The Modalities of RNA Therapeutics
The DNA inside each cell of our body consists of approximately 20,000 protein-coding genes, which accounts for only 1-2% of the entire human genome. From this same DNA come tens of millions of RNA molecules, the fundamental coding sequences for protein synthesis and the regulation of cellular functions. This creates the image that the human cell is simply a bag of RNA, an abstraction that highlights the relative abundance of RNA in cells and its crucial role in understanding health and disease.
This sea of genetic coding material is the frontier of RNA therapeutics, a realm where scientists are no longer just reading the genetic code, but actively rewriting it to combat some of the most challenging diseases we face.
Over the last five years, I’ve worked on a company called Duet BioTherapeutics, where we are developing therapeutics that reprogram the human immune system to weaken the protective mechanisms that allow cancer to grow and spread throughout the body and, subsequently mount a concerted attack against cancer cells. When I first heard about this type of drug called oligonucleotides created by Dr. Marcin Kortylewski, a Professor of Immunology at City of Hope, I recognized that the broad class of RNA therapeutics represented a fundamentally novel approach to treating human disease.
The first RNA drugs have been approved and hundreds more are currently in various stages of clinical development. RNA therapeutics make up a growing category of drugs that will reduce the time needed for development because of their specificity, allow for truly personalized medicine, and make the term “undruggable” a thing of the past.
This will be a three-part series of articles focusing on RNA therapeutics: in this first article, we’ll look at the different modalities and their mechanisms of action; second, we’ll explore the struggle of delivering these delicate molecules in the harsh environment of the human body; and third, we’ll discuss the market opportunity for this class of drug.
Fundamentals of RNA in the Human Body
Before we dive more deeply into RNA therapeutics, it's important to understand the fundamental role of RNA in human cellular biology.
RNA, or ribonucleic acid, is an essential molecule in the synthesis of proteins and the regulation of gene expression. It acts as the messenger that carries instructions from DNA for controlling the synthesis of proteins, which are essential for all living cells and viruses. Each type of RNA, whether it's messenger RNA (mRNA), ribosomal RNA (rRNA), or transfer RNA (tRNA), plays a distinct role in this process. mRNA conveys the genetic blueprints from DNA to the ribosome, where rRNA helps catalyze the assembly of amino acids into protein chains, and tRNA brings the correct amino acid to the ribosome during protein synthesis. The approach of using mRNA to treat human disease was thrust into the spotlight with the rapid development of mRNA-based COVID-19 vaccines, showcasing the speed and scalability of this technology.
Beyond RNA involved in protein synthesis, epigenetic regulatory RNAs like microRNA (miRNA) and small interfering RNA (siRNA) fine-tune gene expression by up-regulating or silencing specific genes. This ability to specifically regulate gene expression is what makes each cell type unique, despite all cells in an organism carrying the same DNA. In essence, it's the differential expression of genes via RNA in various cells that orchestrates the vast diversity of cellular functions and structures found throughout the body.
Understanding how RNA is normally used in the body allows us to appreciate how RNA therapeutics leverage these natural processes to introduce or alter these genetic messages, thereby providing a powerful toolbox to combat disease at its genetic roots. By harnessing the natural roles of RNA, these therapies can mimic, enhance, or suppress these cellular messages, leading to targeted therapeutic effects.
Currently, 30 RNA therapeutics have received approval from the U.S. FDA and other global regulatory agencies, with nine of these approvals occurring in 2023 alone. Note that we did not count approvals for vaccines modified for additional Omicron variants.
List of RNA Therapeutics & Vaccines. This is a comprehensive list of RNA-based drugs market-approved through 2023. You can scroll from left to right for more information about each drug. As you mouse over each drug name, you can click the “open” button to get additional information about the drug in the side view.
Here’s the link to the page that provides a larger view of the RNA Therapeutics & Vaccines database.
RNA therapeutics hinge on a remarkably elegant concept: rather than permanently altering the DNA within a cell's nucleus, these therapies temporarily adjust what proteins are produced by cells. This can be likened to a music conductor directing an orchestra—instead of rewriting the scores of music, the conductor emphasizes or quiets certain sections to shape the performance. In biological terms, RNA therapeutics direct the cellular machinery, enhancing or silencing the expression of genes as needed for therapeutic effect.
The Different Modalities of RNA Therapeutics
In the expanding world of genetic medicine, RNA therapeutics stand out for their versatility and precision in targeting and modulating gene expression.
Each modality offers unique mechanisms and strategies for combating a wide range of diseases, illustrating the innovative scope of RNA as a therapeutic class. This section covers the various modalities of RNA-based treatments in depth, from antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) to the promising realms of microRNAs (miRNAs) and aptamers. If you’d prefer a brief overview of each modality, there is a summary in the conclusions section.
The different modalities of RNA therapeutics. There are six different modalities: RNA aptamers, RNAi (including small interfering RNA, siRNA, and micro RNA or miRNA), antisense oligonucleotides (ASO), messenger RNA (mRNA), CRISPR-Cas9, and circular RNA (circRNA). They all have various functionality that includes inhibition of protein production, protein expression, and genome editing.
Antisense Oligonucleotide (ASO)
Antisense oligonucleotides (ASOs) are short, strands of DNA or RNA that are designed to bind specifically to the messenger RNA (mRNA), the molecules that carry instructions from DNA for making proteins. This binding blocks the mRNA from being used to make proteins, effectively silencing the gene's expression. Unlike other RNA-based therapies like small interfering RNA (siRNA), which typically destroy mRNA via the RNA-induced silencing complex (RISC), ASOs can block protein production without breaking down the mRNA. Additionally, while natural microRNAs regulate many mRNAs at once and non-specifically, ASOs are designed to target very specific mRNAs.
ASOs can be made from various materials, including single-stranded DNA, modified DNA, RNA-like compounds, or specially structured molecules such as locked nucleic acids (LNA) and morpholino oligonucleotides Each type is tailored to bind precisely to its target RNA, blocking its function. This customization allows ASOs to be stable and effective in different therapeutic situations.
ASOs work in three main ways to influence gene expression in the body:
RNA Cutting: ASOs bind to mRNA in a way that recruits an enzyme called RNase H1, which cuts the RNA, silencing the gene. These ASOs are often called "gapmers."
Splicing Modification: ASOs can change how mRNA is processed before it is used to make proteins. By altering this process, ASOs can sometimes increase the production of beneficial proteins, which can help treat diseases like Duchenne muscular dystrophy and spinal muscular atrophy.
Translational Arrest: ASOs can block the machinery that reads mRNA to make proteins by binding to the starting point of the mRNA, directly preventing protein production.
To make ASOs more stable and effective, they can be chemically modified. These modifications protect ASOs from being broken down in the body and improve their ability to bind to their target mRNA.
There are currently 12 ASO-based drugs approved for treating diseases such as spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), and hereditary transthyretin amyloidosis (hATTR). Some of these drugs have become financial blockbusters.
For example, Spinraza, developed by Ionis in partnership with Biogen, treats SMA, a disease caused by mutations in the SMN1 gene. Spinraza generated over $1.7 billion in 2023 alone.
Another example is Exondys 51, developed by Sarepta, which treats Duchenne muscular dystrophy (DMD). DMD is caused by mutations in the DMD gene that produce a non-functional protein called dystrophin. Exondys 51 targets a specific part of the gene (exon 51) to skip over the faulty section, allowing for the production of a shorter but functional version of dystrophin. In 2023, Exondys 51 generated nearly $480 million in revenue.
Small interfering RNAs (siRNAs)
Small interfering RNAs (siRNAs) are short, double-stranded RNA molecules that play a key role in the RNA interference (RNAi) pathway, a natural process used by cells to regulate gene expression and defend against viruses. Unlike antisense oligonucleotides (ASOs), which are typically single-stranded and can block mRNA without degrading it, siRNAs are designed to be double-stranded and about 20-25 nucleotides long to mimic naturally occurring siRNAs. This design helps guide the cellular machinery to the target mRNA, leading to its degradation and suppression of gene expression.
siRNAs work by being incorporated into the protein complex called RISC, described above. Within RISC, one strand of the siRNA, known as the 'sense' strand, is removed, leaving the 'antisense' strand. This remaining strand directs RISC to a specific mRNA molecule that matches its sequence exactly. Once the target mRNA is found, a protein called Argonaute 2 (AGO2) cuts the mRNA, effectively silencing the gene from which the mRNA was transcribed. This precise targeting allows siRNAs to shut down specific genes, making them powerful tools for both scientific research and potential therapeutic applications.
However, siRNAs face challenges such as instability in human blood and the potential to trigger the body's immune system. To address these issues, scientists have developed various chemical modifications to enhance the stability and safety of siRNAs.
One common modification involves altering the ribose sugar in the RNA structure. This can be done by adding a methyl group (2′-O-methyl), a methoxyethyl group (2′-methoxyethyl), or a fluorine atom (2′-fluor). These changes help protect siRNAs from being broken down by enzymes in the bloodstream, thereby extending their functional life. Another approach is to modify the backbone of the siRNA molecule. For example, phosphorothioate modifications replace one of the oxygen atoms in the phosphate backbone with a sulfur atom, enhancing the siRNA's resistance to degradation.
Modifications to the nucleobases themselves, such as adding a methyl group to cytosine (5-methylcytosine), can help reduce unwanted immune responses triggered by siRNAs. These chemical changes collectively improve the therapeutic potential of siRNAs by making them more stable in the body and less likely to cause side effects.
There are currently 6 siRNA-based drugs that have been approved for treating various diseases, including hATTR, hyperoxaluria, and hypercholesterolemia.
Patisiran (marketed as Onpattro by Alnylam Pharmaceuticals) made history in 2018 as the first siRNA-based drug to receive FDA approval. This groundbreaking medication treats polyneuropathy in adults with hATTR, a rare genetic condition that causes abnormal protein deposits in the body's nerves and organs, leading to symptoms like nerve damage. Patisiran works by using the RNA interference (RNAi) mechanism to target and destroy the mRNA responsible for producing transthyretin protein, reducing its harmful accumulation. Despite treating a modest number of patients annually, patisiran generated significant global revenues of $355 million. The subcutaneous version of the siRNA drug, vutrisiran, generated $558 million, bringing the total revenue for Alnylam's TTR drug class to nearly $1 billion in 2023.
MicroRNAs (miRNAs)
MicroRNAs (miRNAs) are tiny, non-coding RNA molecules with a powerful role in controlling gene expression in our cells. They work by regulating the activity of specific mRNAs by preventing mRNAs from being translated into proteins or even by leading to their degradation.
miRNAs are first created from the cell’s DNA as primary miRNAs. These primary structures fold into loops and are then trimmed by an enzyme called Drosha into shorter precursor miRNAs. After being trafficked into the cell's cytoplasm, the enzyme, Dicer, cuts them into even smaller, mature miRNA duplexes, typically 18–25 bases long.
Each miRNA duplex is then loaded into RISC, similar to siRNAs. Here, one strand of the miRNA is removed, leaving the other strand to guide the complex to its target mRNA. The miRNA binds to complementary sequences on the mRNA, usually leading to the suppression of the mRNA's function, either by preventing it from making a protein or by triggering its breakdown.
MicroRNAs (miRNAs) are not just crucial for regulating genes in the body; they are also emerging as potential therapeutic agents in medicine that can be broadly categorized into two types: miRNA mimics and miRNA inhibitors;
miRNA mimics are double-stranded molecules designed to act just like natural miRNAs. By introducing these mimics into the body, scientists aim to boost the levels of specific miRNAs that can help regulate or suppress the activity of disease-related genes, essentially augmenting the body's natural regulatory processes to fight diseases.
miRNA inhibitors, on the other hand, are single-stranded molecules that specifically target and bind to miRNAs, blocking their function. This approach is used when a disease is associated with an overactive miRNA. By inhibiting these miRNAs, the therapy can help restore the normal function of cells.
While there are currently no miRNA-based drugs officially approved and available on the market, several promising candidates are progressing through clinical trials.
One particularly promising miRNA-based therapeutic in development is Cobomarsen (MRG-106), which is being developed by Viridian Therapeutics, formerly known as miRagen Therapeutics. Cobomarsen is designed to target and reduce the levels of miR-155, a microRNA that plays a critical role in the regulation of the immune system and inflammation.
Elevated levels of miR-155 have been linked to various blood cancers, including certain types of lymphoma and leukemia. By specifically targeting and reducing miR-155, Cobomarsen aims to mitigate the abnormal immune responses that contribute to the growth and survival of cancer cells. This targeted approach offers a new avenue for treating blood cancers, potentially providing a more focused and less toxic alternative to traditional therapies that can harm healthy cells as well as cancerous ones.
Aptamers
Aptamers are versatile, single-stranded nucleic acids that act like tiny molecular scouts. Similar to antibodies, these short strands of DNA or RNA are designed to fold into unique three-dimensional shapes that allow them to precisely bind to specific cell-surface target molecules, such as proteins, peptides, carbohydrates, or other molecules. This binding ability is based not just on the sequence of the aptamer but more importantly on its intricate structural configuration. Aptamers are known for their precision and adaptability, making them powerful potential tools in therapeutic applications, disease diagnosis, and targeted drug delivery systems.
Although aptamers hold promise as therapeutics, there are currently no FDA-approved aptamer-based drugs on the market. One aptamer, Macugen (pegaptanib), was approved in 2004 but has since been taken off the market. Macugen specifically targeted and inhibited vascular endothelial growth factor (VEGF), which plays a critical role in the formation of new blood vessels associated with age-related macular degeneration (AMD). Drugs such as ranibizumab (Lucentis) and aflibercept (Eylea), which are also used to treat wet AMD, quickly became preferred over Macugen due to their longer duration of action, better results in enhancing visual acuity, and less frequent administration.
Despite these setbacks, aptamers continue to show promise as alternatives to monoclonal antibodies for both therapeutic and diagnostic uses. They have several appealing advantages: they can be chemically synthesized, making them more cost-effective to produce; they are easier to modify for specific applications; and they typically cause fewer immune reactions compared to antibodies. However, bringing aptamers from the lab to clinical use presents challenges. They are quickly broken down by enzymes in the body and are rapidly cleared by the kidneys, which can limit their effectiveness. Additionally, designing aptamers that effectively target the right molecules is complex and time-consuming, often with low success rates. These hurdles make it difficult to translate the potential of aptamers into practical medical treatments.
Messenger RNA (mRNA)
Messenger RNAs (mRNAs) act as intermediates between the DNA in our genes and the proteins encoded by those genes. Essentially, mRNAs are temporary blueprints of genes that carry instructions from DNA to the cellular machinery that makes proteins.
Synthetic mRNA is a laboratory-made version designed to function just like the natural mRNA produced by our cells. It is a single strand of genetic material that includes a specific region called the "open reading frame," which is the part translated into protein. To help it work effectively in the body, synthetic mRNA has special structures at both ends: a "cap" that starts the translation process and a "tail" made of many A’s (poly(A) tail) that stabilizes the molecule. Once inside a cell, this synthetic mRNA operates in the cytoplasm, directing the production of proteins. It breaks down quickly, usually within minutes to hours, and importantly, it does not integrate into the cell's DNA, meaning it doesn’t make permanent changes to our genetic material.
mRNA is being used in several innovative ways to treat diseases. First, in replacement therapy, mRNA is used to make up for faulty or missing proteins by delivering healthy mRNA to patients, allowing their cells to produce the necessary proteins directly. Second, in vaccination, mRNA that codes for specific disease antigens is administered, prompting the immune system to build a defense against those diseases. Third, in cell therapy, mRNA is introduced into cells outside the patient’s body to change their function or behavior; these modified cells are then returned to the patient’s body to treat or fight disease.
Currently, three prominent biopharmaceutical companies are leading the development of these mRNA therapies: Moderna, based in Boston, Massachusetts; CureVac in Tübingen, Germany; and BioNTech in Mainz, Germany. Each of these companies is at the forefront of pushing the potential of mRNA technology in medicine.
While mRNA has been very successful in vaccines against COVID-19, resulting in FDA approvals of various manufactured vaccines, mRNA has yet to lead to approvals as standalone therapeutics or as part of cell therapy. Despite extensive research and clinical trials exploring its potential beyond vaccines, mRNA faces unique challenges that complicate its use in broader therapeutic contexts. One significant hurdle is the inherent instability of mRNA molecules; they are prone to rapid degradation in the body, which can limit their effectiveness before reaching their target. Additionally, developing efficient and safe delivery systems is crucial yet challenging, as mRNA needs to enter cells to function but must do so without triggering adverse immune responses. These technical obstacles necessitate ongoing innovation in mRNA modification and delivery technologies.
CRISPR-Cas9
Genome editing tools are used to add, remove, or alter genetic material at specific locations in the genome. One of the most notable methods is CRISPR-Cas9, which stands for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9. These RNA-based therapeutics work by using a guide RNA molecule to direct the Cas9 protein to a specific part of a target gene. Once there, Cas9 can make precise cuts, allowing for the removal, addition, or alteration of DNA sections.
This capability has opened up vast possibilities for treating genetic disorders by correcting gene mutations at their source. For example, scientists are exploring CRISPR-Cas9 to potentially treat inherited diseases like cystic fibrosis, sickle cell disease, and even some forms of blindness. This technology could transform medicine by providing more accurate and effective treatments, reducing the burden of disease not just on affected individuals but on entire populations. It ushers in a new era of genetic medicine where the root causes of diseases are directly addressed.
One promising CRISPR-Cas9 therapy in development is EDIT-101, created by Editas Medicine. It is being tested for a rare form of genetic blindness known as Leber Congenital Amaurosis type 10 (LCA10). This condition is caused by a specific genetic mutation in the CEP290 gene, which disrupts normal protein production, affecting the patient's ability to see. EDIT-101 uses precise gene-editing technology to target and correct this mutation. The treatment is delivered directly into the cells of the retina using a safe viral carrier. EDIT-101 aims to cut the faulty DNA segment and utilize the cell's natural repair mechanisms to restore normal function, potentially reversing the effects of the disease. EDIT-101 is currently in a Phase 1/2 clinical trial.
Recently, two CRISPR-based drugs have been approved, marking significant milestones in RNA therapeutics and gene therapy. The first, Casgevy, was approved in December 2023. Developed by Vertex Pharmaceuticals and CRISPR Therapeutics, Casgevy is a groundbreaking gene-editing treatment for sickle cell disease, a disease that affects the body's ability to produce healthy red blood cells. Also in December 2023, the FDA approved Lyfgenia for sickle cell disease, a drug manufactured by Bluebird Bio.
Both of these approved CRISPR-Cas9 drugs use RNA guide strands to target specific genes and a lentiviral vector to deliver the gene-editing components into the cells.
Circular RNA (circRNA)
Circular RNAs (circRNAs) are a unique type of RNA molecule that form a covalently closed loop structure, meaning they have no free ends. This circular shape gives them remarkable stability compared to linear RNAs, making them resistant to degradation by RNA enzymes. This stability makes circRNAs particularly interesting for therapeutic applications.
circRNAs are produced naturally in cells through a process called back-splicing, where a segment of RNA is cut and joined in a loop. They play diverse roles in gene regulation, acting as molecular sponges that bind and sequester microRNAs (miRNAs) or RNA-binding proteins, thereby influencing the expression of other genes. Additionally, some circRNAs can be translated into proteins, adding another layer of functionality.
As of now, the research on circular RNAs (circRNAs) is still in relatively early stages, and there are no circRNA-based therapies that have reached clinical trials. However, the potential of circRNAs in therapeutic applications has generated significant interest in the scientific community and is worth watching. Preclinical studies are actively exploring their use in various diseases, including cancer, neurodegenerative diseases, and cardiovascular conditions.
Conclusion: Pioneering the Future with RNA Therapeutics
RNA therapeutics represent a revolutionary frontier in modern medicine, leveraging the unique properties of RNA molecules to treat a variety of diseases at their genetic roots. Throughout this article, we have explored several key modalities of RNA therapeutics, each offering distinct mechanisms of action and therapeutic potential.
Antisense Oligonucleotides (ASOs) harness the power of synthetic DNA or RNA strands to specifically bind and block messenger RNA (mRNA), effectively silencing disease-causing genes. These versatile molecules can be customized for stability and effectiveness, with several ASO-based drugs already making significant impacts in treating conditions like spinal muscular atrophy and Duchenne muscular dystrophy.
Small Interfering RNAs (siRNAs) utilize a double-stranded RNA approach to guide the cellular machinery in degrading target mRNA, thus preventing the production of harmful proteins. The precision of siRNA technology has led to groundbreaking treatments such as Patisiran for hereditary transthyretin amyloidosis, showcasing the transformative potential of RNA interference.
MicroRNAs (miRNAs) act as natural regulators of gene expression, with therapeutic applications focusing on either mimicking or inhibiting specific miRNAs to restore normal cellular functions. Although no miRNA-based drugs are currently on the market, promising candidates like Cobomarsen are progressing through clinical trials, highlighting the potential of miRNA therapies in treating cancers and other diseases.
Aptamers are short, single-stranded nucleic acids that fold into unique shapes, allowing them to bind precisely to target molecules. Despite challenges in clinical translation, their precision, ease of synthesis, and low immunogenicity position them as promising alternatives to monoclonal antibodies for both therapeutic and diagnostic uses.
Messenger RNA (mRNA) has gained significant attention, particularly through its role in COVID-19 vaccines. These synthetic mRNAs can direct cells to produce specific proteins, offering potential in replacement therapy, vaccination, and cell therapy. While successful in vaccines, mRNA therapeutics face challenges such as stability and delivery, requiring ongoing innovation.
CRISPR-Cas9 represents a cutting-edge approach in genome editing, allowing precise modifications to DNA. This technology has opened new avenues for treating genetic disorders, with recent approvals of CRISPR-based drugs like Casgevy and Exagamglogene autotemcel marking significant milestones in gene therapy.
Circular RNAs (circRNAs) are emerging as a novel class of therapeutics, offering enhanced stability and versatility. Their ability to act as microRNA sponges, protein scaffolds, or even produce therapeutic proteins positions them as a promising tool in genetic medicine, although they are still in early research stages.
In future articles, we will delve deeper into the delivery challenges for RNA therapeutics, exploring the innovative methods being developed to ensure these delicate molecules reach their targets effectively. We will also examine the market opportunity for RNA therapeutics, discussing the economic potential and future growth of this rapidly evolving field.