Advanced Drug Modalities Part 2: Advanced RNA Therapeutic Modalities
Article summary
Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), are rapidly evolving advanced drug modalities with a number of FDA-approved moieties on the market.
ASO and siRNA therapeutics are relatively short oligonucleotides (typically 20-25 nucleotides) that target disease-relevant mRNA molecules, altering the protein products of mRNA translation or targeting mRNA for degradation and eliminating protein expression altogether.
Advantages of ASO and siRNA therapeutics include high specify for their targets, relatively simple structure, standardized chemical synthesis with short development times, the potential for long-lasting effects after a single administration, and the ability to reduce the expression of any protein, including proteins that are typically regarded as “undruggable”.
ASOs and siRNAs offer a precision medicine approach in which the therapy can be customized based on the individual's unique genetic profile. These RNA-based therapeutics can be designed to target various types of genetic variations for the potential treatment of rare genetic disorders.
While ASOs and siRNAs have a number of advantages, they also face challenges and limitations. The main challenges associated with using unmodified ASOs and siRNA in their natural form have to do with the susceptibility of RNA to degradation and its immunogenicity and many of these limitations have been overcome via the use of chemical modifications. Nevertheless, challenges still remain to be addressed for these therapeutic modalities to become more broadly implemented and target a wider range of conditions, including improving systemic circulation, targeting specific organs and tissues beyond the liver, and enhancing intracellular release among others.
RNA Drug Modalities
Drug modalities continuously evolve, from traditional small molecules synthesized for the first time over a century ago to cutting-edge modalities such as cell therapies. In a series of articles we overview drug modalities as a function of their maturity, ranging from established drug modalities like small molecule drugs and monoclonal antibodies, through more advanced modalities like CAR-T cell therapies and antibody-drug conjugates (ADCs), to the emerging modalities including genome and RNA editing approaches and mRNA therapeutics. In this article, we focus on the advanced RNA modalities antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) and review their mechanism of action, FDA-approved moieties, advantages, and limitations.
The last several decades witnessed the emergence of a number of RNA therapeutic modalities, including antisense oligonucleotides (ASOs), aptamers, circular RNAs (circRNAs), small interfering RNAs (siRNAs), microRNAs (miRNAs), anti-microRNAs (antimiRNAs), messenger RNAs (mRNAs), small activating RNAs (saRNAs), long non-coding RNAs (lncRNAs), CRISPR-based genome editing, and ADAR-based RNA editing among others. Depending on their technological maturity and extent of clinical application, RNA therapeutic modalities can be classified as advanced and emerging modalities. ASO and siRNA therapeutics are already in the clinic helping patients while continuing to mature as drug modalities and can thus be classified as advanced modalities. The remaining RNA therapeutic modalities are still undergoing development, with a modest overall number of FDA approvals, and are yet to prove themselves in the clinic.
Antisense Oligonucleotides and Small Interfering RNAs are Leading in the Clinic
The main idea behind the use of ASOs and siRNAs as therapeutics is to identify disease-relevant mRNA molecules and target these molecules with complementary oligonucleotides, altering the protein products of mRNA translation or eliminating the protein expression altogether (Figure 1). Structurally, ASOs and siRNAs therapeutics are both based on relatively short oligonucleotides (typically 20-25 nucleotides). The main difference between these two modalities is that while siRNAs are typically double-stranded RNA (dsRNA), ASOs are single-stranded (ssRNAs).
What is messenger RNA? Messenger RNA (mRNA) is a type of RNA molecule that carries the instructions for protein synthesis from the DNA to the ribosomes. During protein synthesis, the sequence of nucleotides in the mRNA is translated into a corresponding sequence of amino acids by the ribosome, ultimately leading to the production of proteins that carry out the various cellular functions.
How do Antisense Oligonucleotide Therapeutics Work?
ASOs are designed to selectively bind to target mRNA molecules in the cell and thus modulate gene expression through various mechanisms, with two of the most clinically relevant mechanisms being mRNA degradation and modulation of pre-mRNA splicing.
ASOs induce mRNA degradation by specifically binding to complementary mRNA sequences and forming an ASO:mRNA duplex. The duplex formation leads to the recruitment of RNase H, a ribonuclease enzyme that cleaves mRNA, ultimately leading to its degradation and preventing the expression of the corresponding protein (Figure 1, top left).
ASOs alter pre-mRNA splicing by hybridizing with specific intron or exon regions in the pre-mRNA molecule. This interaction can either promote the inclusion or exclusion of particular exons, modifying splicing patterns and allowing for the correction of aberrant splicing events associated with genetic disorders or the generation of alternative splice variants for therapeutic purposes (Figure 1, bottom left).
What is RNA splicing? RNA splicing is a cellular process involved in the maturation of mRNA molecules. Initially, genetic information is transcribed from DNA into a precursor mRNA molecule (pre-mRNA) which contains both exons (protein-coding regions) and introns (non-coding regions). RNA splicing involves the removal of introns and the precise joining of exons. The splicing process ensures that the genetic information is accurately transcribed, allowing for the synthesis of functional proteins during translation in the cytoplasm. Aberrant splicing of pre-mRNA is associated with disease conditions.
Figure 1. Main mechanism of action of antisense oligonucleotides (ASOs, left) and small interfering RNAs (siRNAs, right). Mechanisms are simplified. Top left: ASO RNAse H-mediated RNA degradation. ASOs promote mRNA degradation by recruiting RNAse H after recognition of their mRNA target. Bottom left: ASO pre-mRNA splicing modulation. ASOs modulate pre-mRNA splicing by interacting with various exon and intron regions. Right: siRNAs target mRNAs for degradation . siRNA drugs are typically processed by the nuclease Dicer to generate activated siRNAs. The guide strand of siRNA, together with several proteins, form an RNA-induced silencing complex (RISC). The guide strand acts as a template to recognize target mRNA molecules, which are then cleaved by RISC.
The Evolution of Antisense Oligonucleotides as a Therapeutic Modality
The development of ASOs as therapeutics has achieved significant milestones over several decades. The concept of antisense technology emerged in the 1970s, involving the use of single-stranded nucleic acids to modulate gene expression by binding to complementary RNA sequences. Extensive research in the 1980s identified chemical modifications that improved ASOs stability and selectivity (see below) and enabled their clinical development. The first ASO therapeutic, fomivirsen, was approved by the FDA in 1998 for the treatment of cytomegalovirus retinitis, a groundbreaking milestone for the development of ASO therapeutics. Subsequent years saw the approval of ASO-based therapies for various conditions (Table 1). Ongoing research continues to expand the applications of ASOs to new indications.
Table 1. List of approved antisense oligonucleotide (ASO) therapeutics, together with their mechanism of action, indication, and year of approval.
How do Small Interfering RNA Therapeutics Work?
1998 marked the discovery of RNA interference (RNAi) – the post-translational silencing of genes induced by double-stranded RNA (dsRNA). A few years later, in 2001, followed the discovery that 21-22 dsRNA nucleotides can induce RNAi silencing in mammalian cells, laying the foundation for the development of small interfering RNA (siRNA) therapeutics.
Therapeutic siRNAs, typically 20-25 nucleotides in length, are designed to specifically target and silence the expression of messenger RNA (mRNA) in cells (Figure 1, right). The siRNA duplex consists of two strands: the guide strand, which is selectively loaded into the RNA-induced silencing complex (RISC), and the passenger strand, which is typically degraded. The guide strand is complementary to the target mRNA and guides the RISC using this sequence complementarity. Once bound to its target mRNA, RISC catalyzes the cleavage and degradation of the target mRNA. This process effectively prevents the translation of the mRNA into proteins, offering a precise mechanism for gene silencing.
The Evolution of Small Interfering RNA as a Therapeutic Modality
The development of siRNA therapeutics is marked by several cycles of major milestones and significant drawbacks. The discovery of RNAi by Andrew Fire and Craig Mello and the subsequent Novel Prize in Physiology or Medicine in 2006 fueled enthusiasm for the therapeutic potential of siRNAs. An early breakthrough came in 2004 when the first siRNA-based drug candidate, bevasiranib, entered clinical trials. Despite promising results, the drug failed to demonstrate clinical efficacy. In the following years, it was found that siRNAs could have off-target effects by silencing unintended genes with partial sequence similarity. It was also found that siRNAs could induce unwanted innate immune responses, leading to safety concerns. Developmental challenges related to delivery, safety, and efficacy, and commercial challenges underscored the difficulties in bringing siRNA therapeutics to patients. The field re-gained momentum in 2018 with the FDA approval of patisiran for the treatment of hereditary transthyretin-mediated amyloidosis. This marked a historic achievement as the first-ever siRNA therapeutic to receive FDA approval. Subsequent years saw several FDA approvals of siRNA therapies for various indications (Table 2), further validating the therapeutic potential of siRNAs.
Table 2. List of approved siRNA therapeutics, together with their indication and year of approval.
Understanding the Advantages of Antisense Oligonucleotide and Small Interfering RNA Drug Modalities
ASOs and siRNAs offer a range of advantages, making them versatile modalities addressing challenges encountered by established modalities such as small molecules and protein therapeutics.
Simple Structures. Small molecules and antibodies often require targets with complex structures to bind to and exercise their therapeutic effects. ASOs and siRNAs do not require such complex structures and directly interact with their mRNA targets via base pair formation. Further, while many small molecules have complex structures themselves, and antibodies are large proteins, ASOs and siRNAs are relatively simple oligonucleotides.
High Specificity. ASOs and siRNAs can have higher specificity than small molecules and antibodies. While the later bind to protein surfaces that are not always unique, ASOs and RNAs specificity is solely defined by the often unique sequences of their RNA targets.
Targeting Undruggable Proteins. Many proteins are undruggable by small molecules because they lack well-defined pockets that small molecules typically bind to. ASOs and siRNAs overcome this limitation by design. Genes are transcribed into mRNAs which are subsequently translated into proteins by the ribosome. ASOs and siRNAs induce the cleavage of their target mRNAs, leading to target mRNA degradation and preventing the synthesis of the corresponding proteins altogether.
Target Versatility. The mechanisms of action of ASOs and siRNAs are not limited to targeting protein-coding mRNAs. These modalities can also target non-coding RNAs, many of which carry out or regulate various biological functions and can be associated with various diseases. Thus, ASOs and siRNA modalities can be applied in a wide range of therapeutic areas.
Short Development Times. Because all ASOs and siRNAs are highly similar single-strand or double-strand RNA molecules that mostly differ in their RNA sequence, once the exact RNA sequence, chemical modifications pattern (see below), and method of delivery are defined, the development of these modalities is relatively fast. This is in contrast to small molecule and antibody modalities, which often require years of extensive research and development.
Standardized Synthesis. The synthetic chemistry required for the production of ASOs and siRNAs is relatively standard, contributing to their relatively short development times. This is often in contrast to the synthetic chemistry of small molecule drugs that can be complex and can require development.
Precision Medicine for Rare Diseases. Rare diseases are often associated with unique genetic variations. ASOs and siRNAs offer a precision medicine approach in which the therapy can be customized based on the individual's unique genetic profile. These RNA-based therapeutics can be designed to target various types of genetic variations, including point mutations, insertions, deletions, or splice site mutations. This versatility allows for the potential treatment of a wide range of rare genetic disorders. The relatively short development times for ASOs and siRNAs further contribute to their potential as personalized medicines.
Long-Lasting Effects. When the stability of RNA is improved (e.g., via chemical modifications, see below), ASOs and siRNA modalities can have long-lasting therapeutic effects and a single administration can lead to sustained therapeutic benefits over an extended period. For example, the silencing effect of inclisiran, a siRNA drug (see Table 2), was present for more than six months after a single administration.
Navigating and Overcoming the Limitations of Antisense Oligonucleotides and Small Interfering RNAs
While ASOs and siRNAs have a number of advantages over other drug modalities, they also face unique challenges and limitations. The main challenges associated with using unmodified ASOs and siRNA in their natural form as therapeutics have to do with the inherent susceptibility of RNA to degradation by ribonuclease enzymes, poor cellular uptake, limited systemic circulation, poor tissue and organ selectivity, and the immunogenicity of RNA molecules. Many of these limitations have been overcome, which enabled the development of new FDA-approved therapies, while others are yet to be addressed.
RNA is Susceptible to Degradation. The phosphodiester bond of RNA is inherently susceptible to cleavage by ribonuclease enzymes in physiological conditions. After administration in the body, RNAs get rapidly degraded by these enzymes. The inherent susceptibility of RNA to degradation leads to rapid degradation of unmodified ASOs and siRNAs when administered into patients, limiting their efficacy.
RNA is Immunogenic. When administered in the body, RNA molecules are recognized by immune system receptors such as Toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). The recognition of RNA by the immune system triggers an intense immune response with unintended physiological effects. Because of this immunogenicity, unmodified RNAs in their natural form present potential safety liabilities.
Poor Cellular Uptake. RNA is negatively charged due to the presence of phosphodiester bonds. Because of this charge, unmodified ASOs and siRNAs in their natural form cannot cross the cell endosome membranes effectively, limiting their efficacy.
Rapid Clearance from Circulation. Unmodified siRNAs and ASOs can be rapidly excreted from the body via renal clearance, limiting their exposure to target tissues. This rapid clearance results in a reduced concentration of therapeutic molecules, limiting efficacy.
What is Renal Clarence? Renal clearance is the process by which the kidneys remove molecules from systemic circulation in the body. Renal clearance plays a central role in determining the efficacy of a drug since rapid clearance limits the exposure of target tissues and organs to a drug. Decreasing renal clearance allows a drug to circulate in the body for longer, increasing the likelihood of reaching targets and exerting therapeutic effects.
The inherently susceptibility of RNA to degradation, poor cell membrane crossing, the immunogenicity of RNA molecules, and their rapid clearance from the systemic circulation are limitations that have been overcome via the use of chemical modifications and various delivery systems.
Chemical modifications of RNA’s sugar help reduce immune system activation and improve nuclease resistance. For example, the 2’ position of the sugar can be chemically modified to contain 2’-fluoro (2’-F), 2’-O-methyl (2’-ME), 2’-O-[2-methoxyethyl] (2’-MOE) substitutions.
Chemical alterations of the phosphodiester backbone help reduce the rate of renal clearance, lead to more efficient cellular uptake, and improve ribonuclease resistance. Examples of phosphodiester backbone modifications include the substitution with phosphorothioate (PS) and methylphosphonate (MP) linkages, and peptide nucleic acid (PNA) linkages.
Chemical modification of the base can reduce ASOs and siRNAs immunogenicity while improving nuclease resistance. Notable examples include the substitution for pseudouridine, 2-thiouridine, and N6-methyladenosine.
Conjugation of ligands to ASOs and siRNAs help reduce renal clearance and help target specific organs and tissues. For example, the conjugation of GalNAc results in targeting delivery to the liver (e.g., givosiran and lumasiran) due to the recognition of GalNAc by the specific and highly expressed on hepatocytes asialoglycoprotein receptors.
The use of nanoparticles and polymers for the delivery of ASOs and siRNAs can reduce immune system activation and renal clearance while improving nuclease resistance and cellular uptake. A notable example is the use of lipid nanoparticles (LNPs) as delivery vehicles.
While ASO and siRNA therapeutic modalities already help patients in the clinic, challenges still remain to be address for these therapeutic modalities to become more broadly implemented and target a wider range of conditions. Some of the main remaining challenges include:
Improve vascular escape.
Target specific organs and tissues beyond the liver.
Improve systemic circulation.
Facilitate endosomal escape.
Address safety concerns associated with LNP-based delivery methods.
What is vascular escape? Vascular escape refers to the ability of drugs, after administration in the body, to exit blood vessels and reach target tissues. It is an essential aspect of achieving effective drug delivery to specific tissues, organs, and cell types within the body. The relatively large size and the propensity of ASOs and siRNAs to bind to serum albumin limit their vascular escape, limiting their efficacy.
What is endosomal escape? ASOs and siRNAs cannot effectively cross the cell membrane and are instead typically taken up by cells via endocytosis in endosomes. Endosomes are membrane-bound compartments that are typically involved in sorting and trafficking of materials inside cells. Endosomal escape refers to the process during which therapeutic molecules exit endosomes to enter the cell cytosol and bind their targets. Because endosomes, like cells, are also enveloped by lipid membrane, ASOs and siRNAs typically remain entrapped within endosomes.
Intense research and development is already suggesting ways for these challenges to be overcome. For example, GalNAc conjugation alternatives and antibody conjugates are being assessed for the targeting of specific tissues beyond the liver, peptide conjugates are being evaluated to improve endosomal escape, and exosomes are being explored as alternative delivery vehicles with improved systemic circulation and safety profiles. Overcoming these limitations is expected to improve clinical efficacy and broaden the scope of indications for these RNA drug modalities.
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