Monday Article #72: Antisense Oligonucleotides (ASOs): The Medicine of the Future
Antisense oligonucleotides (ASOs), first discovered back in 1978 have been consistently rising in popularity in tandem with the popularization of precision medicine. These molecules, though tiny, harbor massive potential in the revolutionization of medicine due to their ability to combat a myriad of diseases at their starting point: our genetic source code.
The concept behind ASOs
In our cells’ nucleus, there exists our genetic information in the form of DNA. In order for our genes to carry out their functions, the DNA has to be transcribed into mRNA, which has to be translated into proteins (Clancy and Brown, 2008). Many diseases are influenced by genetic factors through their role in abnormal production of proteins or production of abnormal proteins (SoRelle, 2000).
Understanding the concept of ASOs requires some basic understanding of genetics. Nucleotides are the building blocks of DNA, consisting of a nitrogenous base, a sugar molecule, and a phosphate backbone. DNA has four types of nitrogenous bases, thymine, guanine, cytosine, and adenine. DNA is made up of two strands of nucleotides in an antiparallel fashion whereby cytosine pairs with guanine, while thymine pairs with adenine in a complementary manner. RNA is similar to DNA but is usually single-stranded and has a different sugar molecule, and uracil replaces thymine (Minchin and Lodge, 2019).
Therapeutic ASOs are around 18-30 DNA or RNA base pairs in length and differ in their mechanisms but most act through the same concept. ASOs pair complementarily to mRNA strands and cause changes to the mRNA, leading to modifications in protein formation.
Mechanisms of ASOs
ASOs are flexible molecules as they can work through various mechanisms determined by their structure. Once bound complementary to mRNA, the DNA-ASOs form an RNA-DNA hybrid that becomes a substrate for RNase H1, an enzyme that hydrolyzes the RNA strand of an RNA–DNA duplex, resulting in mRNA degradation (Wu et al., 2004).
ASOs can also block the binding of RNA-binding protein complexes, which stops the translation process from mRNA into proteins (Baker et al., 1997). After the first transcription from DNA to mRNA, the mRNA will be spliced into exons and introns. Introns are non-coding regions whereas exons are coding regions that will be translated into proteins (Clancy, 2014). ASOs can bind to introns splicing silencers to inhibit them. This results in the inclusion of certain exons in the mRNA post-splicing. On the other hand, ASOs can bind to exons splicing enhancers, which results in the exclusion of certain exons (Havens and Hastings, 2016).
Figure 1: Overview of the mechanisms of ASOs. Image taken from Antisense oligonucleotides (Scoles, Minikel and Pulst, 2019).
Evolution of ASOs
The first ASOs to be deployed showed limited clinical potential because of the high susceptibility of ASOs with an unmodified phosphoribose backbone to rapid degradation by endonucleases and exonucleases (Bennett et al., 2017). The large size and charge of the phosphoribose backbone also restrict entry into the cell via passive diffusion (Dowdy, 2017).
Over the years, many modifications have been made to ASOs to increase their efficacy and safety. In phosphorothiorate (PS) ASOs, one of the non-bridging oxygen is replaced with sulfur to form a PS bond, which results in higher resistance to nuclease degradation (Eckstein, 2014). PS-ASOs also bind strongly to albumin which increases its circulation time (Crooke and Bennett, 1996). In phosphorodiamidate morpholino oligomers (PMOs), a different sugar molecule is used to replace the original one (Summerton and Weller, 1997). PMOs are neutral and exert their effects mainly by hindering translation or splicing modulation (Rinaldi and Wood, 2017). Peptide nucleic acids (PNAs) are synthetic nucleic acid mimics that contain neutral N-2-aminoethyl glycine units, with nucleobases connected by a flexible methyl carbonyl linker. PNAs are resistant to enzymatic degradation and possess a strong binding affinity with RNA sequences as compared to unmodified ASOs (Pellestor and Paulasova, 2004). Locked nucleic acids (LNAs) 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 (Braasch and Corey, 2001). Due to the neutral charge, LNAs primarily act by inhibiting translation and modulating splicing (Kaihatsu, Janowski and Corey, 2004). However, gapmers, which consist of LNA-DNA-LNA sequences can also stimulate RNAse H1-based cleavage (Marrosu et al., 2017). Modification of the 2′ position of the sugar molecule such as in MOE-ASOs can also increase resistance to degradation. That being said, MOE-ASOs reduce plasma protein binding, shifting their excretion to the kidneys, which may pose a nephrotoxic effect (Geary et al., 2001). Once again, gapmers that combine MOE-ASOs and unmodified ASOs can strike a balance between efficacy and safety for optimal usage (Hebb and Robertson, 1997).
Figure 2: Modifications of ASOs. Image taken from Antisense oligonucleotides (Scoles, Minikel and Pulst, 2019).
Clinical applications of ASOs
As of writing this blog article, 18 ASOs drugs have been approved by the FDA. Discussing all of them will definitely exceed the scope of this article, so here I will describe the function of a few of said ASOs.
The first-ever approved ASOs is fomivirsen which is indicated for cytomegalovirus retinitis. Fomivirsen is a 21-member oligonucleotide with a phosphorothioate linkage. It blocks the translation of the virus’ mRNA that codes for the protein IE2. It is injected intravitreally (into the organ), so as to prevent toxicity in the systemic circulation (Perry and Barman Balfour, 1999).
Figure 3: Mechanism of fomivirsen. Image taken from Quizlet.
Mipomersen is an MOE-ASO used to treat homozygous familial hypercholesterolemia. Before diving in, some clarification on the terms going to be used. Cholesterol is transported throughout the systemic circulation in lipoproteins such as high-density lipoproteins (HDL), low-density lipoproteins (LDL), and others (Feingold and Grunfeld, 2018). Certain lipoproteins can be retained in the arterial wall and cause plaque buildup, and subsequently atherosclerosis. All these atherogenic lipoproteins carry an apolipoprotein B100, except chylomicrons and chylomicron remnants which have apolipoprotein B48, on their surface to stabilize their structure (Kriško and Etchebest, 2006). Mipomersen is complementary to the apolipoprotein B100 mRNA and when bound to it causes mRNA degradation through RNAse H1-mediated mRNA cleavage. This reduces the amount of apolipoprotein B100 molecules produced, reducing atherogenic lipoprotein production (Hamada and Farzam, 2023).
Figure 4: Mechanism of mipomersen. Image taken from The Role of Antisense Oligonucleotide Therapy in Patients with Familial Hypercholesterolemia: Risks, Benefits, and Management Recommendations (Agarwala, Jones and Nambi, 2014).
Casimersen is an ASO of the PMO subclass used to treat Duchenne muscular dystrophy (DMD). Duchenne muscular dystrophy is characterized by mutations in the dystrophin gene and subsequently protein that results in partial or complete absence of the protein. This protein usually anchors cytoskeleton proteins to proteins of the myofibrillar membrane. When absent, muscular contractions are weak which leads to characteristic symptoms of DMD, namely weakness (Davies and Nowak, 2006). Casimersen binds to the exon splicing enhancer on exon 45, resulting in the exclusion of exon 45 in the dystrophin mRNA. The dystrophin protein produced from this modified mRNA is truncated but is more functionally robust than the mutated version (Zakeri et al., 2022).
Figure 5: Mechanism of casimersen. Image taken from Casimersen for the treatment of Duchenne muscular dystrophy (Zakeri et al., 2022).
In conclusion, ASOs represent a revolution in precision medicine with unbounded potential due to their highly flexible yet selective nature. That being said, we need to have realistic expectations regarding ASOs and more research has to be done regarding hard outcomes, side effects, and other potential applications of ASOs.
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Article prepared by: Jared Ong Kang Jie, R&D Director of MBIOS 2023/2024