What is an Antisense Oligonucleotide?

An antisense oligonucleotide (ASO) is a synthetically constructed single-stranded sequence. It is categorized as a nucleic acid therapy designed to target and bind complementary RNA strands to inhibit protein production in cells. Their mechanisms of action vary depending on their design.

Therapeutic Potential of Antisense Oligonucleotides

Antisense oligonucleotides have the capacity to impact gene expression and the generation of proteins. They manifest their influence via distinct mechanisms, such as impeding the translation of target RNA into protein or fostering its deterioration. They can also regulate alternative splicing, a process enabling the production of multiple protein variations from a single gene. Antisense oligonucleotides present an excellent avenue for addressing genetic disorders by selectively targeting disease-causing genes. They have exhibited therapeutic potential in various conditions, encompassing neurodegenerative diseases, muscular dystrophy, and specific forms of cancer. Antisense oligonucleotides hold the potential for tailored medical interventions, as they can be customized to an individual patient’s condition. Ongoing research and advancements in antisense oligonucleotides technology persistently broaden ASO applications and enhance their efficacy in gene modulation.

Antisense oligonucleotides provide a flexible framework for tackling a broad spectrum of hereditary disorders by modulating gene expression and protein generation. They hold the potential to modulate disease-causing genetic mutations at the RNA level, enabling precise and targeted treatments. Antisense oligonucleotides have displayed encouraging outcomes in preliminary and clinical examinations, exhibiting effectiveness in various conditions, including neurodegenerative disorders, uncommon genetic ailments and specific categories of malignancies. Their adaptability, combined with advancements in delivery systems and the refinement of chemical properties, has elevated antisense oligonucleotides to the forefront of drug discovery, providing novel prospects for formulating innovative and personalized therapies.

Antisense Oligonucleotides Mechanisms of Action

After antisense oligonucleotides enter a cell, they bind to their target pre-mRNA or mRNA with high specificity to prevent the protein encoded by a gene from being expressed. They can induce degradation of the target RNA through RNase H-mediated cleavage or block protein synthesis by preventing mRNA translation. ASOs can also affect gene expression at the transcriptional level by interfering with splicing or inducing chromatin remodeling.

Antisense oligonucleotides can act through several mechanisms, including exon skipping, splicing modulation, mRNA degradation, translational repression and chemical modification, depending on the specific target and design of the antisense oligonucleotides.

Exon Skipping

During exon skipping, an antisense oligonucleotide selectively attaches to a specific exon within the pre-mRNA molecule, leading to the omission of said exon from the ultimate mature mRNA transcript. This sequence of events has the potential to reinstate the proper reading frame of a mutated gene, allowing for the synthesis of a partially operative protein. Exon skipping has exhibited encouraging potential in diverse hereditary conditions, particularly those triggered by distinct mutation types such as nonsense mutations and frame-shift mutations.

Exon skipping has been explored in alternative contexts, such as specific varieties of muscular dystrophy and atrophy (DMD, SMA, and others), beta-thalassemia, and cystic fibrosis. In these instances, targeted exon mutations can be circumvented, thereby reinstating protein functionality or preserving gene expression equilibrium. Continuous research endeavors strive to enhance ASO design, devise effective delivery modalities, and guarantee the safety and efficacy of these approaches across various target tissues.

Splicing Modulation

Splicing modulation serves as a mechanism entailing the interaction between an antisense oligonucleotide and a precise splice site present within the pre-mRNA molecule. This interaction instigates alterations in the splicing patterns, influencing the proportion of alternatively spliced isoforms. By manipulating splicing events, this mechanism presents a potential avenue for rectifying anomalous splicing occurrences linked to diseases.

Splicing modulation can be deployed in the therapeutic management of SMA and other diverse genetic disorders caused by mutations influencing splicing sites (for instance, specific forms of inherited retinal diseases).

Additionally, splicing modulation has exhibited promising implications in conditions such as beta-thalassemia, a hematologic disorder characterized by the abnormal production of hemoglobin. Through the modulation of splicing patterns, antisense oligonucleotides possess the capability to stimulate the incorporation of specific exons, thereby restoring the production of functional hemoglobin.

mRNA Degradation

mRNA degradation, as a mechanism utilized by antisense oligonucleotides, involves the precise binding of an ASO to a specific mRNA molecule. This binding event triggers the recruitment of an enzyme called RNase H, which acts upon the targeted mRNA, inducing its degradation. By facilitating mRNA degradation, this mechanism presents a potential avenue for diminishing the expression of genes implicated in disease pathogenesis.

The application of mRNA degradation through antisense oligonucleotides encompasses a wide array of diseases, encompassing genetic disorders, viral infections, forms of cancer and autoimmune conditions. Continuous research endeavors aim to optimize ASO design and delivery methodologies, striving to augment the specificity and efficacy of mRNA degradation, thereby paving the path for innovative therapeutic interventions.

Translational Repression

Translational repression encompasses the precise binding of an antisense oligonucleotides to a specific mRNA molecule. This binding event culminates in the inhibition of ribosome recruitment and subsequent synthesis of proteins. By hindering the process of translation, this mechanism presents an opportunity to curtail the expression of proteins implicated in disease pathogenesis.

The applications of translational repression through antisense oligonucleotides span various diseases, including genetic disorders, viral infections and neurodegenerative conditions. Optimizing ASO design for enhanced target selection leading to optimal specificity and efficacy of translational repression is a constant process.

The choice of the mechanism depends on the specific disease indication and target gene, as well as considerations for safety and efficacy.

Modulation Mechanisms of Antisense Oligonucleotides in the Nucleus and Cytoplasm

Antisense oligonucleotides are designed to bind to specific mRNAs through complementary base pairing, leading to the modulation of gene expression both in the nucleus and cytoplasm. This binding event blocks the interaction between the mRNA and RNA-binding proteins, such as ribosomes and splicing factors, which play essential roles in gene regulation. By creating a "steric block," ASOs occlude the RNA-binding sites on the mRNA, resulting in reducing mRNA processing and translation efficiency. This effectively silences the gene encoded by the mRNA. The steric blockage mechanism provides a targeted approach to modulating gene expression, allowing for precise silencing of disease-causing genes. These modulation mechanisms occur in both the nucleus and cytoplasm, ensuring comprehensive control over gene expression and offering potential therapeutic benefits.

Effect of Antisense Oligonucleotides on Polyadenylation

Antisense oligonucleotides, depending on their design and target mRNA, can exert various effects on polyadenylation. They have the ability to prevent polyadenylation by binding to the polyadenylation recognition site within the nucleus, inhibiting the addition of the poly(A) tail to the mRNA. This interference disrupts mRNA translation and alters RNA stability, leading to reduced levels of the targeted mRNA and protein. In neurodegenerative diseases, ASOs have shown promise in modifying mRNA polyadenylation and translation, resulting in therapeutic effects. For instance, in neuroblastoma cells, antisense oligonucleotides targeting NEAT1 lncRNA polyadenylation events led to the upregulation of differentiation pathways and suppressed tumorigenesis. Similarly, in spinal muscular atrophy (SMA), ASOs have been utilized to correct cryptic splicing and polyadenylation defects, restoring protein expression and cellular function.

The mechanism of action of ASOs on polyadenylation involves their binding to target RNAs and interfering with the polyadenylation process. In the nucleus, antisense oligonucleotides can bind to the polyadenylation recognition site, preventing polyadenylation by nuclear ribonuclease (RNase) H. Additionally, ASOs can also bind to translation initiation sites of mRNAs in the cytoplasm, inhibiting translation.

It is essential to consider that ASOs' effects on polyadenylation can be influenced by other factors, including translation and the presence of specific RNA-binding proteins. The recruitment of RNase H1 to ASO/mRNA heteroduplexes can be a rate-limiting step, and translating ribosomes may inhibit this process. Moreover, the depletion of U1 snRNP with antisense morpholino oligonucleotides (AMOs) may lead to intronic premature cleavage and polyadenylation (PCPA) of genes.

Effect of Antisense Oligonucleotides on Splicing

Antisense oligonucleotides have been extensively researched for their impact on splicing regulation. They can modulate splicing patterns by targeting specific sequences within exons or introns, leading to changes in exon inclusion or skipping. One notable example is their use in correcting aberrant splicing of the SMN2 gene in spinal muscular atrophy (SMA). ASOs targeting SMN2 exon 7 have been successful in promoting exon inclusion, resulting in increased production of full-length functional SMN protein.

The effectiveness of Antisense oligonucleotides in splicing modulation depends on the design of the antisense portion, which needs to be optimized for the desired splicing outcomes. Various antisense oligonucleotides with different lengths and target positions have been tested to identify the most effective ones. For instance, ASO 02-16 showed a slightly negative effect on exon 7 inclusion in vivo, while ASO 07-21 or 34-48 sequences were predicted to be more efficient in promoting exon inclusion.

Antisense oligonucleotides have also been used to induce splicing defects in animal models, allowing researchers to mimic splicing-associated diseases like SMA. By causing sustained splicing defects with designed Antisense oligonucleotides, scientists gain valuable insights into the pathogenesis of splicing-related disorders and evaluate potential therapeutic approaches.

Beyond SMA, ASOs have been investigated for their impact on splicing in other diseases like cystic fibrosis (CF). antisense oligonucleotides targeting specific mutations in the CFTR gene have shown promise in blocking aberrant splicing and improving chloride secretion in CF patient-derived cells. This highlights the potential of ASOs as a therapeutic strategy for correcting splicing defects in CF and other genetic disorders.

Effects of Antisense Oligonucleotides on Polyadenylation, Splicing, Translation, and Cleavage

Antisense oligonucleotides exert diverse impacts on gene expression processes, encompassing polyadenylation, splicing, translation and cleavage. ASOs possess the ability to influence polyadenylation by obstructing or modifying the interaction between polyadenylation factors and the target mRNA. Antisense oligonucleotides can modulate alternative splicing patterns via binding to specific splice sites, either encouraging or inhibiting the inclusion or exclusion of exons, thereby yielding distinct mRNA isoforms. ASOs can further impede translation by binding to mRNA, preventing ribosome recruitment and subsequently hindering protein synthesis. Additionally, antisense oligonucleotides can instigate mRNA cleavage by engaging enzymes such as RNase H, culminating in the degradation of the targeted mRNA molecule. These versatile effects of antisense oligonucleotides on polyadenylation, splicing, translation, and cleavage furnish a versatile repertoire for gene expression modulation, thereby presenting potential avenues for therapeutic applications.

ASO Delivery Methods and Vehicles

Methods of ASO Delivery

Delivery of antisense oligonucleotides as therapeutics to patients can be achieved through several methods, including

• Direct injection into target tissues
• Systemic administration through intravenous injection or subcutaneous injection
• Local administration through inhalation or topical application.

Delivery Vehicles

Various delivery vehicles, such as liposomes, nanoparticles and viral vectors, have been developed to improve ASO stability, targeting efficiency and cellular uptake. Additionally, chemical modifications of antisense oligonucleotides can enhance their pharmacokinetic properties and reduce their immunogenicity.

The choice of delivery method and vehicle depends on the specific ASO target and the disease indication, as well as considerations for safety, efficacy and manufacturing scalability.

While ASO therapy is effective without additional processing, lipid-based delivery systems have been developed to improve the therapeutic index of antisense oligonucleotides. These delivery systems can increase the intracellular concentration of antisense oligonucleotides, target them to specific cell types and protect them from degradation. Lipid-based delivery systems, including liposomes, can also be used to modulate the activity of ASOs.

Nanoformulation-based delivery systems have also been developed that enable antisense oligonucleotides to penetrate cell membranes and reach their targets intracellularly. These delivery systems typically consist of liposomes or other nanoparticles encapsulating the ASOs and further protecting them from degradation.

Factors to Consider When Designing ASOs

Hybridization Site Selection

When designing antisense oligonucleotides, meticulous consideration should be bestowed upon hybridization site selection to guarantee precise binding to the intended target mRNA while minimizing off-target repercussions.

Stability Enhancements

To enhance ASO stability, modifications such as phosphorothioate backbone or locked nucleic acids (LNAs) can be assimilated to safeguard against nuclease degradation.

Immunostimulation Minimization

During ASO design, immunostimulation should be minimized by eliminating CpG motifs and other immune-stimulatory sequences to diminish the likelihood of undesirable immune responses.

Sequence Length Optimization

Sequence length should be optimized to ensure effective target binding while mitigating non-specific interactions, with thorough consideration of ASO and target mRNA characteristics.

Avoidance of Self-complementarity

Self-complementarity should be minimized to avert the formation of secondary structures that may result in off-target effects or compromised ASO efficacy.

Assessment of G-quartet Structures

The presence of G-quartet structures should be thoughtfully assessed, as they can impact ASO stability and binding specificity, potentially influencing target engagement and efficacy.

Incorporation of Functional Motifs

Functional motifs within the ASO sequence can be incorporated to modulate gene expression or instigate specific desired effects.

Binding Affinity Optimization

Optimizing the binding affinity between the antisense oligonucleotide and the target mRNA is of paramount importance to ensure efficient target engagement and effective modulation of gene expression.

Antisense Oligonucleotide Therapy Applications

Antisense oligonucleotide therapy can potentially be used for many genetic disease applications. Targeting specific genes can lessen the toxicity effects common with traditional drugs. Antisense oligonucleotides can be directly administered with or without the need for liposome-related carriers, which reduces ASO manufacturing time and costs.

Examples of clinically approved uses of ASO therapy include:

Duchenne Muscular Dystrophy (DMD)

Eteplirsen is an ASO that is designed to treat DMD, a genetic disorder characterized by progressive muscle weakness and wasting. DMD is caused by mutations in the dystrophin gene, which leads to a deficiency in the dystrophin protein that is essential for maintaining the structural integrity of muscle cells.

Eteplirsen works by using exon skipping to restore the reading frame of the dystrophin mRNA, allowing for the production of a partially functional dystrophin protein. Specifically, Eteplirsen targets exon 51 of the dystrophin mRNA, which is mutated in approximately 13% of DMD patients. Eteplirsen binds to the dystrophin pre-mRNA at the site of the exon 51 splice acceptor and promotes skipping of exon 51 duringpre-mRNA splicing. This results in the production of a truncated, but partially functional dystrophin protein that can partially restore the structural integrity of muscle cells.

Spinal Muscular Atrophy (SMA)

Nusinersen is an approved ASO treatment for Spinal Muscular Atrophy (SMA). ASO therapy for SMA works by modulating the expression of the SMN2 gene, leading to increased levels of functional SMN protein and reducing the severity of SMA symptoms. This approach provides a new, targeted way to treat SMA and improve the lives of SMA patients.

SMA is caused by mutations in the SMN1 gene, but patients also have a backup gene called SMN2, which produces a smaller and less functional form of the SMN protein. Nusinersen targets the SMN2 gene and increase its expression, effectively increasing the levels of functional SMN protein in SMA patients. It modulates the splicing of the SMN2 gene, effectively converting the SMN2 gene from a low-expressing to a high-expressing form. This leads to increased levels of functional SMN protein, reducing the severity of SMA symptoms.

Familial Amyloid Polyneuropathy (FAP)

Inotersen is an ASO therapeutic that targets transthyretin amyloidosis (ATTR), a rare genetic disease characterized by the accumulation of abnormal transthyretin (TTR) protein in various organs and tissues.

Inotersen works by binding to the TTR mRNA and inducing its degradation through RNase H-mediated cleavage, resulting in reduced production of TTR protein. By reducing the production of abnormal TTR protein, Inotersen can slow the progression of ATTR and improve symptoms in patients with the disease. In a phase 3 clinical trial, Inotersen was shown to reduce TTR protein levels and improve neuropathy symptoms in patients with hereditary ATTR amyloidosis.

Future Applications of Antisense Oligonucleotide Therapy

In the life sciences, ASOs offer a new, targeted approach to modulating gene expression. Current initiatives and possible applications include the following:

Cancer treatment

ASOs can be designed to target cancer-related processes, such as focusing on specific oncogenes responsible for driving cancer growth. Using an ASO therapy may lead to blocking the production of cancer-promoting proteins.

Neuroscience

ASOs have the potential to target mRNAs involved in neurodegenerative diseases such as Alzheimer's, Huntington's and Parkinson's diseases.

Cardiology

Antisense oligonucleotides can be used to investigate target genes involved in heart disease, which may lead to new treatments for conditions such as hypertension, myocardial infarction and heart failure.

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