GeNeViSTA

1 Abstract

Rapid progress in the field of molecular biology has led to the development of numerous genetic therapies. Among these, antisense oligonucleotide (ASO) therapeutics have recently gained momentum due to their application in the spectrum of disorders ranging from neurodegenerative disorders to malignancies. This brief review discusses the principle of ASO based therapies, mechanism of action and their current role in the field of therapeutic medicine.

2 Introduction

Antisense Oligonucleotide (ASO) therapeutics is a well-recognized class of drugs exploiting the Watson and Crick’s base pairing rules to target disease-related RNAs. Although the concept of using synthetic oligonucleotides to modulate RNA function dates back to 1978, the anticipated clinical success was achieved only after recent advances in genomics, chemistry and pharmacology.

Oligonucleotides are unmodified or chemically modified single stranded DNA molecules which are 8-50 bp in length. They hybridize to target RNA and alter its original function through an array of mechanisms. With the knowledge of gene sequences, ASOs directed at specific target sequences are being utilized to understand gene functions. Simplicity of the concept has led to its use in knock-down experiments, target validation, drug therapy, and other applications. The same principle is applicable to the use of ASOs in therapeutics. Another advantage of ASOs is the reversibility of effects as opposed to gene therapy and genomic editing. The current success in the treatment of neuromuscular disorders especially spinal muscular atrophy has proved the potential of ASO based therapies.

The major hurdles in the designing of ASOs include rapid degradation by intracellular exonucleases and endonucleases, inefficient uptake in certain tissues, nonspecific effects and adverse immune responses. However, these issues are being actively addressed to enhance efficacy and specificity of ASOs.

3 Mechanisms of action of ASOs

ASOs modulate the transfer of genetic information to protein in multiple ways-

a)

RNase-H mediated site specific degradation of mRNA: This remains the most utilized antisense mechanism despite major advances in the field of RNA biology (Crooke et al., 2018). RNase-H causes degradation of DNA/RNA heteroduplex when DNA based ASO binds to its RNA targets. Activation of RNase-H is extremely sequence specific. Mismatch of ≥ 3 base pairs results in complete loss of RNase-H activation. Hence extreme caution is required in designing ASO for this action. Nevertheless, this remains the most efficient mechanism which causes 80-95% downregulation of mRNA and protein expression and acts effectively even when targeted at any region of mRNA (Dias et al., 2002)

b)

Steric block of ribosome binding: This includes interruption of RNA translation by preventing the movement of ribosomes onto mRNA thereby inhibiting assembly of 40s, 60s ribosomal subunits.

c)

Modulation of splicing: Some ASOs function by binding to regulatory sequences, masking splicing enhancers or repressor sequences causing exon skipping and forcing inclusion of otherwise alternatively spliced exons. ASOs can also modulate polyadenylation selection in those transcripts with > 1 poly A site at 3’ untranslated region (Vickers et al.,2001) This in turn creates alternative transcript and increases mRNA stability and alters protein expression.

d)

Targeting miRNA and Natural Antisense Transcript (NAT): Recently discovered ASOs are designed in such a way that they can directly bind to miRNA and NAT and prohibit them from binding to their own mRNA specific targets. This in turn causes upregulation of genes targeted by miRNA and NAT (Davis et al., 2009)

3.1 Generations of ASOs

1^st generation ASOs: These compounds have phosphorothioate backbone only, limiting the function to RNase-H degradation. In addition, these compounds have nonspecific interaction with cell surface and intracellular proteins (Kurreck, 2003).

2^nd generation ASOs: These compounds have 2’-sugar modifications like 2’-O-methyl and 2’-O-methoxyethyl (MOE) additions. This makes them resistant to degradation by cell nucleases and increases their affinity and target specificity.

3^rd generation ASOs: These include Locked Nucleic Acid (LNA) and Morpholino modifications. Morpholinos display greater potency in altering splicing and inhibiting translation in vivo but do not activate RNase-H. LNA compounds exhibit enhanced potency and are known for their robust binding improvement and nuclease resistance when compared to other 2’ modified compounds (Swayze et al., 2007).

3.2 Delivery of Oligonucleotides to Cells

Adsorptive endocytosis and fluid phase pinocytosis appear to be the major mechanisms for oligonucleotide internalization. The proportion of internalization depends on the concentration of oligonucleotide. At low concentrations, the likely mechanism of internalization occurs via interaction with membrane bound receptors. At high concentrations, the receptors get saturated and pinocytic process assumes greater importance.

3.3 Applications in the field of therapeutic medicine

The first ASO approved for clinical use was Fomivirsen for cytomegalovirus retinitis. Since then numerous oligonucleotides targeting a wide spectrum of disorders have been studied in various clinical trials. A summary of the various ASOs approved by United States Food and Drug Administration (USFDA) is given in Table I. The recent approvals of Eteplersin for Duchenne muscular dystrophy and Nusinersen for SMA are briefly reviewed.