Delivery Approaches for RNA Therapeutics
One of the significant advantages of oligonucleotides is their precise ability to interact with target molecules via complementary Watson-Crick base pairing. This mechanism allows for easy identification of target sequences, facilitating the design of highly specific compounds based on the primary sequence of a target gene. Lead candidates can be rapidly identified through swift screening processes, offering a stark contrast to the extensive and iterative screening typically required for conventional small-molecule drugs.
Delivering RNA therapeutics effectively to the right cells without triggering the body's immune defenses is one of the biggest challenges in the field. Regardless of their specific functions or mechanisms, all RNA-based treatments need to navigate a complex biological landscape. They must reach the intended tissues, enter the correct cells within those tissues, and escape internal cellular traps called endosomes, all without being destroyed by the body's immune system or other biological processes.
While smaller RNA molecules like antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and ADAR-linked oligonucleotides can be chemically stabilized and delivered using specialized molecular conjugates, larger molecules like mRNA and DNA require a delivery vehicle to enter cells. One of the most successful innovations in this area has been the development of lipid nanoparticles (LNPs). These tiny, fat-based particles can encapsulate RNA, shielding it from degradation while enhancing its delivery into specific cells. This technology was crucial in the development of effective COVID-19 mRNA vaccines and represents a significant advance in the delivery of RNA drugs.
Further enhancing the precision of these treatments, recent developments in targeting ligands allow RNA therapies to home in on specific cell types. These ligands are molecules designed to bind to particular receptors on the surfaces of target cells, ensuring that RNA therapies are delivered directly to diseased cells while healthy cells are left unaffected.
Chemical Modifications of ASOs and siRNAs
Chemical modifications on different parts of the RNA molecule can enhance specificity, improve resistance to degradation by enzymes (i.e., nucleases), and boost delivery to target cells, while also reducing toxicity and immune system activation.
One of the earliest and most common modifications is the phosphorothioate (PS) backbone. In this modification, one of the oxygen atoms in the phosphate group is replaced with sulfur. This change helps the molecules enter cells more easily and increases their stability in the body by making them more hydrophobic and resistant to breakdown by enzymes. It also improves their interaction with proteins in the blood. For example, the PS backbone is used in the drug Mipomersen, which treats familial hypercholesterolemia by lowering cholesterol levels.
Modifying the 2' position of the sugar molecule in the RNA backbone is another key strategy. This includes adding groups like 2'-fluoro (F), 2'-methoxyethyl (MOE), and 2'-O-methyl (O-Me). These modifications enhance the binding strength to the target RNA and raise the temperature at which the RNA strands separate. They also stabilize the RNA against degradation by enzymes and help prevent the immune system from mistakenly attacking the therapeutic RNA. All siRNAs currently in clinical trials have either 2'-F or 2'-O-Me modifications because of these benefits. The drug Patisiran, approved for treating hereditary transthyretin-mediated amyloidosis, utilizes 2'-O-Me modifications to enhance stability and efficacy.
Locked nucleic acids (LNA) and their derivatives, such as constrained ethyl (cEt), are also widely used. These modifications lock the RNA structure into a specific shape, making the molecule more stable and improving its ability to bind to target sequences. This stability is particularly useful for various therapeutic RNA types, including those designed to alter splicing or silence specific genes. While LNA modified oligonucleotides have very high binding affinity for both DNA and RNA targets, they also have the potential to increase liver toxicity.
More complex modifications of LNAs include phosphorodiamidate morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). These modifications completely change the backbone structure while keeping the base-pairing intact, leading to increased stability and effectiveness of the therapeutic RNA. Eteplirsen, a PMO used to treat Duchenne muscular dystrophy, exemplifies how these modifications can enhance drug stability and specificity.
These chemical modifications are crucial for advancing RNA therapeutics, making them more reliable and efficient in treating various diseases.
Chemical Modifications of mRNA, CRISPR-Cas Guide RNAs, and Aptamers
mRNA used for therapeutic purpose is typically produced using in vitro transcription (IVT). These IVT mRNAs are single-stranded RNA, containing several key structures: a 5′ cap, an open reading frame (ORF), 5′ and 3′ untranslated regions (UTRs), and a 3′ poly(A) tail. Optimizing mRNA-based therapeutics often involves tweaking these various RNA structures. This can be done through sequence optimization, nucleoside modification, or substituting sequences in the UTRs to enhance the mRNA’s ability to be translated into proteins. For instance, adding modified nucleosides like pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methylcytidine (m5C) helps minimize immune responses that can be triggered by IVT mRNA. These modifications make the mRNA more stable and less likely to be attacked by the body's immune system.
CRISPR-Cas systems are used to make specific edits within DNA and RNA. To do this, they use guide RNAs to find and bind to the precise location to make these genomic edits. CRISPR-Cas systems face several challenges, including efficient delivery, off-target effects, and potential immune responses. [Chemical modifications similar to those used in ASOs and siRNAs](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729442/#:~:text=Co-delivering chemically modified sgRNAs,as CRISPR-Cas9 (refs.) can be applied to guide RNAs (gRNAs) and Cas9 mRNA to address these issues. For example, modifications like 2′-O-M-3′PS (MS), 2′-O-M, or 2′-OM-3′thioPACE (MSP) can be added to the ends of single-guide RNAs (sgRNAs) to improve genome editing efficiency in human cells. Modifying the ribose-phosphate backbone of gRNAs with 2′-OM-3′-phosphonoacetate (MP) reduces off-target effects while maintaining high accuracy. Additionally, altering the 5′ end of sgRNAs can enhance activity and help evade immune responses. These modifications improve the precision and efficiency of CRISPR-based gene editing.
Aptamers, like ASOs and siRNAs, benefit from chemical modifications that enhance their pharmacokinetic properties. Most clinical aptamers undergo modifications such as adding polyethylene glycol (PEG) at the 5′ end to resist kidney clearance, capping the 3′ end with inverted thymidine to prevent degradation, and substituting the 2′ position on the sugar ring to protect against nuclease activity. These modifications improve the stability and binding affinity of aptamers, making them more effective for therapeutic use.
Lipid-Based Nanoparticles (LNPs) for Delivery
Liposomes are tiny, bubble-like structures formed when phospholipids are mixed with water. Phospholipids have a water-loving (polar) head and a fat-loving (non-polar) tail. In water, they arrange themselves into spheres with their tails pointing inward and their heads facing outward, creating a protective layer around a water-filled core. This unique structure makes liposomes excellent vehicles for delivering RNA therapeutics and other drugs. They can be engineered with different surface features to carry a range of therapeutic substances directly to specific parts of the body, making them highly versatile for different targeted approaches.
One of the earliest therapeutic uses of lipid nanoparticles (LNPs) was Doxil, a liposome-encapsulated version of the chemotherapy drug doxorubicin, developed by Chezy Berenholz. This innovative formulation was designed to treat cancer while significantly reducing the heart toxicity often associated with doxorubicin. This breakthrough demonstrated how nanoparticles can enhance drug distribution in the body, thereby improving safety and efficacy.
Combining liposomes with positively charged molecules known as cationic lipids allows them to effectively encapsulate negatively charged mRNA through electrostatic attraction. Adding cholesterol to the mix enhances this setup, making the liposome-mRNA complex more stable and efficient at delivering the mRNA into cells.
However, liposome delivery systems have several drawbacks. Liposomes can be less stable, leading to fusion or leakage of RNA, resulting in inefficient delivery. They also encapsulate only small amounts of RNA and can become harmful if they undergo oxidation. Additionally, size variability in liposomes can lead to inconsistencies in absorption and distribution in the body, posing a challenge for maintaining consistent quality in production.
Several of these hurdles have been addressed through advances in surface modifications of liposomes. LNPs made of cationic and other lipids, cholesterol, and polyethylene glycol (PEG) with a hydrophilic inner core have improved capacity to carry anionic RNA, protect it from degradation, and prolong its circulation. A notable example is Onpattro (patisiran) from Alnylam Pharmaceuticals, which was discussed above. Another example is the PEG-liposome mRNA vaccines developed by BioNTech and Moderna for COVID-19.
N-Acetylgalactosamine (GalNAc)
RNA can be delivered to specific cells through a method known as "active targeting." This approach involves attaching a ligand—a molecule that binds to a specific receptor—to the delivery system. One highly effective ligand is GalNAc, a carbohydrate-derived molecule that targets the asialoglycoprotein receptor (ASGPR). ASGPR is abundantly present on the surface of a specific type of cell in the liver, hepatocytes, but not on other cell types. When GalNAc binds to ASGPR, it triggers the cell to quickly take in the complex through a process called endocytosis, then return the receptor to the cell surface to be used again. This specificity and efficiency make ASGPR an ideal target for delivering therapeutic RNA directly into liver cells.
The most clinically validated examples of this method are GalNAc–siRNA and GalNAc–ASO conjugates, which have led to FDA-approved drugs like givosiran, lumasiran, and inclisiran. Givosiran (marketed as Givlaari by Alnylam Pharmaceuticals) is the first-ever GalNAc-conjugated siRNA drug, approved by the FDA in November 2019. It is used to prevent acute hepatic porphyria attacks by reducing the production of aminolevulinate synthase 1 mRNA in the liver. This reduction lowers the levels of neurotoxic substances that can lead to severe symptoms such as seizures, paralysis, respiratory failure, and neurological damage, significantly improving patient outcomes.
Similarly, lumasiran and inclisiran, approved by regulatory bodies like the FDA and EMA, demonstrate the effectiveness of GalNAc conjugation for targeting liver cells. These advancements highlight the potential of GalNAc in enhancing the precision and efficacy of RNA-based therapies for liver diseases.
Targeted Delivery Outside the Liver
Small ligands have also been developed for delivery beyond hepatocytes and outside of the liver.
In a study measuring the distribution throughout the body mediated by different siRNA–lipid conjugates, researchers found that hydrophobic lipid conjugates accumulated in the liver, whereas less hydrophobic conjugates accumulated in the kidneys. In the same study, the researchers found that compared to cholesterol (commonly used for targeting liver cells), dichloroacetic acid alone and combined with a phosphocholine head group were better at delivering siRNA to tissues outside the liver, like the lungs and heart, and also somewhat better at reaching skeletal muscle and fat.
A 2021 study demonstrated that cholesterol-functionalized DNA-RNA heteroduplexes could cross the blood-brain barrier in rodents following systemic administration. The study highlighted that the chemical makeup of the lipid and its lipid conjugate played a critical role in targeting specific cells, silencing genes, and influencing drug biodistribution within the body. Additionally, RNA aptamers, which are RNA molecules that fold into precise three-dimensional shapes, have been gaining attention for their potential in targeted drug delivery. For instance, researchers have successfully attached an siRNA that targets STAT3—a crucial protein involved in regulating glioblastoma—to an RNA aptamer that targets the receptor tyrosine kinase PDGFRα. This innovative delivery system was effective in reducing the expression of targeted genes and significantly decreased cell viability in laboratory tests.
A unique approach to targeting specific cell types involves the use of antibodies or antibody fragments. Avidity Biosciences employs this method to target muscle diseases. For instance, small interfering RNA (siRNA) and antisense oligonucleotides (ASO) have been successfully delivered to tissues outside the liver using conjugates made from antibodies linked to siRNA. One notable application involved using an antibody fragment targeted against CD71 to deliver siRNA to the heart and skeletal muscle. This technique resulted in prolonged silencing of the target gene in these tissues, demonstrating the potential of antibody-mediated delivery to enhance the precision and effectiveness of RNA-based therapies.
At Duet BioTherapeutics, we have harnessed the potential of CpG oligodeoxynucleotides (CpG ODNs) to specifically target immune cells within the tumor microenvironment (TME). CpG ODNs activate the immune system by engaging toll-like receptor 9 (TLR9), a critical receptor involved in immune surveillance. These molecules are particularly adept at targeting myeloid cells such as dendritic cells, macrophages, and myeloid-derived suppressor cells (MDSCs)—key players in the TME. They achieve this through rapid uptake by scavenger receptors, which are abundantly expressed on these cell types.
Our innovative therapeutic, DUET-101, links a CpG ODN with a STAT3 antisense molecule. When this conjugate is absorbed by the scavenger receptors and enters the cell via an endosome, the CpG component binds to TLR9. This interaction not only triggers an immune response but also facilitates the release of the molecule into the cell’s cytosol. Once in the cytosol, the STAT3 antisense portion of the molecule binds to STAT3 mRNA, effectively reducing its activity. By knocking down STAT3 expression, our drug lifts the suppressive effects STAT3 has within the TME. This action restores communication between myeloid cells and CD8+ T cells, empowering these T cells to recognize and destroy cancer cells effectively. This targeted approach exemplifies a precision strategy to re-engage and direct the body’s own immune system against cancer.
Conclusion: Delivering RNA Therapeutics
RNA therapeutics hold great promise for treating a wide range of diseases due to their ability to precisely target and modify gene expression. The introduction of chemical modifications, lipid nanoparticles, and ligand conjugates has significantly enhanced the stability, specificity, and delivery of RNA molecules, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and messenger RNAs (mRNAs).
Various chemical modifications, such as phosphorothioate (PS) backbones and 2' sugar modifications, have improved the stability and efficacy of ASOs and siRNAs. These modifications help the molecules resist degradation by nucleases, reduce toxicity, and enhance binding affinity.
In vitro transcribed (IVT) mRNA, along with guide RNAs used in CRISPR-Cas systems, also benefit from chemical modifications. These changes help minimize immune responses and improve the stability and efficiency of these therapeutics. Aptamers, like other RNA-based therapies, have seen improvements through chemical modifications that enhance their pharmacokinetic properties. Modifications such as PEGylation and 2'-substitutions increase their stability and effectiveness in clinical applications.
LNPs, including liposomes, play a crucial role in delivering RNA therapeutics. Liposomes, composed of phospholipids, can encapsulate RNA and protect it from degradation. Despite challenges like stability issues and size variability, innovations in LNP design have led to successful drugs like Onpattro (patisiran) and the COVID-19 mRNA vaccines.
Active targeting using ligands like GalNAc has proven highly effective for delivering RNA to liver cells. GalNAc binds specifically to the asialoglycoprotein receptor (ASGPR) on hepatocytes, facilitating efficient RNA delivery. This method has led to FDA-approved drugs such as givosiran, lumasiran, and inclisiran, which treat conditions like acute hepatic porphyria by targeting specific mRNAs in the liver.
Beyond the liver, RNA can be targeted to other tissues using different ligands and conjugates. For example, hydrophobic lipid conjugates tend to accumulate in the liver, while less hydrophobic conjugates accumulate in the kidneys. Cholesterol-functionalized DNA-RNA heteroduplexes can cross the blood-brain barrier, offering potential for treating neurological conditions. RNA aptamers have also shown promise in targeting specific proteins involved in diseases such as glioblastoma. Antibody-conjugated siRNAs and ASOs, like those developed by Avidity Biosciences, can target muscle tissues, demonstrating prolonged gene silencing.
Overall, advancements in chemical modifications and delivery systems have significantly enhanced the potential of RNA therapeutics. These innovations enable precise targeting, improved stability, and efficient delivery, paving the way for new treatments for a variety of diseases.