Pharmacokinetics and Proceedings in Clinical Application of Nucleic Acid Therapeutics

Abstract

Oligonucleotide therapeutics are a novel promising class of drugs designed to specifically target either coding or non-coding RNA molecules to revolutionize treatment of various diseases. During preclinical development, investigations of the pharmacokinetic characteristics of these oligonucleotide-based drug candidates are essential. Oligonucleotides possess a long history of chemical modifications to enhance their stability and binding affinity, as well as reducing toxicity. Phosphorothioate backbone modifications of oligonucleotides were a hallmark of this development process that greatly enhanced plasma stability and protein binding of these agents. Modifications such as 2′-O-methylation further improved stability, while other modifications of the ribose, such as locked nucleic acid (LNA) modification, significantly increased binding affinity, potency, and tissue half-life. These attributes render oligonucleotide therapeutics able to regulate protein expression in both directions depending on the target RNA. Thus, a growing interest has emerged using these oligonucleotides in the treatment of neurodegenerative and cardiac disorders as well as cancer, since the deregulation of certain coding and non-coding RNAs plays a key role in the development of these diseases. Cutting edge research is being performed in the field of non-coding RNAs, identifying potential therapeutic targets, and developing novel oligonucleotide-based agents that outperform classical drugs. Some of these agents are either in clinical trials showing promising results or are already US Food and Drug Administration (FDA) approved, with more oligonucleotides being developed for therapeutic purposes. This is the advent of mechanism-based next-generation therapeutics for a wide range of diseases.

Graphical Abstract

Oligonucleotide therapeutics are a promising class of drugs designed to target RNA molecules to revolutionize treatment of many diseases, including neurodegenerative and cardiac disorders as well as cancer, since specific targeting of coding and non-coding RNAs may outperform classical drug-based approaches.

Main Text

Chemical Modifications to Increase Nuclease Resistance

The pharmacokinetic (PK) properties of modified single-stranded DNA molecules have been well investigated. This is especially true for phosphorothioate (PS)-modified DNA nucleotides as well as for different ribose modifications.1, 2, 3 However, only few pharmacokinetic data are available for peptide nucleic acids (PNAs), morpholino nucleic acids (MNAs), and locked nucleic acids (LNAs) in part because of limited availability of sensitive assays for quantification.4, 5, 6, 7 A graphical overview of several generations of modified antisense oligonucleotides (ASOs) is shown as Figure 1.⁸

Figure 1.

Different Generations of Oligonucleotide Modifications

Antisense oligonucleotides have been modified over the years. This graphic shows the most common chemical modifications of oligonucleotides, with phosphorothioates (PSs) being the first nominal enhancement in terms of pharmacokinetics. Figure taken from Scoles, D.R., Minikel, E.V., and Pulst, S.M. (2019).⁸

One chemical approach to stabilize ASOs against degradation by exonucleases was described by adding 2′-F and 2′-O-methyl (OMe) ribosugar modifications to small interfering RNAs (siRNAs) and by covalently attaching triantennary N-acetylgalactosamine (GalNAc) ligands, thereby facilitating a targeted delivery to hepatocytes. Two siRNAs of the same sequence but different modification patterns were compared, resulting in different degradation behavior against exonuclease activity. The most prevalent nuclease activity could be observed in endo-lysosomal compartments, with 5′-exonuclease being the most prominent one. Thus, by improving the exonuclease resistance in the 5′ region of siRNAs, the metabolic stability could be further enhanced, resulting in increased gene silencing efficacy and improved liver exposure.⁹

Typically, the phosphate and the ribose 2′-C and 2′-O atoms are preferred sites for chemical modification, but ribose 4′-C substituents lie in proximity of both the 3′- and 5′-adjacent phosphates. In a very recent study, 2′-F and 2′-OMe modifications were combined with 4′-Cα- and 4′-Cβ-OMe, and 2′-F with 4′-Cα-methyl modification, to check for nuclease resistance. In detail, the α- and β-epimers of 4′-C-OMe-uridine and the α-epimer of 4′-C-Me-uridine monomers were incorporated into siRNAs, with the 4′α-epimers improving nuclease stability while having little effect on thermal stability, and the 4′β-epimers displaying absolute resistance against exonuclease activity. Additionally, crystal structures of RNA octamers containing the chain-inverted 4′β-U epimer further underscored this observation.¹⁰

Chemical modifications of ASOs can also affect binding of intracellular proteins, which might affect ASO potency. Work was been done to analyze the structure-activity relationships of ASO-protein interactions where 2′ modifications significantly affected protein binding. Hydrophobic 2′ modifications increase the affinity for proteins in general, although as for certain proteins, e.g., Ku70/Ku80 and TCP1, an affinity increase is much less. A prominent candidate that was found for enhanced protein binding was Hsp90, where binding occurred in the mid-domain. This was especially true for PS-ASOs containing LNA or constrained ethyl bicyclic nucleic acid ((S)-cEt) modifications. The reduction of Hsp90 protein led to a decrease of PS-ASO activities containing a LNA or (S)-cEt modification, but not with a 2′-O-methoxyethyl (2′-MOE) modification on the 5′ terminus. Thus, altering the intracellular protein binding affinity may be utilized to improve the therapeutic performance of ASOs.¹¹

As for thermal stability of ASOs, an interesting study compared various mixmers of the three-way junction (3WJ) of bacteriophage phi29 motor packaging RNA, consisting of (PS-)DNA, RNA, LNA, and 2′-F RNA, respectively. It was found that the thermal stability gradually increased following the order of PS-DNA/PS-DNA < DNA/DNA < DNA/RNA < RNA/RNA < RNA/2′-F RNA < 2′-F RNA/2′-F RNA < 2′-F RNA/LNA < LNA/LNA. Additionally, melting temperatures (Tms) from 21.2°C to more than 95°C could be achieved by mixing these different chemically modified ASOs. The LNA variant was resistant to even boiling temperature and urea denaturation and 50% serum degradation, making LNAs suitable for an improved pharmacokinetics profile.¹²

The mentioned chemical modifications can be combined in various ways to influence pharmacokinetics of ASOs, but all of them can be sorted by six main types of nucleic acid-based drugs (Table 1).

Table 1.

Examples of FDA-Approved, On-Market Drugs Listed Based on Nucleic Acid Types

Type	Drug
Antisense	defibrotide, eteplirsen, fomivirsen, mipomersen, nusinersen, volanesorsen
Aptamers	Pegaptanib
CpG (immunostimulatory)	AVA, CPG 7909 (Engerix-B)
miRNA	none as of yet
Ribozymes	none as of yet
RNAi	givosiran, patisiran

Pharmacokinetics of Nucleic Acid Therapeutics

One parameter modulating ASO pharmacokinetics is the application route. Ideally, a drug is being administered intravenously (i.v.) or subcutaneously (s.c.), whereby the s.c. application usually generates the same bioavailability with a small delay (right-shifted pharmacokinetic curve) compared to an i.v. injection.^3,13 Very meaningful pharmacokinetic relationships between the dose, the route of application, and the plasma half-life of modified oligonucleotides have already been demonstrated for humans and various animals. Typically, oligonucleotides with a PS backbone are more than 90% bound via a non-specific low-affinity binding to plasma proteins such as albumin and thus show a relatively short plasma half-life of 1–2 h. After 12 h, a steady state occurs and less than 1% of the oligonucleotides remain in the plasma. Most of the oligonucleotides are being distributed to the body tissues in the course of these processes, which explains the rapid clearance. Cellular uptake is then accomplished without a transfecting agent by a process called gymnosis, whereby the exact mechanism of uptake still needs to be elucidated.^14,15 One possible explanation might be an uptake by binding to hydrophilic cell surface proteins, which are then internalized together with oligonucleotides via endocytosis. The highest tissue concentration is typically found in the liver, kidney, spleen, and lymph nodes, completely independent of their sequence and across different species.^12,16, 17, 18, 19, 20, 21

Maximum mRNA reduction in the liver of mice could be shown 24–48 h after s.c. administration of ISIS 22023, which is significantly slower than clearance from the plasma, presumably due to the kinetics of transport of the oligonucleotides from the cell surface to their site of action.¹⁷ Inhibition not only of mRNAs, but also of RNAs in general, such as non-coding RNAs (ncRNAs) or microRNAs (miRNAs), has also been reported.22, 23, 24

The clearance of neutrally charged oligonucleotides i.e., PNAs, is significantly faster, which is also reflected in their reduced plasma half-life and tissue concentration.^25,26

However, the oral application of oligonucleotides showed a poor bioavailability of less than 1%, with studies in humans reporting a bioavailability of 10%–12%. However, a pharmacokinetic proof of concept was reported in humans with an apoB-100 antisense inhibitor.^25,27,28

The anti-CMV oligonucleotide ISIS 2922 (fomivirsen) is an example of topical application leading to local distribution and activity and the first of its kind.²⁹ It is complementary to the retinitis causing human cytomegalovirus (CMV) immediate-early RNA and showed specific anti-viral properties in cell cultures.³⁰

Typically, oligonucleotides cannot cross the blood-brain (BBB) barrier due to their polar backbone, but they can be injected into the cerebrospinal fluid (CSF), resulting in a distribution in the brain and spinal cord with long residence times due to a lowered nuclease activity.^31,32

In an interesting study, a translational non-human primate population pharmacokinetic (NHP PopPK) model of nusinersen was developed to predict the pharmacokinetics in pediatric patients with spinal muscular atrophy (SMA). By using initial i.v. dosing data, a two-compartment model was found to accurately describe the plasma pharmacokinetics of nusinersen with subsequent expanding of the model to determine the pharmacokinetics in the CSF, spinal cord, and the brain upon intrathecal (i.t.) administration. Nusinersen immediately distributed into the spinal cord and brain tissues, and the drug concentration in these tissues remained significantly high for a prolonged period of time (4–6 months). Upon allometric scaling, the NHP model was able to predict drug pharmacokinetics for CSF and plasma in pediatric patients with SMA within acceptable limits.³³

The main mechanism for cleavage of target RNAs is ASO-facilitated RNase H-mediated degradation. In an interesting study, the multiple-turnover ability of LNA-based oligonucleotides dependent on the Tm was determined. It was found, that initial reaction rates of ASOs with melting Tms of 40°C–60°C efficiently elicit multiple rounds of RNA scission. Furthermore, an adequate binding affinity is crucial for efficient turnover activities.³⁴

A more recent work investigated the influence of translation on the efficacy of ASO-facilitated RNase H-mediated cleavage of cytoplasmic RNAs. It could be shown that mRNAs associated with ribosomes can be cleaved using ASOs and that translation can alter ASO activity in a way that its translation inhibition activity is increased upon binding to the coding regions of translated mRNAs. This was not the case for nuclear non-coding RNAs or less efficiently translated mRNAs. However, by artificially increasing the level of RNase H1 protein, the enhancing effects of translation inhibition were eliminated. When targeting the 3′ UTRs, ASO activity was not increased by translation inhibition, independent of the translation efficiency of the mRNAs.³⁵

Aptamers represent another interesting chemical class of oligonucleotides. Potentially, these biological ligands can be used as therapeutic agents as well as for analytics and/or diagnostics due to their capability to bind into complex 3D structures.³⁶ This specificity is achieved by a selection method called systematic evolution of ligands by exponential enrichment (SELEX).^37,38

Nuclease resistance is also important for aptamers. By adding side chains to the 5′ position of uracil, the chemical diversity as well as nuclease resistance are increased. By conjugating aptamers to 40-kDa polyethylene glycol, it could be shown that plasma clearance depends on relative hydrophobicity. Hydrophobic side chains lead to increased plasma half-life, while hydrophilic side chains showed the opposite effect. In this manner, plasma clearance is enhanced with increasing side chain number above 28 synthons, but it drastically decreases for shorter sequences.³⁹ The mentioned administration routes, their impact on pharmacokinetics, and the type of nucleic acid-based drugs utlilizing these routes are summarized in Table 2.

Table 2.

Pharmacokinetics of Various Drug Types Dependent on Administration Route

Route	Absorption	Distribution	Metabolization	Elimination	Drug Type
i.v.	zero-order input/direct (blood)	blood plasma/vessels, surrounding tissue and organs (liver, bile, heart, lung, kidney), high bioavailability	exonucleolytic, primarily in liver, blood plasma, various cell types, dependent on applied ASO chemistry	very soon, multiphasic kinetics, long terminal half-life, renal, feces, only metabolites	antisense, aptamer, CpG, miRNA, ribozymes, RNAi
i.p.	delayed, peritoneal blood vessels, gastrointestinal	blood plasma/vessels, surrounding tissue and organs (intestine, ascites fluid, liver, bile, heart, lung, kidney), bioavailability comparable to i.v.	exonucleolytic, gastrointestinal, degradation, in liver (via portal vein, first pass effect), various cell types, dependent on applied ASO chemistry	late, renal, feces, only metabolites	antisense, aptamer, CpG, miRNA, ribozymes, RNAi
i.c.	zero-order input/direct (heart)	blood plasma/vessels, organs (heart, lung, liver, bile, kidney), high bioavailability	exonucleolytic, primarily in liver, blood plasma, various cell types, dependent on applied ASO chemistry	very soon, multiphasic kinetics, long terminal half-life, renal, feces, only metabolites	antisense
p.o.	mouth, stomach, intestine	stomach, intestine, blood vessels, surrounding tissue and organs (liver, bile, heart, lung, kidney), medium bioavailability	degradation by stomach acid, gastric juices, liver (via portal vein)	soon, renal, feces, only metabolites	CpG
i.t.	tumor, blood plasma/vessel	blood plasma/vessels, surrounding tissue and organs (liver, bile, heart, lung, kidney)	exonucleolytic, in liver, blood plasma, various cell types, dependent on applied ASO chemistry	fast from tumor into blood plasma	antisense, CpG, miRNA, RNAi
i.t.	CSF, CNS	CSF, CNS, brain, neurons (including motoneurons), blood plasma	no indication	from blood plasma, multiphasic kinetics, long terminal half-life	miRNA, ribozymes, RNAi
s.c.	delayed, fat, reservoir effect	blood plasma/vessels (delayed), surrounding tissue, organs	exonucleolytic, in blood plasma, various cell types, dependent on applied ASO chemistry	From blood plasma, multiphasic kinetics, long terminal half-life	antisense, aptamer, CpG, miRNA, ribozymes, RNAi
i.m.	delayed, muscle, reservoir effect	muscle, blood plasma/vessels (delayed), surrounding tissue, organs	exonucleolytic, in blood plasma, various cell types, dependent on applied ASO chemistry	from blood plasma, multiphasic kinetics, long terminal half-life	antisense, miRNA

p.o., per os; i.m., intramuscularly.

Absorption of Nucleic Acid Therapeutics

Absorption of nucleic acid therapeutics is not well understood, especially since negative polarity of the ASO backbone is an obstacle for cell entry. Except from an unspecific uptake of ASOs via gymnosis, there are few other proposed mechanisms for targeted nucleic acid uptake.^14,15

One study showed that the exploitation of the asialoglycoprotein receptor (ASGR) found in hepatocytes for cell entry resulted in an approximately 30-fold increase of uptake.⁴⁰ Another study showed that conjugation of ASOs with GalNAc sugars to second-generation ASOs can also be used to enhance the ASGR-mediated uptake in mice. The most efficient delivery was achieved by adding two or three GalNAc conjugates to one ASO, while adding a single GalNAc conjugate enhanced the therapeutic potential in liver.⁴¹

Delivery to other cell types, such as muscle cells, can be accomplished by targeting antibodies or antibody fragments against cell-surface proteins known to be involved in intracellular transport.⁴² Furthermore, transcripts of non-coding RNAs can be packaged into viral vectors and delivered into target cells to mediate their therapeutic effect, while synthetic oligonucleotides, such as miRNA mimics and modified mRNAs, could also serve as potential RNA delivery tools, given their advantages such as ease of dosage control and low immunogenicity.⁴³

A technology for a preformulated delivery system was shown by utilizing the natural transport properties of serum albumin and endogenous binding of gapmer ASOs/albumin constructs. An electrophoretic mobility assay was developed where fatty acid-modified gapmer and human serum albumin (HSA) could self-assemble into constructs that offer favorable pharmacokinetics. The interaction was dependent on fatty acid type, number, and position within the gapmer ASO sequence, as well as PS backbone modifications. Binding correlated with increased blood circulation in mice with an increased plasma half-life from 23 to 49 min for phosphodiester (PO) gapmer ASOs and from 28 to 66 min for PS gapmer ASOs with 2× palmitic acid modification.⁴⁴

Another approach enhancing the uptake of ASOs from blood to muscle tissue was described by increasing albumin binding. This was achieved by synthesizing structurally diverse fatty acids conjugated to ASOs, increasing their hydrophobicity. The binding affinity positively correlated with chain length ranging from 16 to 22 carbon atoms, while degree of unsaturation and conformation of double bonds were reported to show no influence on binding affinity. Palmitic acid conjugation increased the ASO maximum concentration (C_max) in plasma while further improving delivery of ASO to mouse muscle interstitium.⁴⁵

A novel ASO design was reported where ASO monomers to several targets are co-synthesized as heterodimers or multimers via PO linkers that are stable in blood plasma. These multimer ASOs are then taken up by the target cells and release the active ASO monomers upon intracellular cleavage. Compared to single ASOs, these multi-targeting oligonucleotides (MTOs) show increased plasma protein binding and biodistribution to liver, as well as increased in vivo efficacy against single or multiple targets. In vivo, these MTOs provide 4- to 5-fold increased potency and approximately 2-fold increased efficacy, adding an interesting alternative to the repertoire of therapeutic oligonucleotides.⁴⁶

Metabolism of Nucleic Acid Therapeutics

Metabolism of ASOs also plays a role in their degradation after target binding. To evaluate how ASOs might be degraded, eluforsen, a 33-mer 2′-OMe modified PS-ASO targeting the F508del mutation, was incubated with endonucleases and exonucleases and mouse liver homogenates in vitro, whereas mice and monkeys were used to determine in vivo liver and lung metabolism following inhalation. By utilizing a liquid chromatography-mass spectrometry (LC-MS) method for the identification and semi-quantitation of the metabolites of eluforsen, the in vitro and in vivo metabolism was determined. Metabolites of eluforsen could be identified in mouse liver upon 3′ exonuclease treatment and by either 3′ exonuclease or 5′ exonuclease in liver and lung samples of mice and monkeys.⁴⁷

Besides potential metabolization of therapeutic ASOs, cross-reactions with drug molecules at the cytochrome P450 (CYP) enzyme or transporter levels have not been described in detail so far. An interesting study tried to tackle this issue by developing an in vitro assay to investigate the drug-drug interaction (DDI) potential by using 2′-OMe-modified ASOs (ISIS 304801, ISIS 396443, ISIS 420915, and ISIS 681257) containing a single GalNAc₃ modification. In this extensive study, the inhibition on CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two variants of CYP3A4 (midazolam 1′-hydroxylation and testosterone 6β-hydroxylation) and induction on CYP1A2, CYP2B6, and CYP3A4 was assessed utilizing cryopreserved hepatocytes. No significant inhibition or induction events could be observed at a half-maximal inhibitory concentration (IC₅₀) >100 μM, neither on mRNA levels nor on enzymatic phenotypes. Nine major transporters including three hepatic (OCT1, OATP1B1, OATP1B3) as well as renal uptake transporters (OAT1, OAT3, OCT2) and three efflux transporters (P-gp, BCRP, BSEP) were treated with 10 μM of the aforementioned ASOs with no sign of interference or inhibition, leading to the assumption that DDIs via P450 enzyme activity is minimal or even non-existent with nucleic acid therapeutics.⁴⁸

This could lead to the conclusion that exonucleases and endonucleases are the first line responsible for the metabolism of oligonucleotides. Although the PS backbone can slow down this process, it cannot stop it. Exonucleases, which degrade the oligonucleotide by one base at each 3′ end, play a particularly important role. Oligonucleotides that are end-modified are largely exonuclease resistant but are degraded by endonucleases instead. In addition, oligonucleotides are not degraded by the cytochrome P450 oxidase present in the liver, which is why cross-reactions with other substrates of this enzyme are not likely. Smaller oligonucleotides and fragments of larger oligonucleotides, which have no or only low plasma protein binding, are probably filtered directly from the kidney and excreted. This is supported by the fact that no accumulation of these oligonucleotides can be detected in the tissue.^1,49, 50, 51, 52

Clearance of Nucleic Acid Therapeutics

The excretion of most oligonucleotide metabolites is mainly renal and therefore can be detected in urine and to a lesser extent in feces. Experiments with radionucleotides showed that after 1 month only about 50% of radiolabels could be found in urine and feces. From these observations it can be concluded that most radionucleotides are present in their unchanged form enriched in the tissues, while the degraded metabolites can be found in the urine.⁵³

We hope that the issues discussed herein exemplify the key role that pharmacokinetics play in the pre-clinical development of therapeutic oligonucleotides. Degradation of ASOs before entering the target cells is a major hurdle, which can be mitigated by modifying certain regions of ASOs, rendering them exonuclease resistant and less prone to metabolization. This strongly enhances plasma half-life and tissue distribution, whereby potential side effects also need to be put into consideration.

Toxicity of ASOs

Unexpected toxicity is another hurdle in the development of novel drugs in general and specifically for ASOs. Coding or non-coding RNAs may regulate several other mRNAs and thus often complete signaling pathways. Thus, it is important to work with model organisms that are as close as possible to humans in terms of physiology and genetic composition so that the data obtained in animals can be applied to humans. Primates are therefore particularly suitable for advanced preclinical studies, although recently also the minipig has started to be frequently used in oligonucleotide toxicity studies.^54,55

Toxicity can have several causes. A distinction can be made between toxicity resulting from the chemistry used and toxicity resulting from an off-target binding of the drug. Especially, PS-modified antimiRs may trigger an innate immunity response by binding proteins of the tenase complex and thus activate the complement cascade via an alternative pathway.^56,57

This effect is known because viral DNA and RNA are also recognized as foreign and fought by an innate immune response.^54,55 Furthermore, selected LNAs were described to induce acute tubular injury in kidneys with frequent weekly administrations of the experimental drug SPC5001 via s.c. delivery routes. SPC5001 was designed to block proprotein convertase subtilisin/kexin type 9 (PCSK9), which plays a role in reduction of low-density lipoprotein cholesterol levels.⁵⁸ Novel approaches tackling toxicity issues were described, where bioinformatics algorithms were developed to allow oligonucleotide sequences being analyzed in terms of the predictability of their interactions with RNA targets as well as off-targets. These tools enable selection of sequence-specific oligonucleotides where only a few off-targets are expected. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell and animal models before clinical testing. Since potential off-target effects are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are crucial in pre-clinical oligonucleotide drug discovery.⁵⁹

For selected s.c. administered ASOs, specific local reactions have been described.⁶⁰ These ranged from erythema, itching, discomfort, and pain to ulceration or even necrosis. Interestingly, ASO chemistry and the target structure seemed to show no clear association regarding injection site reaction (ISR) incidence or severity. Thus, knowledge of specific skin reactions and their immunostimulatory properties will be necessary to obtain ASOs that are more suitable for clinical applicability.⁶⁰ However, ultimate proof can be only obtained in phase 1 studies, as animal models are seemingly not well predictive for ISRs.

Another study regarding safety, tolerability, and pharmacokinetics was performed for the influenza A drug radavirsen, a phosphorodiamidate morpholino oligomer (PMO). A two-part phase 1 safety and pharmacokinetic study, with one single-ascending-dose study of five cohorts of 8 subjects each with i.v. admission doses ranging from 0.5 to 8 mg/kg or placebo, and one multiple-dose study of 16 subjects receiving 8 mg/kg or placebo once daily for 5 days, was conducted. In 74% of the radavirsen-treated subjects and 93% of the placebo-treated trial subjects, adverse events such as headache and proteinuria were observed and displayed a comparable incidence between these groups. Single dosing of radavirsen up to 8 mg/kg as well as multiple dosing at 8 mg/kg once daily for 5 days were well tolerated in healthy subjects, but further evaluation is needed.⁶¹

One major side effect described upon (chronic) treatment with single-stranded oligonucleotides (SSOs) is thrombocytopenia. The exact mechanisms are still to be elucidated and pre-clinical assessment is hard to conduct. Potential risk factors of thrombocytopenia upon treatment of ASOs were studied by utilizing in vitro assays.⁶² It seems that PS-modified ASOs bind to platelet proteins such as platelet factor 4 (PF4) or platelet collagen receptor glycoprotein VI (GPVI) with subsequent activation of human platelets in vitro. This binding occurs to be positively correlated with ASO length and PS content of the backbone, whereas modifications of the ribose can display enhanced suppression of binding to platelet proteins.

Furthermore, platelet activation might also be triggered by higher PS-ASO concentration, due to its pro-inflammatory potential. Thus, chemical characteristics of ASOs such as backbone modifications or ribosyl modifications should always be considered since these might be associated with a higher risk for thrombocytopenia.⁶²

As discussed before, reducing protein binding curtails unwanted off-target toxicity. It could be shown that PS-ASOs with ribosyl modifications such as LNA, 2′-MOE, or (S)-cEt bind many cellular proteins and, as a consequence, alter function, localization, and stability. RNase H1-dependent delocalization of paraspeckle proteins to nucleoli induces PS-ASO toxicity, and subsequent p53 activation leads to apoptosis. These adverse effects can be mitigated by introducing one 2′-OMe ribosyl modification, which substantially reduces hepatotoxicity. These findings might help advance future ASO design with optimal therapeutic profiles.⁶³

Despite efficacy of SSOs, hepatotoxicity is likely the most severe issue associated with application of SSOs. Unfortunately, aside from large cohort rodent studies, no preclinical models for studying the mechanisms of hepatotoxicity are available. Thus, much effort was invested in the development of in vitro assays. By utilizing primary hepatocytes, an assay could be developed that mimics the hepatotoxic profile of SSOs previously observed in rodents. Hepatotoxicity was monitored by assessing the increase in extracellular lactate dehydrogenase (LDH) and reduction of ATP levels and intracellular glutathione. Furthermore, miR-122 levels were also increased in cell culture supernatants, rendering it as a potential biomarker for hepatotoxicity.⁶⁴

A more comprehensive overview of toxicity in humans or primates based on oligonucleotide chemistry is summarized in Table 3.

Table 3.

Observed Toxicities of Common Oligonucleotide Modifications

Oligonucleotide Chemistry	Observed Toxicity	References
GapmeRs, locked nucleic acid (LNA)	some to no hepatotoxicity, nephrotoxicity, cytotoxicity	58,65, 66, 67, 68, 69
Morpholino	in general little to no toxicity, cardiac arrest in mice due to blood clotting has been reported, no toxicity in brain at low dose	70, 71, 72
2′-O-methoxyethyl (2′-MOE)	low toxicity	73, 74, 75
2′-O-methyl (2′-OMe)	minimal or no toxicity	63,69,76
Peptide nucleic acid (PNA)	low toxicity	74,77,78
Phosphorothioate (PS) DNA	hepatotoxicity (dependent on ribose or base mutation)	69,79
siRNAs	hepatotoxicity, some nephrotoxicity, associated with exaggerated dosing	80, 81, 82, 83

Specific RNA subtypes are often present in a low but finite cellular concentration. A pharmacological effect beyond the saturation of this concentration is not to be expected. Rather, additional toxicity is risked by overdosing, which is why it is of crucial importance to find the right therapeutic dose. If the clearance rate of bound RNAs is also known, a dose can be chosen that is high enough to pose a therapeutic effect, bind newly synthesized RNAs to maintain the therapeutic effect, and avoid toxic side effects due to overdosing.⁸⁴

Potential RNA Targets and Clinical Trials Utilizing ASOs

Many hurdles have to be overcome for the development and approval of a new therapeutic agent. It should be superior in efficacy and safety to existing therapeutics, have superior pharmacokinetic properties, and there should be less pressure that justifies the financial risk for the development of the therapeutic agent. miRNA inhibitors (antimiRs) are a new and promising class of therapeutics, as miRNAs as pharmacological targets have significant advantages over classical targets. They are highly conserved across many species and often regulate several jointly acting signaling pathways in parallel.⁸⁵

So far, a number of different new oligonucleotide chemistries have been developed with the aim to enhance stability and pharmacokinetic parameters and at the same time having only low toxicological side effects.⁸⁴

The lin-4 miRNA was the first non-coding RNA that could be shown to actively inhibit the translation of lin-14 protein by binding to the 3′ UTR of the lin-14 mRNA.^86,87

This discovery in Caenorhabditis elegans was followed by many other miRNAs that play a decisive role in disease genesis in animals and humans.^88,89

The safety of antimiRs plays an important role. An important aspect in the design of antimiRs is their cell permeability. Once in the cell, they should bind their target miRNA sequence specifically so that endogenous miRNA levels are sufficiently reduced. For the effect to induce a biological response, these antimiRs need to have certain stability and must not be excreted too quickly. To guarantee their stability, antimiRs are chemically modified. Oligonucleotide modifications include OMe groups at the 2′-OH of the ribose unit, or LNA, in which the 2′-OH group of the ribose is linked to the 4′-C to force the sugar residue into a C3′ endo conformation, which considerably increases the affinity of the antimiR. It is a truly unique feature of LNA among all of the invented nucleotide analogs that it greatly increases the strength of base pairing. This enables the design of both high-affinity and short ASOs. Short ASOs (12- to 16-mer) show more activity in vivo, presumably due to better cellular uptake. In addition, they are less associated with toxicity due to protein binding.90, 91, 92

The spiking pattern of these modifications with respect to the oligonucleotide sequence represents another dimension of chemical modification.⁹³

The following are selected examples of precursor (pre-)mRNAs, miRNAs, and even proteins whose involvement in human diseases has been demonstrated.

Cancer

eIF4E

For the treatment of colorectal carcinomas, the second-generation ASO ISIS 183750 was shown to block proliferation of colorectal carcinoma cells via mRNA inhibition. When adding irinotecan, an additional antiproliferative effect could be shown where 13 out of 19 patients displayed reduced levels of eIF4E mRNA in peripheral blood, and 5 out of 9 patients in pre- and post-treatment matched tumor biopsies.⁹⁴

Heart Failure

miR-132

miR-132 as part of the miR-212/132 gene family is upregulated under myocardial stress conditions, leading to cardiac hypertrophy in vivo. Cardiomyocyte-specific overexpression of miR-212/132 leads to cardiac hypertrophy, whereas knockout (KO) mutants show no hypertrophy subsequent to transaortic constriction (TAC) wherein the administration of miR-132 inhibitors also inhibits hypertrophy. Mechanistically, this miR-212/132 complex downregulates the FoxO3 transcription factor, resulting in hyperactivation of the pro-hypertrophic calcineurin/NFAT signaling cascade.95, 96, 97 Furthermore, studies were performed prior to the first clinical phase 1B (ClinicalTrials.gov: NCT04045405) studies in patients.98, 99, 100

A LNA (CDR132L) was administered i.v. or intracoronarily (i.c.) to specifically inhibit miR-132 in a clinically highly relevant pig model of heart failure. Furthermore, good pharmacokinetics, tolerability, safety, and dose-dependent pharmacokinetic/pharmacodynamic relationships underlined the high clinical potential of this miRNA inhibitor.⁹⁹ In another large animal study the same antimiR improved both systolic as well as diastolic function in a chronic post-myocardial infarction (MI) pig model after monthly i.v. injection of CDR132L in a blinded, randomized, placebo-controlled fashion. The pigs underwent 90 min of occlusion of the left anterior descending (LAD) artery followed by reperfusion. Cardiac function improved and adverse cardiac remodeling could be reversed. Thus, CDR132L proved to be safe and adequate in a clinically relevant chronic post-MI pig model.¹⁰⁰ In a follow-up study, 28 patients with chronic heart failure of ischemic origin were treated in four cohorts of seven patients (five on verum, two on placebo) with CDR132L in a randomized, placebo-controlled, double-blind, dose-escalation study (ClinicalTrials.gov: NCT04045405). This study showed reliable tolerability of CDR132L without apparent dose-limiting toxicity. Pharmacokinetic/pharmacodynamic dose modeling implied an effective dose of ≥1 mg/kg CDR132L with a dose-dependent miR-132 reduction in plasma and an N-terminal pro-B-type natriuretic peptide (NT-proBNP) median reduction of 23.3%. Beyond that, a significant narrowing of the Q wave, R wave, and S wave (QRS) complex and promising trends for cardiac fibrosis biomarkers could also be observed.¹⁰¹

miR-21

Another miRNA that is regulated very differently in many cardiac pathologies is miR-21. The knockdown of miR-21 with antimiRs also prevents cardiac hypertrophy and additionally can even reverse interstitial fibrosis after TAC.¹⁰² A validation study was done in large animals after MI.¹⁰³

Kidney Disease

miR-21

In the context of kidney diseases, miR-21 was also found to be enriched in the kidney. In one study, 8- to 10-week-old mice (20–30 g) were fasted 4 h prior to intraperitoneal (i.p.) administration of 50 mg/kg streptozotocin (STZ) or sodium citrate (placebo) for 5 days. Blood glucose and body weight were determined. Urine samples were taken with no occurrence of albuminuria or ketonuria. After 8 weeks of hyperglycemia (>16 mmol/L) mice were sacrificed. As for patients, 26 of them and 20 age-matched healthy controls were included. Kidney biopsies were taken for diagnosis of diabetic nephropathy (DN), which could be observed in 11 patients.

Inhibition of miR-21 could decrease mesangial expansion, interstitial fibrosis, macrophage infiltration, albuminuria, podocyte loss, and inflammatory gene expression in general.¹⁰⁴

miR-17

Another miRNA inhibitor was reported for treatment of autosomal dominant polycystic kidney disease (ADPKD). The discovery and characterization of RGLS4326 was a first-in-class, short oligonucleotide inhibitor of miR-17. RGLS4326 enters the kidney and displaces miR-17 from translationally active polysomes, and it de-represses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. RGLS4326 demonstrated a good preclinical safety profile and attenuated cyst growth in human in vitro ADPKD models and multiple PKD mouse models after s.c. administration.¹⁰⁵

miR-27a

The pathogenesis of miR-27a in renal tubulointerstitial fibrosis (TIF) in DN has not been described. It was shown that high levels of glucose increased miR-27a expression, which promoted fibrosis in NRK52E cells. Increased miR-27a leads to repression of PPARγ, activates transforming growth factor (TGF)-β/Smad3 signaling, and thus changes expression of connective tissue growth factor (CTGF), fibronectin, and collagen I, which are key mediators of fibrosis. Thus, targeting miR-27a displays a potential target for nucleic acid therapeutics.¹⁰⁶

Liver Disease

miR-223

miR-223 has been shown to be involved in liver diseases. Thus, the role of miR-223 was investigated in Fas-induced apoptosis of hepatocytes and liver injury in general. i.p. application of 0.5 μg/g body weight anti-Fas antibody Jo2 to wild-type (WT) and miR223 KO mice should give insights in the extent of liver damage and survival. As a result, all WT mice died up to 6 h after antibody administration, whereas all miR-223 KO mice survived. miR-223 KO mice displayed resistance to Fas-induced liver injury, as indicated by fewer apoptotic hepatocytes, less tissue damage, and less elevation of serum transaminases. In conclusion, inhibition of miR-223 by an ASO might protect against Fas-induced liver damage.¹⁰⁷

miR-182, miR-199a-5p, miR-200a-5p, and miR-183

Several miRNAs were found to be upregulated in tissue samples of hepatitis C patients via next-generation sequencing (NGS), whereas serum samples showed no significant alterations in miRNA levels. Patients of late-stage fibrosis (F3 and F4) were compared to patients of early-stage fibrosis (F1 and F2). All of these candidates potentially display potential targets of nucleic acid therapeutics.¹⁰⁸

Neurological Disorders

α-Synuclein

α-Synuclein is upregulated in Parkinson’s disease (PD). By conjugating siRNAs and ASOs that selectively target serotonin (5-HT) or norepinephrine (NE), neurons after intranasal administration with monoamine re-uptake inhibitor indatraline (IND), a selective reduction of α-synuclein expression in the brainstem monoamine nuclei of mice was achieved. α-Synuclein knockdown by 20%–40% did not cause monoaminergic neurodegeneration and enhanced forebrain dopamine (DA) and 5-HT release. This set the stage for the testing of non-viral inhibitory oligonucleotides in α-synuclein models of Parkinson’s disease.¹⁰⁹

OAT3

An interesting application of therapeutic oligonucleotides was shown in a study where the modulation of the BBB function at the molecular level was performed in vivo. A heteroduplex oligonucleotide (HDO), composed of an ASO and its complementary RNA, conjugated to α-tocopherol as a delivery agent, reduced the expression of the organic anion transporter 3 (OAT3) gene in brain microvascular endothelial cells in mice. This approach could serve as a novel platform to advance the biology and pathophysiology of the BBB in vivo and also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders.¹¹⁰

Nucleotide Repeat Disorders

Another potential field for the application of ASOs has been pointed out for nucleotide repeat disorders (NRDs) such as Huntington’s disease, myotonic dystrophies, and spinocerebellar ataxias. NRDs often adopt hairpin, cruciform, and triplex structures. For such gain-of-function disorders, therapeutic oligonucleotides can be used to target either transcripts or duplex DNA.¹¹¹

Prion Protein

ASOs could also be used lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA. This is of potential interest, since ASOs were reported to interact with PrP in cell culture, but in a sequence-unspecific fashion. In a most recent study, the anti-prion properties of certain ASOs were assessed in vitro and in cell culture, with binding affinities being in the low and middle nanomolar range independent of the chemical modifications and oligonucleotide sequence. Interestingly, the interaction of ASOs and PrP was characterized by large aggregate formation, explaining the sequence independence. This was assured by utilizing state-of-the-art analytical tools such as nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), dynamic light scattering, and visual inspection. However, PrP concentrations in human CSF were not influenced upon administration of ASOs. It was hypothesized that the inefficacy of some non-PrP-lowering ASOs in vivo may be caused by a drop below the effective ASO concentration necessary for sequence-unspecific aggregation.¹¹²

Pediatric Disease

MALAT1

A rather new application of ASOs is in the field of pediatric diseases. These are to a large part caused by genetic disorders, which can be diagnosed via prenatal screening. By administration of ASOs in utero, a safe and more effective fetal therapy can be established, without harming the fetus and providing a constant supply of pharmacotherapy by exploiting the amniotic cavity surrounding the fetus. It could be shown that an ASO targeting the MALAT1 RNA reduced its expression up to 4 weeks when administered by transuterine microinjection into the mouse amniotic cavity at embryonic day 13–13.5. Additionally, another ASO administered by the same injection route corrected gene expression by targeting a splice site mutation causing the Usher syndrome, thus rendering transuterine administration of ASOs a promising platform for further treatment of pediatric diseases.¹¹³

Virus-Induced Diseases

VP24 and NPC1

Also in the field of virology, oligonucleotides can be of therapeutic use. A LNA-modified ASO was developed to target the critical Ebola nucleoprotein VP24 and the human intracellular host protein NPC1. VP24 inhibits a proper immune response and thus promotes virus replication whereas NPC1 is required for viral entry.¹¹⁴

Viral SREs

LNA mixmers also can affect the viral splicing pattern of human immunodeficiency virus 1 (HIV-1). The viral splicing regulatory elements (SREs) G_I3-2 and ESE_tat are key in the generation of viral vif, vpr, and tat mRNAs. Thus, altering the splicing of pre-mRNAs by affecting these SREs can be therapeutically exploited. By masking these SREs with LNA mixmers, the viral splicing pattern and viral particle production are impaired. In a recent study, LNA mixmers targeting G_I3-2 and ESE_tat were administered into HIV-1-infected Jurkat/PM1 cells as well as in peripheral blood mononuclear cell (PBMCs) via gymnosis. These mixmers accumulated in the cytoplasm, associating with P-bodies, which are known mRNA-decay foci, and were degrading mRNAs containing the corresponding target sequence with subsequent abrogation of viral replication.¹⁴

This is just a small selection, but it shows chemically modified oligonucleotides being interesting and very potent candidates for the inhibition of various RNAs de-regulated in human diseases. The establishment of lead compounds is an important process in which several hundred to thousand molecules have to be synthesized and screened. In the past, chemically modified oligonucleotides have been shown to successfully be translatable into clinical human studies.115, 116, 117, 118

However, for ASOs there is a performance discrepancy between in vivo and in vitro, so it is reasonable to screen directly for their in vivo efficacy. Although the use of chemical modifications theoretically increases the number of possible permutations, it is precisely these modifications that limit the applicability of the modification due to its chemical properties and design. In any case, it makes more sense to develop and select ASO chemistries based on in vivo data, as these make the true pharmacological profile of ASOs accessible, although it is often easier to determine the optimal parameters for binding and cellular uptake of ASOs in vitro. It is important to investigate possible downstream effects in animal models showing certain disease patterns to obtain the necessary pharmacokinetic/dynamic data, since these are not observable under baseline conditions.⁸⁴

In Table 4, various ASOs enrolled in completed clinical studies are listed, with 3 more trials being active and 15 being in the status of recruiting. This compilation underlines the tremendous potential of therapeutic oligonucleotides in the treatment of RNA-driven diseases.

Table 4.

Completed Clinical Trials of ASOs

Study Title	Conditions	Disease Type	Interventions
Phase I dose escalation study to investigate the safety of ISTH0036 in subjects with glaucoma undergoing trabeculectomy	primary open angle glaucoma	cancer	drug: TGF-β2 antisense oligonucleotide
Infusional C-myb ASODN in advanced hematologic malignancies	hematologic malignancies	cancer	drug: C-myb ASODN
Phase 1 study of EZN-2968 weekly in adult patients with advanced solid tumors or lymphoma	Carcinoma	cancer	drug: intravenous EZN-2968 (anti-HIF-1α LNA AS-ODN)
	Lymphoma
A pilot study of EZN-2968, an antisense oligonucleotide inhibitor of HIF-1alpha, in adults with advanced solid tumors with liver metastases	neoplasms	cancer	drug: EZN-2968
	liver metastases
Study to determine the maximum tolerated dose of LErafAON in patients with advanced cancer	neoplasms	cancer	drug: LErafAON-ETU
Pilot immunotherapy trial for recurrent malignant gliomas	malignant glioma of brain	cancer	drug: IGF-1R/AS ODN
			device: biodiffusion chamber
Safety study of an antisense product in prostate, ovarian, NSCL, breast or bladder cancer	neoplasms	cancer	drug: OGX-427
			drug: docetaxel
Phase 1/2, open-label, dose-escalation study of IONIS-STAT3Rx, administered to patients with advanced cancers	advanced cancers	cancer	drug: IONIS-STAT3Rx
	diffuse large B cell lymphoma
	lymphoma
Bcl-2 antisense oligodeoxynucleotide G3139 and paclitaxel in treating patients with recurrent small cell lung cancer	lung cancer	cancer	biological: oblimersen sodium
			drug: paclitaxel
OGX-011 and docetaxel in treating patients with metastatic or locally recurrent solid tumors	bladder cancer	cancer	drug: dustirsen sodium
	breast cancer		drug: docetaxel
	kidney cancer		other: pharmacological study
Oblimersen and interferon alfa in treating patients with metastatic renal cell cancer	recurrent renal cell cancer	cancer	biological: recombinant interferon alfa
	stage IV renal cell cancer		biological: oblimersen sodium
			other: pharmacological study
Phase I/II study of genasense in patients with chronic lymphocytic leukemia	chronic lymphocytic leukemia	cancer	drug: oblimersen sodium, G3139
OGX-011 and docetaxel in treating women with locally advanced or metastatic breast cancer	breast cancer	cancer	drug: custirsen sodium
			drug: docetaxel
Combination chemotherapy plus oblimersen in treating patients with previously untreated extensive-stage small cell lung cancer	extensive stage small cell lung cancer	cancer	biological: oblimersen sodium
			drug: carboplatin
			drug: etoposide
Dacarbazine with or without oblimersen (G3139) in treating patients with advanced malignant melanoma	melanoma (skin)	cancer	biological: oblimersen sodium
			drug: dacarbazine
OGX-427 in castration resistant prostate cancer patients	castration resistant	cancer	drug: OGX-427
	prostate cancer		drug: prednisone
Oblimersen, rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone in treating patients with stage II, stage III, or stage IV diffuse large B cell lymphoma	lymphoma	cancer	biological: oblimersen sodium
			biological: rituximab
			drug: cyclophosphamide
Genasense® (oblimersen sodium), fludarabine, and rituximab in subjects with chronic lymphocytic leukemia	chronic lymphocytic leukemia	cancer	drug: oblimersen-rituximab-fludarabine
Dexamethasone with or without oblimersen in treating patients with relapsed or refractory multiple myeloma	multiple myeloma and plasma cell neoplasm	cancer	biological: oblimersen sodium
			drug: dexamethasone
Docetaxel with or without oblimersen in treating patients with hormone-refractory adenocarcinoma (cancer) of the prostate	prostate cancer	cancer	biological: oblimersen sodium
			drug: docetaxel
Fludarabine and cyclophosphamide with or without oblimersen in treating patients with relapsed or refractory chronic lymphocytic leukemia	leukemia	cancer	biological: filgrastim
			biological: oblimersen sodium
			drug: cyclophosphamide
			drug: fludarabine phosphate
Hormone therapy and OGX-011 before radical prostatectomy in treating patients with prostate cancer	prostate cancer	cancer	drug: buserelin
			drug: custirsen sodium
			drug: flutamide
A phase I study of G3139 subcutaneous in solid tumors	tumors	cancer	drug: G3139, oblimersen sodium, Bcl-2 antisense oligonucleotide
Oblimersen and irinotecan in treating patients with metastatic or recurrent colorectal cancer	colorectal cancer	cancer	biological: oblimersen sodium
			drug: irinotecan hydrochloride
Chemotherapy in treating women with previously treated metastatic breast cancer	breast cancer	cancer	biological: ISIS 3521
			drug: ISIS 5132
Phase 2 study of docetaxel ± OGX-427 in patients with relapsed or refractory metastatic bladder cancer	bladder cancer	cancer	drug: OGX-427
	urothelial carcinoma		drug: docetaxel
Combination of capecitabine and GTI-2040 in the treatment of renal cell carcinoma	carcinoma, renal cell	cancer	drug: GTI-2040
	metastases, neoplasm
Phase I dose-escalation study of AZD4785 in patients with advanced solid tumors	non-small cell lung cancer	cancer	drug: AZD4785
	advanced solid tumors
Phase I open label dose escalation study to investigate the safety & pharmacokinetics of AZD5312 in patients with androgen receptor tumors	advanced solid tumors with androgen receptor pathway as a potential factor	cancer	drug: AZD5312
Studying cells collected through ductal lavage in women undergoing surgery for ductal carcinoma in situ or other breast cancer	breast cancer	cancer	genetic: RNA analysis
			genetic: polymerase chain reaction
			genetic: protein expression analysis
A phase I/Ib study of AZD9150 (ISIS-STAT3Rx) in patients with advanced/metastatic hepatocellular carcinoma	advanced adult hepatocellular carcinoma	cancer	drug: AZD9150
	hepatocellular carcinoma metastatic
MEDI4736 alone and in combination with tremelimumab or AZD9150 in adult subjects with relapsed/refractory DLBCL (D4190C00023)	diffuse large B cell lymphoma	cancer	drug: MEDI4736
			drug: tremelimumab
			drug: AZD9150
Genasense as a 2-hour intravenous infusion in subjects with solid tumors	solid tumors	cancer	drug: oblimersen (genasense)
Chemotherapy and bone marrow transplantation in treating patients with chronic myelogenous leukemia	leukemia	cancer	biological: c-myb antisense oligonucleotide G4460
			biological: filgrastim
			drug: busulfan
Combination chemotherapy plus oblimersen in treating patients with advanced solid tumors	unspecified adult solid tumor, protocol specific	cancer	biological: oblimersen sodium
			drug: paclitaxel
			other: laboratory biomarker analysis
			other: pharmacological study
Safety and dose study of GRN163L to treat patients with chronic lymphoproliferative disease (CLD)	chronic lymphoproliferative diseases	cancer	drug: GRN163L
A study of GRN163L with paclitaxel and bevacizumab to treat patients with locally recurrent or metastatic breast cancer	breast cancer	cancer	drug: GRN163L
Study of GRN163L with paclitaxel and carboplatin in patients with advanced or metastatic non-small cell lung cancer	lung cancer	cancer	drug: imetelstat sodium (GRN163L)
Safety and dose study of GRN163L and Velcade to treat patients with refractory or relapsed myeloma	multiple myeloma	cancer	drug: imetelstat sodium (GRN163L)
Safety and dose study of GRN163L administered to patients with refractory or relapsed solid tumor malignancies	solid tumor malignancies	cancer	drug: imetelstat sodium (GRN163L)
A study inhibiting telomerase to reverse trastuzumab resistance in HER2+ breast cancer	breast neoplasms	cancer	drug: GRN163L in combination with trastuzumab
Imetelstat sodium in treating patients with primary or secondary myelofibrosis	primary myelofibrosis	cancer	drug: imetelstat
	secondary myelofibrosis
	myeloid malignancies
Open label study with imetelstat to determine effect of imetelstat in patients w/ previously treated multiple myeloma	multiple myeloma	cancer	drug: imetelstat (7.5 mg/kg)
			drug: lenalidomide standard of care
			drug: imetelstat (9.4 mg/kg)
Imetelstat in combination with paclitaxel (with or without bevacizumab) in patients with locally recurrent or metastatic breast cancer	locally recurrent or metastatic breast cancer	cancer	drug: imetelstat sodium
			drug: bevacizumab
			drug: paclitaxel
Imetelstat as maintenance therapy after initial induction chemotherapy in non-small cell lung cancer (NSCLC)	non-small cell lung cancer	cancer	drug: imetelstat
			drug: bevacizumab
Open label study to evaluate the activity of imetelstat in patients with essential thrombocythemia or polycythemia vera	essential thrombocythemia	cancer	drug: imetelstat
	polycythemia vera
Study to evaluate activity of 2 dose levels of imetelstat in participants with intermediate-2 or high-risk myelofibrosis (MF) previously treated with Janus kinase (JAK) inhibitor	primary myelofibrosis	cancer	drug: imetelstat 9.4 mg/kg
			drug: imetelstat 4.7 mg/kg
Stereotactic body radiotherapy and radiofrequency ablation for lung tumors near central airways	lung cancer	cancer	radiation: stereotactic body radiation
			radiation: radiofrequency ablation
Trebananib with or without low-dose cytarabine in treating patients with acute myeloid leukemia	adult acute megakaryoblastic leukemia (M7)	cancer	biological: trebananib
	adult acute minimally differentiated myeloid leukemia (M0)		drug: cytarabine
	adult acute monoblastic leukemia (M5a)		other: laboratory biomarker analysis
	adult acute monocytic leukemia (M5b)		other: pharmacological study
	adult acute myeloblastic leukemia with maturation (M2)
	adult acute myeloblastic leukemia without maturation (M1)
	adult acute myeloid leukemia with 11q23 (MLL)
	adult acute myeloid leukemia with del(5q)
	adult acute myeloid leukemia with inv(16)(p13;q22)
	adult acute myeloid leukemia with t(16;16)(p13;q22)
	adult acute myeloid leukemia with t(8;21)(q22;q22)
	adult acute myelomonocytic leukemia (M4)
	adult erythroleukemia (M6a)
	adult pure erythroid leukemia (M6b)
	recurrent adult acute myeloid leukemia
	untreated adult acute myeloid leukemia
Phase II trial of carboplatin and pemetrexed ± OGX-427 in untreated stage IV non-squamous-non-small cell lung cancer	non-squamous non-small cell lung cancer	cancer	drug: OGX-427
			drug: placebo
A phase 2 study comparing chemotherapy in combination with OGX-427 or placebo in patients with bladder cancer	urologic neoplasms	cancer	drug: OGX-427 (600 mg)
	metastatic bladder cancer		drug: OGX-427 (1,000 mg)
	urinary tract neoplasms		drug: placebo
			drug: gemcitabine
			drug: cisplatin
			drug: carboplatin
Safety of adding IMO-2055 to erlotinib + bevacizumab in second-line treatment for patients with NSCLC	non-small cell lung cancer	cancer	drug: IMO-2055
Study of IMO-2055 in metastatic or locally recurrent clear cell renal carcinoma	renal cell carcinoma	cancer	drug: IMO-2055
EMD 1201081 in combination with cetuximab in second-line cetuximab-naive subjects with recurrent or metastatic squamous cell carcinoma of the head and neck	squamous cell carcinoma of the head and neck cancer	cancer	drug: cetuximab
			drug: EMD 1201081
Safety of adding IMO-2055 to erlotinib + bevacizumab in 2nd line treatment for patients with NSCLC	non-small cell lung cancer	cancer	drug: IMO-2055
Study of IMO-2055 in metastatic or locally recurrent clear cell renal carcinoma	renal cell carcinoma	cancer	drug: IMO-2055
XIAP antisense AEG35156 in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC)	advanced hepatocellular carcinoma	cancer	drug: AEG35156 antisense i.v. infusion
			drug: sorafenib
AEG35156 and docetaxel in treating patients with solid tumors	unspecified adult solid tumor, protocol specific	cancer	drug: AEG35156
			drug: docetaxel
AEG35156 and docetaxel in treating patients with locally advanced, metastatic, or recurrent solid tumors	unspecified adult solid tumor, protocol specific	cancer	drug: AEG35156
			drug: docetaxel
			genetic: protein expression analysis
			genetic: reverse transcriptase-polymerase chain reaction
			other: flow cytometry
			other: immunoenzyme technique
			other: immunohistochemistry staining method
			other: laboratory biomarker analysis
Study of XIAP antisense given with chemotherapy for refractory/relapsed AML	leukemia, myelomonocytic, acute	cancer	drug: XIAP antisense
A study of PNT2258 in patients with advanced solid tumors	cancer	cancer	drug: PNT2258
	lymphoma
	prostate cancer
	melanoma
Study of PNT2258 for treatment of relapsed or refractory non-Hodgkin’s lymphoma	lymphoma, non-Hodgkin’s	cancer	drug: PNT2258
PNT2258 for treatment of patients with r/r DLBCL (Wolverine)	lymphoma, diffuse large B cell	cancer	drug: PNT2258
ISIS 183750 with irinotecan for advanced solid tumors or colorectal cancer	colorectal neoplasms	cancer	drug: ISIS 183750
	colorectal
	carcinoma
	colorectal tumors
LY2275796 in advanced cancer	advanced cancers	cancer	drug: LY2275796
Safety and tolerability study of ISIS EIF4E Rx in combination with carboplatin and paclitaxel	non-small cell lung cancer	cancer	drug: ISIS EIF4E Rx
			drug: paclitaxel
			drug: carboplatin
Safety and tolerability study of ISIS EIF4E Rx in combination with docetaxel and prednisone (CRPC)	castrate-resistant prostate cancer	cancer	drug: ISIS EIF4E Rx
			drug: prednisone
			drug: docetaxel
ISIS 104838, an inhibitor of tumor necrosis factor, for active rheumatoid arthritis	rheumatoid arthritis	chronic diseases	drug: ISIS 104838
Safety, efficacy, pharmacokinetic, and pharmacodynamic characteristics of orally inhaled SB010 in male patients with mild asthma (multiple dose)	asthma	chronic diseases	drug: SB010
			drug: placebo (phosphate-buffered saline)
Multiple dose ASM8 in mild asthmatics	asthma	chronic diseases	drug: ASM8
Alicaforsen (ISIS 2302) in patients with active Crohn’s disease	Crohn’s disease	chronic diseases	drug: alicaforsen
Efficacy, pharmacokinetics, tolerability, safety of SB012 intrarectally applied in active ulcerative colitis patients	colitis, ulcerative	chronic diseases	drug: SB012
			drug: placebo
Efficacy, safety, tolerability, pharmacokinetics and pharmacodynamics study of the topical formulation SB011 applied to lesional skin in patients with atopic eczema	mild to moderate atopic dermatitis	chronic diseases	drug: SB011, 2% (water/oil/water) emulsion of hgd40
			drug: multiple water/oil/water formulation, active ingredient-free vehicle
A study of RG-012 in subjects with Alport syndrome	Alport syndrome	genetic diseases	drug: RG012
Safety and efficacy study of antisense oligonucleotides in Duchenne muscular dystrophy	Duchenne muscular dystrophy (DMD)	genetic diseases	drug: AVI-4658 (PMO)
Study to evaluate QR-110 in subjects with Leber’s congenital amaurosis (LCA) due to the c.2991+1655A > G mutation (p.Cys998X) in the CEP290 gene	Leber’s congenital amaurosis	genetic diseases	drug: QR-110
Exploratory study to evaluate QR-010 in subjects with cystic fibrosis ΔF508 CFTR mutation	cystic fibrosis	genetic diseases	drug: QR-010
Dose escalation Study of QR-010 in Homozygous ΔF508 Cystic Fibrosis Patients	cystic fibrosis	genetic diseases	drug: QR-010
			drug: placebo
Safety and tolerability of WVE-210201 in patients with Duchenne muscular dystrophy	Duchenne muscular dystrophy	genetic diseases	drug: WVE-210201
			drug: placebo
Safety study of eteplirsen to treat advanced stage Duchenne muscular dystrophy	Duchenne muscular dystrophy	genetic diseases	drug: eteplirsen
Study of eteplirsen in DMD patients	Duchenne muscular dystrophy	genetic diseases	drug: eteplirsen
Safety study of eteplirsen to treat early stage Duchenne muscular dystrophy	Duchenne muscular dystrophy	genetic diseases	drug: eteplirsen
Efficacy, safety, and tolerability rollover study of eteplirsen in subjects with Duchenne muscular dystrophy	Duchenne muscular dystrophy	genetic diseases	drug: AVI-4658 (Eteplirsen)
Efficacy study of AVI-4658 to induce dystrophin expression in selected duchenne muscular dystrophy patients	Duchenne muscular dystrophy	genetic diseases	drug: AVI4658 (eteplirsen)
			other: placebo
Dose-ranging study of AVI-4658 to induce dystrophin expression in selected Duchenne muscular dystrophy (DMD) patients	Duchenne muscular dystrophy	genetic diseases	drug: AVI-4658
Safety and efficacy study of antisense oligonucleotides in Duchenne muscular dystrophy	Duchenne muscular dystrophy	genetic diseases	drug: AVI-4658 (PMO)
An open-label safety and tolerability study of nusinersen (ISIS 396443) in participants with spinal muscular atrophy (SMA) who previously participated in ISIS 396443-CS2 (ClinicalTrials.gov: NCT01703988) or ISIS 396443-CS10 (ClinicalTrials.gov: NCT01780246)	spinal muscular atrophy	genetic diseases	drug: nusinersen
An open-label safety, tolerability and dose-range finding study of multiple doses of nusinersen (ISIS 396443) in participants with spinal muscular atrophy	spinal muscular atrophy	genetic diseases	drug: nusinersen
An open-label safety and tolerability study of nusinersen (ISIS 396443) in participants with spinal muscular atrophy who previously participated in ISIS 396443-CS1 (ClinicalTrials.gov: NCT01494701)	spinal muscular atrophy	genetic diseases	drug: nusinersen
A study to assess the efficacy, safety and pharmacokinetics of nusinersen (ISIS 396443) in infants with spinal muscular atrophy (SMA)	spinal muscular atrophy	genetic diseases	drug: nusinersen
A study to assess the efficacy and safety of nusinersen (ISIS 396443) in participants with later-onset spinal muscular atrophy (SMA)	spinal muscular atrophy	genetic diseases	drug: nusinersen
			procedure: sham
An open-label safety, tolerability, and dose-range finding study of nusinersen (ISIS 396443) in participants with spinal muscular atrophy (SMA)	spinal muscular atrophy	genetic diseases	drug: nusinersen
The study of an investigational drug, revusiran (ALN-TTRSC), for the treatment of transthyretin (TTR)-mediated amyloidosis in patients whose disease has continued to worsen following liver transplant	transthyretin-mediated amyloidosis	genetic diseases	drug: revusiran
	familial amyloidotic polyneuropathy (FAP)
	ATTR amyloidosis
	familial amyloid neuropathies
A extension study to evaluate revusiran (ALN-TTRSC) in patients with transthyretin (TTR) cardiac amyloidosis	TTR-mediated amyloidosis	genetic diseases	drug: revusiran (ALN-TTRSC)
Phase 2 study to evaluate ALN-TTRSC (revusiran) in patients with transthyretin (TTR) cardiac amyloidosis	TTR-mediated amyloidosis	genetic diseases	drug: ALN-TTRSC (revusiran) for subcutaneous administration
A phase 1, single- and multi-dose, dose escalation study to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of subcutaneously administered ALN-TTRSC (revusiran) in healthy volunteers	TTR-mediated amyloidosis	genetic diseases	drug: ALN-TTRSC (revusiran)
			drug: sterile normal saline (0.9% NaCl)
ENDEAVOUR: phase 3 multicenter study of revusiran (ALN-TTRSC) in patients with transthyretin (TTR) mediated familial amyloidotic cardiomyopathy (FAC)	TTR-mediated familial amyloidotic cardiomyopathy	genetic diseases	drug: revusiran (ALN-TTRSC)
	amyloidosis, hereditary		drug: sterile normal saline (0.9% NaCl)
	amyloid neuropathies, familial
Safety and tolerability of patisiran (ALN-TTR02) in transthyretin (TTR) amyloidosis	TTR-mediated amyloidosis	genetic diseases	drug: patisiran
APOLLO: the study of an investigational drug, patisiran (ALN-TTR02), for the treatment of transthyretin (TTR)-mediated amyloidosis	TTR-mediated amyloidosis	genetic diseases	drug: patisiran (ALN-TTR02)
	amyloidosis, hereditary		drug: sterile normal saline (0.9% NaCl)
	amyloid neuropathies, familial
The study of ALN-TTR02 (Patisiran) for the treatment of transthyretin (TTR)-mediated amyloidosis in patients who have already been treated with ALN-TTR02 (patisiran)	TTR-mediated amyloidosis	genetic diseases	drug: ALN-TTR02 (patisiran) administered by i.v. infusion
A study of the safety, tolerability and pharmacokinetics of ALN-TTR02 in Japanese healthy volunteers	TTR-mediated amyloidosis	genetic diseases	drug: patisiran (ALN-TTR02)
			drug: sterile normal saline (0.9% NaCl)
Trial to evaluate safety, tolerability, and pharmacokinetics of ALN-TTR02 in healthy volunteer subjects	TTR-mediated amyloidosis	genetic diseases	drug: ALN-TTR02
			drug: sterile normal saline (0.9% NaCl)
Efficacy and safety of inotersen in familial amyloid polyneuropathy	familial amyloid polyneuropathy	genetic diseases	drug: inotersen
	TTR amyloidosis		drug: placebo

Taken from https://clinicaltrials.gov (November 8, 2020).

Conclusions

Taken together, RNAs and especially miRNAs represent excellent drug targets due to their small size and, in the case of miRNAs, genetic conservation. Due to the fact that a miRNA often has several target mRNAs, it is potentially possible to regulate entire signaling cascades. In addition, many miRNAs have homologous sequences that are identical or very similar, so that an ASO can regulate even more potential miRNAs and their downstream targets. The downside of this potency, however, includes potential off-target effects that can cause toxic side effects. Also, the chemical modifications used in these miRNA inhibitors still need to be thoroughly investigated with regard to possible toxic side effects, and the complete characterization of their pharmacokinetics and efficacy profile is and will continue to be the subject of intensive research. Nevertheless, numerous applications of ASOs as therapeutic agents were shown with a significant number being enrolled in clinical trials, proving the potential of becoming therapeutic oligonucleotides while others are already listed as US Food and Drug Administration (FDA)-approved drugs.