siRNA Delivery Methods

Small interfering RNA (siRNA) is the initiator of RNA interference, which was first discovered in mammalian cells in 1998, and the Fire team delivered long fragments of dsRNA to C. elegans, and the results showed that dsRNA could effectively silence target gene expression. Stimulation of target mRNA silencing is of great significance for gene regulation and disease treatment. However, the efficient delivery of siRNAs to target cells remains a major challenge. This article discusses the main design strategies of siRNA delivery methods, and describes their mechanisms, benefits, and applications.

What is siRNA Delivery?

Small interfering RNAs (siRNAs), also known as silencing RNAs, short interfering RNAs, or non-coding RNAs. siRNA is a small fragment of RNA with a specific length and sequence generated by cleaving double-stranded RNA (dsRNA) expressed by foreign invasive genes, and the specific length of siRNA is 21~25 bp. siRNA delivery refers to the process of directing small interfering RNA (siRNA) into the target cell so that it can perform its function inside the cell.

Common siRNA delivery methods

siRNA has a short half-life, poor plasma stability, and is easily degraded by nucleases, and the siRNA is not easily negatively charged to enter tumor cells through the negatively charged cell membrane and is easily degraded by intracellular lysosomes. Therefore, it is difficult for siRNA to be directly used as a drug for disease treatment, and certain means need to be taken to achieve its effective delivery in living organisms and achieve the purpose of treating diseases.

Lipid-based delivery system

Lipid nanoparticles (LNPs)

Lipid nanoparticles (LNPs) are one of the most well-established siRNA delivery methods and have excellent biocompatibility and stability. LNPs encapsulate siRNAs to form nanoparticles, protecting the siRNA from degradation and facilitating its entry into target cells. Recent studies have shown that siRNA delivery efficiency and gene silencing can be significantly improved by optimizing lipid composition and regulating the size of nanoparticles. LNP-mediated delivery was one of the first methods to demonstrate effective gene silencing in humans and was the basis for the first approved siRNA drug, Patisiran.

Fig.1 Delivery mechanism of LNPs. (Suzuki Yuta, et al., 2021)

Table 1. siRNA conjugation service at BOC Sciences.

Cationic lipids

It is suitable for uncharged siRNAs, which adsorb to the surface of liposomes through electrostatic interactions, increasing uptake in cells. Utilizing the ability of polycationic lipids to electrostatically bind siRNAs to form nano-sized complexes, thereby enhancing cellular absorption.

Glucide-based delivery systems

GalNAc conjugates

N-acetylgalactosamine (GalNAc) conjugation is currently the most commonly used small nucleic acid drug delivery system. It is used to improve the specific distribution of siRNA in the liver and is suitable for RNA interference therapy. For instance, Givlaari (givosiran), the world's second RNAi drug approved by the FDA in 2019, is also the world's first approval of GalNAc-conjugated RNA therapy for the treatment of adult patients with acute hepatic porphyria (AHP).

Fig.2 Mechanism of action of GalNAc-conjugated siRNAs. (An Guohua., 2024)

Polymer-based delivery systems

Polymer nanoparticles

Polymeric nanoparticles are another common siRNA delivery vehicle. Molecular-defined nanostructures can be designed to the appropriate shape and size to bind siRNA strands with a high degree of programmability. The researchers have developed siRNA delivery systems based on clinically approved mPEG-b-PLGA, cationic lipids, and ionizable lipids. By adjusting the mass ratios of these components, the formation of nanoparticles is optimized, resulting in efficient gene silencing.

Other delivery systems

Antibody-mediated delivery systems

Antibody-mediated siRNA delivery systems take advantage of the specific binding capacity of antibodies to target the delivery of siRNAs to specific cell types. The antibody-siRNA binding platform's state-of-the-art clinical program uses monoclonal antibodies targeting transferrin receptor 1 (TfR1) for bone, heart, and smooth muscle delivery. A Phase I/II study of amyotrophic myelitis with anti-TfR1-siRNA-targeted myotonin-protein kinase (DMPK) in human patients demonstrated significant improvements in disease outcomes.

Peptide conjugates

Peptide conjugates are a promising siRNA delivery pathway due to their ability to simplify the development process through chemical synthesis. For example, the use of glucagon-like peptide 1 (GLP1) as a ligand for the GLP1 receptor has been successful in delivering antisense oligonucleotides (ASOs) into pancreatic islet cells. Although information on peptide conjugates for extrahepatic siRNA delivery is limited in the public domain, many academic institutions and pharmaceutical companies are actively investigating focused and unbiased selection strategies to further explore the potential of peptide conjugates.

RNA aptamer-mediated delivery

RNA aptamers are an emerging siRNA delivery method with the advantages of low immunogenicity and ease of production. Aptamers can form complexes with siRNAs to enable efficient siRNA delivery by specifically binding to receptors on the surface of target cells. For example, researchers use aptamers targeting the ERBB2 receptor to deliver siRNAs into cells expressing the receptor to achieve specific gene silencing.

siRNA delivery materials

The materials for siRNA delivery mainly include viral and non-viral vectors. Each material has its own unique advantages and disadvantages, and choosing the right delivery material depends on the specific application and cell type of interest.

Viral vectors

Adenovirus: It has a highly efficient gene transduction ability and can infect a wide range of cell types, but may cause an immune response, leading to toxicity and inflammation.

Lentivirus: It can be integrated into the host genome and provide a long-term gene silencing effect, but the production process is complex and there is a potential risk of gene integration.

Adeno-associated Virus (AAV): It has low immunogenicity and high safety profile, but has limited packaging capacity and is not suitable for the delivery of large gene fragments.

Non-viral vectors

Liposomes: These materials can effectively encapsulate and protect siRNA and promote cellular uptake, but may cause cytotoxicity and immune responses. Such as 1,2-dioleoyl-3-trimethylammonium chloride-propenyl ester (DOTAP), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

Polymers: These materials have high gene transduction performance and protect siRNA from degradation, but may cause cytotoxicity at high concentrations. Such as PLGA, polyethylene glycol (PEG) modified polyethylene imide (PEI).

Nanoparticles: The functionalization of nanoparticles is easy and binds siRNA stably, but can cause cytotoxicity and inflammation. Such as PLGA nanoparticles, gold nanoparticles, and magnetic nanoparticles (such as iron oxide nanoparticles).

Direction of application

Due to the effectiveness and selectivity of RNA interference, it has become the method of choice for silencing specific gene expression in mammalian cells. Silencing therapy with specific RNA interference has a wide range of applications in viral infections, cancers, familial genetic diseases, and autoimmune diseases. Since the Nobel Prize was awarded in 2006 for research on RNA interference, Big Pharma has poured billions of dollars into the development of human gene silencing therapies. Since then, siRNA research has entered a new field and begun a stage of rapid development.

Genetic disorders

For some genetic diseases, siRNA delivery systems can correct gene dysfunction by silencing mutant genes. For example, the use of fusion peptide-mediated siRNA nanocomplexes can efficiently deliver siRNAs that silence disease-causing genes and thus alleviate disease symptoms. For example, in 2018, the FDA approved Patisiran (Onpattro, also known as ALN-TTR02), the first RNA interference-based drug developed by Alnylam Pharmaceuticals in the United States, which brings new hope to patients with hereditary transthyretin amyloidosis with unmet medical needs.

Cancer treatment

siRNA delivery systems have shown great potential in cancer therapy. By delivering siRNAs in a targeted manner, genes associated with tumor growth and metastasis can be specifically silenced, thereby inhibiting tumor progression. For example, researchers at Vanderbilt University's Hoogenboezem team achieved in situ binding to albumin by optimizing bivalent lipid-coupled siRNAs. This structure shows significant knockout and in vivo effects of tumor growth inhibition by silencing the oncogene MCL1.

Viral infections

siRNA delivery systems are also showing potential in antiviral therapeutics. By targeting viral genes, viral replication can be effectively inhibited and infection mitigated. For example, using an antibody-mediated siRNA delivery system, researchers successfully inhibited viral gene expression in HIV-infected cells.

Summary

The continuous innovation and optimization of siRNA delivery methods have opened up new prospects for its clinical application. By combining different nanocarriers and delivery mechanisms, researchers have made significant progress. In the future, with the further optimization of delivery systems and the development of new technologies, siRNAs are expected to play an important role in the treatment of more diseases.