Advances in Non-Viral Vector Delivery Systems for Nucleic Acid Drugs

Nucleic acid drugs are mainly divided into two major categories: small nucleic acid drugs and mRNA. Small nucleic acid drugs, also known as oligonucleotide drugs, include antisense nucleotides (ASO), small interfering RNA (siRNA), microRNA, nucleic acid aptamers, and others. mRNA products can be further categorized into mRNA vaccines and mRNA drugs.

Characteristics of Nucleic Acid Drugs

Nucleic acid drugs offer distinct advantages. Traditional small molecule and antibody drugs primarily function by binding to target proteins, but their development is often constrained by the druggability of these target proteins. In contrast, nucleic acid drugs modulate genes associated with protein expression, enabling them to regulate both intracellular and extracellular proteins, as well as membrane-bound proteins. Moreover, most nucleic acid drugs operate on the principle of base-pairing complementarity, making sequence design relatively straightforward once the target gene's base sequence is known.

However, limitations such as instability, immunogenicity, low cellular uptake efficiency, and difficulties in escaping endocytic pathways have restricted the development of nucleic acid drugs. To address the challenges associated with the molecules themselves, the development of delivery carrier systems that facilitate the uptake of nucleic acids into target cells has become crucial. These delivery carriers need to overcome barriers both extracellularly and intracellularly, withstand nucleases in the bloodstream, enhance and assist in cellular uptake of nucleic acid drugs, and promote intracellular escape of the nucleic acid drugs once inside the cell.

Nucleic Acid Drug Delivery System

In the early stages of research, viruses were commonly used as carriers for delivering nucleic acids. Virus carriers utilized in clinical trials included adenovirus (Ad), adeno-associated virus (AAV), lentivirus (LV), herpes simplex virus (HSV), and others. However, some virus carriers exhibited undesirable characteristics, such as potential carcinogenicity and high immunogenicity, which led to serious clinical adverse events and hindered the research on their clinical applications. With the development of materials and preparation techniques, non-viral carriers that are cost-effective, easily synthesized, purified, and possess high transfection efficiency with low immunogenicity have emerged as the "optimal candidates" for delivering nucleic acid drugs.

1. GalNac Modification

GalNac (N-acetylgalactosamine) conjugate modification is the most commonly used nucleic acid drug delivery system today. GalNac is a lactose analog covalently attached to the 3' end of nucleic acids in a trivalent form.

Following subcutaneous injection of GalNac-siRNA conjugates, they can rapidly enter the liver through the circulatory system. Subsequently, they are rapidly internalized by liver cells through ASPGR receptor-mediated uptake, accumulate in lysosomes, and are slowly released, continuously loading onto the RNA-induced silencing complex (RISC), thus achieving long-lasting inhibitory effects.

Currently, drugs modified with GalNAc primarily include GalNAc-antisense oligonucleotides (ASO) and GalNAc-siRNA. Since effective ASOs for therapy have already undergone extensive modifications, a delivery carrier is not necessary. siRNAs, on the other hand, are prone to degradation on their own, so carrier delivery technologies are often employed.

1. Nanoparticles

(1) Lipid Nanoparticle

Lipid Nanoparticle (LNP), is one of the important technologies in lipid carrier drug delivery system. The main components are categorized into the following four types:

• Ionic lipids that can be ionized are the most crucial excipients and serve as determinants of delivery and transfection efficiency. Due to their relatively easy uptake by antigen-presenting cells, they are commonly used in vaccines.
• Neutral helper lipids, typically saturated lipids, can increase the phase transition temperature of cationic liposomes, support the formation of a lamellar lipid bilayer structure, and stabilize its structural arrangement.
• Cholesterol, possessing strong membrane fusion capabilities, facilitates intracellular uptake and cytoplasmic entry of mRNA.
• PEGylated lipids, located on the surface of lipid nanoparticles, enhance their hydrophilicity, prevent rapid clearance by the immune system, prevent particle aggregation, and increase stability.

(2) Polymer Nanoparticles

Cationic polymers have become another major type of non-viral gene delivery vector due to their ease of synthesis and flexibility. Polymers can bind with nucleic acids to form polycomplexes at physiological pH, facilitating gene delivery. Typically, polymer nanoparticles contain positively charged units that promote electrostatic binding with nucleic acids. Additionally, covalent linkage between nucleic acids and polymers can be achieved using degradable linkers. Common polymer materials include polyethylenimine (PEI) and chitosan (CS).

Another class of polymers used for RNA delivery is dendrimers. These large molecules have a core molecule at their center and are synthesized through repeated growth reactions, resulting in highly branched polymers. Dendrimers carrying cationic groups can form complexes with RNA. Research has shown that they can deliver RNA to the central nervous system and siRNA to liver endothelial cells. Modifying the structure of dendrimers can protect nucleotides from enzymatic degradation.

siG12D-LODER is a biodegradable polymer matrix containing siRNA targeting KRASG12D. Currently, Novartis is conducting Phase II clinical trials for siG12D-LODER to assess its effectiveness in combination with chemotherapy drugs like gemcitabine and paclitaxel in treating locally advanced pancreatic cancer patients.

(3) Inorganic Nanoparticles

• Gold nanoparticles have unique optical properties, ease of synthesis and surface functionalization, and can be selectively and synergistically modified with nucleic acids through covalent or non-covalent affixation. Nucleic acid chains are covalently attached to the gold nanoparticle core (typically 13-15 nm) via thiol groups. This strategy can be used for DNA and siRNA, which can be directly attached to gold cores or polymer-modified gold cores.
• Spherical nucleic acids (SNA) are composed of nucleic acids arranged on the surface of small spherical gold nanoparticles. The platform drug, NU-0129, is currently being studied in a clinical phase 1 study in recurrent glioma.Once NU-0129 crosses the blood-brain barrier and enters the tumor, the nucleic acid component is able to target a gene called Bcl2L12. The researchers believe that targeting the Bcl2L12 gene with NU-0129 will help stop the growth of gliomas.
• Silica nanoparticles (100-250 nm in diameter), are used for nucleic acid delivery due to their good biocompatibility and tunability. Typically, nucleic acid molecules are loaded into silica nanoparticles through weak non-covalent interactions. Small pore (2.5-5 nm) silica nanoparticles are suitable for delivery of small siRNA.
• Iron oxide nanoparticles (consisting of Fe₃O₄or Fe₂O₃) have superparamagnetic properties of a certain size and show success as delivery carriers and magnetothermal based therapies. Cationic iron oxide nanoparticles and anionic nucleic acid drugs, bind to each other by electrostatic forces. 50-100 nm lipid-coated iron oxide nanoparticles show optimal siRNA delivery activity.

1. New Delivery Systems

(1) Exosomes

Extracellular vesicles, derived from endosomes and released into the extracellular space through multivesicular body fusion with the cell membrane, are membrane-bound vesicular structures with diameters ranging between 50 and 150 nm. These extracellular vesicles contain various biological macromolecules, including proteins, nucleic acids, and lipids. Extracellular vesicles can transfer a variety of biological macromolecules between cells, making them a drug delivery method with significant inherent advantages.

• Firstly, extracellular vesicles are "naturally tamed" nanocarriers that inherently contain multiple active components, allowing them to carry a wide range of drug types, including small molecules, nucleic acids, and recombinant proteins.
• Secondly, as endogenous nanoparticles, extracellular vesicles have low immunogenicity, resulting in high safety.
• Thirdly, extracellular vesicles can circulate through all the body's cavities, exhibiting good tissue selectivity.
• Finally, complex engineering modifications can be applied to extracellular vesicles through genetic or chemical approaches, allowing precise control over their composition and biological functions, thus better serving our therapeutic purposes.

(2) Peptides

The current technologies used in nucleic acid drug delivery that are on the market today do not effectively address the issue of tissue-specific targeting. Peptides offer solutions to many problems that other delivery systems cannot resolve. Chemical modifications of non-natural amino acids have greatly improved the half-life of peptide drugs in the body. The development of cyclization techniques has increased the rigidity of peptide structures, significantly enhancing their affinity for target proteins. Peptide-drug conjugates (PDCs) exhibit strong tumor penetration, low immunogenicity, and renal metabolism, among other characteristics.

Currently, companies like Ionis, Alnylam, Entrada Therapeutics, and others are actively establishing platforms for peptide-nucleic acid conjugate drugs. The future looks promising for peptide-nucleic acid conjugates.

(3) Others

Renowned scientist Zhang Feng's company, Aera Therapeutics, has introduced a novel delivery platform called Protein Nanoparticles (PNP), which utilizes endogenous human proteins to address the limitations of current delivery technologies.

Altamira Therapeutics, a biotechnology company focused on RNA therapy, has announced the development of an innovative peptide-based SemaPhore^TM nanoparticle technology platform. This delivery platform is designed for safe and effective systemic or local administration of oligonucleotides such as siRNA and mRNA to target cells. Currently, the company has established two preclinical siRNA projects on this platform for the treatment of KRAS-driven cancers and rheumatoid arthritis.