treatment. Eteplirsen binds to exon 51 of DMD, which restores the reading frame of the dystrophin mRNA, translating short, but functional dystrophin [105,106]. In a preliminary study, two DMD patients were treated with Eteplirsen, and five patients from the placebo group were given saline as control. The patients receiving Eteplirsen showed an increase in dystrophin levels as quantified by immunohistochemistry-based endpoint analysis [107]. The clinical efficacy of Eteplirsen was evaluated in a small cohort of patients, wherein a marginal elevation from 0.16% at baseline to 0.48% at 48 weeks in dystrophin protein levels was observed [108]. Eteplirsen received FDA approval after considering the lack of available treatment options, severity, and progressive nature of the DMD disease. Additional post-approval clinical trial studies to validate the efficacy of Eteplirsen in decelerating the progression of DMD disease are ongoing as mandated by the FDA [109].

5.7. Golodirsen (Vyondys 53TM)

Golodirsen is developed by Sarepta Therapeutics for the treatment of DMD. Golodirsen utilizes a similar therapeutic strategy (PMO chemistry) as the one described for Eteplirsen. Golodirsen however, addresses patients with a different deletion in the dystrophin gene. It hybridizes with DMD pre-mRNA and cause exon 53 skipping leading to the production of short but functional dystrophin protein required for muscle activity. The safety and efficacy of Golodirsen was evaluated in a randomized, double-blind, placebo-controlled study in two phases. In the first phase, all 25 patients treated with increasing doses of Golodirsen for 48 weeks showed a 16-fold increase in dystrophin protein levels over baseline as compared to the placebo group. Golodirsen was found to be well-tolerated in patients after weekly IV infusion for 48 weeks. Muscle biopsies also indicated an increase in dystrophin production in the Golodirsen treated group as compared to the placebo group [110].

5.8. Givosiran (Givlaari®)

Acute hepatic porphyria (AHP) is a genetic disorder in which the enzyme delta-aminolevulinate synthase 1 (ALAS1) is produced in excess. The excess of ALAS1 enzyme accumulates neurotoxins, aminolevulinic acid (ALA), and porphobilinogen (PBG), which results in intense abdominal pain, nausea, and seizures [111]. Givosiran, developed by Alnylam Pharmaceuticals, is the second FDA approved siRNA drug for the treatment of AHP (Table 2). This siRNA is conjugated to GalNAc to achieve liver-specific delivery, where it leads to decreased ALAS1 enzyme, eventually reducing the levels of ALA and PBG [112]. A clinical study of Givosiran consisting of a total of 94 patients, showed that 74% of the patients receiving Givosiran experienced fewer incidences of porphyria. Givosiran is given by a subcutaneous route once a month. Common side effects include nausea and reactions at the injection site. Liver and kidney functions and allergic reactions are required to be monitored during and after treatment. The pharmacokinetics and long-term safety of Givosiran in acute intermittent porphyria treatment (AIP) is under clinical investigation [113].

5.9. Milasen

Milasen is a personalized ASO that was specifically designed to treat a six-year-old child suffering from neuronal ceroid lipofuscinosis 7 (CLN7) that affects the central nervous system (Table 2). This rare and fatal neurodegenerative condition, also known as Batten disease, leads to loss of vision, dysarthria, and dysphagia. Whole-genome sequencing of the patient identified the insertion of SVA (SINE-VNTR-Alu) retrotransposon in the MFSD8 gene (also called CLN7) that alters the splicing of transcripts. A series of ASOs were tested in patient fibroblasts to target cryptic splice sites in the MFSD8 pre-mRNA and restore normal (exon 6–7 ) splicing. The lead ASO, Milasen, is 22-nucleotides long and was developed using the same chemical modifications as present in Nusinersen. Milasen did not show any toxicity during pre-clinical studies in rats. Based on promising preliminary pre-clinical results, Milasen was granted an expedited N-of-1 approval for use in this patient. Excitingly, Milasen reduced the frequency of the seizures and effectively improved the quality of life of the patient [114]. Development of Milasen for targeting Batten disease in an N-of-1 patient is a promising example of RNA-based personalized medicine.

6. Potential Drug Candidates in Clinical Trials

The list of the antisense formulations in clinical trials is expanding with many candidates successfully reaching phase III clinical trials (Table 3). Here, we discuss a few selected investigational drugs that showed promise in phase III clinical trials.

6.1. Tominersen

Huntington disease (HD) is an autosomal dominant, progressive neurodegenerative disorder characterized by dystonia, cognitive dysfunction, and behavioral difficulties [115]. In HD, the CAG trinucleotide repeat expansion results in mutant Huntington (mHTT) protein with extended polyglutamine tract [116]. mHTT accumulates in neurons, thereby affecting their normal function [117]. Tominersen (RG6042) is an ASO developed by Ionis Pharmaceuticals for targeting mHTT mRNA to prevent protein production. The disease-modifying potential of Tominersen was evident in phase I and II clinical trials evaluated in 46 patients with early-stage HD. Patients received a monthly dose of Tominersen (10, 30, 60, 90, 120 mg) or a placebo, administered intrathecally for three months. An average 40% reduction in mHTT proteins was observed in the cerebrospinal fluid at a dose of 90 and 120 mg. The drug candidate showed good tolerability in patients and showed no adverse effects at high doses.

6.2. Tofersen

Superoxide dismutase (SOD1) enzyme plays an essential role in scavenging free radicals generated in the body. Mutation in the SOD1 gene results in the production of dysfunctional SOD1 that accumulates as toxic protein in the cells and results in familial amyotrophic lateral sclerosis (ALS). Tofersen is an ASO designed to target mutated SOD1 mRNA to prevent protein production, thereby slowing ALS progression. A phase I and II double-blind, randomized, placebo-controlled trial showed a reduction in dysfunctional cerebrospinal fluid SOD1 concentrations in the Tofersen treated group [118]. Tofersen treatment also results in an improvement in respiratory and muscle function as compared to placebo control. Tofersen is developed by Biogen and a phase III trial is ongoing to examine the clinical efficacy of Tofersen in SOD1-ALS patients.

6.3. Volanesorsen (Waylivra®)

Familial chylomicronemia syndrome (FCS) is caused by mutations in the lipoprotein lipase (LPL) gene. This condition impairs the usual break down of fats resulting in increased accumulation of fats in the blood. Abdominal pain, eruptive xanthoma, pancreatitis, hepatosplenomegaly, and lipemia retinalis are serious manifestations of FCS. Volanesorsen, developed by Ionis Pharmaceuticals, inhibits hepatic APOC3 mRNA, resulting in reduced plasma triglyceride and apolipoprotein C-III levels. In a 53-week placebo-controlled clinical trial, Volanesorsen was given once weekly subcutaneously to 66 patients with FCS. The total triglyceride levels reduced by 76.5%, and the apolipoprotein C-III levels reduced by 84.2% from baseline at three months in patients treated with Volanesorsen [119]. During the trial, two patients showed a low platelet count of 25,000 platelets per microliter of blood (normal platelet count is 140,000–450,000 platelets per microliter of blood). The platelet count returned to normal levels in the two patients 33 days after discontinuing the drug. Mild to moderate injection site reactions were observed in 20 patients receiving Volanesorsen. Due to significant lipid-lowering potential, Volanesorsen received conditional marketing authorization in Europe to treat patients with genetic FCS and patients at high risk of acute pancreatitis in whom lipid-lowering therapy has not been effective.

6.4. Alicaforsen

Pouchitis is inflammation caused in the lining of a pouch created during complications in surgery or ulcerative colitis. Intercellular adhesion molecule-1 (ICAM-1), is a cell surface receptor that directs the white blood cells in circulation to sites of inflammation and increases the inflammatory response in pouchitis. Alicaforsen is an ASO developed by Atlantic Healthcare that hybridizes with ICAM-1 mRNA and reduces its levels. In clinical trials, treatment with Alicaforsen reduces inflammation and stool frequency, thereby improving the quality of life. Endoscopy showed an improvement in the underlying tissue as well [120].

6.5. Vutrisiran

Vutrisiran is an investigational drug developed by Alnylam Pharmaceuticals for the treatment of hATTR [121]. The mechanism of action of this drug is the same as Patisiran. Vutrisiran is administered by the subcutaneous route (25 mg every 3 months for 2 years) and phase III clinical studies are ongoing to determine its therapeutic effect.

6.6. Fitusiran

Hemophilia is an X-linked bleeding disorder caused due to deficiency of blood clotting factor VIII or factor IX [122]. Fitusiran, an siRNA-based investigational drug developed by Alnylam Pharmaceuticals, acts by inhibiting antithrombotic (AT) mRNA in the liver. In phase II clinical trials, 25 Hemophilia A and B patients were given Fitusiran (50 mg) once a month. Interestingly, 81% knockdown of AT mRNA corresponded to 49–100% reduction in bleeding frequency. The annualized bleeding rate (ABR) of patients improved from 12 to 1.7 after treatment with Fitusiran for 13 months [123].

6.7. Inclisiran

Patients with heterozygous familial hypercholesterolemia (HeFH) have high serum LDL levels, which predisposes them to atherosclerotic cardiovascular disease [124]. Though monoclonal antibodies targeting PCSK9 reduce LDL levels by 50%, these antibodies require weekly administration and only target circulating PCSK9. Inclisiran is a siRNA drug developed by The Medicines Company for targeting PCSK9 mRNA for the treatment of HeFH in patients. In a phase III clinical trial, Inclisiran was administered to 242 patients, compared to 240 patients receiving placebo subcutaneously on days 1, 90, 270, and 450. Patients receiving Inclisiran showed a 39.7% decrease in LDL, while the placebo group showed an increase of 8.2% after 16 months of treatment [125]. The LDL levels were below 100 mg per deciliter in about 65% of the patients treated with Inclisiran. Inclisiran only requires two doses a year, which is an advantage over weekly monoclonal antibody therapy. Following the promising results of Inclisiran in phase III clinical trials, Novartis acquired The Medicines Company and Inclisiran rights in January 2020.

7. ASOs Targeting microRNA

miRNAs are 20–25 nucleotides long ncRNAs that play critical roles in the development and establishment of cell identity, and atypical expression of miRNAs leads to various malignant and non-malignant disorders. Miravirsen was developed by Roche for targeting miR-122 for hepatitis C virus (HCV) infection [165]. Miravirsen contains LNA as well as PS-based chemistry. Severe side effects, however, halted clinical trials of Miravirsen. Similarly, Regulus Therapeutics developed antimiR-21 (RG-012) for Alport syndrome to decrease the rate of progression of renal fibrosis [166]. RG-012 received orphan drug status in the US and Europe. MicroRNAs that promote carcinogenesis and metastasis have also been targeted by the ASOs [167]. One important example is miR-155, which has been shown to be up-regulated in many subtypes of lymphoma, including diffuse large B-cell lymphoma (DLBCL) [168,169]. Cobomarsen (MRG-106) is an LNA-based miR-155 inhibitor developed by miRagen Therapeutics and is currently in phase II trials to treat cutaneous T-cell lymphoma as well as adult T-cell lymphoma and leukemia [170]. The biogenesis and mechanism of action of miRNA are illustrated below (Figure 3).

8. Conclusions

The excitement surrounding antisense oligonucleotide-based drug discovery and development had previously waned due to lack of clinical efficacy even at high ASO doses [171]. Incredible research efforts, however, were made at the interface of chemistry and biology to improve the biophysical and biological attributes of the ASOs [172]. This advanced their in vivo efficacy with minimal off-target effects, leading to several FDA and EMA approvals of ASO-based drugs. The revelation of new molecular targets has also invigorated medical and biological sciences to exploit different classes of ASOs to treat rare and genetic disorders which were previously deemed untreatable by conventional small-molecule-based therapies. In addition, different classes of ASOs are being tested successfully to target the same disease (Patisiran and Inotersen are used to treat hATTR, and Golodirsen and Eteplirsen for treating DMD) to broaden their clinical application based on patient history and clinical outcome.
Overall, ASO-based investigational drugs possess numerous benefits; flexibility in design due to Watson–Crick base recognition, optimized synthesis protocols as well as broad quality control during the development phase. Since ASO-based drugs target the root cause of specific diseases, they have to be administered weekly (Mipomersen) or once every four months (Nusinersen) to establish their efficacy as compared to small molecule-based drugs, which have to be administered daily in most cases. Excitingly, this field has gathered momentum in the last several years as ASO-based drugs received FDA and EMA approval for clinical applications. In addition, the rapid translation of Milasen from proof-of-concept to clinical use within months is an exceptional and enthusiastic example of the development of an ASO-based precision medicine that can improve a patient’s quality of life by reducing disease severity. Overall, ASOs can be employed as personalized medicine (Milasen), as well as for the treatment of a sizeable patient population (Inclisiran).
However, several challenges need to be overcome to broaden the clinical use of ASOs, principal among them being their delivery. ASOs typically accumulate in organs like kidney, liver, and spleen, resulting in toxicity. Since several delivery platforms have been developed, a more concerted effort needs to be employed to explore delivery strategies that can deliver ASOs in maximum effective dose to target organs without off-target accumulation. Optimized delivery platforms will help reduce ASO dose, increase efficiency, and allow tissue-specific targeting that can further minimize their toxicity. In considering the delivery platforms, it is also critical to be mindful of scalability and reproducibility at the industrial scale. Similar caution must be used when designing ASOs given their potential for self-hybridization and off-target binding.
In addition to rare and genetic diseases, several attempts have been made in the past to develop novel antiviral-based ASO drugs [173]. The world is witnessing a devastating pandemic of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus, SARS-CoV-2. Several ASO-based drugs are under evaluation by RNA-based companies like Alnylam Pharmaceuticals to target SARS-CoV-2. Since the viral genome is known, multiple ASOs can be designed and tested to target different viral regions; spike (S) protein, membrane (M) protein, envelop (E) protein, and nucleocapsid (N) for impairing viral function [174]. In addition, ASO therapies are in process to provide symptomatic relief by suppressing cytokines. A few studies are centered on the development of novel ASOs to target toll-like receptor (TLR) and trigger the production of protective interferons to combat the virus [175]. In summary, ASOs have seen tremendous progress in recent years and gained stature in the pharmaceutical market parallel to small molecule-based drugs. Hence, it is not too early to envision that in the future, in conjunction with novel delivery platforms, ASO-based drugs will be used for treating widespread diseases and disorders with maximum efficacy and minimal toxicity.

Author Contributions

Writing, editing and reviewing were done by K.D., C.B., E.Q., H.P., A.G., A.V., and R.B. All authors have read and agreed to the published version of the manuscript.

Funding

This work in R.B. lab was supported by St. Baldrick Foundation Jack’s Pack—We Still Have His Back-a St. Baldrick’s Hero Fund, Charles H. Hood Foundation Child Health Research Award and NIH R01 (1R01CA241194-01A1 and 1R01HL147028-01A1) grant.

Acknowledgments

The figures were created with Biorender.com.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Dá Mesquita, S.; Ferreira, A.C.; Sousa, J.C.; Correia-Neves, M.; Sousa, N.; Marques, F. Insights on the pathophysiology of Alzheimer’s disease: The crosstalk between amyloid pathology, neuroinflammation and the peripheral immune system. Neurosci. Biobehav. Rev. 2016, 68, 547–562. [Google Scholar] [CrossRef] [PubMed][Green Version]
  2. Gibson, R.L.; Burns, J.L.; Ramsey, B.W. Pathophysiology and Management of Pulmonary Infections in Cystic Fibrosis. Am. J. Respir. Crit. Care Med. 2003, 168, 918–951. [Google Scholar] [CrossRef] [PubMed]
  3. Tabrizi, S.J.; Leavitt, B.R.; Landwehrmeyer, G.B.; Wild, E.J.; Saft, C.; Barker, R.A.; Blair, N.F.; Craufurd, D.; Priller, J.; Rickards, H.; et al. Targeting huntingtin expression in patients with Huntington’s disease. N. Engl. J. Med. 2019, 380, 2307–2316. [Google Scholar] [CrossRef] [PubMed]
  4. Kato, G.J.; Steinberg, M.H.; Gladwin, M.T. Intravascular hemolysis and the pathophysiology of sickle cell disease View project Sickle Cell Anemia View project. J. Clin. Investig. 2017, 127, 750–760. [Google Scholar] [CrossRef] [PubMed]
  5. Wightman, B.; Ha, I.; Ruvkun, G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993, 75, 855–862. [Google Scholar] [CrossRef]
  6. Giegé, R. Toward a more complete view of tRNA biology. Nat. Struct. Mol. Biol. 2008, 15, 1007–1014. [Google Scholar] [CrossRef]
  7. Pink, R.C.; Wicks, K.; Caley, D.P.; Punch, E.K.; Jacobs, L.; Carter, D.R.F. Pseudogenes: Pseudo-functional or key regulators in health and diseasě. RNA 2011, 17, 792–798. [Google Scholar] [CrossRef][Green Version]
  8. Girard, A.; Sachidanandam, R.; Hannon, G.J.; Carmell, M.A. A germline-specific class of small RNAs binds mammalian Piwi proteins. Nature 2006, 442, 199–202. [Google Scholar] [CrossRef]
  9. Yao, R.W.; Wang, Y.; Chen, L.L. Cellular functions of long noncoding RNAs. Nat. Cell Biol. 2019, 21, 542–551. [Google Scholar] [CrossRef]
  10. Kristensen, L.S.; Andersen, M.S.; Stagsted, L.V.W.; Ebbesen, K.K.; Hansen, T.B.; Kjems, J. The biogenesis, biology and characterization of circular RNAs. Nat. Rev. Genet. 2019, 20, 675–691. [Google Scholar] [CrossRef]
  11. Kaczmarek, J.C.; Kowalski, P.S.; Anderson, D.G. Advances in the delivery of RNA therapeutics: From concept to clinical reality. Genome Med. 2017, 9, 60. [Google Scholar] [CrossRef] [PubMed][Green Version]
  12. Sharma, V.K.; Sharma, R.K.; Singh, S.K. Antisense oligonucleotides: Modifications and clinical trials. Medchemcomm 2014, 5, 1454–1471. [Google Scholar] [CrossRef]
  13. Bennett, C.F.; Baker, B.F.; Pham, N.; Swayze, E.; Geary, R.S. Pharmacology of Antisense Drugs. Annu. Rev. Pharmacol. Toxicol. 2017, 57, 81–105. [Google Scholar] [CrossRef] [PubMed]
  14. Dowdy, S.F. Overcoming cellular barriers for RNA therapeutics. Nat. Biotechnol. 2017, 35, 222–229. [Google Scholar] [CrossRef]
  15. Furdon, P.J.; Dominski, Z.; Kole, R. RNase H cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds. Nucleic Acids Res. 1989, 17, 9193–9204. [Google Scholar] [CrossRef][Green Version]
  16. Eckstein, F. Phosphorothioate oligodeoxynucleotides: What is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev. 2000, 10, 117–121. [Google Scholar] [CrossRef]
  17. Eckstein, F. Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther. 2014, 24, 374–387. [Google Scholar] [CrossRef]
  18. Crooke, S.T.; Bennett, C.F. Progress in antisense oligonucleotide therapeutics. Annu. Rev. Pharmacol. Toxicol. 1996, 36, 107–129. [Google Scholar] [CrossRef]
  19. Agrawal, S.; Temsamani, J.; Tang, J.Y. Pharmacokinetics, biodistribution, and stability of oligodeoxynucleotide phosphorothioates in mice. Proc. Natl. Acad. Sci. USA 1991, 88, 7595–7599. [Google Scholar] [CrossRef][Green Version]
  20. Agrawal, S.; Teamani, J.; Galbraith, W.; Tang, J. Pharmacokinetics of Antisense Oligonucleotides. Clin. Pharmacokinet. 1995, 28, 7–16. [Google Scholar] [CrossRef]
  21. Chiasson, B.J.; Armstrong, J.N.; Hooper, M.L.; Murphy, P.R.; Robertson, H.A. The application of antisense oligonucleotide technology to the brain: Some pitfalls. Cell. Mol. Neurobiol. 1994, 14, 507–521. [Google Scholar] [CrossRef] [PubMed]
  22. Summerton, J.; Weller, D. Morpholino antisense oligomers: Design, preparation, and properties. Antisense Nucleic Acid Drug Dev. 1997, 7, 187–195. [Google Scholar] [CrossRef] [PubMed][Green Version]
  23. Hudziak, R.M.; Barofsky, E.; Barofsky, D.F.; Weller, D.L.; Huang, S.; Weller, D.D. Resistance of Morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acid Drug Dev. 1996, 6, 267–272. [Google Scholar] [CrossRef] [PubMed]
  24. Devi, G.R.; Beer, T.M.; Corless, C.L.; Arora, V.; Weller, D.L.; Iversen, P.L. In vivo bioavailability and pharmacokinetics of a c-MYC antisense phosphorodiamidate morpholino oligomer, AVI-4126, in solid tumors. Clin. Cancer Res. 2005, 11, 3930–3938. [Google Scholar] [CrossRef][Green Version]
  25. Pellestor, F.; Paulasova, P. The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics. Eur. J. Hum. Genet. 2004, 12, 694–700. [Google Scholar] [CrossRef] [PubMed]
  26. Lundin, P.; Johansson, H.; Guterstam, P.; Holm, T.; Hansen, M.; Langel, Ü.; Andaloussi, S.E.L. Distinct uptake routes of cell-penetrating peptide conjugates. Bioconjug. Chem. 2008, 19, 2535–2542. [Google Scholar] [CrossRef] [PubMed]
  27. Dragulescu-Andrasi, A.; Rapireddy, S.; Frezza, B.M.; Gayathri, C.; Gil, R.R.; Ly, D.H. A simple γ-backbone modification preorganizes peptide nucleic acid into a helical structure. J. Am. Chem. Soc. 2006, 128, 10258–10267. [Google Scholar] [CrossRef]
  28. Zhou, P.; Wang, M.; Du, L.; Fisher, G.W.; Waggoner, A.; Ly, D.H. Novel binding and efficient cellular uptake of guanidine-based peptide nucleic acids (GPNA). J. Am. Chem. Soc. 2003, 125, 6878–6879. [Google Scholar] [CrossRef]
  29. Swenson, C.S.; Heemstra, J.M. Peptide nucleic acids harness dual information codes in a single molecule. Chem. Commun. 2020, 56, 1926–1935. [Google Scholar] [CrossRef]
  30. Braasch, D.A.; Corey, D.R. Locked nucleic acid (LNA): Fine-tuning the recognition of DNA and RNA. Chem. Biol. 2001, 8, 1–7. [Google Scholar] [CrossRef][Green Version]
  31. Marrosu, E.; Ala, P.; Muntoni, F.; Zhou, H. Gapmer Antisense Oligonucleotides Suppress the Mutant Allele of COL6A3 and Restore Functional Protein in Ullrich Muscular Dystrophy. Mol. Ther. Nucleic Acids 2017, 8, 416–427. [Google Scholar] [CrossRef] [PubMed][Green Version]
  32. Hagedorn, P.H.; Persson, R.; Funder, E.D.; Albæk, N.; Diemer, S.L.; Hansen, D.J.; Møller, M.R.; Papargyri, N.; Christiansen, H.; Hansen, B.R.; et al. Locked nucleic acid: Modality, diversity, and drug discovery. Drug Discov. Today 2018, 23, 101–114. [Google Scholar] [CrossRef] [PubMed][Green Version]
  33. Kurreck, J. Design of antisense oligonucleotides stabilized by locked nucleic acids. Nucleic Acids Res. 2002, 30, 1911–1918. [Google Scholar] [CrossRef] [PubMed][Green Version]
  34. Juliano, R.L. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016, 44, 6518–6548. [Google Scholar] [CrossRef] [PubMed]
  35. Freier, S. The ups and downs of nucleic acid duplex stability: Structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 1997, 25, 4429–4443. [Google Scholar] [CrossRef] [PubMed][Green Version]
  36. Hebb, M.O.; Robertson, H.A. End-capped antisense oligodeoxynucleotides effectively inhibit gene expression in vivo and offer a low-toxicity alternative to fully modified phosphorothioate oligodeoxynucleotides. Mol. Brain Res. 1997, 47, 223–228. [Google Scholar] [CrossRef]
  37. Shen, W.; De Hoyos, C.L.; Sun, H.; Vickers, T.A.; Liang, X.H.; Crooke, S.T. Acute hepatotoxicity of 2 fluoro-modified 5–10–5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins. Nucleic Acids Res. 2018, 46, 2204–2217. [Google Scholar] [CrossRef][Green Version]
  38. Krieg, A.M. Antiinfective applications of toll-like receptor 9 agonists. Proc. Am. Thorac. Soc. 2007, 4, 289–294. [Google Scholar] [CrossRef]
  39. Ortega, J.A.; Blas, J.R.; Orozco, M.; Grandas, A.; Pedroso, E.; Robles, J. Binding affinities of oligonucleotides and PNAs containing phenoxazine and G-clamp cytosine analogues are unusually sequence-dependent. Org. Lett. 2007, 9, 4503–4506. [Google Scholar] [CrossRef]
  40. Chenna, V.; Rapireddy, S.; Sahu, B.; Ausin, C.; Pedroso, E.; Ly, D.H. A simple cytosine to G-clamp nucleobase substitution enables chiral γ-PNAs to invade mixed-sequence double-helical B-form DNA. ChemBioChem 2008, 9, 2388–2391. [Google Scholar] [CrossRef]
  41. Rapireddy, S.; Bahal, R.; Ly, D.H. Strand invasion of mixed-sequence, double-helical B-DNA by γ-peptide nucleic acids containing g-clamp nucleobases under physiological conditions. Biochemistry 2011, 50, 3913–3918. [Google Scholar] [CrossRef] [PubMed][Green Version]
  42. Stephenson, M.L.; Zamecnik, P.C. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA 1978, 75, 285–288. [Google Scholar] [CrossRef] [PubMed][Green Version]
  43. De Mesmaeker, A.; HÄner, R.; Martin, P.; Moser, H.E. Antisense Oligonucleotides. Acc. Chem. Res. 1995, 28, 366–374. [Google Scholar] [CrossRef]
  44. Ward, A.J.; Norrbom, M.; Chun, S.; Bennett, C.F.; Rigo, F. Nonsense-mediated decay as a terminating mechanism for antisense oligonucleotides. Nucleic Acids Res. 2014, 42, 5871–5879. [Google Scholar] [CrossRef] [PubMed][Green Version]
  45. Rinaldi, C.; Wood, M.J.A. Antisense oligonucleotides: The next frontier for treatment of neurological disorders. Nat. Rev. Neurol. 2018, 14, 9–21. [Google Scholar] [CrossRef] [PubMed]
  46. Wu, H.; Lima, W.F.; Zhang, H.; Fan, A.; Sun, H.; Crooke, S.T. Determination of the Role of the Human RNase H1 in the Pharmacology of DNA-like Antisense Drugs. J. Biol. Chem. 2004, 279, 17181–17189. [Google Scholar] [CrossRef][Green Version]
  47. Yin, W.; Rogge, M. Targeting RNA: A Transformative Therapeutic Strategy. Clin. Transl. Sci. 2019, 12, 98–112. [Google Scholar] [CrossRef][Green Version]
  48. Crooke, S.T. Molecular mechanisms of action of antisense drugs. Biochim. Biophys. Acta Gene Struct. Exp. 1999, 1489, 31–44. [Google Scholar] [CrossRef]
  49. Zamore, P.D.; Tuschl, T.; Sharp, P.A.; Bartel, D.P. RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000, 101, 25–33. [Google Scholar] [CrossRef][Green Version]
  50. Matranga, C.; Tomari, Y.; Shin, C.; Bartel, D.P.; Zamore, P.D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 2005, 123, 607–620. [Google Scholar] [CrossRef][Green Version]
  51. Rand, T.A.; Petersen, S.; Du, F.; Wang, X. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 2005, 123, 621–629. [Google Scholar] [CrossRef] [PubMed][Green Version]
  52. Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D.W.Y. RNAi therapeutics: A potential new class of pharmaceutical drugs. Nat. Chem. Biol. 2006, 2, 711–719. [Google Scholar] [CrossRef]
  53. Dias, N.; Dheur, S.; Nielsen, P.E.; Gryaznov, S.; Van Aerschot, A.; Herdewijn, P.; Hélène, C.; Saison-Behmoaras, T.E. Antisense PNA tridecamers targeted to the coding region of Ha-ras mRNA arrest polypeptide chain elongation. J. Mol. Biol. 1999, 294, 403–416. [Google Scholar] [CrossRef]
  54. Bennett, C.F.; Swayze, E.E. RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic Platform. Annu. Rev. Pharmacol. Toxicol. 2010, 50, 259–293. [Google Scholar] [CrossRef] [PubMed]
  55. Catalanotto, C.; Cogoni, C.; Zardo, G. MicroRNA in control of gene expression: An overview of nuclear functions. Int. J. Mol. Sci. 2016, 17, 1712. [Google Scholar] [CrossRef] [PubMed][Green Version]
  56. Crooke, S.T.; Witztum, J.L.; Bennett, C.F.; Baker, B.F. RNA-Targeted Therapeutics. Cell Metab. 2018, 27, 714–739. [Google Scholar] [CrossRef]
  57. Qi, L.L.; Rabinowitz, A.; Yun, C.C.; Yokota, T.; Yin, H.F.; Alter, J.; Jadoon, A.; Bou-Gharios, G.; Partridge, T. Systemic delivery of antisense oligoribonucleotide restorers dystrophin expression in body-wide skeletal muscles. Proc. Natl. Acad. Sci. USA 2005, 102, 198–203. [Google Scholar] [CrossRef][Green Version]
  58. Svasti, S.; Suwanmanee, T.; Fucharoen, S.; Moulton, H.M.; Nelson, M.H.; Maeda, N.; Smithies, O.; Kole, R. RNA repair restores hemoglobin expression in IVS2-654 thalassemic mice. Proc. Natl. Acad. Sci. USA 2009, 106, 1205–1210. [Google Scholar] [CrossRef][Green Version]
  59. Passini, M.A.; Bu, J.; Richards, A.M.; Kinnecom, C.; Sardi, S.P.; Stanek, L.M.; Hua, Y.; Rigo, F.; Matson, J.; Hung, G.; et al. Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy. Sci. Transl. Med. 2011, 3. [Google Scholar] [CrossRef][Green Version]
  60. Siva, K.; Covello, G.; Denti, M.A. Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases. Nucleic Acid Ther. 2014, 24, 69–86. [Google Scholar] [CrossRef][Green Version]
  61. van der Wal, E.; Bergsma, A.J.; Pijnenburg, J.M.; van der Ploeg, A.T.; Pijnappel, W.W.M.P. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease. Mol. Ther. Nucleic Acids 2017, 7, 90–100. [Google Scholar] [CrossRef] [PubMed][Green Version]
  62. Pagani, F.; Baralle, F.E. Genomic variants in exons and introns: Identifying the splicing spoilers. Nat. Rev. Genet. 2004, 5, 389–396. [Google Scholar] [CrossRef] [PubMed]
  63. Venables, J.P. Aberrant and alternative splicing in cancer. Cancer Res. 2004, 64, 7647–7654. [Google Scholar] [CrossRef] [PubMed][Green Version]
  64. Havens, M.A.; Duelli, D.M.; Hastings, M.L. Targeting RNA splicing for disease therapy. Wiley Interdiscip. Rev. RNA 2013, 4, 247–266. [Google Scholar] [CrossRef]
  65. Bauman, J.; Jearawiriyapaisarn, N.; Kole, R. Therapeutic potential of splice-switching oligonucleotides. Oligonucleotides 2009, 19, 1–13. [Google Scholar] [CrossRef]
  66. Dominski, Z.; Kole, R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 1993, 18, 8673–8677. [Google Scholar] [CrossRef][Green Version]
  67. Clark, A.J.; Davis, M.E. Increased brain uptake of targeted nanoparticles by adding an acid-cleavable linkage between transferrin and the nanoparticle core. Proc. Natl. Acad. Sci. USA 2015, 112, 12486–12491. [Google Scholar] [CrossRef][Green Version]
  68. Lorenzer, C.; Dirin, M.; Winkler, A.M.; Baumann, V.; Winkler, J. Going beyond the liver: Progress and challenges of targeted delivery of siRNA therapeutics. J. Control. Release 2015, 203, 1–15. [Google Scholar] [CrossRef][Green Version]
  69. Suk, J.S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L.M. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv. Drug Deliv. Rev. 2016, 99, 28–51. [Google Scholar] [CrossRef][Green Version]
  70. Huang, Y. Preclinical and Clinical Advances of GalNAc-Decorated Nucleic Acid Therapeutics. Mol. Ther. Nucleic Acids 2017, 17, 116–132. [Google Scholar] [CrossRef][Green Version]
  71. Shi, B.; Abrams, M.; Sepp-Lorenzino, L. Expression of Asialoglycoprotein Receptor 1 in Human Hepatocellular Carcinoma. J. Histochem. Cytochem. 2013, 61, 901–909. [Google Scholar] [CrossRef] [PubMed][Green Version]
  72. Zhu, X.; Xu, Y.; Solis, L.M.; Tao, W.; Wang, L.; Behrens, C.; Xu, X.; Zhao, L.; Liu, D.; Wu, J.; et al. Long-circulating siRNA nanoparticles for validating Prohibitin1-targeted non-small cell lung cancer treatment. Proc. Natl. Acad. Sci. USA 2015, 112, 7779–7784. [Google Scholar] [CrossRef] [PubMed][Green Version]
  73. Schiffelers, R.M.; Ansari, A.; Xu, J.; Zhou, Q.; Tang, Q.; Storm, G.; Molema, G.; Lu, P.Y.; Scaria, P.V.; Woodle, M.C. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res. 2004, 32, e149. [Google Scholar] [CrossRef] [PubMed]
  74. Benjaminsen, R.V.; Mattebjerg, M.A.; Henriksen, J.R.; Moghimi, S.M.; Andresen, T.L. The possible “proton sponge “ effect of polyethylenimine (PEI) does not include change in lysosomal pH. Mol. Ther. 2013, 21, 149–157. [Google Scholar] [CrossRef] [PubMed][Green Version]
  75. Danhier, F.; Ansorena, E.; Silva, J.M.; Coco, R.; Le Breton, A.; Préat, V. PLGA-based nanoparticles: An overview of biomedical applications. J. Control. Release 2012, 161, 505–522. [Google Scholar] [CrossRef]
  76. Cai, C.; Xie, Y.; Wu, L.; Chen, X.; Liu, H.; Zhou, Y.; Zou, H.; Liu, D.; Zhao, Y.; Kong, X.; et al. PLGA-based dual targeted nanoparticles enhance miRNA transfection efficiency in hepatic carcinoma. Sci. Rep. 2017, 7, 46250. [Google Scholar] [CrossRef]
  77. Rehman, Z.U.; Hoekstra, D.; Zuhorn, I.S. Mechanism of polyplex- and lipoplex-mediated delivery of nucleic acids: Real-time visualization of transient membrane destabilization without endosomal lysis. ACS Nano 2013, 28, 3767–3777. [Google Scholar] [CrossRef]
  78. Mahmoodi Chalbatani, G.; Dana, H.; Gharagouzloo, E.; Grijalvo, S.; Eritja, R.; Logsdon, C.D.; Memari, F.; Miri, S.R.; Rad, M.R.; Marmari, V. Small interfering RNAs (siRNAs) in cancer therapy: A nano-based approach. Int. J. Nanomed. 2019, 2019, 3111–3128. [Google Scholar] [CrossRef][Green Version]
  79. Kumar, V.; Qin, J.; Jiang, Y.; Duncan, R.G.; Brigham, B.; Fishman, S.; Nair, J.K.; Akinc, A.; Barros, S.A.; Kasperkovitz, P.V. Shielding of lipid nanoparticles for siRNA delivery: Impact on physicochemical properties, cytokine induction, and efficacy. Mol. Ther. Nucleic Acids 2014, 3, e210. [Google Scholar] [CrossRef]
  80. Azad, R.F.; Driver, V.B.; Tanaka, K.; Crooke, R.M.; Anderson, K.P. Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region. Antimicrob. Agents Chemother. 1993, 37, 1945–1954. [Google Scholar] [CrossRef][Green Version]
  81. Geary, R.S.; Henry, S.P.; Grillone, L.R. Fomivirsen: Clinical pharmacology and potential drug interactions. Clin. Pharmacokinet. 2002, 41, 255–260. [Google Scholar] [CrossRef] [PubMed]
  82. Hutcherson, S.L.; Lanz, R. A randomized controlled clinical trial of intravitreous fomivirsen for treatment of newly diagnosed peripheral cytomegalovirus retinitis in patients with aids. Am. J. Ophthalmol. 2002, 133, 467–474. [Google Scholar] [CrossRef]
  83. Goldberg, A.C.; Hopkins, P.N.; Toth, P.P.; Ballantyne, C.M.; Rader, D.J.; Robinson, J.G.; Daniels, S.R.; Gidding, S.S.; De Ferranti, S.D.; Ito, M.K.; et al. Familial hypercholesterolemia: Screening, diagnosis and management of pediatric and adult patients: Clinical guidance from the National Lipid Association Expert Panel on Familial Hypercholesterolemia. J. Clin. Lipidol. 2011, 5, S1–S8. [Google Scholar] [CrossRef]
  84. Raal, F.J.; Santos, R.D.; Blom, D.J.; Marais, A.D.; Charng, M.J.; Cromwell, W.C.; Lachmann, R.H.; Gaudet, D.; Tan, J.L.; Chasan-Taber, S.; et al. Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: A randomised, double-blind, placebo-controlled trial. Lancet 2010, 375, 998–1006. [Google Scholar] [CrossRef]
  85. Hair, P.; Cameron, F.; McKeage, K. Mipomersen sodium: First global approval. Drugs 2013, 73, 487–493. [Google Scholar] [CrossRef] [PubMed]
  86. Lefebvre, S.; Bürglen, L.; Reboullet, S.; Clermont, O.; Burlet, P.; Viollet, L.; Benichou, B.; Cruaud, C.; Millasseau, P.; Zeviani, M.; et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell 1995, 80, 155–165. [Google Scholar] [CrossRef][Green Version]
  87. Kolb, S.J.; Kissel, J.T. Spinal muscular atrophy: A timely review. Arch. Neurol. 2011, 68, 979–984. [Google Scholar] [CrossRef][Green Version]
  88. Hua, Y.; Liu, Y.H.; Sahashi, K.; Rigo, F.; Frank Bennett, C.; Krainer, A.R. Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models. Genes Dev. 2015, 29, 288–297. [Google Scholar] [CrossRef][Green Version]
  89. Rigo, F.; Chun, S.J.; Norris, D.A.; Hung, G.; Lee, S.; Matson, J.; Fey, R.A.; Gaus, H.; Hua, Y.; Grundy, J.S.; et al. Pharmacology of a central nervous system delivered 2′-O-methoxyethyl- modified survival of motor neuron splicing oligonucleotide in mice and nonhuman primates. J. Pharmacol. Exp. Ther. 2014, 350, 46–55. [Google Scholar] [CrossRef][Green Version]
  90. Mercuri, E.; Darras, B.T.; Chiriboga, C.A.; Day, J.W.; Campbell, C.; Connolly, A.M.; Iannaccone, S.T.; Kirschner, J.; Kuntz, N.L.; Saito, K.; et al. Nusinersen versus sham control in later-onset spinal muscular atrophy. N. Engl. J. Med. 2018, 378, 625–635. [Google Scholar] [CrossRef]
  91. Finkel, R.S.; Mercuri, E.; Darras, B.T.; Connolly, A.M.; Kuntz, N.L.; Kirschner, J.; Chiriboga, C.A.; Saito, K.; Servais, L.; Tizzano, E.; et al. Nusinersen versus sham control in infantile-onset spinal muscular atrophy. N. Engl. J. Med. 2017, 377, 1723–1732. [Google Scholar] [CrossRef] [PubMed][Green Version]
  92. Wurster, C.D.; Winter, B.; Wollinsky, K.; Ludolph, A.C.; Uzelac, Z.; Witzel, S.; Schocke, M.; Schneider, R.; Kocak, T. Intrathecal administration of nusinersen in adolescent and adult SMA type 2 and 3 patients. J. Neurol. 2019, 266, 183–194. [Google Scholar] [CrossRef] [PubMed]
  93. ClinicalTrials.gov. Effect of Nusinersen on Adults with Spinal Muscular Atrophy. Available online: https://clinicaltrials.gov/ct2/show/NCT03878030 (accessed on 19 March 2020).
  94. Sekijima, Y. Transthyretin (ATTR) amyloidosis: Clinical spectrum, molecular pathogenesis and disease-modifying treatments. J. Neurol. Neurosurg. Psychiatry 2015, 86, 1036–1043. [Google Scholar] [CrossRef] [PubMed]
  95. Richardson, S.J. Cell and Molecular Biology of Transthyretin and Thyroid Hormones. Int. Rev. Cytol. 2007, 258, 137–193. [Google Scholar] [CrossRef] [PubMed]
  96. Adams, D.; Koike, H.; Slama, M.; Coelho, T. Hereditary transthyretin amyloidosis: A model of medical progress for a fatal disease. Nat. Rev. Neurol. 2019, 15, 387–404. [Google Scholar] [CrossRef] [PubMed]
  97. Kulkarni, J.A.; Cullis, P.R.; Van Der Meel, R. Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility. Nucleic Acid Ther. 2018, 28, 146–157. [Google Scholar] [CrossRef] [PubMed][Green Version]
  98. Akinc, A.; Maier, M.A.; Manoharan, M.; Fitzgerald, K.; Jayaraman, M.; Barros, S.; Ansell, S.; Du, X.; Hope, M.J.; Madden, T.D.; et al. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 2019, 14, 1084–1087. [Google Scholar] [CrossRef]
  99. Adams, D.; Gonzalez-Duarte, A.; O’Riordan, W.D.; Yang, C.C.; Ueda, M.; Kristen, A.V.; Tournev, I.; Schmidt, H.H.; Coelho, T.; Berk, J.L.; et al. Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N. Engl. J. Med. 2018, 379, 11–21. [Google Scholar] [CrossRef]
  100. ClinicalTrials.gov. Patisiran in Patients with Hereditary Transthyretin-Mediated Amyloidosis (hATTR Amyloidosis) Disease Progression Post-Liver Transplant. Available online: https://clinicaltrials.gov/ct2/show/NCT03862807 (accessed on 19 March 2020).
  101. ClinicalTrials.gov. The Study of an Investigational Drug, Patisiran (ALN-TTR02), for the Treatment of Transthyretin (TTR)-Mediated Amyloidosis in Patients Who Have Already been Treated with ALN-TTR02 (Patisiran). Available online: https://clinicaltrials.gov/ct2/show/NCT02510261 (accessed on 19 March 2020).
  102. ClinicalTrials.gov. HELIOS-A: A Study of Vutrisiran (ALN-TTRSC02) in Patients with Hereditary Transthyretin Amyloidosis (hATTR Amyloidosis). Available online: https://clinicaltrials.gov/ct2/show/NCT03759379 (accessed on 19 March 2020).
  103. Benson, M.D.; Waddington-Cruz, M.; Berk, J.L.; Polydefkis, M.; Dyck, P.J.; Wang, A.K.; Planté-Bordeneuve, V.; Barroso, F.A.; Merlini, G.; Obici, L.; et al. Inotersen treatment for patients with Hereditary transthyretin amyloidosis. N. Engl. J. Med. 2018, 379, 22–31. [Google Scholar] [CrossRef]
  104. Bushby, K.; Finkel, R.; Birnkrant, D.J.; Case, L.E.; Clemens, P.R.; Cripe, L.; Kaul, A.; Kinnett, K.; McDonald, C.; Pandya, S.; et al. Diagnosis and management of Duchenne muscular dystrophy, part 1: Diagnosis, and pharmacological and psychosocial management. Lancet Neurol. 2010, 9, 77–93. [Google Scholar] [CrossRef]
  105. McClorey, G.; Moulton, H.M.; Iversen, P.L.; Fletcher, S.; Wilton, S.D. Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD. Gene Ther. 2006, 13, 1373–1381. [Google Scholar] [CrossRef] [PubMed]
  106. Lim, K.R.Q.; Maruyama, R.; Yokota, T. Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug Des. Devel. Ther. 2017, 11, 533–545. [Google Scholar] [CrossRef] [PubMed][Green Version]
  107. Kinali, M.; Arechavala-Gomeza, V.; Feng, L.; Cirak, S.; Hunt, D.; Adkin, C.; Guglieri, M.; Ashton, E.; Abbs, S.; Nihoyannopoulos, P.; et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: A single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol. 2009, 8, 918–928. [Google Scholar] [CrossRef][Green Version]
  108. Aartsma-Rus, A.; Krieg, A.M. FDA Approves Eteplirsen for Duchenne Muscular Dystrophy: The Next Chapter in the Eteplirsen Saga. Nucleic Acid Ther. 2017, 27, 1–3. [Google Scholar] [CrossRef] [PubMed][Green Version]
  109. ClinicalTrials.gov. Study of Eteplirsen in Young Patients with DMD Amenable to Exon 51 Kkipping. Available online: https://clinicaltrials.gov/ct2/show/NCT03218995 (accessed on 19 March 2020).
  110. Frank, D.E.; Schnell, F.J.; Akana, C.; El-Husayni, S.H.; Desjardins, C.A.; Morgan, J.; Charleston, J.S.; Sardone, V.; Domingos, J.; Dickson, G.; et al. Increased dystrophin production with golodirsen in patients with Duchenne muscular dystrophy. Neurology 2020, 94, e2270–e2282. [Google Scholar] [CrossRef] [PubMed][Green Version]
  111. Bissell, D.M.; Anderson, K.E.; Bonkovsky, H.L. Porphyria. N. Engl. J. Med. 2017, 377, 862–872. [Google Scholar] [CrossRef] [PubMed]
  112. Scott, L.J. Givosiran: First Approval. Drugs 2020, 80, 335–339. [Google Scholar] [CrossRef]
  113. ClinicalTrials.gov. A Study to Evaluate Long-Term Safety and Clinical Activity of Givosiran (ALN-AS1) in Patient with Acute Intermittent Porphyria (AIP). Available online: https://clinicaltrials.gov/ct2/show/NCT02949830 (accessed on 19 March 2020).
  114. Kim, J.; Hu, C.; El Achkar, C.M.; Black, L.E.; Douville, J.; Larson, A.; Pendergast, M.K.; Goldkind, S.F.; Lee, E.A.; Kuniholm, A.; et al. Patient-customized oligonucleotide therapy for a rare genetic disease. N. Engl. J. Med. 2019, 381, 1644–1652. [Google Scholar] [CrossRef]
  115. Walker, F.O. Huntington’s disease. Lancet 2007, 369, 218–228. [Google Scholar] [CrossRef]
  116. Finkbeiner, S. Huntington’s disease. Cold Spring Harb. Perspect. Biol. 2011, 3, a007476. [Google Scholar] [CrossRef][Green Version]
  117. MacDonald, M.E.; Ambrose, C.M.; Duyao, M.P.; Myers, R.H.; Lin, C.; Srinidhi, L.; Barnes, G.; Taylor, S.A.; James, M.; Groot, N.; et al. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes. Cell 1993, 72, 971–983. [Google Scholar] [CrossRef]
  118. Biogen. Biogen to Present New Interim Data from its Phase 1/2 Clinical Stidy of Tofersen (BIIB067) for the Potential Treatment of a Subtype of Familial Amyotrophic Lateral Sclerosis (ALS). Available online: https://investors.biogen.com/news-releases/news-release-details/biogen-present-new-interim-data-its-phase-12-clinical-study (accessed on 11 May 2020).
  119. Witztum, J.L.; Gaudet, D.; Freedman, S.D.; Alexander, V.J.; Digenio, A.; Williams, K.R.; Yang, Q.; Hughes, S.G.; Geary, R.S.; Arca, M.; et al. Volanesorsen and triglyceride levels in familial chylomicronemia syndrome. N. Engl. J. Med. 2019, 381, 531–542. [Google Scholar] [CrossRef] [PubMed]
  120. Van Deventer, S.J.H.; Wedel, M.K.; Baker, B.F.; Xia, S.; Chuang, E.; Miner, P.B. A Phase II dose ranging, double-blind, placebo-controlled study of alicaforsen enema in subjects with acute exacerbation of mild to moderate left-sided ulcerative colitis. Aliment. Pharmacol. Ther. 2006, 23, 1415–1425. [Google Scholar] [CrossRef] [PubMed][Green Version]
  121. Shilling, R.; Karsten, V.; Silliman, N.; Chen, J.; Li, W.; Vest, J. Study design and rationale of HELIOS-B: A Phase 3 study to evaluate the clinical efficacy and safety of Vutrisiran in patients with ATTR amyloidosis with cardiomyopathy. J. Am. Coll. Cardiol. 2020, 75, 3579. [Google Scholar] [CrossRef]
  122. Mannucci, P.M.; Tuddenham, E.G.D. The hemophilias—From royal genes to gene therapy. N. Engl. J. Med. 2001, 344, 1773–1779. [Google Scholar] [CrossRef] [PubMed]
  123. Machin, N.; Ragni, M.V. An investigational RNAi therapeutic targeting antithrombin for the treatment of hemophilia A and B. J. Blood Med. 2018, 9, 135–140. [Google Scholar] [CrossRef][Green Version]
  124. Bruikman, C.S.; Hovingh, G.K.; Kastelein, J.J.P. Molecular basis of familial hypercholesterolemia. Curr. Opin. Cardiol. 2017, 32, 262–266. [Google Scholar] [CrossRef]
  125. Raal, F.J.; Kallend, D.; Ray, K.K.; Turner, T.; Koenig, W.; Wright, R.S.; Wijngaard, P.L.J.; Curcio, D.; Jaros, M.J.; Leiter, L.A.; et al. Inclisiran for the treatment of heterozygous familial hypercholesterolemia. N. Engl. J. Med. 2020, 382, 1520–1530. [Google Scholar] [CrossRef]
  126. ClinicalTrials.gov. ARRx in Combination with Enzalutamide in Metastatic Castration Resistant Prostate Cancer. Available online: https://clinicaltrials.gov/ct2/show/NCT03300505 (accessed on 19 March 2020).
  127. ClinicalTrials.gov. A Study of RG-012 in Subjects With Alport Syndrome. Available online: https://clinicaltrials.gov/ct2/show/NCT03373786 (accessed on 19 March 2020).
  128. ClinicalTrials.gov. Exploratory Study to Evaluate QR-010 in Subjects with Cystic Fibrosis ΔF508 CFTR Mutation. Available online: https://clinicaltrials.gov/ct2/show/NCT02564354 (accessed on 19 March 2020).
  129. ClinicalTrials.gov. Phase I dose Escalation Study to Investigate the Safety of ISTH0036 in Subjects sith Glaucoma Undergoing Trabeculectomy. Available online: https://clinicaltrials.gov/ct2/show/NCT02406833 (accessed on 19 March 2020).
  130. ClinicalTrials.gov. Study of ARO-APOC3 in Healthy Volunteers, Hypertriglyceridemic Patients and Patients with Familial Chylomicronemia Syndrome (FCS). Available online: https://clinicaltrials.gov/ct2/show/NCT03783377 (accessed on 19 March 2020).
  131. ClinicalTrials.gov. Study of ARO-ANG3 in Healthy Volunteers and in Dyslipidemic Patients. Available online: https://clinicaltrials.gov/ct2/show/NCT03747224 (accessed on 19 March 2020).
  132. ClinicalTrials.gov. Safety Study of a Single IVT Injection of QPI-1007 in Chronic Optic Nerve Atrophy and Recent Onset NAION Patients. Available online: https://clinicaltrials.gov/ct2/show/NCT01064505 (accessed on 19 March 2020).
  133. ClinicalTrials.gov. A Study of ALN-AAT02 in Healthy Participants and Participants with ZZ Type alpha-1 Antitrypsin Deficiency Liver Disease. Available online: https://clinicaltrials.gov/ct2/show/NCT03767829 (accessed on 19 March 2020).
  134. ClinicalTrials.gov. A Study of MEDI1191 in Sequential and Concurrent Combination with Durvalumab in Subjects with Advanced Solid Tumors. Available online: https://clinicaltrials.gov/ct2/show/NCT03946800 (accessed on 19 March 2020).
  135. ClinicalTrials.gov. A Safety, Tolerability, PK, and PD Study of Once Weekly ISIS-FGFR4RX SC in Obese Patients (FGFR4-CS2). Available online: https://clinicaltrials.gov/ct2/show/NCT02476019 (accessed on 19 March 2020).
  136. ClinicalTrials.gov. Safety, Tolerability, and Pharmacodynamics of IONIS-DGAT2Rx in Adult Patients with Type 2 Diabetes. Available online: https://clinicaltrials.gov/ct2/show/NCT03334214 (accessed on 19 March 2020).
  137. ClinicalTrials.gov. An Extension Study of IONIS-PKK-LRx in Participants with Hereditary Angioedema. Available online: https://clinicaltrials.gov/ct2/show/NCT04307381 (accessed on 19 March 2020).
  138. ClinicalTrials.gov. Safety, Tolerability and Efficacy of ISIS-GCGRRx in Patients with Type 2 Diabetes. Available online: https://clinicaltrials.gov/ct2/show/NCT02583919 (accessed on 19 March 2020).
  139. ClinicalTrials.gov. Evaluation of Safety and Feasibility of OGX-011 in Combination with 2nd-line Chemotherapy in Patients with HRPC. Available online: https://clinicaltrials.gov/ct2/show/NCT00327340 (accessed on 19 March 2020).
  140. ClinicalTrials.gov. A Phase 2 Study Comparing Chemotherapy in Combination with OGX-427 or Placebo in Patients with Bladder Cancer. Available online: https://clinicaltrials.gov/ct2/show/NCT01454089 (accessed on 19 March 2020).
  141. ClinicalTrials.gov. Phase 2 Study of ISIS 681257 (AKCEA-APO(a)-LRx) in Patients with Hyperlipoproteinemia(a) and Cardiovascular Disease. Available online: https://clinicaltrials.gov/ct2/show/NCT03070782 (accessed on 19 March 2020).
  142. ClinicalTrials.gov. Study of AKCEA-ANGPTL3-LRX (ISIS 703802) in Patients with Familial Partial Lipodystrophy (FPL). Available online: https://clinicaltrials.gov/ct2/show/NCT03514420 (accessed on 19 March 2020).
  143. ClinicalTrials.gov. Study of ISIS 678354 (AKCEA-APOCIII-LRx) in Patients with Hypertriglyceridemia and Established Ardiovascular Disease (CVD). Available online: https://clinicaltrials.gov/ct2/show/NCT03385239 (accessed on 19 March 2020).
  144. ClinicalTrials.gov. Danvatirsen and Durvalumab in Treating Patients with Advanced and Refractory Pancreatic, Non-Small Cell Lung Cancer, and Mismatch Repair Deficient Colorectal Cancer. Available online: https://clinicaltrials.gov/ct2/show/NCT02983578 (accessed on 19 March 2020).
  145. ClinicalTrials.gov. SOLAR: Efficacy and Safety of Cobomarsen (MRG-106) vs. Active Comparator in Subjects with Mycosis Fungoides (SOLAR). Available online: https://clinicaltrials.gov/ct2/show/NCT03713320 (accessed on 19 March 2020).
  146. ClinicalTrials.gov. Efficacy, Safety, and Tolerability of Remlarsen (MRG-201) Following Intradermal Injection in Subjects with a History of Keloids. Available online: https://clinicaltrials.gov/ct2/show/NCT03601052 (accessed on 19 March 2020).
  147. ClinicalTrials.gov. A Study of Cemdisiran in Adults with Immunoglobulin A Nephropathy (IgAN). Available online: https://clinicaltrials.gov/ct2/show/NCT03841448 (accessed on 19 March 2020).
  148. ClinicalTrials.gov. Assessment of Changes in a Novel Histological Activity Scale in Response to ARO-AAT. Available online: https://clinicaltrials.gov/ct2/show/NCT03946449 (accessed on 19 March 2020).
  149. ClinicalTrials.gov. PF-04523655 dose Escalation Study, and Evaluation of PF-04523655 with/without Ranibizumab in Diabetic Macular Edema (DME) (MATISSE). Available online: https://clinicaltrials.gov/ct2/show/NCT01445899 (accessed on 19 March 2020).
  150. ClinicalTrials.gov. AZD8601 Study in CABG Patients. Available online: https://clinicaltrials.gov/ct2/show/NCT03370887 (accessed on 19 March 2020).
  151. ClinicalTrials.gov. Study of DS-5141b in Patients with Duchenne Muscular Dystrophy. Available online: https://clinicaltrials.gov/ct2/show/NCT02667483 (accessed on 19 March 2020).
  152. ClinicalTrials.gov. Safety, Efficacy, PK, and PD Characteristics of Orally Inhaled SB010 in Male Patients with Mild Asthma (Multiple Dose). Available online: https://clinicaltrials.gov/ct2/show/NCT01743768 (accessed on 19 March 2020).
  153. ClinicalTrials.gov. Miravirsen Study in Null Responder to Pegylated Interferon Alpha Plus Ribavirin Subjects with Chronic Hepatitis C. Available online: https://clinicaltrials.gov/ct2/show/NCT01727934 (accessed on 19 March 2020).
  154. ClinicalTrials.gov. Clinical Trial of BP1001 (Liposomal Grb2 Antisense Oligonucleotide) in Combination with Dasatinib in Patients with Ph + CML Who Have Failed TKI, Ph+ AML, Ph+ MDS. Available online: https://clinicaltrials.gov/ct2/show/NCT02923986 (accessed on 19 March 2020).
  155. ClinicalTrials.gov. An Open-Label Extension Study to Evaluate Long-Term Safety and Tolerability of RO7234292 (RG6042) in Huntington’s Disease Patients Who Participated in Prior Roche and Genentech Sponsored Studies. Available online: https://clinicaltrials.gov/ct2/show/NCT03842969 (accessed on 19 March 2020).
  156. ClinicalTrials.gov. An Efficacy, Safety, Tolerability, Pharmacokinetics and Pharmacodynamics Study of BIIB067 in Adults with Inherited Mmyotrophic Lateral Sclerosis (ALS) (VALOR (Part C)). Available online: https://clinicaltrials.gov/ct2/show/NCT02623699 (accessed on 19 March 2020).
  157. ClinicalTrials.gov. Open-Label Extension Assessing Long Term Safety and Efficacy of IONIS-TTR Rx in Familial Amyloid Polyneuropathy (FAP). Available online: https://clinicaltrials.gov/ct2/show/NCT02175004 (accessed on 19 March 2020).
  158. ClinicalTrials.gov. The Approach Open Label Study: A Study of Volanesorsen (Formerly IONIS-APOCIIIRx) in Patients with Familial Chylomicronemia Syndrome. Available online: https://clinicaltrials.gov/ct2/show/NCT02658175 (accessed on 19 March 2020).
  159. ClinicalTrials.gov. CARDIO-TTRansform: A Study to Evaluate the Efficacy and Safety of AKCEA-TTR-LRx in Participants with Transthyretin-Mediated Amyloid Cardiomyopathy (ATTR CM). Available online: https://clinicaltrials.gov/ct2/show/NCT04136171 (accessed on 19 March 2020).
  160. ClinicalTrials.gov. Efficacy of Alicaforsen in Pouchitis Patients Who Have Failed to Respond to at Least One Course of Antibiotics. Available online: https://clinicaltrials.gov/ct2/show/NCT02525523 (accessed on 19 March 2020).
  161. ClinicalTrials.gov. HELIOS-B: A Study to Evaluate Vutrisiran in Patients with Transthyretin Amyloidosis with Cardiomyopathy. Available online: https://clinicaltrials.gov/ct2/show/NCT04153149 (accessed on 19 March 2020).
  162. ClinicalTrials.gov. A Study of Fitusiran in Severe Hemophilia A and B Patients Previously Receiving Factor or Bypassing Agent Prophylaxis (ATLAS-PPX). Available online: https://clinicaltrials.gov/ct2/show/NCT03549871 (accessed on 19 March 2020).
  163. ClinicalTrials.gov. QPI-1002 Phase 3 for Prevention of Major Adverse Kidney Events (MAKE) in Subjects at High Risk for AKI Following Cardiac Surgery. Available online: https://clinicaltrials.gov/ct2/show/NCT03510897 (accessed on 19 March 2020).
  164. ClinicalTrials.gov. Trial to Assess the Effect of Long Term Dosing of Inclisiran in Subjects with High CV Risk and Elevated LDL-C (ORION-8). Available online: https://clinicaltrials.gov/ct2/show/NCT03814187 (accessed on 19 March 2020).
  165. Ottosen, S.; Parsley, T.B.; Yang, L.; Zeh, K.; Van Doorn, L.J.; Van Der Veer, E.; Raney, A.K.; Hodges, M.R.; Patick, A.K. In Vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122. Antimicrob. Agents Chemother. 2015, 59, 599–608. [Google Scholar] [CrossRef][Green Version]
  166. Gomez, I.G.; MacKenna, D.A.; Johnson, B.G.; Kaimal, V.; Roach, A.M.; Ren, S.; Nakagawa, N.; Xin, C.; Newitt, R.; Pandya, S.; et al. Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways. J. Clin. Investig. 2015, 125, 141–156. [Google Scholar] [CrossRef]
  167. Shah, M.Y.; Ferrajoli, A.; Sood, A.K.; Lopez-Berestein, G.; Calin, G.A. microRNA Therapeutics in Cancer—An Emerging Concept. EBioMedicine 2016, 12, 34–42. [Google Scholar] [CrossRef] [PubMed][Green Version]
  168. Eis, P.S.; Tam, W.; Sun, L.; Chadburn, A.; Li, Z.; Gomez, M.F.; Lund, E.; Dahlberg, J.E. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc. Natl. Acad. Sci. USA 2005, 102, 3627–3632. [Google Scholar] [CrossRef][Green Version]
  169. Slack, F.J.; Chinnaiyan, A.M. The Role of Non-coding RNAs in Oncology. Cell 2019, 179, 1033–1055. [Google Scholar] [CrossRef] [PubMed]
  170. Seto, A.G.; Beatty, X.; Lynch, J.M.; Hermreck, M.; Tetzlaff, M.; Duvic, M.; Jackson, A.L. Cobomarsen, an oligonucleotide inhibitor of miR-155, co-ordinately regulates multiple survival pathways to reduce cellular proliferation and survival in cutaneous T-cell lymphoma. Br. J. Haematol. 2018, 183, 428–444. [Google Scholar] [CrossRef] [PubMed][Green Version]
  171. Khvorova, A.; Watts, J.K. The chemical evolution of oligonucleotide therapies of clinical utility. Nat. Biotechnol. 2017, 35, 238–248. [Google Scholar] [CrossRef]
  172. Watts, J.; Corey, D. Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic. J. Pathol. 2012, 226, 365–379. [Google Scholar] [CrossRef][Green Version]
  173. Spurgers, K.B.; Sharkey, C.M.; Warfield, K.L.; Bavari, S. Oligonucleotide antiviral therapeutics: Antisense and RNA interference for highly pathogenic RNA viruses. Antivir. Res. 2008, 78, 26–36. [Google Scholar] [CrossRef]
  174. Liu, C.; Zhou, Q.; Li, Y.; Garner, L.V.; Watkins, S.P.; Carter, L.J.; Smoot, J.; Gregg, A.C.; Daniels, A.D.; Jervey, S.; et al. Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases. ACS Cent. Sci. 2020, 6, 315–331. [Google Scholar] [CrossRef]
  175. Rossi, J.J.; Rossi, D. Oligonucleotides and the COVID-19 Pandemic: A Perspective. Nucleic Acid Ther. 2020, 30, 129–132. [Google Scholar] [CrossRef][Green Version]
Figure 1. Antisense oligonucleotide (ASO) design. Chemically modified ASOs provide nuclease resistance and improved binding affinity to their target. Full length modified design represents chemical modifications throughout the sequence. Gapmer design includes a central region consist of DNA nucleotides and a stretch of LNA or 2′ modifications or PS nucleotides flanking both terminals of the sequence. Mixmer design contains LNA (or 2′ modifications) and DNA nucleotides present sequentially.
Figure 1. Antisense oligonucleotide (ASO) design. Chemically modified ASOs provide nuclease resistance and improved binding affinity to their target. Full length modified design represents chemical modifications throughout the sequence. Gapmer design includes a central region consist of DNA nucleotides and a stretch of LNA or 2′ modifications or PS nucleotides flanking both terminals of the sequence. Mixmer design contains LNA (or 2′ modifications) and DNA nucleotides present sequentially.
Jcm 09 02004 g001
Figure 2. Mechanism of action of antisense oligonucleotides (ASOs): ASOs act by either causing (1) RNA cleavage or (2) RNA blockage. (1a) RNase H1 mediated cleavage, (1b) RNA interference (RNAi), (2a) Steric hindrance, and (2b) Splice modulation. (1a) ASO-mRNA heteroduplex recruits RNase H1 enzyme and this enzyme cleaves the target mRNA. (1b) mRNA degradation by siRNA associated with RNA inducing silencing complex (RISC). (2a) ASO-mRNA complex sterically blocks and prevents the interaction of mRNA with ribosomes for protein translation. (2b) is an example of splice switching oligonucleotides (SSO). Rectangles depict the coding exon regions separated by a curve depicting the non-coding intron region of the pre-mRNA. The red square represents the mutated region of the exon. The dashed line represents the splicing pattern of pre-mRNA. RNase H1 mediated cleavage, RNA interference, and steric hindrance mechanisms produce less protein, while splice modulation produce the correct form of protein. Phosphorothioate (PS) and 5′methylcytosine base modification induces mRNA cleavage. Peptide nucleic acids (PNA), 2′-O-methyl (2′-O-Me) and 2′-O-methoxyethyl (2′-O-MOE) modifications, phosphorodiamidate morpholino (PMO), locked nucleic acid (LNA) act on mRNA to sterically block its translation or these ASOs can act as SSO to modulate splicing pattern.
Figure 2. Mechanism of action of antisense oligonucleotides (ASOs): ASOs act by either causing (1) RNA cleavage or (2) RNA blockage. (1a) RNase H1 mediated cleavage, (1b) RNA interference (RNAi), (2a) Steric hindrance, and (2b) Splice modulation. (1a) ASO-mRNA heteroduplex recruits RNase H1 enzyme and this enzyme cleaves the target mRNA. (1b) mRNA degradation by siRNA associated with RNA inducing silencing complex (RISC). (2a) ASO-mRNA complex sterically blocks and prevents the interaction of mRNA with ribosomes for protein translation. (2b) is an example of splice switching oligonucleotides (SSO). Rectangles depict the coding exon regions separated by a curve depicting the non-coding intron region of the pre-mRNA. The red square represents the mutated region of the exon. The dashed line represents the splicing pattern of pre-mRNA. RNase H1 mediated cleavage, RNA interference, and steric hindrance mechanisms produce less protein, while splice modulation produce the correct form of protein. Phosphorothioate (PS) and 5′methylcytosine base modification induces mRNA cleavage. Peptide nucleic acids (PNA), 2′-O-methyl (2′-O-Me) and 2′-O-methoxyethyl (2′-O-MOE) modifications, phosphorodiamidate morpholino (PMO), locked nucleic acid (LNA) act on mRNA to sterically block its translation or these ASOs can act as SSO to modulate splicing pattern.
Jcm 09 02004 g002
Figure 3. miRNA biogenesis and mechanism of action. miRNA is transcribed by RNA polymerase II (RNAP II) to form double stranded hairpin loop structure called pri-miRNA, which gets cleaved by nuclease Drosha to form pre-miRNA. Exportin transports the pre-miRNA to the cytoplasm where it is further processed by Dicer to form a single stranded mature miRNA. The mature miRNA is uploaded in the RNA induced silencing complex (RISC) where it associates with Argonaute 2 protein. This miRNA-RISC complex interacts with the seed region of the mRNA and regulates the mRNA translation by either mRNA cleavage or by steric hindrance.
Figure 3. miRNA biogenesis and mechanism of action. miRNA is transcribed by RNA polymerase II (RNAP II) to form double stranded hairpin loop structure called pri-miRNA, which gets cleaved by nuclease Drosha to form pre-miRNA. Exportin transports the pre-miRNA to the cytoplasm where it is further processed by Dicer to form a single stranded mature miRNA. The mature miRNA is uploaded in the RNA induced silencing complex (RISC) where it associates with Argonaute 2 protein. This miRNA-RISC complex interacts with the seed region of the mRNA and regulates the mRNA translation by either mRNA cleavage or by steric hindrance.
Jcm 09 02004 g003
Table 1. Chemical modifications of antisense oligonucleotides (ASO).
Table 1. Chemical modifications of antisense oligonucleotides (ASO).
NameStructureMechanismProperties
Phosphate modification
Phosphorothioate (PS) Jcm 09 02004 i001RNase H1 cleavageEnzymatic stability
Sugar phosphatemodification
Phosphorodiamidate morpholino (PMO) Jcm 09 02004 i002Steric hindrance/splice modulationImproved aqueous solubility, higher binding affinity
Peptide nucleic acid (PNA) Jcm 09 02004 i003Steric hindrance/splice modulationEnzymatic stability, higher binding affinity, no immune activation
Sugar modification
Locked nucleic acid (LNA) Jcm 09 02004 i004Steric hindrance/RNase H1 cleavageHigher binding affinity, enzymatic stability
2′-O-methyl (2′-O-Me) Jcm 09 02004 i005Steric hindrance/splice modulationHigher binding affinity, enzymatic stability, reduced immune stimulation
2′-O-methoxyethyl (2′-O-MOE) Jcm 09 02004 i006Steric hindrance/splice modulationHigher binding affinity, enzymatic stability, reduced immune stimulation
2′fluoro (2′ F) Jcm 09 02004 i007Steric hindrance/splice modulationHigher binding affinity
NucleoBase modification
5′methylcytosine Jcm 09 02004 i008RNase H1 cleavageHigher binding affinity, no immune stimulation
G-clamp Jcm 09 02004 i009Steric hindranceHigher binding affinity
Table 2. FDA approved drugs.
Table 2. FDA approved drugs.
DrugChemistryRouteTargetIndicationApproval DesignationCompany
Fomivirsen (VitraveneTM)PSIVTCMV mRNACMV infectionFDA (1998)-Ionis
Mipomersen (KynamroTM)2′-O-MOE, PS, 5-methyl cytosineSCapo-B-100 mRNA HoFHFDA (2013)OrphanGenzyme
Nusinersen (Spinraza®)2′-O-MOE, PS, 5-methyl cytosineITHSMN2 pre-mRNA SMAFDA (2016), EMA (2017)OrphanBiogen
Patisiran (Onpattro®)siRNAIVTTR mRNAhATTRFDA (2018), EMA (2018)OrphanAlnylam
Inotersen (Tegsedi®)2′-O-MOE, PSSCTTR mRNAhATTRFDA (2018), EMA (2018)OrphanIonis
Eteplirsen (Exondys 51®)PMOIVexon 51DMDFDA (2016), EMA (2018)OrphanSarepta
Golodirsen (Vyondys 53TM)PMOIVDMD pre-mRNADMDFDA (2019)OrphanSarepta
Givosiran (Givlaari®)siRNASCALS1 mRNAAHPFDA (2019), EMA (2020)OrphanAlnylam
Milasen2′-O-MOE, PS, 5-methyl cytosineITHintron 6 spice acceptor cryptic siteCLN7FDA * (2018)OrphanBoston Children’s Hospital
* Milasen is a personalized medicine developed for a single patient. IV—Intravenous, SC—Subcutaneous, IVT—intravitreal, ITH—Intrathecal.
Table 3. Potential drug candidates in clinical trials.
Table 3. Potential drug candidates in clinical trials.
Drug Candidate NCT IDChemistry/DeliveryTargetMOARouteCompanyIndicationRef.
Phase I status
ARRx
03300505
cEt gapmerAndrogen receptor mRNARNase H1IVRogel Cancer CenterProstate cancer[126]
RG-012
03373786
-miR-21antimiRSCGenzymeAlport syndrome[127]
QR-010
02564354
-CFTR mRNASplice modulationINProQRCystic fibrosis[128]
ISTH0036
02406833
LNATGF beta 2RNase H1IVTIsarnaPrimary open angle glaucoma[129]
ARO-APOC3
03783377
siRNA-GalNAcApoC-III mRNARNAiSCArrow headHTG, FCS[130]
ARO-ANG3
03747224
siRNA-GalNAcAngiopoietin-like protein 3 mRNARNAiSCArrow headDyslipidemias, FH, HTG[131]
QPI-1007
01064505
siRNACaspase 2 mRNARNAiIVTQuarkAnterior ischemic optic neuropathy, glaucoma[132]
ALN-AAT02
03767829
siRNA-GalNAcAlpha-1 antitrypsin mRNARNAiSCAlnylamAlpha-1 antitrypsin deficiency liver disease[133]
MEDI1191
03946800
mRNA LNPIL-12coding mRNAITMed ImmuneSolid tumors[134]
Phase II status
ISIS-FGFR4RX
02476019
2′-O-MOE-PSFGFR4 mRNARNase H1SCIonisObesity[135]
IONIS DGAT2Rx
03334214
2′-O-MOE-PSDGAT 2 mRNARNase H1SCIonisHepatic steatosis[136]
IONIS-PKK Rx
04307381
2′-O-MOE-PSPre kallikrein mRNARNase H1SCIonisHereditary angioedema[137]
ISIS-GCGRRx
02583919
2′-O-MOE GalNAcGlucagon receptor mRNARNase H1SCIonisType 2 diabetes[138]
Custirsen
00327340
siRNAClusterin mRNARNase H1IVAchieve Life SciencesProstate cancer[139]
OGX-427
01454089
LNAHsp27 mRNARNase H1IVAchieve Life SciencesMetastatic bladder cancer, urinary tract neoplasms[140]
ISIS681257
03070782
2′-O-MOE-PSLp(a) mRNARNase H1SCAkceaElevated lipoprotein (a), cardiovascular disease[141]
AKCEA-ANGPTL3-LRx
03514420
cEt gapmerANGPTL3 mRNARNase H1SCAkceaFamilial partial lipodystrophy[142]
ISIS678354
03385239
GalNAc-ASOApoC-III mRNAmRNA inhibitorSCAkceaHTG, cardiovascular diseases[143]
Danvatirsen
02983578
2′-O-MOE-PSSTAT3 mRNARNase H1IVM.D. Anderson Cancer CenterRefractory pancreatic, NSCLC, colorectal cancer[144]
Cobomarsen (MRG106)
03713320
LNAmiR-155antimiRITmiRagenCutaneous T-cell lymphoma[145]
Remlarsen
03601052
2′-O-MOEmiR-29miRNA mimicIDmiRagenKeloid[146]
Cemdisiran
03841448
siRNA-GalNAcC5 mRNARNAiSCAlnylamIgA nephropathy glomerulo nephritis[147]
ARO-AAT
03946449
siRNA-GalNAcAlpha-1 antitrypsin mRNARNAiSCArrow headAlpha 1-antitrypsin deficiency[148]
PF-655
01445899
siRNARTP801RNAiIVTQuarkDiabetic macular edema[149]
AZD8601
03370887
mRNA
LNP
VEGF-A mRNAcoding mRNAEIAstra ZenecaHeart failure[150]
DS-5141b
02667483
ENADystrophin mRNA exon 45Splice modulationSCDaiichi SankyoDMD[151]
SB010
01743768
-GATA-3DNAzymeISterna Bio.Asthma[152]
Miravirsen
01727934
LNAmiR-122antimiRSCSantarisHepatitis C[153]
BP1001
02923986
LNAGrb2-IVBio-Path HoldingsLeukemia[154]
Phase III status
Tominersen
03842969
2′-O-MOE-PSHTT mRNARNase H1ITHIonisHuntington’s disease[155]
Tofersen
02623699
2′-O-MOE-PSSOD1 mRNARNase H1ITHIonisAmyotrophic lateral sclerosis[156]
IONIS-TTR RX
02175004
2′-O-MOE-PSTTR mRNARNase H1SCIonisFamilial amyloid poly neuropathy[157]
Volanesorsen
02658175
2′-O-MOE-PSApoC-III mRNARNase H1SCIonisFCS hyperlipo proteinemia type 1[158]
AKCEA-TTR-LRx
04136171
siRNA GalNAcTTR mRNARNase H1SCIonisATTR cardio myopathy[159]
Alicaforsen
02525523
PSICAM-1 mRNARNase H1EAtlanticPouchitis[160]
Vutrisiran
04153149
siRNA-GalNAcTTR mRNARNAiSCAlnylamATTR with cardio myopathy[161]
Fitusiran
03549871
siRNA-GalNAcAnti-thrombin mRNARNAiSCGenzymeHemophilia[162]
QPI-1002
03510897
siRNAp53 mRNARNAiIVQuarkCardiac surgery[163]
Inclisiran
03814187
siRNA-GalNAcPCSK9 mRNARNAiSCThe Medicines CompanyHeterozygous FH[164]
MOA—mechanism of action, IV—Intravenous, SC—Subcutaneous, IN—Intranasal, IVT—intravitreal, IT—Intratumoral, ID—Intradermal, EI—Epicardial, ITH—Intrathecal, I—Inhalation, E—Enema, cEt—Constrained ethyl, CFTR—Cystic fibrosis transmembrane conductance regulator, TGF—Transforming growth factor, Apo—Apolipoprotein, HTG—Hypertriglyceridemia, FCS—Familial chylomicronemia syndrome, IL—Interleukin, FGFR4—Fibroblast growth factor receptor 4, DGAT—Diacylglycerol transferase, Hsp—Heat shock protein, Lp(a)—Lipoprotein (a), ANGPTL3—Angiopoietin-like protein 3, STAT3—Signal transducer and activator of transcription 3, NSCLC—Non-small cell lung cancer, C5—Complement C5, VEGF—Vascular endothelial growth factor, ENA—Ethylene-bridged nucleic acid, DMD—Duchenne muscular dystrophy, HTT—Huntingtin, ICAM—Intercellular adhesion molecule, ATTR—Transthyretin amyloidosis, p53—Tumor protein, FH—Familial hypercholesterolemia.

Share and Cite

MDPI and ACS Style

Dhuri, K.; Bechtold, C.; Quijano, E.; Pham, H.; Gupta, A.; Vikram, A.; Bahal, R. Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. J. Clin. Med. 2020, 9, 2004. https://doi.org/10.3390/jcm9062004

AMA Style

Dhuri K, Bechtold C, Quijano E, Pham H, Gupta A, Vikram A, Bahal R. Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development. Journal of Clinical Medicine. 2020; 9(6):2004. https://doi.org/10.3390/jcm9062004

Chicago/Turabian Style

Dhuri, Karishma, Clara Bechtold, Elias Quijano, Ha Pham, Anisha Gupta, Ajit Vikram, and Raman Bahal. 2020. "Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development" Journal of Clinical Medicine 9, no. 6: 2004. https://doi.org/10.3390/jcm9062004

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop