The ability to make synthetic DNA and RNA by chemical or enzymatic means has revolutionized genetic research and drug discovery. Antisense technology employs short, chemically synthesized oligonucleotides (antisense oligonucleotides, or ASOs) that are made in an “antisense orientation” to the target cellular RNA so that they will bind or anneal to that RNA and be used to alter gene expression. The method was first described in the late 1970s by Lasker Award winner Paul Zamecnik, PhD, and Mary Stephenson, PhD, who used unmodified DNA ASOs to inhibit viral RNA translation in the Rous sarcoma virus.
The next decade saw rapid innovation and improvement in antisense methods, largely through incorporation of chemical modifications into the ASO that impart nuclease stability and increase binding affinity, both of which improve potency. Based on the pattern of chemical modification employed, antisense has split into two primary modalities:
1) ASOs that direct degradation of the target RNA (typically an mRNA) and
2) Those that function as steric blockers and do not lead to RNA degradation.
Degradative ASOs typically contain a stretch of DNA residues; when the ASO anneals to a messenger RNA (mRNA), an RNA/DNA heteroduplex structure is formed that is a substrate for degradation by cellular RNase H. This application of antisense leads to functional down-regulation of gene expression by removing an mRNA species, thereby reducing protein translation. The ability to specifically reduce gene expression has been an important research tool used in understanding gene function. It allows for the development of antisense therapeutics, where the ASO is a drug that lowers expression of a gene to treat diseases that are caused by the presence of a mutant gene product or by gene overexpression.
Steric blocking ASOs typically are made entirely of chemically modified residues that are both resistant to degradation and have high binding affinity; without a DNA domain, these compounds do not lead to RNA degradation. If binding affinity is sufficiently high, annealing of the antisense compound can block interaction of the RNA with proteins, changing the biological function of that RNA. One significant application of steric-blocking antisense is targeting specific sites within nuclear hnRNA (unspliced pre-mRNA) to alter splice patterns so that the final mRNA is changed to either delete or include a specific exon. This class of antisense compounds are called SSOs (splice shifting oligonucleotides).
Not surprisingly, antisense methods have been a significant area of drug development research in the 45 years since its discovery. The first FDA-approved ASO therapeutic was Fomivirsen (Vitravene), which was introduced in 1998 to treat CMV retinitis in patients with AIDs. (The drug was withdrawn in 2001; introduction of HAART antiviral therapy reduced the incidence of AIDS CMV retinitis and Fomivirsen was no longer needed.) After that, a series of ASO clinical trials failed, often due to insufficient efficacy. As with any new therapeutic drug modality, it took years for researchers to optimize the compounds and understand what the best gene targets and diseases were for success.
ASOs on the rebound
I joined Integrated DNA Technologies (IDT) in the early 2000s, when enthusiasm for ASO therapeutics was waning, largely due to efficacy and safety concerns. At that time, a new modality for suppressing gene expression (i.e., gene knockdown) was discovered, RNA interference (RNAi). RNAi exploits an entirely different mechanism of action, yet, like antisense, can be mediated by synthetic oligonucleotide compounds and target any gene based on sequence. A sizeable fraction of drug development energy shifted from antisense to RNAi, but, like antisense, this even newer therapeutic drug modality required years to fully optimize compounds and understand suitable targets and diseases.
ASO research and drug development efforts have made a comeback as knowledge of optimal reagent design (e.g., chemical modification) and target selection has improved. Further, certain advantages were observed for ASOs when targeting RNA species in the cell nucleus, especially for long-noncoding-RNAs, which are responsible for regulating gene transcription and post-translational modifications and may be novel targets. During the 2010s, several ASO-class therapeutics were approved by the FDA, including Nusinersen for spinal muscular atrophy, and Eteplirsen and Casimersen for Duchenne muscular dystrophy (all three are SSO class drugs, a pathway not available using RNAi). Importantly, patents covering some of the key chemical modifications employed to make the most safe and effective ASO compounds expired, including 2′-O-(2-methoxyethyl) and LNA, encouraging more groups to join the effort to develop antisense therapeutics.
ASO therapeutics are particularly valuable in nano-rare genetic disorders. The nonprofit n-Lorem Foundation has a mission to develop and deliver ASO therapeutics to patients at no cost. It was founded in 2020 by Stanley Crooke, PhD, who pioneered the development of ASO therapeutics as CEO of Ionis Pharmaceuticals. More ASOs have been given to humans in either clinical trials or as an approved therapeutic than any other class of oligonucleotide drug. As such, there is a greater understanding of the safety profile and risks of ASOs, making them ideal as the first line therapy in the quest for treatment of rare genetic disorders, where extensive testing prior to patient administration is not feasible.
The future of ASOs and oligonucleotide-based drugs
Today, 45 years and 25 years after the discovery of antisense and RNAi, respectively, 13 drugs of this type have been approved. This includes nine ASOs (four degradative ASOs and five SSOs) and four siRNAs.
In spite of these successes, more work is needed. Like any next-generation therapeutic, balancing cellular uptake and potency with minimum off-target effects (OTEs) and toxicity remains a challenge. Finding the optimal target ASO-RNA binding site that fulfills the above criteria requires a combination of methods. ASO-RNA hybridization can be hindered by many factors, from RNA secondary structures to their interactions with intracellular nucleic acids and proteins, which can impact ASO accessibility to the target RNA. Candidates for ASO drug development must be screened both in cells and in animal model systems to identify suitable compounds, and hundreds to even thousands of ASOs are screened before moving lead compounds forward.
Verdict: ASOs—a mainstay in genetic medicines
Today, our “gene targeting arsenal” includes many options ranging from ASO and RNAi to CRISPR-Cas9 and newer genome editing platforms. Each has its advantages and disadvantages. As the founding technology of this class of agents, ASOs have the best characterized safety profile in humans. I am hopeful that the combination of synthetic chemistry, bioinformatics, and computational tools will help pave the way for potent, selective, and non-toxic ASOs as a cornerstone of future genetic medicines.
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