Self-Amplifying RNA Delivery Systems

Self-amplifying RNA (saRNA) is a novel nucleic acid technology that has emerged as a promising platform for vaccination and gene therapy. Unlike conventional messenger RNA (mRNA), saRNA molecules contain both the coding sequence of the desired protein and the replication machinery of an RNA virus, allowing them to amplify themselves and produce high levels of protein expression in the host cells. This feature confers several advantages to saRNA, such as low dose requirement, prolonged duration of action, and enhanced immunogenicity. However, saRNA delivery also faces significant challenges and limitations, mainly due to the inherent instability and vulnerability of RNA molecules in the biological environment. saRNA molecules are susceptible to degradation by nucleases, clearance by the reticuloendothelial system, and recognition by the innate immune system, resulting in reduced bioavailability, distribution, and efficacy. Therefore, the development of effective delivery systems that can protect, transport, and deliver saRNA molecules to the target cells and tissues is essential for the successful application of saRNA technology.

Delivery Systems for saRNA

The delivery of saRNA molecules to the target cells and tissues is a critical challenge for the successful application of saRNA technology. Due to the inherent instability and vulnerability of RNA molecules in the biological environment, saRNA molecules require effective delivery systems that can protect them from degradation, clearance, and immune recognition, as well as facilitate their transport, uptake, and release into the cytoplasm. In the last three decades, a broad range of non-viral delivery systems for RNA have been investigated, including lipid nanoparticles, polymeric nanoparticles, cationic nanoemulsions, and electroporation.

Fig.1 Non-viral saRNA delivery systems.¹

Lipid nanoparticles (LNPs) are one of the most widely used and successful delivery systems for saRNA. LNPs are composed of a lipid bilayer that encapsulates the saRNA molecules and typically contain four types of lipids: an ionizable lipid that interacts with the negatively charged saRNA and facilitates endosomal escape, a helper lipid that stabilizes the bilayer, a cholesterol that enhances the rigidity and stability of the LNP, and a polyethylene glycol (PEG) lipid that prolongs the circulation time and reduces the immunogenicity of the LNP. LNPs have several advantages as saRNA delivery systems, such as high encapsulation efficiency, protection from nuclease degradation, scalability, and versatility. LNPs can also be modified with targeting ligands or antibodies to enhance the specificity and uptake of saRNA by the target cells.

Polymeric nanoparticles (PNPs) are another type of delivery system for saRNA that are composed of biodegradable and biocompatible polymers that form complexes with saRNA molecules. PNPs can be classified into two categories: cationic PNPs and pH-responsive PNPs. Cationic PNPs are formed by electrostatic interactions between positively charged polymers, such as polyethylenimine (PEI), chitosan, or poly(lactic-co-glycolic acid) (PLGA), and negatively charged saRNA molecules. Cationic PNPs can protect saRNA from degradation and enhance their cellular uptake, but they may also cause cytotoxicity and immunogenicity due to their high charge density and non-specific interactions with cells and tissues. pH-responsive PNPs are formed by polymers that undergo conformational changes or degradation in response to the acidic pH of the endosomes. pH-responsive PNPs can facilitate the endosomal escape and release of saRNA into the cytoplasm, but they may also have low stability and complex synthesis.

Cationic nanoemulsions (CNEs) are composed of oil-in-water emulsions that contain cationic surfactants that interact with saRNA molecules. CNEs can protect saRNA from degradation and enhance their cellular uptake, but they may also cause cytotoxicity and immunogenicity due to their high charge density and non-specific interactions with cells and tissues. CNEs have some advantages over LNPs and PNPs, such as lower cost, simpler synthesis, and higher stability.

Electroporation is a physical method of saRNA delivery that involves the application of an electric field to the target cells or tissues, creating transient pores in the cell membrane that allow the entry of saRNA molecules. Electroporation can achieve high transfection efficiency and protein expression, but it may also cause damage to the cells or tissues, pain, inflammation, and scarring. Electroporation has some advantages over other delivery systems, such as simplicity, versatility, and scalability.

Table 1. Comparison of Different Delivery Systems for saRNA

Cellular Mechanisms of saRNA Nanoparticle Uptake and Entry

The cellular uptake and entry of saRNA nanoparticles are crucial steps for the successful delivery and expression of saRNA molecules in the target cells. The cellular uptake and entry of saRNA nanoparticles involve various molecular and cellular processes, such as endocytosis, endosomal escape, and cytoplasmic release, which are influenced by the physicochemical properties of the nanoparticles, such as size, shape, charge, surface modification, and composition, as well as the cell type and physiological conditions.

The cellular uptake of saRNA nanoparticles is mainly mediated by endocytosis, which is a process of internalization of extracellular materials into membrane-bound vesicles called endosomes. There are different types of endocytosis, such as clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, and others, which differ in their mechanisms, kinetics, and specificity. The type of endocytosis that is involved in the uptake of saRNA nanoparticles depends on the characteristics of the nanoparticles and the target cells. For example, LNPs and PNPs are mainly taken up by clathrin-mediated endocytosis, while CNEs are mainly taken up by caveolae-mediated endocytosis. The endosomal escape of saRNA nanoparticles is a critical step for the delivery and expression of saRNA molecules in the cytoplasm. The endosomal escape of saRNA nanoparticles is facilitated by the interaction of the nanoparticles with the endosomal membrane, which can result in membrane destabilization, fusion, or rupture. The mechanism of endosomal escape of saRNA nanoparticles depends on the composition and properties of the nanoparticles and the delivery systems. For example, LNPs and PNPs use ionizable lipids and polymers, respectively, that can change their charge and conformation in response to the acidic pH of the endosomes, and thus disrupt the endosomal membrane and release the saRNA molecules into the cytoplasm. CNEs use cationic surfactants that can interact with the endosomal membrane and induce membrane fusion and release of the saRNA molecules into the cytoplasm. Electroporation uses an electric field that can create transient pores in the cell membrane and allow the direct entry of saRNA molecules into the cytoplasm.

The uptake and entry of saRNA nanoparticles can be influenced by various factors, such as the size, shape, charge, surface modification, and composition of the nanoparticles, as well as the cell type, density, cycle, and physiological conditions. These factors can affect the efficiency, specificity, and kinetics of the uptake and entry of saRNA nanoparticles, and thus the protein expression and therapeutic outcome of saRNA delivery. Therefore, it is important to study and quantify the uptake and entry of saRNA nanoparticles under different conditions and parameters. The methods and techniques for studying and quantifying the uptake and entry of saRNA nanoparticles include various imaging, flow cytometry, and PCR methods. Imaging methods, such as fluorescence microscopy, confocal microscopy, and electron microscopy, can provide qualitative and quantitative information on the localization, distribution, and morphology of saRNA nanoparticles and their interaction with cells and tissues. Flow cytometry methods, such as fluorescence-activated cell sorting (FACS), can provide quantitative information on the percentage, intensity, and kinetics of saRNA nanoparticle uptake by the cells. PCR methods, such as reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR), can provide quantitative information on the amount, amplification, and expression of saRNA molecules in the cells and tissues.

Routes of Administration and Biodistribution of saRNA Nanoparticles

The routes of administration and biodistribution of saRNA nanoparticles are important factors that affect the delivery and expression of saRNA molecules in the target cells and tissues. The routes of administration and biodistribution of saRNA nanoparticles depend on the characteristics of the nanoparticles, such as size, shape, charge, surface modification, and composition, as well as the delivery systems, such as LNPs, PNPs, CNEs, and electroporation.

Table 2. Different Routes of Administration of saRNA Nanoparticles

Intravenous (IV) administration is a common route of administration for saRNA nanoparticles that involves the injection of the nanoparticles directly into the bloodstream. IV administration can achieve rapid and systemic delivery of saRNA nanoparticles to the target cells and tissues, especially the liver, spleen, and bone marrow, which are rich in phagocytic cells that can take up the nanoparticles. IV administration can also avoid the degradation and clearance of saRNA nanoparticles by the gastrointestinal tract and the first-pass metabolism by the liver. However, IV administration also has some limitations and challenges, such as the potential toxicity and immunogenicity of saRNA nanoparticles in the blood, the rapid clearance and elimination of saRNA nanoparticles by the kidneys and the reticuloendothelial system, and the difficulty in targeting specific organs or tissues.

Intramuscular (IM) administration is another common route of administration for saRNA nanoparticles that involves the injection of the nanoparticles into the muscle tissue. IM administration can achieve local and sustained delivery of saRNA nanoparticles to the muscle cells, which can act as a depot and a factory for saRNA expression and protein secretion. IM administration can also induce strong immune responses by activating the antigen-presenting cells and the lymph nodes in the muscle tissue. However, IM administration also has some limitations and challenges, such as the low transfection efficiency and protein expression of saRNA nanoparticles in the muscle cells, the variability and pain of the injection site, and the difficulty in targeting other organs or tissues.

Intranasal (IN) administration is an emerging route of administration for saRNA nanoparticles that involves the delivery of the nanoparticles into the nasal cavity. IN administration can achieve direct and efficient delivery of saRNA nanoparticles to the respiratory tract, which is a major site of infection and inflammation for many respiratory diseases. IN administration can also induce mucosal and systemic immune responses by activating the nasal-associated lymphoid tissue and the draining lymph nodes. However, IN administration also has some limitations and challenges, such as the low retention and penetration of saRNA nanoparticles in the nasal mucosa, the degradation and clearance of saRNA nanoparticles by the nasal enzymes and cilia, and the potential toxicity and irritation of saRNA nanoparticles to the nasal epithelium.

Innate Immune Recognition and Response to saRNA Nanoparticles

The innate immune system is the first line of defense against foreign nucleic acids, such as saRNA. The innate immune system can recognize and respond to saRNA nanoparticles by various receptors and pathways, such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and the cGAS-STING pathway. The innate immune recognition and response to saRNA nanoparticles can have both positive and negative effects on the delivery and expression of saRNA molecules, as well as the therapeutic outcome of saRNA delivery.

The innate immune receptors and pathways for saRNA nanoparticles are mainly located in the endosomes and the cytoplasm, where saRNA nanoparticles are delivered and released. The endosomal receptors include TLR3, TLR7, and TLR8, which can sense double-stranded RNA (dsRNA) and single-stranded RNA (ssRNA), respectively. The cytoplasmic receptors include RLRs, such as RIG-I and MDA5, which can sense dsRNA and ssRNA with 5'-triphosphate, and cGAS, which can sense cytosolic DNA that may be generated from saRNA replication. These receptors can activate downstream signaling pathways, such as NF-κB, IRF3, and IRF7, which can induce the production of type I interferons (IFNs) and other pro-inflammatory cytokines. The innate immune recognition and response to saRNA nanoparticles can have both beneficial and detrimental effects on the delivery and expression of saRNA molecules, as well as the therapeutic outcome of saRNA delivery. On one hand, the innate immune recognition and response to saRNA nanoparticles can enhance the immunogenicity and efficacy of saRNA vaccines, by inducing the maturation and activation of antigen-presenting cells, such as dendritic cells and macrophages, and the priming and expansion of adaptive immune cells, such as B cells and T cells. On the other hand, the innate immune recognition and response to saRNA nanoparticles can also impair the stability and functionality of saRNA molecules, by inducing the degradation and clearance of saRNA by nucleases, such as RNase L and Dicer, and the inhibition and silencing of saRNA by microRNAs, such as miR-155 and miR-146. Moreover, the innate immune recognition and response to saRNA nanoparticles can also cause adverse effects, such as inflammation, toxicity, and autoimmunity, by inducing the production of excessive or inappropriate cytokines, such as TNF-α, IL-6, and IFN-α.

Reference

1. Blakney, Anna K., Shell Ip, and Andrew J. Geall. "An update on self-amplifying mRNA vaccine development." Vaccines 9.2 (2021): 97.