Synthetic antisense oligonucleotides are a novel and efficient laboratory tool to regulate the expression of specific genes. These are synthetic single-stranded DNA analogs; usually 15-30 base pairs in length, whose sequence (3' to 5') is antisense and complementary to the sense sequence of the target nucleotide (mRNA) hence called antisense oligonucleotides [2]. They selectively bind to specific pre-messenger ribonucleic acid (pre-mRNA)/mRNA sequences and alter protein synthesis by several mechanisms. First studied in the late 1970s to inhibit oncogenic viral production [3], further research led to designing of highly modified ASOs with more targeted delivery, tolerability, safety, with a prominent role in treating life-threatening diseases that were previously incurable [4].

The mechanism of action of ASOs may briefly be summarized in three sequential steps, as follows [5]:

Pre-hybridization phase is the phase in which the ASO enters the cell, distributes within the cell, to achieve sufficient concentrations at the target RNA site. The internalization of the ASO within the cell by carrier protein-mediated endocytosis is a complex process – further, the ASO needs to escape the cellular endosomal pathway to reach the target site, which is a rate-limiting process [6].

Hybridization phase is the phase in which ASO sorts through the cellular nucleic acid sequence space to hybridize to its target RNA site. This is a complex process that involves interactions with proteins, such as Ago2, or other cellular components [5].

Post hybridization phase: After binding to the mRNA site depending on the chemical design of the ASO, a variety of events may be induced that alter the target RNA to achieve the desired pharmacological outcome. There are two main mechanisms: the common mechanism is by induction of endogenous RNAse H activity (ASO-RNase H) that cleaves the mRNA-ASO hetero-duplex which leads to degradation of the target toxic mRNA and leaves the ASO intact. The second mechanism includes binding to the RNA and causing translational inhibition by steric hindrance, exon skipping, exon inclusion, destabilization of pre-mRNA in the nucleus, or targeting the destruction of microsomal RNAs that control the expression of other genes [2,7]. For example, ASOs can bind to mRNA structures and prevent the 5’-mRNA cap formation or, alternatively, they modify the polyadenylation site to prevent mRNA translation or alter RNA stability. Moreover, ASOs can directly stick to the mRNA and sterically block the 40S and 60S ribosomal subunits from attaching or running along the mRNA transcript during translation. Other ASOs bind on pre-mRNA intron/exon junctions and directly modulate splicing by masking splicing enhancers and repressor sequences, skipping exons, or forcing the inclusion of otherwise alternatively spliced exons. These actions are independent of RNase activity, as with Eteplirsen.

Fig. 1 RNAse H independent mechanisms to prevent mRNA translation by ASO. Step 1. Binds to 5’ cap; Step 2. Binds to poly A tail; Step 3. Stearic hindrance; and Step 4. Modifies exon splicing.

The mechanism of ASO action is shown in Fig. 1. There are many hurdles to incorporating ASOs in therapeutic use, because of their mechanism of cellular uptake and action (Box 1). Thus, modifications are needed on the native ASOs to overcome these disadvantages, and this has led to various versions for clinical use.

These are obtained by replacing one of the non-bridging oxygen atoms in the phosphate group of nucleotide with either sulfur groups (phosphorothioates), methyl groups (methylphosphonates) or amines (posphoroamidates). Phosphorothioate substitution was the earliest and the most commonly used modification that renders the inter-nucleotide linkage resistant to nuclease degradation, supports endogenous RNAse H activity to degrade the target mRNA, improves the pharmacokinetic characteristics by their binding with plasma proteins which alter the half-life and increases the availability of ASO to the target site. The other type is thiophosphoramidate substitution, though it had many side-effects in animal models and　in vitro　experiments [2,8].

Second generation ASOs were developed to overcome the shortcomings of first-generation ASOs. These second-generation antisense agents, contain a Phosphorothioate backbone and replacement of the 2-hydroxyl by many different groups but most commonly by 2’-O-methoxy (OMe), 2’-O-methoxyethyl (MOE), and locked nucleic acid (LNA). 2’-OMe modifications are commonly used in a ‘gapmer’ design, which is a chimeric oligo nucleotide comprising a DNA sequence core with flanking 2’-MOEnucleotides.2-Methoxyethyl is probably the most common one used in trials. Their mechanism of action is RNAse independent. The advantages of these ASOs are improved nuclease resistance, target-binding affinity, increased thermal stability of complementary hybridization, encourages tighter binding and allowing use of shorter oligonucleotides [2,4,7].

These are modified ASOs which help in their intracellular uptake and effective delivery to the target. These ASOs are covalently bound to a carrier or ligand, such as lipid particles, liposomes, nanoparticles, and, more recently, the sugar N-acetyl galactosamine to enhance safer delivery to the target site [2,9,10].

The rationale for understanding the pharmacokinetics of ASOs is to correlate effective dose with clearance rates to optimize effectiveness while reducing potentially harmful side-effects [6]. The route of administration of these drugs is parenteral – intravenous, subcutaneous and intrathecal because oral bioavailability is less than 1% [11].

Absorption: The pharmacokinetic properties of oligonucleotides following parenteral administration are predominantly studied in phosphorothioate oligo-nucleotides, which after parenteral administration, bind to plasma proteins at >90% and transfer rapidly from blood to tissues with a distribution half-life of 1-2 hour. By 12 hour after dosing, <1% of the administered dose remains in circulation. At the same time, <5% of the administered dose is recovered in urine and feces over the first day, and this broad distribution to tissues causes the rapid disappearance of compound from blood. The highest tissue accumulation has been observed in kidney, liver, spleen, lymph nodes, adipocytes and bone marrow. In marked contrast, those oligonucleotides lacking charge and/or binding to plasma proteins (peptide nucleic acids, morpholinos) are rapidly cleared from plasma, with substantially higher excretion in urine in the first day resulting in lower overall tissue accumulation and ultimate target bioavailability [12].These drugs do not cross the blood-brain barrier and poorly distribute in skeletal muscle, heart, and lung.

Distribution: Although distribution out of plasma to tissue is rapid, the ultimate distribution to the active site within cells following a single dose is maximally realized at 24-48 h. Thus, the onset of action for antisense oligonucleotides is slower than the distribution out of plasma, which is explained due to the intracellular uptake and the kinetics for transport from the cell surface to the nucleus. Once distributed to cells, they are slowly cleared with tissue half-life ranging from 2-4 weeks [11,13].

Metabolism: These drugs are metabolized by endonucleases, which are expressed in most tissues hence liver dysfunction does not appear to affect their action. These drugs are not substrates for cytochrome P450 enzymes, and hence a very low drug-drug interaction is known [11].

Excretion: Excretion is predominantly renal and fecal; biliary uptake is minimal.

The most common adverse reactions of nusinersen are (>10%) upper and lower-respiratory tract infections (39%-43%), atelectasis (14%), constipation (30%), headache (50%), back pain (41%), and post-lumbar puncture syndrome (41%). Its high cost and route of administration are its major limitations [22,26].

Mipomersen: Mipomersen is the first FDA-approved systemically-delivered ASO in 2013 for familial hypercholesterolemia [16]. This is a disease caused loss of function mutations in both LDL-receptor genes which results in the reduced liver uptake of plasma LDL cholesterol leading to a very high plasma concentration of low-density lipoprotein. The core protein of the LDL particle is apolipoprotein B [27]. Mipomersen is targeted to the coding region of the apoB mRNA which effectively reduces plasma LDL and cholesterol levels with less deleterious effects on HDL. Side effects are injection site reactions and liver toxicity [28].

Other drugs include Defibrotide for severe hepatic veno-occlusive disease occurring after high dose chemotherapy and autologous bone marrow transplantation [29], and Revusiran, Patisiran and Inotersen for Transthyretin amyloidosis [30-32].

Oligonucleotides are prone to a diverse array of off-target interactions because of their size, negative charge, and potential to be synthetic [4]. Thus, despite a good overall safety profile, a few adverse reactions are encountered due to their off-target effects [33]. However, the number of studies and the sample size included are too small to determine the general side effect profile, the dose relationship and class effect for these drugs.

Binding of nucleic acid to cell surface proteins or to proteins inside cells -oligonucleotides can bind serine/threonine protein kinase PKR or Toll-like receptors (TLRs) and activate the innate immune response/alternate complement pathway. These can also bind to dRNA and DNA by complementary base-pairing. Vasculitis or glomerulonephritis are rare manifestations of immune activation [9]. All ASOs and dsRNAs will be at least partially complementary to DNA or RNA sequences inside cells that are not their intended targets and thus can modify the actions of genes on these mRNA/DNA, which could be harmful [4].

Thrombocytopenia is one of the most common side-effects seen [34]. Two forms of thrombocytopenia are studied. The more common form is milder, transient and dose-dependent wherein bleeding episodes are very rare. In humans, thrombocytopenia has been reported in cancer studies with a number of first-generation ASOs and occasionally with second-generation ASOs such as Mipomersen [35]. Other is rarer and severe form with bleeding episodes [36].

Despite remarkable progress in the development of ASOs for clinical use, the cost remains a major limitation for widespread use. For Eteplirsen, the costs were estimated at US$ 57,600 (INR 38,59,000) per month [37]. For Nusinersen, the cost of treatment of a patient with SMA amounts to US$ 750,000 (INR 5,02,50,000) for the first year, and half of that every year afterward [38,39].Thus the high costs are a major setback to any healthcare system plus the poor validity of the clinical trials in showing efficacy and adverse effects do not give a definitive risk-benefit advantage.

The invention of ASOs represents a therapeutic milestone in those diseases for which we do not have a definitive cure by modifying the disease pathways. ASOs are under development or have already been tested in clinical trials for the treatment of many other diseases like myotonic dystrophy, Huntington chorea, Amyotrophic lateral sclerosis, Hemophilia A, Hereditary neuropathies. There have been some demands from individual patients and patient-support groups in LMICs (including India) to permit use of these drugs through publicly-funded programs. Although these drugs have good safety and tolerability, their high cost, route of administration, localized target (not applicable to all variants of disease), and lack of significant clinical trials describing mechanism of action, target sites, efficacy, and side effects are the major limitations. Hence, further research is required to better elucidate these important aspects, before wide-spread use would be a possibility.

Contributors: Both authors were involved in planning the manuscript and review of literature. Initial draft: AVK; Final manuscript: AVK, DM. Both authors approved the final manuscript.

Funding: None; Competing interest: None stated.

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